S. cerevisiae

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 452 - 5
{Studies on synthesis of glutathione by E . coli BL21 (pTrc-gsh) coupled with Saccharomyces cerevisiae}; Shen LX et al.; During synthesis of GSH by the engineered strain E . coli BL21(pTrc-gsh) coupled with Saccharomyces cerevisiae producing ATP from adenosin, the inconsistency of two systems in the concentration of phosphate buffer was solved by decreasing concentration to 250 mmol/L . The conditions under 250 mmol/L phosphate buffer were optimized and the yield of GSH was 1.6 g/L, which was higher than that of summation by two systems under the same conditions respectively . Addition of glycine later after glutamate and cysteine weakened the inhibition of GSH to GSHI . It made the yield of GSH reach to 2.13 g/L which was 30.7% higher than the control.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 410 - 3
{Expression and characterization of human pulmonary surfactant-associated protein A1 in Saccharomyces cerevisiae}; Lan HK et al.; The cDNA encoding pulmonary surfactant-associated protein A1 (SP-A1) derived from healthy adult's lung was cloned into the pVT102U/alpha, expression vector of Saccharomyces cerevisiae, which contains the yeast alpha-factor signal sequence, leading to the secretion of expressed protein, and then transformed into Saccharomyces cerevisiae S-78 (leu2, ura3, rep4) by electroporation . After 2-3 days culture in adequate pH, the expressed SP-A1 accumulated up to 400 mg/L in supernatant . The pure proteins were obtained by Sephadex G-25, G-75, Sepharose 4B . The expressed recombinant products, 62 kD and 32 kD, reacted to specific antibody using ELISA and Western blot . The SP-A1 protein expressed in Saccharomyces cerevisiae was efficient in enhancing the phagocytosis of E . coli J5 by alveolar macrophages.

Science, 2001 Dec 21, 294(5551), 2552 - 6 Epub 2001 Nov 08.
A DNA microarray-based genetic screen for nonhomologous end-joining mutants in Saccharomyces cerevisiae; Ooi SL et al.; We describe a microarray-based screen performed by imposing different genetic selections on thousands of yeast mutants in parallel, representing most genes in the yeast genome . The presence or absence of mutants was detected by oligonucleotide arrays that hybridize to 20-nucleotide "barcodes." We used this method to screen for components of the nonhomologous end-joining (NHEJ) pathway . Known components of the pathway were identified, as well as a gene not previously known to be involved in NHEJ, NEJ1 . Nej1 protein interacts with the amino terminus of LIF1/XRCC4, a recently recognized "guardian of the genome" against cancer.

Anal Biochem, 2001 Nov 15, 298(2), 283 - 92
High-performance liquid chromatography analysis of molecular species of sphingolipid-related long chain bases and long chain base phosphates in Saccharomyces cerevisiae after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; Lester RL et al.; The molecular species of dihydrosphingosines and phytosphingosines and their 1-phosphates with carbon chain lengths from 16 to 20 have been tagged with the fluorescent amino group reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate . All these derivatives could be resolved by reversed phase HPLC on a C18 column . A convenient one-pot method is described whereby lipid extracts from strains of Saccharomyces cerevisiae containing carbon chain length homologs of sphingolipid long chain bases and their phosphorylated derivatives were directly reacted with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, ester lipids were deacylated, and the reaction mixtures were subjected to liquid chromatography . Five molecular species of both sphingolipid long chain bases and their phosphorylated derivatives are for the first time separated and analyzed . The procedure is quite sensitive, requiring only approximately 10(8) wild-type cells .

BMC Mol Biol . 2001;2(1):11 . Epub 2001 Oct 08.
Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae; Maiti AK et al.; BACKGROUND: Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes . These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes . RESULTS: A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed . The sequence was inserted within a nucleosome positioned upstream of the beta-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10 Upstream Activating Sequences (UASg) . Upon derepression with galactose, beta-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences . This reduction in expression is correlated with reduced transcription . Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells . CONCLUSION: We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription . Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells.

Biochemistry, 2001 Nov 13, 40(45), 13699 - 709
Identification of the gene and characterization of the activity of the trans-aconitate methyltransferase from Saccharomyces cerevisiae; Cai H et al.; We have identified the yeast open reading frame YER175c as the gene encoding the trans-aconitate methyltransferase of Saccharomyces cerevisiae . Extracts of a yeast strain with a disrupted YER175c gene demonstrate a complete loss of activity toward the methyl-accepting substrates trans-aconitate, cis-aconitate, DL-isocitrate, and citrate . Reintroduction of the YER175c gene on a plasmid results in an overexpression of the activity toward each of these methyl-accepting substrates . We now designate this gene TMT1 for trans-aconitate methyltransferase . We examined the methyl-accepting substrate specificity of this enzyme in extracts from overproducing cells . We found that trans-aconitate was the best substrate with a Km of 0.66 mM . Other substrates were recognized much more poorly, including cis-aconitate with a Km of 74 mM and the decarboxylation product itaconate with a Km of 44 mM . The ratio of the maximal velocity to the Km of these substrates was only 0.24% and 0.9% that of trans-aconitate; for other substrates including citrate and other tricarboxylate and dicarboxylate derivatives, this ratio ranged from 0.0003% to 0.062% that of trans-aconitate . We then asked if any of these compounds were present endogenously in yeast extracts . We were able to identify trans-aconitate 5-methyl ester as well as additional unidentified radiolabeled products when S-adenosyl-L-{methyl-3H}methionine was mixed with TMT1+ extracts (but not with tmt1- extracts), suggesting that there may be additional substrates for this enzyme . We showed that the product 5-methyl ester of trans-aconitate is not readily metabolized in yeast extracts . Finally, we demonstrated that the activity of the yeast trans-aconitate methyltransferase is localized in the cytosol and increases markedly as cells undergo the metabolic transition at the diauxic shift.

Nat Genet, 2001 Dec, 29(4), 482 - 6
Correlation between transcriptome and interactome mapping data from Saccharomyces cerevisiae; Ge H et al.; Genomic and proteomic approaches can provide hypotheses concerning function for the large number of genes predicted from genome sequences . Because of the artificial nature of the assays, however, the information from these high-throughput approaches should be considered with caution . Although it is possible that more meaningful hypotheses could be formulated by integrating the data from various functional genomic and proteomic projects, it has yet to be seen to what extent the data can be correlated and how such integration can be achieved . We developed a 'transcriptome-interactome correlation mapping' strategy to compare the interactions between proteins encoded by genes that belong to common expression-profiling clusters with those between proteins encoded by genes that belong to different clusters . Using this strategy with currently available data sets for Saccharomyces cerevisiae, we provide the first global evidence that genes with similar expression profiles are more likely to encode interacting proteins . We show how this correlation between transcriptome and interactome data can be used to improve the quality of hypotheses based on the information from both approaches . The strategy described here may help to integrate other functional genomic and proteomic data, both in yeast and in higher organisms.

Mol Biol Cell, 2001 Nov, 12(11), 3428 - 38
The target of rapamycin signaling pathway regulates mRNA turnover in the yeast Saccharomyces cerevisiae; Albig AR et al.; The target of rapamycin (TOR) signaling pathway is an important mechanism by which cell growth is regulated by nutrient availability in eukaryotes . We provide evidence that the TOR signaling pathway controls mRNA turnover in Saccharomyces cerevisiae . During nutrient limitation (diauxic shift) or after treatment with rapamycin (a specific inhibitor of TOR), multiple mRNAs were destabilized, whereas the decay of other mRNAs was unaffected . Our findings suggest that the regulation of mRNA decay by the TOR pathway may play a significant role in controlling gene expression in response to nutrient depletion . The inhibition of the TOR pathway accelerated the major mRNA decay mechanism in yeast, the deadenylation-dependent decapping pathway . Of the destabilized mRNAs, two different responses to rapamycin were observed . Some mRNAs were destabilized rapidly, while others were affected only after prolonged exposure . Our data suggest that the mRNAs that respond rapidly are destabilized because they have short poly(A) tails prematurely either as a result of rapid deadenylation or reduced polyadenylation . In contrast, the mRNAs that respond slowly are destabilized by rapid decapping . In summary, the control of mRNA turnover by the TOR pathway is complex in that it specifically regulates the decay of some mRNAs and not others and that it appears to control decay by multiple mechanisms.

Mol Biol Cell, 2001 Nov, 12(11), 3402 - 16
Cell cycle-dependent degradation of the Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdh1) and a bipartite destruction sequence; Hildebrandt ER et al.; Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly . Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase . Cin8p degradation required the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p . Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates . We carried out an extensive mutagenesis study to define the cis-acting sequence required for Cin8p degradation in vivo . The C terminus of Cin8p contains two elements required for its degradation: 1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and 2) a nuclear localization signal . The bipartite destruction sequence appears in other BimC kinesins as well . Expression of nondegradable Cin8p showed very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles.

Pharmacogenetics, 2001 Nov, 11(8), 709 - 18
Functional evaluation of cytochrome P450 2D6 with Gly42Arg substitution expressed in Saccharomyces cerevisiae; Tsuzuki D et al.; A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms . The protein levels of G42R, P34S and P34S/S486T in microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices . The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum . In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation . The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution . These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution.

Curr Genet, 2001 Sep, 40(2), 91 - 109
Sensors of extracellular nutrients in Saccharomyces cerevisiae; Forsberg H et al.; It has been known for a long time that yeast are capable of making rapid metabolic adjustments in response to changing extracellular nutrient conditions . Until recently it was thought that yeast, in contrast to mammalian cells, primarily monitored nutrient availability through the activity of intracellular sensors . Recent advances in our understanding of nutrient sensing indicate that yeast cells possess several nutrient-sensing systems localized in the plasma membrane that transduce information regarding the presence of extracellular amino acids, ammonium . and glucose . Strikingly, the transmembrane components of several of these sensors, Ssylp, Mep2p, Snf3p . and Rgt2p, are unique members of nutrient-transport protein families . Perhaps with the exception of Mep2p, the ability of these transporter homologues to transduce nutrient-(ligand)-induced signals across the plasma membrane appears to be independent of nutrient uptake; and thus these sensor components may function analogously to traditional ligand-dependent receptors . Additionally, the G protein-coupled receptor Gpr1p has been shown to exhibit properties consistent with it being a sensor . These recent advances indicate that yeast cells obtain information regarding their growth environments using sensing systems that are more similar to those present in mammalian cells than previously thought . The fact that yeast plasma membrane nutrient sensors have only recently been discovered reveals how little is understood regarding the molecular signals that enable eukaryotic cells to adapt to changing environments.

Mol Aspects Med, 2001 Aug-Oct, 22(4-5), 217 - 46
Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases; Costa V et al.; In yeast, as in higher eukaryotes, reactive oxygen species are produced as normal by-products of cellular metabolism . Under physiological conditions, the cell defence mechanisms are able to avoid molecular damages . This balance is disturbed when yeast cells are exposed to diverse environmental stress conditions, such as the presence of oxidants, heat shock, ethanol and metal ions . The increased production of reactive oxygen species is sensed by the cell, leading to the induction of defence mechanisms - the oxidative stress response . The present review discusses the mechanisms by which reactive oxygen species are sensed and the signalling pathways that are coupled with changes in genomic expression programs . Yeast has been used as an eukaryotic cell system to characterise the molecular mechanisms underlying the oxidative stress response . Furthermore, yeast has been utilised to elucidate the role of oxidative stress in ageing, apoptosis, and diseases, such as familial amyotrophic lateral sclerosis and Friedreich's ataxia.

Gene, 2001 Aug 22, 274(1-2), 151 - 6
A genomic screen identifies AUT8 as a novel gene essential for autophagy in the yeast Saccharomyces cerevisiae; Barth H et al.; Autophagy is a starvation-induced transport pathway delivering parts of the cytosol into the lysosome (vacuole) for degradation . Autophagy significantly differs from other transport pathways by using double membrane layered transport intermediates . Based on the identification of autophagy genes in Saccharomyces cerevisiae, which served as a pacemaker for higher cells, our mechanistic knowledge of autophagy notably increased over the past few years . We here identify AUT8 as a novel gene essential for autophagy by screening a collection of approximately 5000 yeast deletion strains, each containing a defined deletion in an individual gene . This collection is a result of the world-wide Saccharomyces deletion project and covers the non-essential genes of the whole yeast genome . Homozygous aut8 Delta cells are impaired in maturation of proaminopeptidase I, and they fail to undergo the cell differentiation process of sporulation . The essential function of AUT8 for autophagy is further demonstrated by the lack of accumulation of autophagic vesicles in the vacuoles of aut8 Delta cells starved of nitrogen in the presence of the proteinase B inhibitor phenylmethylsulfonyl fluoride.

Gene, 2001 Aug 22, 274(1-2), 15 - 25
Nonsense-mediated mRNA decay in Saccharomyces cerevisiae; Gonzalez CI et al.; Cell survival depends on the precise and correct production of polypeptides . Eukaryotic cells have evolved conserved proofreading mechanisms to get rid of incomplete and potentially deleterious proteins . The nonsense-mediated mRNA decay (NMD) pathway is an example of a surveillance mechanism that monitors premature translation termination and promotes degradation of aberrant transcripts that code for nonfunctional or even harmful proteins . In this review we will describe our current knowledge of the NMD pathway, analyzing primarily the results obtained from the yeast Saccharomyces cerevisiae, but establishing functional comparisons with those obtained in higher eukaryotes . Based on these observations, we present two related working models to explain how this surveillance pathway recognizes and selectively degrades aberrant mRNAs.

J Biol Chem, 2001 Dec 28, 276(52), 49244 - 50 Epub 2001 Oct 22.
Three cell wall mannoproteins facilitate the uptake of iron in Saccharomyces cerevisiae; Protchenko O et al.; Analysis of iron-regulated gene expression in Saccharomyces cerevisiae using cDNA microarrays has identified three putative cell wall proteins that are directly regulated by Aft1p, the major iron-dependent transcription factor in yeast . FIT1, FIT2, and FIT3 (for facilitator of iron transport) were more highly expressed in strains grown in low concentrations of iron and in strains in which AFT1-1(up), a constitutively active allele of AFT1, was expressed . Northern blot analysis confirmed that FIT1, FIT2, and FIT3 mRNA transcript levels were increased 60-230-fold in response to iron deprivation in an Aft1p-dependent manner . Fit1p was localized exclusively to the cell wall by indirect immunofluorescence . Deletion of the FIT genes, individually or in combination, resulted in diminished uptake of iron bound to the siderophores ferrioxamine B and ferrichrome, without diminishing the uptake of ferric iron salts, or the siderophores triacetylfusarinine C and enterobactin . FIT-deletion strains exhibited increased expression of Aft1p target genes as measured by a FET3-lacZ reporter gene or by Arn1p Western blotting, indicating that cells respond to the absence of FIT genes by up-regulating systems of iron uptake . Aft1p activation in FIT-deleted strains occurred when either ferrichrome or ferric salts were used as sources of iron during growth, suggesting that the FIT genes enhance uptake of iron from both sources . Enzymatic digestion of the cell wall resulted in the release of significant amounts of iron from cells, and the relative quantity of iron released was reduced in FIT-deletion strains . Fit1p, Fit2p, and Fit3p may function by increasing the amount of iron associated with the cell wall and periplasmic space.

Biochem J, 2001 Nov 1, 359(Pt 3), 631 - 7
gamma-Glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione; Mehdi K et al.; In the yeast Saccharomyces cerevisiae, the enzyme gamma-glutamyl transpeptidase (gamma-GT; EC 2.3.2.2) is a glycoprotein that is bound to the vacuolar membrane . The kinetic parameters of GSH transport into isolated vacuoles were measured using intact vacuoles isolated from the wild-type yeast strain Sigma 1278b, under conditions of gamma-GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions) . Vacuoles devoid of gamma-GT displayed a K(m) (app) of 18+/-2 mM and a V(max) (app) of 48.5+/-5 nmol of GSH/min per mg of protein . Vacuoles containing gamma-GT displayed practically the same K(m), but a higher V(max) (app) (150+/-12 nmol of GSH/min per mg of protein) . Vacuoles prepared from a disruptant lacking gamma-GT showed no increase in V(max) (app) with nitrogen starvation . From a comparison of the transport data obtained for vacuoles isolated from various reference and mutant strains, it appears that the yeast cadmium factor 1 (YCF1) transport system accounts for approx . 70% of the GSH transport capacity of the vacuoles, the remaining 30% being due to a vacuolar (H(+)) ATPase-coupled system . The V(max) (app)-increasing effect of gamma-GT concerns only the YCF1 system . gamma-GT in the vacuolar membrane activates the Ycf1p transporter, either directly or indirectly . Moreover, GSH accumulating in the vacuolar space may exert a feedback effect on its own entry . Excretion of glutamate from radiolabelled GSH in isolated vacuoles containing gamma-GT was also measured . It is proposed that gamma-GT and a L-Cys-Gly dipeptidase catalyse the complete hydrolysis of GSH stored in the central vacuole of the yeast cell, prior to release of its constitutive amino acids L-glutamate, L-cysteine and glycine into the cytoplasm . Yeast appears to be a useful model for studying gamma-GT physiology and GSH metabolism.

Biotechnol Bioeng, 2001 Nov, 76(3), 259 - 68
Cell cycle-dependent protein secretion by Saccharomyces cerevisiae; Frykman S et al.; Synchronized Saccharomyces cerevisiae cell populations were used to examine secretion rates of a heterologous protein as a function of cell cycle position . The synchronization procedure had a profound effect on the type and quality of data obtained . When cell synchrony was induced by cell cycle-arresting drugs, a significant physiological perturbation of cells was observed that obscured representative secretion data . In contrast, synchronization with centrifugal elutriation resulted in synchronized first-generation daughter cells with undetectable perturbation of the physiological state . The synchronized cells did not secrete significant amounts of protein until they reached cell division, suggesting that the secretion process in these cells is strongly cell cycle dependent . However, the maximum secretion rate of the synchronized culture (7-14 molecules/cell/second) was significantly lower than that of an asynchronous culture (29-51 molecules/cell/second) . This result indicates that young daughter cells isolated in the synchronization process exhibit different protein secretion behavior than older mother cells that are absent in the synchronized cell population but present in the asynchronous culture .

Genetika, 2001 Sep, 37(9), 1213 - 24
{New mutation in Saccharomyces cerevisiae SRM genes and some features of their phenotypic effects}; Koltovaia NA et al.; The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17 on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiae cells and on cell sensitivity to ionizing radiation were studied . The srm8 mutation caused instability of chromosome maintenance in diploid cells . In yeast cells with the intact mitochondrial genome, all examined srm mutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element . Mutations srm12, srm15, and srm17 also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8 mutation did not markedly affect the maintenance of this plasmid . Mutations srm8, srm12, and srm17 were shown to increase cell sensitivity to gamma-ray irradiation . The SRM8 gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.

Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12608 - 13 Epub 2001 Oct 16.
A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity; Birrell GW et al.; The recent completion of the deletion of essentially all of the ORFs in yeast is an important new resource for identifying the phenotypes of unknown genes . Each ORF is replaced with a cassette containing unique tag sequences that allow rapid parallel analysis of strains in a pool by using hybridization to a high-density oligonucleotide array . We examined the utility of this system to identify genes conferring resistance to UV irradiation by using a pool of 4,627 individual homozygous deletion strains (representing deletions of all nonessential genes) . We identified most of the nonessential genes previously shown to be involved in nucleotide excision repair, in cell cycle checkpoints, in homologous recombination, and in postreplication repair after UV damage . We also identified and individually confirmed, by replacing the genes, three new genes, to our knowledge not previously reported to confer UV sensitivity when deleted . Two of these newly identified genes have human orthologs associated with cancer, demonstrating the potential of this system to uncover human genes affecting sensitivity to DNA-damaging agents and genes potentially involved in cancer formation.

Genetics, 2001 Oct, 159(2), 487 - 97
Genetic interactions of Spt4-Spt5 and TFIIS with the RNA polymerase II CTD and CTD modifying enzymes in Saccharomyces cerevisiae; Lindstrom DL et al.; Genetic and biochemical studies have identified many factors thought to be important for transcription elongation . We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it . We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1 . We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD . Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II . We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS . In contrast, an spt16 mutation did not show these interactions . These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function.

Genetics, 2001 Oct, 159(2), 453 - 70
Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae; Biggins S et al.; Accurate chromosome segregation requires the precise coordination of events during the cell cycle . Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase . At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles . To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome . Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified . We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior . We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation . We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication . We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.

Genetics, 2001 Oct, 159(2), 441 - 52
Direct estimate of the mutation rate and the distribution of fitness effects in the yeast Saccharomyces cerevisiae; Wloch DM et al.; Estimates of the rate and frequency distribution of deleterious effects were obtained for the first time by direct scoring and characterization of individual mutations . This was achieved by applying tetrad analysis to a large number of yeast clones . The genomic rate of spontaneous mutation deleterious to a basic fitness-related trait, that of growth rate, was U = 1.1 x 10(-3) per diploid cell division . Extrapolated to the fruit fly and humans, the per generation rate would be 0.074 and 0.92, respectively . This is likely to be an underestimate because single mutations with selection coefficients s < 0.01 could not be detected . The distribution of s > or = 0.01 was studied both for spontaneous and induced mutations . The latter were induced by ethyl methanesulfonate (EMS) or resulted from defective mismatch repair . Lethal changes accounted for approximately 30-40% of the scored mutations . The mean s of nonlethal mutations was fairly high, but most frequently its value was between 0.01 and 0.05 . Although the rate and distribution of very small effects could not be determined, the joint share of such mutations in decreasing average fitness was probably no larger than approximately 1%.

EMBO Rep, 2001 Nov, 2(11), 1035 - 9 Epub 2001 Oct 17.
Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway; Einwachter H et al.; Import of peroxisomal matrix proteins is essential for peroxisome biogenesis . Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process . Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms . We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case . Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms . These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins . We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import.

Int J Biochem Cell Biol, 2001 Dec, 33(12), 1202 - 7
UGA4 gene encoding the gamma-aminobutyric acid permease in Saccharomyces cerevisiae is an acid-expressed gene; Moretti MB et al.; BACKGROUND AND AIMS: biological processes in all organisms are controlled by environmental conditions, however, information concerning the molecular responses to external pH is scarce . In this work we studied the pH response of UGA4 gene encoding delta-aminolevulinic acid and gamma-aminobutyric acid permease in Saccharomyces cerevisiae . METHODS: we analyzed the effect of pH on the expression of UGA4 gene measuring beta-galactosidase activity in cells carrying a UGA4::lacZ fusion gene . RESULTS: results indicate that UGA4 expression is higher at acidic pH . The expression of UGA3 and UGA35 genes, which encode two positive transcription factors, is not regulated by external pH, while the expression of UGA43 gene encoding a repressor of UGA4 transcription is dependent on pH . Using a strain lacking Uga43p we clearly showed that the effect of ambient pH on UGA4 expression is not a secondary effect of the pH regulation on UGA43 . We have also demonstrated that the effect of pH can only be detected when UGA4 gene is not subject to a strong repression by Uga43p nor to GABA induction . CONCLUSION: here, we demonstrate that UGA4 is an acid-expressed gene . This regulation is probably mediated by Rim101p through the consensus site 5'-GCCARG-3' at 237 bp preceding the UGA4 coding sequence (201).

Res Microbiol, 2001 Sep, 152(7), 605 - 12
Protein-based inheritance in Saccharomyces cerevisiae: {URE3} as a prion form of the nitrogen regulatory protein Ure2; Edskes HK; The {URE3} element of the yeast Saccharomyces cerevisiae results from the presence of an altered form of the nitrogen regulatory protein Ure2 . This altered form acts as an infectious protein (prion) . Genes affecting {URE3} initiation and propagation should give valuable information about prion diseases as well as other conformational diseases.

Prikl Biokhim Mikrobiol, 2001 Sep-Oct, 37(5), 578 - 81
{Effect of culture media on the composition of free amino acids in Saccharomyces cerevisiae yeast}; Khalilova EA et al.; The quantitative and qualitative compositions of free amino acids of the yeast Saccharomyces cerevisiae Y-503 cultivated in different nutrient media were studied by liquid chromatography . The yeast grown in the medium containing geothermal water was shown to accumulate more amino acids . During lyophilization, the stabilization of the physiological activity of the yeast in this nutrient medium was observed . The increased biological value of dry yeast was shown to depend on the content of free amino acids, including essential amino acids: arginine, histidine, leucine, isoleucine, lysine, threonine, serine and phenylalanine.

Mol Cell Biol, 2001 Nov, 21(22), 7663 - 72
Peripheral mitochondrial inner membrane protein, Mss2p, required for export of the mitochondrially coded Cox2p C tail in Saccharomyces cerevisiae; Broadley SA et al.; Cytochrome oxidase subunit 2 (Cox2p) is synthesized on the matrix side of the mitochondrial inner membrane, and its N- and C-terminal domains are exported across the inner membrane by distinct mechanisms . The Saccharomyces cerevisiae nuclear gene MSS2 was previously shown to be necessary for Cox2p accumulation . We have used pulse-labeling studies and the expression of the ARG8(m) reporter at the COX2 locus in an mss2 mutant to demonstrate that Mss2p is not required for Cox2p synthesis but rather for its accumulation . Mutational inactivation of the proteolytic function of the matrix-localized Yta10p (Afg3p) AAA-protease partially stabilizes Cox2p in an mss2 mutant but does not restore assembly of cytochrome oxidase . In the absence of Mss2p, the Cox2p N terminus is exported, but Cox2p C-terminal export and assembly of Cox2p into cytochrome oxidase is blocked . Epitope-tagged Mss2p is tightly, but peripherally, associated with the inner membrane and protected by it from externally added proteases . Taken together, these data indicate that Mss2p plays a role in recognizing the Cox2p C tail in the matrix and promoting its export.

Mol Cell Biol, 2001 Nov, 21(22), 7576 - 86
Overproduction of PDR3 suppresses mitochondrial import defects associated with a TOM70 null mutation by increasing the expression of TOM72 in Saccharomyces cerevisiae; Koh JY et al.; Most mitochondrial proteins are synthesized with cleavable amino-terminal targeting signals that interact with the mitochondrial import machinery to facilitate their import from the cytosol . We previously reported that the presequence of the F(1)-ATPase beta subunit precursor (pre-F(1)beta) acts as an intramolecular chaperone that maintains the precursor in an import-competent conformation prior to import (P . Hajek, J . Y . Koh, L . Jones, and D . M . Bedwell, Mol . Cell . Biol . 17:7169-7177, 1997) . We also found that a mutant form of pre-F(1)beta with a minimal targeting signal (Delta 1,2 pre-F(1)beta) is inefficiently imported into mitochondria because it rapidly folds into an import-incompetent conformation . We have now analyzed the consequences of reducing the pre-F(1)beta targeting signal to a minimal unit in more detail . We found that Delta 1,2 pre-F(1)beta is more dependent upon the Tom70p receptor for import than WT pre-F(1)beta is, resulting in a growth defect on a nonfermentable carbon source at 15 degrees C . Experiments using an in vitro mitochondrial protein import system suggest that Tom70p functions to maintain a precursor containing the Delta 1,2 pre-F(1)beta import signal in an import-competent conformation . We also identified PDR3, a transcriptional regulator of the pleiotropic drug resistance network, as a multicopy suppressor of the mitochondrial import defects associated with Delta 1,2 pre-F(1)beta in a tom70 Delta strain . The overproduction of PDR3 mediated this effect by increasing the import of Delta 1,2 pre-F(1)beta into mitochondria . This increased the mitochondrial ATP synthase activity to the extent that growth of the mutant strain was restored under the selective conditions . Analysis of the transcription patterns of components of the mitochondrial outer membrane import machinery demonstrated that PDR3 overproduction increased the expression of TOM72, a little studied TOM70 homologue . These results suggest that Tom72p possesses overlapping functions with Tom70p and that the pleiotropic drug resistance network plays a previously unappreciated role in mitochondrial biogenesis.

J Biol Chem, 2001 Dec 21, 276(51), 48539 - 48 Epub 2001 Oct 15.
Phosphatidylethanolamine has an essential role in Saccharomyces cerevisiae that is independent of its ability to form hexagonal phase structures; Storey MK et al.; Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn) . Mitochondrial Psd1p provides approximately 90% of total cellular phosphatidylserine decarboxylase activity . When the PSD1 gene is deleted, the resultant strain (psd1Delta) grows normally at 30 degrees C in glucose and in the absence of exogenous choline or ethanolamine . However, at elevated temperature (37 degrees C) or on the nonfermentable carbon source lactate, the growth of psd1Delta strains is minimal without ethanolamine supplementation . The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid . These results suggest that there is a critical level of PtdEtn required to support growth . This theory is supported by growth data revealing that a psd1Delta psd2Delta dpl1Delta strain can only grow in the presence of ethanolamine . In contrast, a psd1Delta psd2Delta strain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine . psd1Delta psd2Delta cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn) . PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn . The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content . The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by (31)P NMR.

J Biol Chem, 2001 Dec 14, 276(50), 47556 - 62 Epub 2001 Oct 15.
Manganese superoxide dismutase in Saccharomyces cerevisiae acquires its metal co-factor through a pathway involving the Nramp metal transporter, Smf2p; Luk EE et al.; Eukaryotes express both copper/zinc (SOD1)- and manganese (SOD2)-requiring superoxide dismutase enzymes that guard against oxidative damage . Although SOD1 acquires its copper through a specific copper trafficking pathway, nothing is known regarding the intracellular manganese trafficking pathway for SOD2 . We demonstrate here that in Saccharomyces cerevisiae cells delivery of manganese to SOD2 in the mitochondria requires the Nramp metal transporter, Smf2p . SOD2 activity is greatly diminished in smf2Delta mutants, even though the mature SOD2 polypeptide accumulates to normal levels in mitochondria . Treating smf2Delta cells with manganese supplements corrected the SOD2 defect, as did elevating intracellular manganese through mutations in PMR1 . Hence, manganese appears to be inaccessible to mitochondrial SOD2 in smf2 mutants . Cells lacking SMF2 also exhibited defects in manganese-dependent steps in protein glycosylation and showed an overall decrease in steady-state levels of accumulated manganese . By comparison, mutations in the cell surface Nramp transporter, Smf1p, had very little impact on manganese accumulation and trafficking . Smf2p resides in intracellular vesicles and shows no evidence of plasma membrane localization, even in an end4 mutant blocked for endocytosis . We propose a model in which Smf2p-containing vesicles play a central role in manganese trafficking to the mitochondria and other cellular sites as well.

Nucleic Acids Res, 2001 Oct 15, 29(20), 4134 - 43
Efficient repair of large DNA loops in Saccharomyces cerevisiae; Corrette-Bennett SE et al.; Small looped mispairs are efficiently corrected by mismatch repair . The situation with larger loops is less clear . Repair activity on large loops has been reported as anywhere from very low to quite efficient . There is also uncertainty about how many loop repair activities exist and whether any are conserved . To help address these issues, we studied large loop repair in Saccharomyces cerevisiae using in vivo and in vitro assays . Transformation of heteroduplexes containing 1, 16 or 38 nt loops led to >90% repair for all three substrates . Repair of the 38 base loop occurred independently of mutations in key genes for mismatch repair (MR) and nucleotide excision repair (NER), unlike other reported loop repair functions in yeast . Correction of the 16 base loop was mostly independent of MR, indicating that large loop repair predominates for this size heterology . Similarities between mammalian and yeast large loop repair were suggested by the inhibitory effects of loop secondary structure and by the role of defined nicks on the relative proportions of loop removal and loop retention products . These observations indicate a robust large loop repair pathway in yeast, distinct from MR and NER, and conserved in mammals.

J Radiat Res (Tokyo), 2001 Jun, 42(2), 217 - 27
Effect of a hypoxic radiosensitizer, AK 2123 (Sanazole), on yeast Saccharomyces cerevisiae; Pasupathy K et al.; Sanazole/DNA repair/Hypoxic radiosensitization/DNA polymerases/Saccharomyces cerevisiae Yeast Saccharomyces cerevisiae can exist in two physiological states, namely anaerobic and aerobic . They differ in their response to gamma- radiation and radiomodification . We report hereon our results concerning radiosensitization by Sanazole (AK-2123), a well-known hypoxic radio sensitizer, whose mechanism of action has been studied extensively . The results have revealed that Sanazole (1 mM) when present during irradiation could specifically sensitize wild-type anaerobic yeast cells with a DMF of 2.4 . In a radiation-sensitive mutant which lacks a DNA repair pathway specific for the recovery from gamma-radiation induced DNA damage, the extent of sensitization was considerably lower and the DMF was only 1.3 . Studies on the liquid holding recovery of cells of both wild- type and rad52 yeast cells exposed to radiation in presence of Sanazole revealed that sensitization by Sanazole is due to a preferential increase in the DNA damage, and not by impairing DNA repair . This system thus holds promise for screening potential hypoxic chemical radiosensitizers.

Mol Biol Cell, 2001 Oct, 12(10), 3295 - 306
The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p; Fraering P et al.; Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins . After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes . These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c) . Gpi16p is an essential N-glycosylated transmembrane glycoprotein . Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif . Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins . Gpi8p and Gpi16p are unstable if either of them is removed by depletion . Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable . Overexpression of Gpi8p cannot compensate for the lack of Gpi16p . Homologues of Gpi16p are found in all eucaryotes . The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively . When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.

Environ Toxicol Chem, 2001 Oct, 20(10), 2353 - 60
Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium; Momose Y et al.; DNA microarray technology enables genome-wide detection of cell response at the transcriptional level . We are planning to make bioassay systems that can detect environmental chemicals to screen for potential bioreactive agents . To develop a DNA microarray for our purposes, the changes in gene expression underlying the yeast stress response to cadmium were analyzed by a microarray of total mRNA . Cadmium is a potent cell poison known to cause oxidative stress by changing intracellular glutathione levels . We report here that not only the glutathione synthesis gene (GSH1) but also almost all transcripts of the enzymes involved in the sulfur amino acid metabolism, especially MET14 and MET17, were greatly induced after exposure to cadmium . While several common stress-responsive genes, such as HSP26, GRE1, HSP12, and DDR48, were up-regulated more than almost fourfold by cadmium, there were also 42 other genes up-regulated more than fourfold . Based on these results, we concluded that DNA microarrays are very useful instruments for creating new bioassay systems and finding genetic promoters of stress indicators.

Physiol Genomics, 2001 Oct 10, 7(1), 27 - 34
Two-hybrid analysis of the Saccharomyces cerevisiae 26S proteasome; Cagney G et al.; A two-hybrid screen against an activation domain array of Saccharomyces cerevisiae proteins was carried out for 31 yeast proteasome proteins . Fifty-five putative interactions were identified: 21 between components of the proteasome complex and 34 between proteasome proteins and other proteins . Many of these latter interactions involved either proteins of the ubiquitin pathway, cell cycle proteins, protein kinases or a translation initiation factor subunit . The role of eleven proteins associated with proteasome function by these screens was analyzed by examining the corresponding deletion strains for temperature sensitivity and canavanine sensitivity and for the stability of a ubiquitin-beta-galactosidase fusion protein . These assays additionally implicated three proteins, Bim1, Ump1, and YKL171W, in proteasome function . This study demonstrates the utility of genome-wide two-hybrid assays as an entry point for the further analysis of a large protein complex.

FEBS Lett, 2001 Oct 5, 506(2), 149 - 56
Functional characterization of the Bag7, Lrg1 and Rgd2 RhoGAP proteins from Saccharomyces cerevisiae; Roumanie O et al.; Rho proteins are down-regulated in vivo by specific GTPase activating proteins (RhoGAP) . We have functionally studied three Saccharomyces cerevisiae putative RhoGAP . By first identifying Rho partners with a systematic two-hybrid approach and then using an in vitro assay, we have demonstrated that the Bag7 protein stimulated the GTPase activity of the Rho1 protein, Lrg1p acted on the Cdc42 and Rho2 GTPases and we showed that Rgd2p has a GAP activity on both Cdc42p and Rho5p . In addition, we brought the first evidence for the existence of a sixth functional Rho in yeast, the Cdc42/Rac-like GTPase Rho5.

Biotechnol Bioeng, 2001 Nov 5, 75(3), 334 - 44
Statistical reconciliation of the elemental and molecular biomass composition of Saccharomyces cerevisiae; Lange HC et al.; A systematic mathematical procedure capable of detecting the presence of a gross error in the measurements and of reconciling connected data sets by using the maximum likelihood principle is applied to the biomass composition data of yeast . The biomass composition of Saccharomyces cerevisiae grown in a chemostat under glucose limitation was analyzed for its elemental and for its molecular composition . Both descriptions initially resulted in conflicting results concerning the elemental composition, molecular weight, and degrees of reduction . The application of the statistical reconciliation method, based on elemental balances and equality relations, is used to obtain a consistent biomass composition . Simultaneously, the error margins of the data sets are significantly reduced in the reconciliation process . On the basis of statistical analysis it was found that inclusion of about 4% water in the list of biomass constituents is essential to adequately describe the dry biomass and match both set of measurements . The reconciled carbon content of the biomass varied 4% from the ones obtained from the molecular analysis . The proposed method increases the accuracy of biomass composition data of its elements and its molecules by providing a best estimate based on all available data and thus provides an improved and consistent basis for metabolic flux analysis as well as black box modeling approaches .

Eur J Biochem, 2001 Oct, 268(19), 5209 - 14
The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons; Nett JH et al.; The selection of the site for initiation of translation for the Saccharomyces cerevisiae NFS1 gene was examined using mutated AUG1, AUG2 and AUG3 codons . When AUG1 of the yeast NFS1 gene was mutated to UUG and the resulting mRNA was translated in vitro using a reticulocyte system, initiation from the mutated codon was abolished and occurred instead at downstream codons at increased rates . When the same mRNA was translated using a yeast extract, translation initiated at the mutated codon, albeit at a reduced rate, and there was no increased translation at downstream AUG codons . The NFS1 gene in which AUG1 was replaced by UUG was also able to substitute for the wild-type gene in vivo in yeast . Western blots confirmed that the encoded protein was the same size as that encoded by the wild-type gene and that both the wild-type and mutated proteins localized to mitochondria . This is apparently the first example of a yeast protein where mutagenesis of AUG1 does not lead to alternate use of a downstream AUG.

Eur J Biochem, 2001 Oct, 268(19), 5057 - 65
Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae; Boubekeur S et al.; This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates . Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol . Therefore we constructed mutants in two parental strains (YPH499 and W303-1a) . Some differences appeared in the growth characteristics of mutants obtained from these two parental strains . For these experiments we used ethanol, pyruvate or lactate as substrates . Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway . The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain . The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent . To understand these differences, we measured the enzyme content of these different strains . We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase . Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation . Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient . However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.

Mol Genet Genomics, 2001 Sep, 266(1), 20 - 7
Overexpression of Bud5p can suppress mutations in the Gsp1p guanine nucleotide exchange factor Prp20p in Saccharomyces cerevisiae; Clement M et al.; The gene product Prp20p, which is located in the nucleus, serves as the nucleotide exchange factor (GEF) for the small nuclear G protein Gsp1p in Saccharomyces cerevisiae, and catalyses the replacement of Gsp1-bound GDP by GTP . These proteins are involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules, cell cycle progression, DNA replication and maintenance of chromosome structure/stability . It is believed that in order to complete a full GDP/GTP cycle, Gsp1p has to shuttle between the nucleus and the cytoplasm, where its GTPase Activating Protein (GAP) Rna1p is located . Here, we report on the ability of Bud5p, the exchange factor for Rsr1p, to suppress conditional prp20 mutants when an extra copy of GSP1 is present . This suppression by BUD5 can be reversed by simultaneous overexpression of RNA1, and is not Rsr1p-dependent, nor allele-specific . We also show that Bud5p can physically interact with Gsplp, both in vitro and in vivo . These,findings raise the possibility that Bud5p could act as a cytoplasmic exchange factor for Gsp1p and, therefore, that a complete GDP/GTP cycle could take place in the cytoplasm.

Mol Genet Genomics, 2001 Sep, 266(1), 142 - 55
Mutations in WSC genes for putative stress receptors result in sensitivity to multiple stress conditions and impairment of Rlm1-dependent gene expression in Saccharomyces cerevisiae; Zu T et al.; Intracellular signaling by mitogen-activated protein (MAP) kinase cascades plays an essential role in the cellular response to environmental stress . In the yeast Saccharomyces cerevisiae, the PKC1-regulated, stress-activated MAP kinase pathway, the MPK1 cascade, is activated by heat and by a decrease in osmolarity . The genes WSC1, WSC2 and WSC3 encode putative receptors that maintain cell wall integrity under conditions of heat stress . Genetic studies place the function of the WSC genes upstream of the MPK1 kinase cascade . To further define the role of the WSC family in the stress response we determined whether: (1) the wscdelta mutants are sensitive to other environmental stress conditions, in addition to heat shock; (2) expression from four transcriptional control elements, known to be activated by stress, is impaired in wscdelta mutants; and (3) Wsc4, a Wsc homolog, has functions that overlap with those of the other Wsc family members . We report here that deletion of WSC and PKC1 causes hypersensitivity to ethanol, hydrogen peroxide and DNA-damaging drugs . In wscdelta mutants expression of beta-galactosidase from the AP-1 response element (ARE), the heat shock response element (HSE) or the stress response element (STRE) is not reduced . In contrast, expression of a reporter gene placed under the control of the Rlm1 (transcription factor)-dependent response element is significantly reduced in wscdelta mutants . This suggests that the lysis defect of wscdelta mutants is at least in part caused by a defect in transcriptional regulation by Rlm1 . Phenotypic analysis of the effect of deleting WSC4 in a wsc1delta mutant show that, unlike WSC2 or WSC3, deletion of WSC4 does not exacerbate the lysis defect of a wsc1delta strain . In contrast, deletion of WSC4 enhances the sensitivity of the wsc1delta mutant to heat shock, ethanol, and a DNA-damaging drug, suggesting that WSC4 plays a role in the response to environmental stress but that its function may differ from those of the other WSC family members.

Mol Genet Genomics, 2001 Sep, 266(1), 115 - 25
Role of the kinetochore protein Ndc10 in mitotic checkpoint activation in Saccharomyces cerevisiae; Fraschini R et al.; Mitotic checkpoints delay cell cycle progression in response to alterations in the mitotic apparatus, thus ensuring correct chromosome segregation . While improper spindle orientation activates the Bub2/Bfa1-dependent checkpoint in budding yeast, delaying exit from mitosis, lack of bipolar kinetochore-microtubule attachment activates a signal transduction cascade that prevents both anaphase onset and exit from mitosis by inhibiting the Cdc20/APC (Anaphase Promoting Complex)-mediated proteolysis of securin and inactivation of mitotic cyclin-dependent kinases (CDKs), respectively . Proteolysis of the securin Pdsl is necessary to liberate the separase Esp1, which then triggers sister chromatid separation, whereas inactivation of mitotic CDKs is a prerequisite for exit from mitosis and for starting a new round of DNA replication in the next cell cycle . In budding yeast, this latter checkpoint response involves the proteins Mad1, 2, 3, Bub1 and Bub3, whose vertebrate counterparts localize to unattached kinetochores . Mutations that alter other kinetochore proteins result in mitotic checkpoint activation, while the ndc10-1 mutation not only impairs kinetochore function, but also disrupts the checkpoint response, indicating a role for Ndc10 in this process . Here we present evidence that Ndc10 is not part of the Bub2/Bfa1-dependent pathway, and its role in the checkpoint response might also be different from that of the other Mad and Bub proteins . Indeed, Ndc10, unlike other mitotic checkpoint proteins, is not required for the mitotic block induced by overexpression of the Mpsl protein kinase, which is implicated in mitotic checkpoint control . Furthermore, the delay in mitotic exit caused by non-degradable Pds1, which does not require Mad and Bub proteins, depends on Ndc10 function . We propose that a pathway involving Ndc10 might monitor defects in the mitotic apparatus independently of the Mad and Bub proteins . Since the Espl separase is required for exit from mitosis in both ndc10-1 and nocodazole-treated mad2delta cells, the two signal transduction cascades might ultimately converge on the inactivation of Esp1.

Methods Enzymol, 2001, 342, 159 - 67
Purification and characterization of Saccharomyces cerevisiae Rnt1p nuclease; Lamontagne B et al.; In this article, we have described methods used to purify Rnt1p and study its biochemical properties . Rnt1p can be easily purified from bacteria as N-terminal His6-tagged protein and its activity may be monitored in vitro . Rnt1p cleaves the RNA by binding to a cleavage site followed by hydrolysis and product release . The kinetic parameters of Rnt1p are similar to those of other nucleases, including bacterial RNase III . The ability of Rnt1p to bind substrate without cleaving it in the absence of divalent metal ions provides a convenient means to study RNA recognition and binding independent of catalysis . The gel mobility shift and in-the-gel cleavage assays described here reveal the formation of two Rnt1p-RNA complexes with different cleavage activities, suggesting that the protein may bind the substrate in two different forms or through a two-step binding reaction.

J Biol Chem, 2001 Dec 14, 276(50), 47671 - 4 Epub 2001 Oct 03.
Binding and partial denaturing of G-quartet DNA by Cdc13p of Saccharomyces cerevisiae; Lin YC et al.; The protein Cdc13p binds telomeres in vivo and is essential for the maintenance of the telomeres of Saccharomyces cerevisiae . In addition, Cdc13p is known to bind single-stranded TG(1-3) DNA in vitro . Here we have shown that Cdc13p also binds DNA quadruplex, G-quartet, formed by TG(1-3) DNA . Moreover, the binding of Cdc13p causes a partial denaturing of the G-quartet DNA . Formation of DNA quadruplexes may involve the intermolecular association of TG(1-3) DNA and inhibit the extension of telomeres by telomerase . Thus, our finding suggests that Cdc13p may disrupt telomere association and facilitate telomere replication.

Cancer Res, 2001 Oct 1, 61(19), 6982 - 6
SKY1 is involved in cisplatin-induced cell kill in Saccharomyces cerevisiae, and inactivation of its human homologue, SRPK1, induces cisplatin resistance in a human ovarian carcinoma cell line; Schenk PW et al.; The therapeutic potential of cisplatin, one of the most active and widely used anticancer drugs, is severely limited by the occurrence of cellular resistance . In this study, using budding yeast Saccharomyces cerevisiae as a model organism to identify novel drug resistance genes, we found that disruption of the yeast gene SKY1 (serine/arginine-rich protein-specific kinase from budding yeast) by either transposon insertion or one-step gene replacement conferred cellular resistance to cisplatin . Heterologous expression of the human SKY1 homologue SRPK1 (serine/arginine-rich protein-specific kinase) in SKY1 deletion mutant yeast cells restored cisplatin sensitivity, suggesting that SRPK1 is a cisplatin sensitivity gene, the inactivation of which could lead to cisplatin resistance . Subsequently, we investigated the role of SRPK1 in cisplatin sensitivity and resistance in human ovarian carcinoma A2780 cells using antisense oligodeoxynucleotides . Treatment of A2780 cells with antisense oligodeoxynucleotides directed against the translation initiation site of SRPK1 led to down-regulation of SRPK1 protein and conferred a 4-fold resistance to cisplatin . The human SRPK1 gene has not been associated with drug resistance before . Our new findings strongly suggest that SRPK1 is involved in cisplatin-induced cell kill and indicate that SRPK1 might potentially be of importance for studying clinical drug resistance.

FEMS Microbiol Lett, 2001 Sep 25, 203(2), 223 - 7
Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro; Cytrynska M et al.; Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and PKA-2) . The presence of PKA in both fractions was confirmed by immunoblotting with anti-Bcy1 antibodies . Yeast pyruvate kinase Pyk1 identified by amino acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodies copurified with the PKA-1 but not the -2 fraction . Pyk1 can be phosphorylated by yeast PKA in vitro in the presence of cAMP and cGMP . Two-dimensional gel electrophoretic analysis revealed four phosphorylated forms of Pyk1 modified by PKA . In phosphorylation of Pyk1 mainly the Tpk2 catalytic subunit of yeast PKA was involved.

Mol Microbiol, 2001 Sep, 41(6), 1311 - 26
Lre1 affects chitinase expression, trehalose accumulation and heat resistance through inhibition of the Cbk1 protein kinase in Saccharomyces cerevisiae; Versele M et al.; The addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae triggers activation of the cAMP pathway with a rapid drop in stress resistance as a consequence . We have isolated the LRE1 gene as a multicopy suppressor of glucose-induced loss of heat resistance . Overexpression of LRE1 in a wild-type strain causes the same phenotype as observed in strains with reduced activity of the cAMP-PKA pathway: higher heat resistance and enhanced trehalose levels . Deletion of LRE1 results in the opposite phenotypes . Epistasis analysis indicates that these effects are independent of cAMP and PKA, of the protein kinases Yak1, Sch9 and Rim15 and of the transcription factors Msn2 and Msn4 . Lre1 has recently been isolated in a two-hybrid screen using the conserved protein kinase Cbk1 as a bait . Cbk1 controls the expression of CTS1 (encoding chitinase) through the transcription factor Ace2 . We demonstrate here that overexpression of LRE1 represses CTS1 whereas deletion of LRE1 induces the expression of CTS1 . Repression of CTS1 results in deficient cell separation as a result of inefficient degradation of the chitin ring after cytokinesis . Neither deletion nor overexpression of LRE1 has any effect on CTS1 expression in a cbk1Delta mutant, indicating that Lre1 inhibits Cbk1 . In addition, we show that increased trehalose accumulation and increased heat resistance caused by overexpression of LRE1 are also the result of inhibition of Cbk1, revealing a novel control pathway for certain targets affected by PKA . The yeast genome contains a homologue of LRE1, YDR528w, which we have called HLR1 (for homologue of Lre1) . Deletion and overexpression of HLR1 causes similar but less pronounced effects compared with LRE1.

Microbiology, 2001 Oct, 147(Pt 10), 2849 - 55
New aspects of the glucose activation of the H(+)-ATPase in the yeast Saccharomyces cerevisiae; Souza MA et al.; The glucose-induced activation of plasma membrane ATPase from Saccharomyces cerevisiae was first described by Serrano in 1983 . Many aspects of this signal transduction pathway are still obscure . In this paper, evidence is presented for the involvement of Snf3p as the glucose sensor related to this activation process . It is shown that, in addition to glucose detection by Snf3p, sugar transport is also necessary for activation of the ATPase . The participation of the G protein, Gpa2p, in transducing the internal signal (phosphorylated sugars) is also demonstrated . Moreover, the involvement of protein kinase C in the regulation of ATPase activity is confirmed . Finally, a model pathway is presented for sensing and transmission of the glucose activation signal of the yeast H(+)-ATPase.

Nucleic Acids Res, 2001 Oct 1, 29(19), 4043 - 51
Specific distribution of the Saccharomyces cerevisiae linker histone homolog HHO1p in the chromatin; Freidkin I et al.; In virtually all eukaryotic organisms, linker DNA between nucleosomes is associated with a histone termed linker histone or histone H1 . In Saccharomyces cerevisiae, HHO1 encodes a putative linker histone with very significant homology to histone H1 . The encoded protein is expressed in the nucleus, but has not been shown to affect global chromatin structure, nor has its deletion shown any detectable phenotype . In vitro chromatin assembly experiments with recombinant HHO1p have shown that it is able to complex with dinuncleosomes in a similar manner to histone H1 . Here we report that while disruption of HHO1 has little affect on RNA levels of most cellular transcripts, there are numerous exceptions . Measurement of HHO1p concentration in the wild-type cell showed a stoichiometry of about one HHO1p molecule per 37 nucleosomes . Localization of HHO1p in the chromatin, using an immunoprecipitation technique, showed preferential HHO1p binding to rDNA sequences . These results suggest that HHO1p may play a similar role to linker histones, but at restricted locations in the chromatin.

Biochem Biophys Res Commun, 2001 Oct 5, 287(4), 837 - 42
Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae; Berhe A et al.; The derepressible Pho84 high-affinity phosphate permease of Saccharomyces cerevisiae, encoded by the PHO84 gene belongs to a family of phosphate:proton symporters (PHS) . The protein contains 12 native cysteine residues of which five are predicted to be located in putative transmembrane regions III, VI, VIII, IX, and X, and the remaining seven in the hydrophilic domains of the protein.Here we report on the construction of a Pho84 transporter devoid of cysteine residues (C-less) in which all 12 native residues were replaced with serines using PCR mutagenesis and the functional consequences of this . Our results clearly demonstrate that the C-less Pho84 variant is able to support growth of yeast cells to the same extent as the wild-type Pho84 and is stably expressed under derepressible conditions and is fully active in proton-coupled phosphate transport across the yeast plasma membrane .

Yeast, 2001 Oct, 18(14), 1317 - 30
The HTL1 gene (YCR020W-b) of Saccharomyces cerevisiae is necessary for growth at 37 degrees C, and for the conservation of chromosome stability and fertility; Lanzuolo C et al.; A small 78 codon ORF, named HTL1 (Chen et al., unpublished results), situated between loci MAK31 and HSP30 on chromosome III of Saccharomyces cerevisiae, is required for growth at 37 degrees C . In this communication, we characterize the ORF and show that disruption of HTL1, besides preventing growth at 37 degrees C, causes genetic and/or epigenetic instability at 26 degrees C: ploidy increases in about 10% of cells grown from individual disruptants and a fraction of disruptant clones are predestined to a rapid and progressive loss of fertility during growth at 26 degrees C .

Yeast, 2001 Oct, 18(14), 1309 - 16
Human pancreatic glucokinase (GlkB) complements the glucose signalling defect of Saccharomyces cerevisiae hxk2 mutants; Mayordomo I et al.; Human pancreatic glucokinase (GlkB, hexokinase IV) has been expressed in Saccharomyces cerevisiae . The recombinant protein showed similar enzyme kinetics to those described for the original enzyme . When expressed in hxk2 yeast mutants, GlkB complemented both the glucose induction and the glucose repression defects present in the mutant . It was also functional in regulating the activity of the Snf1 kinase complex in response to glucose, participating in the regulation of the Reg1/Glc7 phosphatase complex, as its yeast counterpart .

BMC Mol Biol . 2001;2(1):9 . Epub 2001 Aug 24.
Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae; Urakov VN et al.; BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3 . eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl - dependent manner . Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough . RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state . This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes . Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons . Itt1p interacts in vitro with both eRFl and eRF3 . Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p . CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast . This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.

J Biol Chem, 2001 Nov 23, 276(47), 43824 - 8 Epub 2001 Sep 21.
Structure of DNA polymerase delta from Saccharomyces cerevisiae; Johansson E et al.; DNA polymerase delta (Pol delta) from Saccharomyces cerevisiae consists of three subunits, Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa), present at a 1:1:1 stoichiometry in purified preparations . Previously, based on gel filtration studies of Pol delta, we suggested that the enzyme may be a dimer of catalytic cores, with dimerization mediated by the Pol32 subunit (Burgers, P . M., and Gerik, K . J . (1998) J . Biol . Chem . 273, 19756-19762) . We now report on extensive gel filtration, glycerol gradient sedimentation, and analytical equilibrium centrifugation studies of Pol delta and of several subassemblies of Pol delta . The hydrodynamic parameters of these assemblies indicate that (i) Pol32 is a rod-shaped protein with a frictional ratio f/f(0) = 2.22; (ii) any complex containing Pol32 also has an extremely asymmetric shape; (iii) the results of these studies are independent of concentration (varied between 0.1-20 microm); (iv) all complexes are monomeric under the conditions studied (up to 20 microm) . Moreover, a two-hybrid analysis of the Pol32 subunit did not detect a Pol32-Pol32 interaction in vivo . Therefore, we conclude that the assembly structure of Pol delta is that of a monomer.

Biomol Eng, 2001 Oct 15, 18(3), 135 - 42
The major exoglucanase secreted by Saccharomyces cerevisiae as a model to study protein glycosylation; Larriba G et al.; The major yeast exoglucanase (ExgIb) consists of a 408 amino acid polypeptide carrying two short N-linked oligosaccharides attached to asparagines 165 (Asn(165)) and 325 (Asn(325)) . These oligosaccharides are very similar, in both length and composition, to those present in the vacuolar protease carboxypeptidase Y . Minor glycoforms of exoglucanase arise by underglycosylation of the protein precursor (Exg(165) and Exg(325)) or by elongation of the second oligosaccharide (ExgIa) . The fact that these glycoforms can be readily separated and identified by HPLC and/or Western blots converts ExgI in an excellent model to study the role of the several components or branches of the precursor oligosaccharide in the efficiency and selectivity of the oligosaccharidyl transferase in vivo . We have found that the presence of a single glucose attached to Dol-PP-GlcNAc(2)-Man(9) increases the efficiency of transfer of that oligosaccharide to the protein acceptor . Also, the glucotriose unit appears to be involved in the selection of the sequons to be occupied, in such a way that its absence results in a bias towards the glycosylation of a particular sequon . Finally, we have shown the transfer of GlcNAc(2) from Dol-PP-GlcNAc(2) to exoglucanase, an indication that this intermediate is able to translocate from the cytoplasmic to the lumenal face of the endoplasmic reticulum membrane.

Biochim Biophys Acta, 2001 Sep 28, 1533(2), 81 - 5
Degradation of conjugated linoleic acid isomers in the yeast Saccharomyces cerevisiae; Gurvitz A et al.; Propagation of Saccharomyces cerevisiae cells in conjugated linoleic acid (CLA) medium resulted in activation of the transcriptional machinery that responds to fatty acids . Cells utilized efficiently trans-10,cis-12 CLA, but not the corresponding cis-9,trans-11 isomer, probably due to the formation of cis-3,trans-5-dienoyl-CoA intermediates that are recalcitrant to beta-oxidation.

Electrophoresis, 2001 Aug, 22(14), 2955 - 68
Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry; Schafer H et al.; The identification of peroxisomal membrane proteins is very important to understand the import mechanisms of substrates and proteins into these organelles and the pathogenesis of human peroxisomal disorders like the Zellweger Syndrom . Peroxisomal membrane proteins were identified after separation by gel electrophoresis, tryptic digestion and mass spectrometric analysis . Using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), it was possible to identify 45 proteins of isolated yeast peroxisomal membranes.

Biochem J, 2001 Oct 1, 359(Pt 1), 193 - 201
Regulatory elements in the FBP1 promoter respond differently to glucose-dependent signals in Saccharomyces cerevisiae; Zaragoza O et al.; In Saccharomyces cerevisiae expression of the fructose-1,6-bisphosphatase-encoding gene, FBP1, is controlled by glucose through the upstream activating sequences UAS1 and UAS2 and the upstream repressing sequence URS1 in its promoter . We have studied the regulation of the proteins that could bind to these elements . We have investigated the role of the putative transcription factors Cat8 and Sip4 in the formation of specific DNA-protein complexes with UAS1 and UAS2, and in the expression of UAS1-lacZ and UAS2-lacZ . The expression of CAT8-lacZ and SIP4-lacZ has been also measured in mig1, tup1 or hxk2 mutants, partially refractory to catabolite repression . We conclude that there is no strict correlation between Cat8 and Sip4 expression or in vitro formation of DNA-protein complexes and expression of UAS1-lacZ and UAS2-lacZ . The URS1 element binds the regulatory protein Mig1, which blocks transcription by recruiting the proteins Cyc8 and Tup1 . The pattern of complexes of URS1 with nuclear extracts was dependent on the carbon source and on Cyc8, but not on Tup1; it was also affected by the protein kinase Snf1 and by the exportin Msn5 . The repression caused by URS1 in a fusion gene was dependent on Mig1, Cyc8 and Tup1, and on the carbon source in the medium; in a snf1 strain the repression observed was independent of the carbon source . Expression of Mig1 could occur in the absence of Snf1 and was moderately sensitive to glucose . We present data showing that different elements of the regulatory system controlling FBP1 responded differently to the concentration of glucose in the medium.

Mol Cells, 2001 Aug 31, 12(1), 77 - 83
Characterization of the CDC10 product and the timing of events of the budding site of Saccharomyces cerevisiae; Jeong JW et al.; Budding cells of the yeast Saccharomyces cerevisiae possess a ring of septin filaments of unknown biochemical nature that lies under the inner surface of the plasma membrane in the neck that connects the mother cell to its bud . Mutants, defective in any of the four genes (CDC3, CDC10, CDC11, CDC12), lack these septin filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis . The cloned CDC10 was fused to bacterial genes to generate antibodies specific for the CDC10 product was a constituent of the septin filaments . Cdc10p-specific antibodies for septin staining and actin-specific rhodamine-phalloidine were used to investigate the timing of the localization of septin and actin at the budding site using the immunofluorescence microscopic technique . In wild-type cells, the timing of the appearance and disappearance of these proteins was indistinguishable . In addition, the cdc10 mutant did not prevent actin localization at the budding site . The mutant that was blocked in the actin function also did not prevent the septin localization of the Cdc10p . This result may suggest an organizational independence between these proteins in the bud formation . Finally, the localization of septin and actin in the cdc24 mutant cell was examined . It was found that the CDC24 function was necessary for the organization of septin and actin at the budding site.

Yeast, 2001 Sep 30, 18(13), 1207 - 16
A PBS2 homologue from Debaryomyces hansenii shows a differential effect on calcofluor and polymyxin B sensitivity in Saccharomyces cerevisiae; Bansal PK et al.; The PBS2 gene encodes a MAP kinase kinase that plays a pivotal role in osmosensing signal-transduction pathway in the yeast Saccharomyces cerevisiae . Mutation in the PBS2 gene has a pleotropic effect . Besides being osmosensitive, pbs2 mutants show altered sensitivity to polymyxin B and calcofluor . Recent studies revealed that Pbs2p plays a different role in osmoadaptation and calcofluor sensitivity . We have isolated a gene homologous to PBS2 from the highly salt-tolerant yeast Debaryomyces hansenii by phenotypic complementation . DNA sequencing of the clone revealed that the gene encoded a protein of 683 amino acid residues . Like Pbs2p, this protein also has a proline-rich motif . Further characterization revealed that this gene could complement polymyxin B sensitivity but did not affect calcofluor sensitivity . Thus, it appeared that Pbs2p also has an independent role in these two physiological processes . The GenBank Accession No . of this sequence is AF371315 .

Genetics, 2001 Sep, 159(1), 77 - 89
The Ras/PKA signaling pathway of Saccharomyces cerevisiae exhibits a functional interaction with the Sin4p complex of the RNA polymerase II holoenzyme; Howard SC et al.; Saccharomyces cerevisiae cells enter into the G(0)-like resting state, stationary phase, in response to specific types of nutrient limitation . We have initiated a genetic analysis of this resting state and have identified a collection of rye mutants that exhibit a defective transcriptional response to nutrient deprivation . These transcriptional defects appear to disrupt the control of normal growth because the rye mutants are unable to enter into a normal stationary phase upon nutrient deprivation . In this study, we examined the mutants in the rye1 complementation group and found that rye1 mutants were also defective for stationary phase entry . Interestingly, the RYE1 gene was found to be identical to SIN4, a gene that encodes a component of the yeast Mediator complex within the RNA polymerase II holoenzyme . Moreover, mutations that affected proteins within the Sin4p module of the Mediator exhibited specific genetic interactions with the Ras protein signaling pathway . For example, mutations that elevated the levels of Ras signaling, like RAS2(val19), were synthetic lethal with sin4 . In all, our data suggest that specific proteins within the RNA polymerase II holoenzyme might be targets of signal transduction pathways that are responsible for coordinating gene expression with cell growth.

Genetics, 2001 Sep, 159(1), 65 - 75
Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities; Greene CN et al.; The accumulation of frameshift mutations during DNA synthesis is determined by the rate at which frameshift intermediates are generated during DNA polymerization and the efficiency with which frameshift intermediates are removed by DNA polymerase-associated exonucleolytic proofreading activity and/or the postreplicative mismatch repair machinery . To examine the relative contributions of these factors to replication fidelity in Saccharomyces cerevisiae, we determined the reversion rates and spectra of the lys2 Delta Bgl +1 frameshift allele . Wild-type and homozygous mutant diploid strains with all possible combinations of defects in the exonuclease activities of DNA polymerases delta and epsilon (conferred by the pol3-01 and pol2-4 alleles, respectively) and in mismatch repair (deletion of MSH2) were analyzed . Although there was no direct correlation between homopolymer run length and frameshift accumulation in the wild-type strain, such a correlation was evident in the triple mutant strain lacking all repair capacity . Furthermore, examination of strains defective in one or two repair activities revealed distinct biases in the removal of the corresponding frameshift intermediates by exonucleolytic proofreading and/or mismatch repair . Finally, these analyses suggest that the mismatch repair machinery may be important for generating some classes of frameshift mutations in yeast.

Genetics, 2001 Sep, 159(1), 35 - 45
Roles for internal and flanking sequences in regulating the activity of mating-type-silencer-associated replication origins in Saccharomyces cerevisiae; Sharma K et al.; ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette . Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own . We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins . We could not detect consistent differences in sequence requirements for the two types of origins . Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity . Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase . In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p.

Genetics, 2001 Sep, 159(1), 17 - 33
Mutations in SID2, a novel gene in Saccharomyces cerevisiae, cause synthetic lethality with sic1 deletion and may cause a defect during S phase; Jacobson MD et al.; SIC1 encodes a nonessential B-type cyclin/CDK inhibitor that functions at the G1/S transition and the exit from mitosis . To understand more completely the regulation of these transitions, mutations causing synthetic lethality with sic1 Delta were isolated . In this screen, we identified a novel gene, SID2, which encodes an essential protein that appears to be required for DNA replication or repair . sid2-1 sic1 Delta strains and sid2-21 temperature-sensitive strains arrest preanaphase as large-budded cells with a single nucleus, a short spindle, and an approximately 2C DNA content . RAD9, which is necessary for the DNA damage checkpoint, is required for the preanaphase arrest of sid2-1 sic1 Delta cells . Analysis of chromosomes in mutant sid2-21 cells by field inversion gel electrophoresis suggests the presence of replication forks and bubbles at the arrest . Deleting the two S phase cyclins, CLB5 and CLB6, substantially suppresses the sid2-1 sic1 Delta inviability, while stabilizing Clb5 protein exacerbates the defects of sid2-1 sic1 Delta cells . In synchronized sid2-1 mutant strains, the onset of replication appears normal, but completion of DNA synthesis is delayed . sid2-1 mutants are sensitive to hydroxyurea indicating that sid2-1 cells may suffer DNA damage that, when combined with additional insult, leads to a decrease in viability . Consistent with this hypothesis, sid2-1 rad9 cells are dead or very slow growing even when SIC1 is expressed.

Eur J Biochem, 2001 Sep, 268(18), 4860 - 7
Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor; Leplatois P et al.; Heterologous expression of the human neurotensin receptor type I (hNT1-R) has been achieved in the yeast Saccharomyces cerevisiae . Immunoanalysis of membranes prepared from cells expressing a c-myc-tagged version of hNT1-R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT1-R was glycosylated in yeast . High-affinity binding sites for 125I-labeled-{monoiodo-Tyr3}neurotensin ({125I-Tyr3}NT) were detected on hNT1-R-expressing cells with Kd and Bmax values of 3.2 nM and of 500 receptors per cell, respectively . Competition binding studies of neurotensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT1-R, indicated that the yeast-produced recombinant receptor displayed the same pharmacological properties as hNT1-R expressed in mammalian cells . Interestingly, neurotensin activated the pheromone pathway in hNT1-R-expressing cells in a dose-dependent fashion, as revealed by a beta-galactosidase activity assay with a pheromone-responsive Fus1:lacZ construct . Mutational inactivation of the SST2 and STE2 genes increased the level of beta-galactosidase activity in response to neurotensin by twofold . Recombinant hNT1-R-producing cells, which lacked the endogenous G-protein-coupled receptor for the alpha pheromone, mated with wild-type MATalpha haploid cells in response to neurotensin, leading to bona fide diploid zygote formation . This is the first report of a mammalian receptor that can replace the endogenous pheromone receptor when produced in yeast, by signaling a fully effective, agonist-induced, mating process.

Mikrobiologiia, 2001 Jul-Aug, 70(4), 531 - 5
{Heat shock-induced changes in the respiration of the yeast Saccharomyces cerevisiae}; Rikhvanov EG et al.; The incubation of Saccharomyces cerevisiae at elevated temperature (45 degrees C) stimulated the respiration of yeast cells and decreased their survival rate . The respiration-deficient mutant of this yeast was found to be more tolerant to the elevated temperature than the wild-type strain . At the same time, the cultivation of the wild-type strain in an ethanol-containing medium enhanced the respiration, catalase activity, and thermotolerance of yeast cells, as compared with their growth in a glucose-containing medium . It is suggested that the enhanced respiration of yeast cells at 45 degrees C leads to an intense accumulation of reactive oxygen species, which may be one of the reasons for the heat shock-induced cell death.

J Biol Chem, 2001 Nov 23, 276(47), 43548 - 56 Epub 2001 Sep 13.
The membrane proteins, Spt23p and Mga2p, play distinct roles in the activation of Saccharomyces cerevisiae OLE1 gene expression . Fatty acid-mediated regulation of Mga2p activity is independent of its proteolytic processing into a soluble transcription activator; Chellappa R et al.; The Saccharomyces OLE1 gene encodes the Delta-9 fatty acid desaturase, an enzyme that converts saturated fatty acyl-CoAs into cis-Delta-9 unsaturated fatty acids . OLE1 gene expression is regulated by unsaturated fatty acids, which repress transcription and destabilize the OLE1 mRNA . Expression of OLE1 is activated by N-terminal proteolytic fragments of two homologous endoplasmic reticulum membrane proteins, Spt23p and Mga2p . Disruption of either gene does not significantly affect cell growth or fatty acid metabolism; cells that contain null alleles of both genes, however, are unsaturated fatty acid auxotrophs . An analysis of spt23Delta and mga2Delta strains shows that Spt23p and Mga2p differentially activate and regulate OLE1 transcription . In glucose-grown cells, both genes activate transcription to similar levels of activity . Expressed alone, Mga2p induces high levels of OLE1 transcription in cells exposed to cobalt or grown in glycerol-containing medium . Spt23p expressed alone activates OLE1 transcription to levels similar to those in wild type cells . OLE1 expression is strongly repressed by unsaturated fatty acids in spt23Delta or mga2Delta cells, under all growth conditions . To test if OLE1 expression is controlled by fatty acids at the level of membrane proteolysis, soluble N-terminal fragments of Spt23p and Mga2p that lack their membrane-spanning regions (Deltatm) were expressed under the control of their native promoters in spt23Delta;mga2Delta cells . Under those conditions, Mga2pDeltatm acts as a powerful transcription activator that is strongly repressed by unsaturated fatty acids . By comparison, the Spt23pDeltatm polypeptide weakly activates transcription and shows little regulation by unsaturated fatty acids . Co-expression of the two soluble fragments results in activation to levels observed with the Mga2pDeltatm protein alone . The fatty acid repression of transcription under those conditions is attenuated by Spt23Deltatm, however, suggesting that the two proteins may interact to modulate OLE1 gene expression.

Arch Biochem Biophys, 2001 Sep 15, 393(2), 207 - 14
The functional role of conserved acidic residues of the Qcr7 protein of the cytochrome bc(1) complex in Saccharomyces cerevisiae; Lee SY et al.; The 14-kDa Qcr7 protein represents one of the 10 subunits that are components of a functional cytochrome bc(1) complex in Sacharomyces cerevisiae . Previous studies have shown that the N-terminus of the Qcr7 protein may be involved in the assembly of the cytochrome bc(1) complex and its C-terminus by interacting with cytochrome b and QCR8 proteins . It has also been suggested that Qcr7 protein may be involved in proton pumping . The coding sequence for two highly conserved aspartate residues, D46 and D47, in the QCR7 gene was altered by site-directed mutagenesis and the mutated genes expressed in cells lacking a functional QCR7 gene . Mutants D46E, D46G, D46N, and D47E were comparable to wild type in growth phenotype on nonfermentable carbon sources . Mutants D47G and D47N were respiratory deficient and analysis of complex components by immunoblotting and spectral analysis of cytochrome b suggests defective assembly . Despite being respiratory competent and having normal electron transport rates in broken mitochondria, the mutant D46G had markedly reduced ATP synthesis from electron transport reactions catalyzed by complexes II plus III of the respiratory chain . This suggests that the geometry of proton uptake by the bc(1) complex is disturbed by the mutation in D46 .

Hum Mol Genet, 2001 Sep 1, 10(18), 1889 - 900
Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae; Ellison AR et al.; Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease caused by defects in the process of DNA mismatch repair (MMR), and mutations in the hMLH1 or hMSH2 genes are responsible for the majority of HNPCC . In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice variants, genetic screening has revealed a large number of missense codons with less obvious functional consequences . The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HNPCC where the opportunity exists for early diagnosis and preventive intervention . In this study, quantitative in vivo DNA MMR assays in the yeast Saccharomyces cerevisiae were performed to determine the functional significance of amino acid replacements observed in the human population . Missense codons previously observed in human genes were introduced at the homologous residue in the yeast MLH1 or MSH2 genes . This study also demonstrated feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins . Three classes of missense codons were found: (i) complete loss of function, i.e . mutations; (ii) variants indistinguishable from wild-type protein, i.e . silent polymorphisms; and (iii) functional variants which support MMR at reduced efficiency, i.e . efficiency polymorphisms . There was a good correlation between the functional results in yeast and available human clinical data regarding penetrance of the missense codon . The results reported here raise the intriguing possibility that differences in the efficiency of DNA MMR exist between individuals in the human population due to common polymorphisms.

Genes Cells, 2001 Sep, 6(9), 775 - 88
PKC1, a protein kinase C homologue of Saccharomyces cerevisiae, participates in microtubule function through the yeast EB1 homologue, BIM1; Hosotani T et al.; BACKGROUND: RSC is a chromatin-remodelling complex of Saccharomyces cerevisiae and essential for growth . Its catalytic subunit is encoded by the NPS1/STH1 gene . At the present time, little is known regarding the cellular function of RSC . RESULTS: To identify genes with functions related to NPS1, we screened high-copy suppressor genes for the temperature- and thiabendazole (TBZ)-sensitive mutant allele of NPS1, nps1-105 . Amongst the suppressors we cloned PKC1/STT1 and BIM1 that encoded a homologue of mammalian protein kinase C and a conserved microtubule binding protein homologous to human EB1, respectively . Both the temperature sensitive mutation of PKC1, stt1, and the bim1 null mutation caused synthetic growth defects with nps1-105 . A genetic analysis of the functional relationships between these genes revealed that PKC1 suppressed the defect of nps1-105 through the BIM1 function but not by the activation of the MPK1/MAPK pathway . The stt1 mutation alone showed TBZ sensitivity and delayed the G2-phase progression at semi-permissive temperatures . Both of these stt1 phenotypes were suppressed by the over-expression of BIM1 . In addition, stt1 as well as nps1-105, mis-segregated a mini-chromosome at frequencies higher than the wild-type at a permissive temperature . The mis-segregation was enhanced in the nps1-105 stt1 double mutant . CONCLUSION: These results suggest that Pkc1p plays a role which is relevant to microtubule functions and that this role is mediated by a hitherto unknown PKC signalling pathway and by Bim1p

Radiat Res, 2001 Oct, 156(4), 379 - 87
Cell cycle arrest determines the intensity of the global transcriptional response of Saccharomyces cerevisiae to ionizing radiation; De Sanctis V et al.; Whole-genome analysis was performed using DNA microarrays to define the changes in the gene expression patterns occurring in Saccharomyces cerevisiae cells exposed to ionizing radiation . The effects of sublethal dose on wild-type, rad53 (enhanced sensitivity to radiation and impaired in a cell cycle damage checkpoint), and rad6 (enhanced sensitivity to radiation and functional cell cycle block by radiation) mutant backgrounds and of a higher dose on the wild-type and G(2)-phase-arrested cells were analyzed . Several gene pathways were identified as being implicated in the response to radiation . In particular, the cell cycle blockage that occurred in the wild-type strain after a high radiation dose and in the rad6 mutant after a lower dose entailed modifications of defined gene expression patterns, which are described here and are compared with the gene modulation patterns observed in the rad53 strain in the absence of efficient blockage . Loss of the RAD53 function caused a major increase in the number of genes modulated by radiation . Given that Rad53-Sad1p, the protein encoded by RAD53, has functions other than those directly connected to cell cycle arrest, we determined the gene patterns that were modulated upon irradiation of rad53 cells that had been forced to arrest in G(2) phase by nocodazole treatment . These differential whole-genome analyses shed light on the multiplicity of functions of the pivotal Rad53-Sad1p protein . The results obtained describe how the cells respond to different irradiation conditions by modulating important gene classes, including those associated with stress defense, ribosomal proteins, histones, ergosterol and GCR1-controlled sugar metabolism.

Mol Biol Cell, 2001 Sep, 12(9), 2646 - 59
ADY1, a novel gene required for prospore membrane formation at selected spindle poles in Saccharomyces cerevisiae; Deng C et al.; ADY1 is identified in a genetic screen for genes on chromosome VIII of Saccharomyces cerevisiae that are required for sporulation . ADY1 is not required for meiotic recombination or meiotic chromosome segregation, but it is required for the formation of four spores inside an ascus . In the absence of ADY1, prospore formation is restricted to mainly one or two spindle poles per cell . Moreover, the two spores in the dyads of the ady1 mutant are predominantly nonsisters, suggesting that the proficiency to form prospores is not randomly distributed to the four spindle poles in the ady1 mutant . Interestingly, the meiosis-specific spindle pole body component Mpc54p, which is known to be required for prospore membrane formation, is localized predominantly to only one or two spindle poles per cell in the ady1 mutant . A partially functional Myc-Pfs1p is localized to the nucleus of mononucleate meiotic cells but not to the spindle pole body or prospore membrane . These results suggest that Pfs1p is specifically required for prospore formation at selected spindle poles, most likely by ensuring the functionality of all four spindle pole bodies of a cell during meiosis II.

Mol Biol Cell, 2001 Sep, 12(9), 2614 - 28
Aux1p/Swa2p is required for cortical endoplasmic reticulum inheritance in Saccharomyces cerevisiae; Du Y et al.; In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus . The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event . To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p) . This approach has revealed that AUX1/SWA2 plays a role in ER inheritance . The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles . Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells . These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance . In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes.

Curr Biol, 2001 Sep 4, 11(17), 1328 - 35
The role of the Mre11-Rad50-Xrs2 complex in telomerase- mediated lengthening of Saccharomyces cerevisiae telomeres; Tsukamoto Y et al.; BACKGROUND: The Saccharomyces Mre11p, Rad50p, and Xrs2p proteins form a complex, called the MRX complex, that is required to maintain telomere length . Cells lacking any one of the three MRX proteins and Mec1p, an ATM-like protein kinase, undergo telomere shortening and ultimately die, phenotypes characteristic of cells lacking telomerase . The other ATM-like yeast kinase, Tel1p, appears to act in the same pathway as MRX: mec1 tel1 cells have telomere phenotypes similar to those of telomerase-deficient cells, whereas the phenotypes of tel1 cells are not exacerbated by the loss of a MRX protein . RESULTS: The nuclease activity of Mre11p was found to be dispensable for the telomerase-promoting activity of the MRX complex . The association of the single-stranded TG1-3 DNA binding protein Cdc13p with yeast telomeres occurred efficiently in the absence of Tel1p, Mre11p, Rad50p, or Xrs2p . Targeting of catalytically active telomerase to the telomere suppressed the senescence phenotype of mec1 mrx or mec1 tel1 cells . Moreover, when telomerase was targeted to telomeres, telomere lengthening was robust in mec1 mrx and mec1 tel1 cells . CONCLUSIONS: These data rule out models in which the MRX complex is necessary for Cdc13p binding to telomeres or in which the MRX complex is necessary for the catalytic activity of telomerase . Rather, the data suggest that the MRX complex is involved in recruiting telomerase activity to yeast telomeres.

J Biol Chem, 2001 Nov 9, 276(45), 41906 - 12 Epub 2001 Sep 10.
Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA; Hong EL et al.; Dmc1 and Rad51 are eukaryotic RecA homologues that are involved in meiotic recombination . The expression of Dmc1 is limited to meiosis, whereas Rad51 is expressed in mitosis and meiosis . Dmc1 and Rad51 have unique and overlapping functions during meiotic recombination . Here we report the purification of the Dmc1 protein from the budding yeast Saccharomyces cerevisiae and present basic characterization of its biochemical activity . The protein has a weak DNA-dependent ATPase activity and binds both single-strand DNA (ssDNA) and double-strand DNA . Electrophoretic mobility shift assays suggest that DNA binding by Dmc1 is cooperative . Dmc1 renatures linearized plasmid DNA with first order reaction kinetics and without requiring added nucleotide cofactor . In addition, Dmc1 catalyzes strand assimilation of ssDNA oligonucleotides into homologous supercoiled duplex DNA in a reaction promoted by ATP or the non-hydrolyzable ATP analogue AMP-PNP.

Epidemiol Mikrobiol Imunol, 2001 Aug, 50(3), 131 - 5
{Initial experience with detection of Saccharomyces cerevisiae antibodies in patients with primary nonspecific inflammatory bowel disease}; Malickova K et al.; INTRODUCTION: Inflammatory bowel diseases (IBD), with Crohn's disease (CD) and ulcerative colitis (UC) as the two main disorders, is a heterogeneous group of diseases of unknown etiology . Actually we have no ideal disease marker, to identify people at risk of the disease, which can differentiate CD from UC, be highly specific for CD or UC and easily applicable in routine laboratory praxis . AIMS: Determine the clinical significance of serological testing p-ANCA and ASCA in patients with IBD . METHODS: P-ANCA in IgG isotype were detected by indirect fluorescence assay on human ethanol-fixed granulocytes, ASCA antibodies in IgG and IgA isotypes were determined by ELISA with mannan as a target antigen . RESULTS: P-ANCA and ASCA were studied in a group of 86 patients (38 CD, 26 UC, 3 non-inflammatory gastrointestinal disorder, 19 health controls) . P-ANCA was associated with UC in 46% . ASCA was associated with CD in 76% . Specificity of ANCA for UC compared to healthy controls was 100%, specificity of ASCA for CD compared to healthy controls was 89.5% . CONCLUSION: Although the sensitivity of ASCA and p-ANCA is low, their specificity is high, especially when combining these two markers . We think that combined assay for ASCA and p-ANCA is more useful in IBD.

J Biomed Sci, 2001 Sep, 8(5), 416 - 20
Functional correlation between the nuclear localization of Fht1p and its flocculation and heat tolerance activities in budding yeast Saccharomyces cerevisiae; Iha H et al.; Fht1p is involved in the flocculation and heat tolerance machinery of budding yeast Saccharomyces cerevisiae . Despite knowledge of its involvement in those phenotypes, a precise mechanism has yet to be discovered . To this end, we monitored the relationship between subcellular localization of Fht1p and its flocculation or heat tolerance function using newly developed expression vectors with a recombinant green fluorescent protein (GFP; S65T/S147P) of Aequorea victoria added at both the N- and C-terminus of Fht1p . The main fluorescent signal of the GFP tagged with either a wild-type Fht1p or mutants which preserve their flocculation function was detected in the nucleus, whereas signals of functionless mutants were dispersed to the cytoplasm .

Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 513 - 6
Coupling effects of osmotic pressure and temperature on the viability of Saccharomyces cerevisiae; Beney L et al.; The osmotic tolerance of cells of Saccharomyces cerevisiae as a function of glycerol concentration and temperature has been investigated . Results show that under isothermal conditions (25 degrees C) cells are resistant (94% viability) to hyperosmotic treatment at 49.2 MPa . A thigher osmotic pressure, cell viability decreases to 25% at 99 MPa . Yeast resistance to high osmotic stress (99 Mpa) is enhanced at low temperatures (5-11 degrees C) . Therefore, the temperature at which hyperosmotic pressure is achieved greatly affects cell viability . These results suggest that temperature control is a suitable means of enhancing cell survival in response to osmotic dehydration.

J Biol Chem, 2001 Nov 9, 276(45), 41769 - 81 Epub 2001 Sep 06.
Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p; Dodt G et al.; PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1) . Although PEX5 is not involved in the import of PTS2-targeted proteins in yeast, it is essential for PTS2 protein import in mammalian cells . Human cells generate two isoforms of PEX5 through alternative splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long . Only one isoform, PEX5L, is involved in PTS2 protein import, and PEX5L physically interacts with PEX7, the import receptor for PTS2-containing proteins . In this report we map the regions of human PEX5L involved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes . These studies revealed that amino acids 1-230 of PEX5L are required for PTS2 protein import, amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for targeting to peroxisomes . We also identified a 21-amino acid-long peptide motif of PEX5L, amino acids 209-229, that overlaps the regions sufficient for full PTS2 rescue activity and PEX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p, two yeast peroxins that act only in PTS2 protein import in yeast . A mutation in PEX5 that changes a conserved serine of this motif abrogates PTS2 protein import in mammalian cells and reduces the interaction of PEX5L and PEX7 in vitro . This peptide motif also lies within regions of Pex18p and Pex21p that interact with yeast PEX7 . Based on these and other results, we propose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import . This hypothesis may explain the essential role of PEX5L in PTS2 protein import in mammalian cells and its lack of importance for PTS2 protein import in yeast.

Int J Food Microbiol, 2001 Aug 15, 68(1-2), 155 - 60
Release of nitrogen compounds to the extracellular medium by three strains of Saccharomyces cerevisiae during induced autolysis in a model wine system; Martinez-Rodriguez AJ et al.; To detect differences among three strains of Saccharomyces cerevisiae used in the manufacture of sparkling wines and to study the changes in nitrogen compounds during autolysis, a model wine system was used . Significant differences were observed between the mean values of the autolytic capacity of the three strains . The amount of nitrogen (total, protein, peptide and amino) present in the autolysates and the concentration of most free amino acids was significantly affected by the strain . These findings suggest that the strain of yeast used in the manufacture of sparkling wines can play an important role in the aging process and can affect final composition.

J Biol Chem, 2001 Nov 9, 276(45), 41710 - 6 Epub 2001 Sep 05.
The initial step of the glycerolipid pathway: identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae; Zheng Z et al.; The initial step of phospholipid biosynthesis in yeast is carried out through the acylation of glycerol 3-phosphate (G-3-P) and dihydroxyacetone phosphate by stereospecific sn-1 acyltransferases . Here we report the identification of two key fatty acyltransferases of the glycerolipid biosynthesis pathway in Saccharomyces cerevisiae . Disruption of the open reading frame YBL011w, corresponding to a gene previously identified as a choline transporter suppressor (SCT1), resulted in a substantial decrease of total cellular G-3-P acyltransferase activity . A yeast strain disrupted at the open reading frame YKR067w, which encodes a protein closely related to Sct1p, also exhibited a dramatic reduction in G-3-P acyltransferase activity . Molecular characterizations of the genes revealed that a missense mutation in YKR067w accounted for a defect in the activities of the G-3-P acyltransferase in the yeast mutant strain TTA1 . Heterologous expression of YKR067w in Escherichia coli further confirmed its enzyme activity . These results indicate that YKR067w and YBL011w, designated herein as GAT1 and GAT2(SCT1), respectively, are yeast G-3-P acyltransferase genes . Furthermore, biochemical results are presented to show that both Gat1p and Gat2p(Sct1p) are G-3-P/dihydroxyacetone phosphate dual substrate-specific sn-1 acyltransferases . The fatty acyl specificity of Gat1p is similar to that of the mammalian microsomal G-3-P acyltransferase, as it can effectively utilize a broad range of fatty acids as acyl donors . In contrast, Gat2p(Sct1p) displayed preference toward 16-carbon fatty acids . The most notable of the altered phospholipid compositions of the gat1Delta and gat2(sct1)Delta strains are a decreased phosphatidic acid pool and an increased phosphatidylserine/phosphatidylinositol ratio . This did not appear to affect the mutants as no growth defect was found . However, null mutations of both GAT1 and GAT2(SCT1) are synthetically lethal to yeast.

Yeast, 2001 Sep 15, 18(12), 1155 - 71
Functional analysis of the Saccharomyces cerevisiae YFR021w/YGR223c/YPL100w ORF family suggests relations to mitochondrial/peroxisomal functions and amino acid signalling pathways; Georgakopoulos T et al.; Saccharomyces cerevisiae YFR021w, YGR223c and YPL100w are paralogous ORFs of unknown function . Phenotypic analysis of overexpression, single-, double- and triple-ORF deletion strains under various growth conditions indicated mitochondria-related functions for all three ORFs . Two-hybrid screens of a yeast genomic library identified potentially interacting proteins for the three ORFs . Among these, the transcriptional activator Rtg3p interacted with both Yfr021wp and Ypl100wp and both ORF single deletions reduced the constitutive expression of the RTG-regulated CIT2 and DLD3 genes and caused typical retrograde response of CIT2 and DLD3 under growth conditions requiring functional mitochondria, indicating that YFR021w and YPL100w are also involved in unidentified mitochondrial functions . Ptr3p, a component of the amino acid sensor Ssy1p/Ptr3p, was also found as a two-hybrid interactant of Yfr021wp . Of the three single-ORF deletions, ypl100w Delta exhibited ptr3 Delta-similar phenotypes . These findings, combined with the fact that RTG-dependent expression is modulated by specific amino acids, suggested possible relations of Yfr021wp and Ypl100wp to amino acid signalling pathways . Under most conditions examined, the effects of the single- and double-ORF deletions indicated that YFR021w, YPL100w and YGR223c are not parts of the same pathway . We found no unique phenotype attributed to the deletion of YGR223c . However, its function interferes with the function of the other two ORFs, as revealed by the effects of double- and triple-ORF deletions .

Oncogene, 2001 Aug 30, 20(38), 5279 - 90
Toxicity of human adenovirus E4orf4 protein in Saccharomyces cerevisiae results from interactions with the Cdc55 regulatory B subunit of PP2A; Roopchand DE et al.; The E4orf4 protein of human adenovirus induces p53-independent apoptosis, a process that may promote cell death and viral spread . When expressed alone, E4orf4 kills transformed cells but not normal human cells . The only clear target of E4orf4 in mammalian cells is the Balpha (B55) subunit of protein phosphatase 2A (PP2A), a member of one of three classes of regulatory B subunits . Here we report the effects of E4orf4 in Saccharomyces cerevisiae, which encodes two PP2A regulatory B subunits, CDC55 and RTS1, that share homology with mammalian B and B' subunits, respectively . E4orf4 expression was found to be toxic in yeast, resulting in the accumulation of cells in G2/M phase that failed to grow upon removal of E4orf4 . E4orf4-expressing yeast also displayed an elongated cell morphology similar to cdc55 deletion strains . E4orf4 required CDC55 to elicit its effect, whereas RTS1 was dispensable . The recruitment of the PP2A holoenzyme by E4orf4 was entirely dependent on Cdc55 . These studies indicate that E4orf4-induced apoptosis in mammalian cells and cell death in yeast require functional interactions with B-type subunits of PP2A . However, some inhibition of growth by E4orf4 was observed in the cdc55 strain and with an E4orf4 mutant that fails to interact with Cdc55, indicating that E4orf4 may possess a second Cdc55-independent function affecting cell growth.

Biochemistry, 2001 Sep 11, 40(36), 10800 - 9
Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae; Shi W et al.; Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine . It is the sole catalyst for adenine recycling in most eukaryotes . The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues . Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 A, respectively . Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded beta-sheet surrounded by five alpha-helices, a structural theme found in all known purine phosphoribosyltransferases . The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form . Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket . Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively . Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-fold, implicating altered dynamics for the catalytic site loop.

Mol Cell Biol, 2001 Oct, 21(19), 6559 - 73
Intrachromatid excision of telomeric DNA as a mechanism for telomere size control in Saccharomyces cerevisiae; Bucholc M et al.; We have previously identified a process in the yeast Saccharomyces cerevisiae that results in the contraction of elongated telomeres to wild-type length within a few generations . We have termed this process telomeric rapid deletion (TRD) . In this study, we use a combination of physical and genetic assays to investigate the mechanism of TRD . First, to distinguish among several recombinational and nucleolytic pathways, we developed a novel physical assay in which HaeIII restriction sites are positioned within the telomeric tract . Specific telomeres were subsequently tested for HaeIII site movement between telomeres and for HaeIII site retention during TRD . Second, genetic analyses have demonstrated that mutations in RAD50 and MRE11 inhibit TRD . TRD, however, is independent of the Rap1p C-terminal domain, a central regulator of telomere size control . Our results provide evidence that TRD is an intrachromatid deletion process in which sequences near the extreme terminus invade end-distal sequences and excise the intervening sequences . We propose that the Mre11p-Rad50p-Xrs2p complex prepares the invading telomeric overhang for strand invasion, possibly through end processing or through alterations in chromatin structure.

Mol Cell Biol, 2001 Oct, 21(19), 6395 - 405
Upc2p and Ecm22p, dual regulators of sterol biosynthesis in Saccharomyces cerevisiae; Vik A et al.; Sterol levels affect the expression of many genes in yeast and humans . We found that the paralogous transcription factors Upc2p and Ecm22p of yeast were sterol regulatory element (SRE) binding proteins (SREBPs) responsible for regulating transcription of the sterol biosynthetic genes ERG2 and ERG3 . We defined a 7-bp SRE common to these and other genes, including many genes involved in sterol biosynthesis . Upc2p and Ecm22p activated ERG2 expression by binding directly to this element in the ERG2 promoter . Upc2p and Ecm22p may thereby coordinately regulate genes involved in sterol homeostasis in yeast . Ecm22p and Upc2p are members of the fungus-specific Zn{2}-Cys{6} binuclear cluster family of transcription factors and share no homology to the analogous proteins, SREBPs, that are responsible for transcriptional regulation by sterols in humans . These results suggest that Saccharomyces cerevisiae and human cells regulate sterol synthesis by different mechanisms.

Mol Microbiol, 2001 Aug, 41(3), 717 - 30
Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects; Alonso-Monge R et al.; The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature . Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22 . Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37 degrees C . This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition . The suppression of the temperature-induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall-degrading enzymes . LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants . In addition, the isolated multicopy suppressor genes suppress temperature-induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high-osmolarity response routes.

Genes Cells, 2001 Aug, 6(8), 677 - 87
Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae; Pouliot JJ et al.; BACKGROUND: The covalent linkage between DNA and the active site tyrosine of topoisomerase I can be stabilized by chemotherapeutic agents, adjacent DNA lesions, or mutational defects in the topoisomerase itself . Following collision with a replication fork, the covalent complex can be converted to a double-strand break . Tdp1, an enzyme that can hydrolyse the bond between topoisomerase I and DNA, is thought to be involved in the repair of these lesions, but little is known about how such repair is accomplished . RESULTS: Reaction kinetics with model substrates reveal that the catalytic efficiency of Saccharomyces cerevisiae Tdp1 is relatively poor when the scissile bond is located in the middle of a duplex, but much better when it is located at the end of a structure . Survival of yeast after induction of a toxic topoisomerase is substantially reduced by inactivation of the TDP1 gene . Comparison of survival of single and double mutants places TDP1 and RAD52 in the same epistasis group but TDP1 and RAD9 in different epistasis groups . In the absence of RAD9, inactivation of TDP1 has a significant effect on the survival of cells following exposure to camptothecin but is without consequence for the survival of agents that do not target topoisomerase I . CONCLUSIONS: Tdp1 acts as a specific repair enzyme for topoisomerase I lesions . Rather than working at their earliest occurrence, the enzyme acts after covalent complexes have been converted to DSBs . A second repair pathway also exists that functions independently of Tdp1 but requires RAD9 function to efficiently repair topoisomerase I-linked DSBs . The efficiency of these pathways differs for complexes induced with the chemotherapeutic agent camptothecin vs . those accumulated by mutant forms of topoisomerase I.

Biochem Cell Biol, 2001, 79(4), 517 - 24
Contribution of leucine 85 to the structure and function of Saccharomyces cerevisiae iso-1 cytochrome c; Parrish JC et al.; Cytochrome c is a small electron-transport protein whose major role is to transfer electrons between complex III (cytochrome reductase) and complex IV (cytochrome c oxidase) in the inner mitochondrial membrane of eukaryotes . Cytochrome c is used as a model for the examination of protein folding and structure and for the study of biological electron-transport processes . Amongst 96 cytochrome c sequences, residue 85 is generally conserved as either isoleucine or leucine . Spatially, the side chain is associated closely with that of the invariant residue Phe82, and this interaction may be important for optimal cytochrome c activity . The functional role of residue 85 has been examined using six site-directed mutants of Saccharomyces cerevisiae iso-1 cytochrome c, including, for the first time, kinetic data for electron transfer with the principle physiological partners . Results indicate two likely roles for the residue: first, heme crevice resistance to ligand exchange, sensitive to both the hydrophobicity and volume of the side chain; second, modulation of electron-transport activity through maintenance of the hydrophobic character of the protein in the vicinity of Phe82 and the exposed heme edge, and possibly of the ability of this region to facilitate redox-linked conformational change.

Acta Crystallogr D Biol Crystallogr, 2001 Sep, 57(Pt 9), 1321 - 3 Epub 2001 Aug 23.
Crystallization of the catalytic subunit of Saccharomyces cerevisiae acetohydroxyacid synthase; Pang SS et al.; Acetohydroxyacid synthase (AHAS; E.C . 4.1.3.18) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids isoleucine, leucine and valine . It is a thiamin diphosphate-dependent enzyme which catalyses the decarboxylation of pyruvate and its condensation with either 2-ketobutyrate or a second molecule of pyruvate to give 2-aceto-2-hydroxybutyrate or 2-acetolactate, respectively . The enzyme is the target of sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors . Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Saccharomyces cerevisiae AHAS is reported . Data to 2.7 A resolution have been collected using synchrotron radiation (Advanced Photon Source, Chicago) . Crystals have unit-cell parameters a = 95.8, b = 110.0, c = 178.9 A and belong to the space group P2(1)2(1)2(1) . Preliminary analysis indicates there is one dimer located in each asymmetric unit.

Curr Genet, 2001 Jul, 39(5-6), 284 - 90
Genome-wide expression analysis of a Saccharomyces cerevisiae strain deleted for the Tup1p-interacting protein Cdc73p; Kerkmann K et al.; Tuplp is a general corepressor in Saccharomyces cerevisiae . We performed a split-ubiquitin screen with Tup1p as bait, in order to understand how Tuplp mediates repression . Cdc73p was the only component of a holoenzyme of transcription isolated as a Tuplp-interacting protein in this screen . Expression analysis of a strain deleted for CDC73 on a genome-wide scale revealed that Cdc73p is involved in the positive and negative regulation of transcription . Our results show that a subset of genes is regulated by both Cdc73p and Tup1p . Furthermore, our results indicate that Cdc73p plays a role in the repression of telomere-proximal genes, which are not repressed by Tup1p.

Curr Genet, 2001 Jul, 39(5-6), 273 - 83
The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions; Aguilera J et al.; The enzyme aldose reductase plays an important role in the osmo-protection mechanism of diverse organisms . Here, we show that yeast aldose reductase is encoded by the GRE3 gene . Expression of GRE3 is carbon-source independent and up-regulated by different stress conditions, such as NaCl, H2O2, 39 degrees C and carbon starvation . Measurements of enzyme activity and intracellular sorbitol in wild-type cells also indicate that yeast aldose reductase is stress-regulated . Overexpression of GRE3 increases methylglyoxal tolerance in Saccharomyces cerevisiae . Furthermore, high expression of GRE3 complements the deficiency of the glyoxalase system of a glo1delta mutant strain . Consistent with this, in vitro and in vivo assays of yeast aldose reductase activity indicate that methylglyoxal is an endogenous substrate of aldose reductase . Furthermore, addition of NaCl or H2O2 to exponential-phase cells triggers an initial transient increase in the intracellular level of methylglyoxal, which is dependent on the Gre3p and Glo1p function . These observations indicate that the metabolism of methylglyoxal is stimulated under stress conditions; and they support a methylglyoxal degradative pathway, in which this compound is metabolised by the action of aldose reductase.

Mol Genet Genomics, 2001 Jul, 265(5), 930 - 40
Functional cloning, based on azole resistance in Saccharomyces cerevisiae, and characterization of Rhizopus nigricans redox carriers that are differentially involved in the P450-dependent response to progesterone stress; Kunic B et al.; The filamentous fungus Rhizopus nigricans responds to treatment with progesterone by inducing P450-associated redox carriers . Selection for azole resistance following expression of a cDNA library constructed with RNA from progesterone-treated R . nigricans in the yeast Saccharomyces cerevisiae led to the identification of CPR1-FL and CYB5-1 cDNAs, which code for functionally competent NADPH-cytochrome P450 reductase and cytochrome b5, respectively . The central region (CPR2-CS) of an additional reductase gene sharing 66% identity with CPR1-FL was cloned from progesterone-induced mRNA by RT-PCR, using primers based on consensus sequences . Northern analysis of the 2.1-kb transcripts revealed that, of the two cloned reductase genes, only CPR1-FL mRNA was strongly induced by progesterone; transcription of CYBS-1 and CPR2-CS mRNAs was not significantly affected . Analysis of the subcellular localization and function of the R . nigricans reductase in yeast indicated that the CPR1-FL cDNA and a derivative (CPR1-S) truncated at the first ATG codon gave rise to functionally equivalent products that were found in both cytosolic and microsomal fractions . In contrast, addition of an in-frame initiation codon at the 5' end of the CPR1-FL sequence resulted in localization of the activity mainly to the microsomes, and improved ketoconazole resistance but decreased NADPH-cytochrome c reductase activity in the host strain . These findings suggest that, of the three genes for P450-associated redox carriers investigated, only CPR1-FL is associated with the progesterone response and that its major transcript encodes a reductase that shows an unusual pattern of subcellular localization.

Mol Genet Genomics, 2001 Jul, 265(5), 873 - 82
Metal transporters that contribute copper to metallochaperones in Saccharomyces cerevisiae; Portnoy ME et al.; Copper metallochaperones represent a new family of soluble, low-molecular-weight proteins that function to deliver copper to specific sites within a cell . How the metallochaperones acquire their copper, however, is not known . In this study, we have conducted a survey of known metal ion transporters in bakers' yeast, Saccharomyces cerevisiae, to identify those that contribute copper to pathways involving the metallochaperones Atxlp and Lys7p . The results indicatethat, in addition to the well known Ctr1p and Ctr3p high-affinity copper transporters, the metallochaperones can acquire their copper through pathways involving the relatively non-specific divalent metal ion transporter Fet4p and the putative low-affinitycopper transporter Ctr2p . We have examined the localization of Ctr2p using an epitope tagged version of the protein and find that Ctr2p does not localize to the cell surface but may operate at the level of the vacuole to mobilize intracellular copper . Inaddition to Ctrlp, Ctr2p, Ctr3p and Fet4p, other metal transport systems can act as upstream donors of copper for the metallochaperones when copper availability in the medium is increased . Although the nature of these auxiliary systems is unknown, they do not appear to involve the yeast members of the Nramp family of divalent transporters, or uptake mechanisms that involve endocytosis . Since vastly different metal transporters located at either the cell surface or intracellular sites can all contribute copper to metallochaperones, it is unlikely that the metallochaperones directly interact with the metal transporters to obtain the metal.

Mol Genet Genomics, 2001 Jul, 265(5), 826 - 36
Transcription of mutS- and mutL-homologous genes during meiosis in Saccharomyces cerevisiae and identification of a regulatory cis-element for meiotic induction of MSH2; Meyer C et al.; We have analysed the levels of mRNA transcripts of the mutS- and mutL-homologous genes of the yeast Saccharomyces cerevisiae during the course of meiosis, by quantitative RT-PCR . We found that all mutS homologues (MSH1-6) were induced during meiosis, whereas no evidence for regulation of the mutL homologues (PMS1, MLH1-3) was obtained . Temporal expression patterns indicative of co-regulation were observed for the gene pairs MSH4/MSH5 and MSH2/ SPO11 . Sequence comparisons of the 5' flanking regions revealed similar sequence stretches in the respective gene pairs, which may constitute regulatory elements . Similar sequences were also found in the 5' flanking regions of the pairs MSH1/MSH3 and MSH1/MSH6 . Upstream of MSH2 three closely spaced sequences similar to UASH elements were found, which - surprisingly are located within the coding region of SPO21 . Deletion of these elements resulted in loss of meiotic induction of MSH2 . Genetic analysis of homozygous deletion mutants did not reveal any differences from wild type with respect to genetic distance estimates, aberrant segregation, or suppression of homoeologus recombination in an interspecies cross with Saccharomyces paradoxus.

Mol Genet Genomics, 2001 Aug, 265(6), 1087 - 96
Synergism between base excision repair, mediated by the DNA glycosylases Ntg1 and Ntg2, and nucleotide excision repair in the removal of oxidatively damaged DNA bases in Saccharomyces cerevisiae; Gellon L et al.; In Saccharomyces cerevisiae, inactivation of the two DNA N-glycosylases Ntg1p and Ntg2p does not result in a spontaneous mutator phenotype, whereas simultaneous inactivation of Ntglp, Ntg2p and Radlp or Rad14p, both of which are involved in nucleotide excision repair (NER), does . The triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 show 15- and 22-fold increases, respectively, in spontaneous forward mutation to canavanine resistance (CanR) relative to the wild-type strain (WT) . In contrast, neither of these triple mutants shows an increase in the incidence of Lys+ revertants of the lys1-1 ochre allele . Furthermore, the rad1 ntg1 ntg2 mutant is hypersensitive to the lethal effect of H2O2 relative to WT, rad1 and ntg1 ntg2 mutant strains . Moreover, the rad1 ntg1 ntg2 strain is hypermutable (CanR and Lys+) upon exposure to H2O2, relative to WT, rad1 and ntg1 ntg2 strains . Mutagen sensitivity and enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant, relative to the other strains tested, were also observed upon exposure to oxidizing agents such as tertbutylhydroperoxide and menadione . In contrast, the sensitivity of the rad1 ntg1 ntg2 triple mutant to gamma-irradiation does not differ from that of the WT . However, the triple mutant shows an increase in the frequency of Lys+ revertants recovered after gamma-irradiation . The results reported in this study demonstrate that base excision repair (BER) mediated by Ntglp and Ntg2p acts synergistically with NER to repair endogenous or induced lethal and mutagenic oxidative DNA damage in yeast . The substrate specificity of Ntg1 p and Ntg2p, and the spectrum of lesions induced by the DNA-damaging agents used, strongly suggest that oxidized DNA bases, presumably oxidized pyrimidines, represent the major targets of this repair pathway.

Mol Genet Genomics, 2001 Aug, 265(6), 1076 - 86
Proteins that genetically interact with the Saccharomyces cerevisiae transcription factor Gal11p emphasize its role in the initiation-elongation transition; Badi L et al.; The GAL11 gene encodes a transcription factor that is a component of the SRB/Mediator sub-complex of the RNA polymerase II holoenzyme in the yeast Saccharomyces cerevisiae . In agreement with this biochemical characterization, Gal11p has been found to be required for optimal production of mRNA from many yeast promoters, and recessive mutations in GAL11 have been shown to cause pleiotropic defects . Despite this progress, the role of Gal11p in gene regulation remains largely unknown . In a multicopy suppressor analysis of a gal11delta mutation we have identified genes encoding proteins that are part of, or can interact with, the RNA polymerase II transcription complex, as well as factors involved in cell cycle regulation . Among the suppressors that are clearly related to the transcriptional apparatus, Gal11p genetically interacts with components of the SRB/Mediator complex, as well as with factors such as TFIIE and TFIIH that are required for promoter clearance and transcription elongation by RNA polymerase II . These findings, taken together with published results of biochemical and genetic analyses, suggest a role for Galllp at the interface between the SRB/Mediator complex and the general transcription factors TFIIE and TFIIH, which modulate, via phosphorylation of the CTD, the activity of the RNA polymerase II during the transition between initiation and elongation.

Nucleic Acids Res, 2001 Sep 1, 29(17), 3631 - 7
Rpm2p: separate domains promote tRNA and Rpm1r maturation in Saccharomyces cerevisiae mitochondria; Stribinskis V et al.; Rpm2p is a protein subunit of yeast mitochondrial RNase P and is also required for the maturation of Rpm1r, the mitochondrially-encoded RNA subunit of the enzyme . Previous work demonstrated that an insertional disruption of RPM2, which produces the C-terminally truncated protein Rpm2-DeltaCp, supports growth on glucose but cells lose some or all of their mitochondrial genome and become petite . These petites, even if they retain the RPM1 locus, lose their ability to process the 5'-ends of mitochondrial tRNA . We report here that if strains containing the truncated RPM2 allele are created and maintained on respiratory carbon sources they have wild-type mitochondrial genomes, and a significant portion of tRNA transcripts are processed . In contrast, precursor Rpm1r transcripts accumulate and mature Rpm1r is not made . These data show that one function of the deleted C-terminal region is in the maturation of Rpm1r, and that this region and mature Rpm1r are not absolutely required for RNase P activity . Finally, we demonstrate that full activity can be restored if the N-terminal and C-terminal domains of Rpm2p are supplied in trans.

Biochim Biophys Acta, 2001 Aug 17, 1506(2), 89 - 102
Analysis of suppressor mutation reveals long distance interactions in the bc(1) complex of Saccharomyces cerevisiae; Brasseur G et al.; Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b(L) and b(H), of the bc(1) complex . The conserved glycine 131 is involved in the packing of heme b(L) and is separated by only 3 A from this heme in the bc(1) complex structure . The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc(1) complex . An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine . This respiratory competent revertant exhibited a low bc(1) complex activity and was affected in the electron transfer at the Q(P) site . The k(min) for the substrate DBH(2) was diminished by an order of magnitude and EPR spectra showed a partially empty Q(P) site . However, the binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain . Optical spectroscopy revealed that heme b(L) is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values . Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc(1) complex and the insertion of heme b(L) despite the presence of the serine at position 131 . Surprisingly, reversion at position 260 was located 13 A away from the original mutation and revealed a long distance interaction in the yeast bc(1) complex.

Int J Food Microbiol, 2001 Aug 5, 67(3), 241 - 6
Involvement of osmotic cell shrinkage on the proton extrusion rate in Saccharomyces cerevisiae; Martinez de Maranon I et al.; Saccharomyces cerevisiae has been subjected to hyperosmotic shocks by using permeating (sorbitol, xylitol, glycerol, NaCl) and nonpermeating (PEG 600) solutes . The proton extrusion rate decreased as the osmotic pressure increased, whichever solute was used . However, the total inhibition of the cellular H+ extrusion depended on the solute used . A total inhibition was observed at about 20 MPa with glycerol, xylitol and sorbitol . With PEG 600, a total inhibition of extracellular acidification was obtained at 8.5 MPa . NaCl, with an extracellular pressure of 37.8 MPa (near saturation), did not completely inhibit the extracellular acidification . These results showed that the total inhibition of proton extrusion, involving probably the membrane H+-ATPase . was not correlated to the hydric state of the external medium but was strictly linked to the degree of permeation of solutes across the plasma membrane . The extracellular acidification was totally inhibited by a critical final cell volume reached after the osmotic shrinkage, whichever solute was used . This critical final cell volume represented 50% of the initial cell volume . This result suggests that the final cell volume reached after an osmotic stress represents a key thermodynamic parameter for cell osmoregulation in which H+-ATPase would be implicated.

Genetika, 2001 Jun, 37(6), 762 - 9
{Genetic control of growth and development of yeast Saccharomyces cerevisiae cells . Phenotypic selection of mutants among strains of the Peterhof genetic collection}; Chitavichius D; Results of identifying phenotypes intrinsic to mutations of genes that regulate the activity of the signal transduction pathway of RAS-cyclic adenosinemonophosphate in six strains belonging to the Peterhof genetic collection of the yeast Saccharomyces cerevisiae are presented: an increase or decrease in the amount of the accumulated glycogen, resistance or sensitivity to heat shock and nitrogen starvation, and the growth and viability in media containing unfermentable carbon sources (potassium acetate, ethanol, and glycerol) at temperatures 30 and 70 degrees C . Collectively these phenotypic characteristics in five examined yeast strains can be interpreted as indicating disturbances in the activity of the RAS/cAMP pathway . However, the discrepancies revealed between cellular phenotypes in these strains and in the strains with a decreased or increased activity of the RAS/cAMP pathway did not allow them to be assigned to a particular functional activity group . The inconsistencies between phenotypes detected in this study may be prerequisites for the identification of new genes responsible for this signal transduction pathway or new mutations in the known genes that determine other phenotypic combinations.

Genetika, 2001 Jun, 37(6), 754 - 61
{Detection of concealed "illegitimate" nuclei in tetrad analysis of the diploid progeny of heterokaryons in Saccharomyces cerevisiae}; Nevzgliadova OV et al.; In this work, the studies on the previously detected phenomenon of concealed heterokaryosis in Saccharomyces cerevisiae were continued . In genetic and Southern blotting experiments, one of the nuclei in the heterokaryon was shown to be active (capable of division and ensuring the corresponding cell phenotype), whereas the other was not expressed until the heterokaryotic clone was transferred to the medium selective for this concealed nucleus . Moreover, the concealed nucleus was able to assume the active state after fusion with the second parental nucleus . It was analyzed whether the nuclei with new marker combinations occurring in meiosis can behave as exceptional nuclei . Tetrad analysis of hybrids carrying the kar1 mutation in their nuclei revealed the relatively high percentage of exceptional tetrads (more than 10%) . One spore in these tetrads usually formed diploid cells capable of sporulation . The presented data of genetic and molecular biological studies testify in favor of the assumption that abnormal spores contain two nuclei, which form an "illegitimate" hybrid after fusion . An extraneous spore (termed x) has usually a genotype close to that of one of the spores in this tetrad . Thus, it was assumed that the additional DNA replication round occurs in the absence of cell division during one of meiotic divisions . Results of cytological analysis conducted by the method of specific DNA staining confirmed the existence of exceptional tetrads, one spore of which contains two nuclei.

Biosci Biotechnol Biochem, 2001 Jul, 65(7), 1589 - 95
A novel screening for inhibitors of a pleiotropic drug resistant pump, Pdr5, in Saccharomyces cerevisiae; Hiraga K et al.; Yeast is an excellent model system of eukaryotes for the study of molecular mechanisms of ATP-binding cassette transporters . Pdr5 protein is a yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs . Here, we described a novel drug screening system designated to detect compounds that inhibit the function of Pdr5 . An indicator strain with increased drug sensitivity was constructed with an ergosterol-deficient background (delta syr1/erg3 null mutation) . The sensitivity of the indicator strain (delta syr1/erg3 delta pdr5 delta snq2) to the Pdr5 substrates, cycloheximide and cerulenin, was increased 16-fold and 4-fold against wild type, respectively . The screening system is mainly based on the growth inhibition of the PDR5-overexpressed indicator strain with the combination of a sample and cycloheximide or cerulenin . The effect of an mdr inhibitor, FK506 on the screening system was clearly detected even at a low concentration (approximately 0.5 microg/ml) . In addition, accumulation of rhodamine 6G in the cells was detected as a result of Pdr5 inhibition by FK506 . These results indicated that the screening system is useful for a sensitive screening of Pdr5-specific inhibitors with low toxicity.

Biosci Biotechnol Biochem, 2001 Jul, 65(7), 1511 - 8
1-Aminocyclopropane-1-carboxylate synthase of Penicillium citrinum: primary structure and expression in Escherichia coli and Saccharomyces cerevisiae; Kakuta Y et al.; A plant hormone, ethylene, is formed through 1-aminocyclopropane-1-carboxylic acid (ACC) . A fungus, Penicillium citrinum, was found to synthesize ACC and to degrade ACC into 2-oxobutyrate and ammonia . ACC synthase, responsible for ACC synthesis in P . citrinum, was characterized on the molecular level by sequencing of N terminal and proteolytic peptides of the enzyme, and cloning and sequencing of its cDNA . The ACC synthase from P . citrinum had 430 amino acid residues and a shorter C terminal than the plant enzyme . The enzyme purified from Escherichia coli transformed with ACC-synthase-encoding DNA showed similar properties to those of the purified enzyme from P . citrinum . Saccharomyces cerevisiae with ACC synthase accumulated ACC in the medium with increasing time of incubation . The sequence of ACC synthase from P . citrinum was compared with that of the plant enzyme with discussion about important residues for catalysis.

Arch Latinoam Nutr, 2001 Mar, 51(1), 72 - 80
{Use of diet containing yeast protein (Saccharomyces cerevisiae): effects upon pregnancy, lactation and development in rats}; de Oliveira SR et al.; The nutritive value of manioc flour (Manihot esculenta) enriched with yeast protein (Saccharomyces cerevisiae) added to a food mixture most frequently consumed by low-income populations was assessed in female Wistar rats (n = 30; 100-120 days old) . Animals were divided into three groups, mated and had free access to diets and water . Diets were as follows: beans, rice, yeast-enriched manioc flour (BRYMF17); beans, rice, manioc flour (BRMF13); casein (17% protein) (CAS17) . Body weight gains and food consumption were recorded during pregnancy and lactation . At the parturition, the number of pups per litter was recorded and offspring were uniformly distributed (7 pups per litter) . Weight gains were determined until weaning (21 days) . At weaning two youngs were selected from each litter and individually housed . Weight gains, food consumption and the length of the tail were measured until rats were 70 days old . Rats had their liver and brain removed for protein determination and wet and relative weights . Liver samples were histologically examined . Blood hemoglobin, hematocrit and proteins, as well as the Food Efficiency Ratio (FER), were determined . ANOVA and Tukey's test were used . The experimental diet had not significant effect on pregnant and lactating dams . Values for the investigated parameters were higher in experimental youngs than in their controls and lower than in the standard group . This yeast protein-enriched manioc flour proved to be valid in terms of dietary supplementation.

Mol Biol Cell, 2001 Aug, 12(8), 2519 - 33
Cnm67p is a spacer protein of the Saccharomyces cerevisiae spindle pole body outer plaque; Schaerer F et al.; In Saccharomyces cerevisiae, the spindle pole body (SPB) is the functional homolog of the mammalian centrosome, responsible for the organization of the tubulin cytoskeleton . Cytoplasmic (astral) microtubules essential for the proper segregation of the nucleus into the daughter cell are attached at the outer plaque on the SPB cytoplasmic face . Previously, it has been shown that Cnm67p is an integral component of this structure; cells deleted for CNM67 are lacking the SPB outer plaque and thus experience severe nuclear migration defects . With the use of partial deletion mutants of CNM67, we show that the N- and C-terminal domains of the protein are important for nuclear migration . The C terminus, not the N terminus, is essential for Cnm67p localization to the SPB . On the other hand, only the N terminus is subject to protein phosphorylation of a yet unknown function . Electron microscopy of SPB serial thin sections reveals that deletion of the N- or C-terminal domains disturbs outer plaque formation, whereas mutations in the central coiled-coil domain of Cnm67p change the distance between the SPB core and the outer plaque . We conclude that Cnm67p is the protein that connects the outer plaque to the central plaque embedded in the nuclear envelope, adjusting the space between them by the length of its coiled-coil.

J Bacteriol, 2001 Sep, 183(18), 5402 - 13
Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization; Scherz R et al.; Recently we reported an unusual multicellular organization in yeast that we termed stalk-like structures . These structures are tall (0.5 to 3 cm long) and narrow (1 to 3 mm in diameter) . They are formed in response to UV radiation of cultures spread on high agar concentrations . Here we present an anatomical analysis of the stalks . Microscopic inspection of cross sections taken from stalks revealed that stalks are composed of an inner core in which cells are dense and vital and a layer of cells (four to six rows) that surrounds the core . This outer layer is physically separated from the core and contains many dead cells . The outer layer may form a protective shell for the core cells . Through electron microscopy analysis we observed three types of cells within the stalk population: (i) cells containing many unusual vesicles, which might be undergoing some kind of cell death; (ii) cells containing spores (usually one or two spores only); and (iii) familiar rounded cells . We suggest that stalk cells are not only spatially organized but may undergo processes that induce a certain degree of cell specialization . We also show that high agar concentration alone, although not sufficient to induce stalk formation, induces dramatic changes in a colony's morphology . Most striking among the agar effects is the induction of growth into the agar, forming peg-like structures . Colonies grown on 4% agar or higher are reminiscent of stalks in some aspects . The agar concentration effects are mediated in part by the Ras pathway and are related to the invasive-growth phenomenon.

Genetics, 2001 Aug, 158(4), 1825 - 7
Identification of a mutant locus by noncomplementation of a transposon insertion library in Saccharomyces cerevisiae; Wiatrowski HA et al.; We describe a new approach for identifying the gene corresponding to a mutation in Saccharomyces cerevisiae . A library of mTn-lacZ/LEU2 insertions is tested for failure to complement the mutation, and the noncomplementing insertion is used to obtain sequence . This approach offers an alternative to cloning by complementation with a plasmid library.

Genetics, 2001 Aug, 158(4), 1457 - 76
Meiotic recombination involving heterozygous large insertions in Saccharomyces cerevisiae: formation and repair of large, unpaired DNA loops; Kearney HM et al.; Meiotic recombination in Saccharomyces cerevisiae involves the formation of heteroduplexes, duplexes containing DNA strands derived from two different homologues . If the two strands of DNA differ by an insertion or deletion, the heteroduplex will contain an unpaired DNA loop . We found that unpaired loops as large as 5.6 kb can be accommodated within a heteroduplex . Repair of these loops involved the nucleotide excision repair (NER) enzymes Rad1p and Rad10p and the mismatch repair (MMR) proteins Msh2p and Msh3p, but not several other NER (Rad2p and Rad14p) and MMR (Msh4p, Msh6p, Mlh1p, Pms1p, Mlh2p, Mlh3p) proteins . Heteroduplexes were also formed with DNA strands derived from alleles containing two different large insertions, creating a large "bubble"; repair of this substrate was dependent on Rad1p . Although meiotic recombination events in yeast are initiated by double-strand DNA breaks (DSBs), we showed that DSBs occurring within heterozygous insertions do not stimulate interhomologue recombination.

Genetics, 2001 Aug, 158(4), 1431 - 44
SPO14 separation-of-function mutations define unique roles for phospholipase D in secretion and cellular differentiation in Saccharomyces cerevisiae; Rudge SA et al.; In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid . SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion) . We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation . Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct . Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis . Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein . Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth . These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis.

Biochem J, 2001 Sep 1, 358(Pt 2), 447 - 55
Subnuclear localization and mitotic phosphorylation of HIRA, the human homologue of Saccharomyces cerevisiae transcriptional regulators Hir1p/Hir2p; De Lucia F et al.; The HIRA gene encodes a nuclear protein with histone-binding properties that have been conserved from yeast to humans . Hir1p and Hir2p, the two HIRA homologues in Saccharomyces cerevisiae, are transcriptional co-repressors whose action resides at the chromatin level and occurs in a cell-cycle-regulated fashion . In mammals, HIRA is an essential gene early during development, possibly through the control of specific gene-transcription programmes, but its exact function remains to be deciphered . Here we report on the subnuclear distribution and cell-cycle behaviour of the HIRA protein . Using both biochemical and immunofluorescence techniques, a minor fraction of HIRA was found tightly associated with the nuclear matrix, the material that remains after nuclease treatment and high-salt extraction . However, most HIRA molecules proved extractable . In non-synchronized cell populations, extraction from chromatin necessitated 300 mM NaCl whereas 150 mM was sufficient in mitotic cells . Immunofluorescence staining and microscopic examination of mitotic cells revealed HIRA as excluded from condensed chromosomes, confirming a lack of association with chromatin during mitosis . Western-blot analysis indicated that HIRA molecules were hyper-phosphorylated at this point in the cell cycle . Metabolic labelling and pulse-chase experiments characterized HIRA as a stable protein with a half-life of approx . 12 h . The mitotic phosphorylation of HIRA could provide the dividing cell with a way to retarget HIRA-containing multi-protein complexes to different chromatin regions in daughter compared with parental cells.

Planta Med, 2001 Aug, 67(6), 501 - 4
Inhibitory activity for chitin synthase II from Saccharomyces cerevisiae by tannins and related compounds; Hwang EI et al.; In the course of search for potent inhibitors of chitin synthase II from natural resources, seven tannins and related compounds were isolated from the aerial part of Euphorbia pekinensis and identified as gallic acid (1), methyl gallate (2), 3-O-galloyl-(-)-shikimic acid (3), corilagin (4), geraniin (5), quercetin-3-O-(2"-O-galloyl)-beta-D-glucoside (6), and kaempferol-3-O-(2"-O-galloyl)-beta-D-glucoside (7) . These and nine related compounds, (-)-quinic acid (8), (-)-shikimic acid (9), ellagic acid (10), kaempferol (11), quercetin (12), quercitrin (13), rutin (14), quercetin-3-O-(2"-O-galloyl)-beta-D-rutinoside (15) and 1,3,4,6-tetra-O-galloyl-beta-D-glucose (16), were evaluated for the inhibitory activity against chitin synthase II and III . They inhibited chitin synthase II with IC(50) values of 18-206 microM, except for two organic acids, (-)-quinic acid (8) and (-)-shikimic acid (9) . Among them, 3-O-galloyl-(-)-shikimic acid (3) was the most potent inhibitor against chitin synthase II of Saccharomyces cerevisiae with an IC(50) value of 18 microM . The inhibition appears to be selective for chitin synthase II, as they did not appreciably inhibit chitin synthase III.

Biochimie, 2001 Jun, 83(6), 505 - 14
Depletion and deletion analyses of eucaryotic translation initiation factor 1A in Saccharomyces cerevisiae; Kainuma M et al.; Translation initiation factor eIF1A is a highly conserved, small, acidic protein that is required for cell growth in yeast . Biochemical studies in vitro implicate eIF1A in dissociating ribosomes, promoting methionyl-tRNA(i) binding to 40S ribosomal subunits, scanning of mRNAs and recognizing the AUG initiation codon . To elucidate the pleiotropic functions of eIF1A in vivo, the factor was depleted by placing its gene behind the repressible GAL1 promoter . After Saccharomyces cerevisiae cells were shifted to glucose medium, depletion of eIF1A was seen after 3-4 generations, corresponding with cessation of cell growth . Polysome profiles of the depleted strain showed ribosome run-off from mRNAs, indicating that eIF1A is involved in the initiation phase of translation . A decrease in free 40S ribosomes and an apparent increase in free 60S ribosomes were attributed to the formation of 40S subunit dimers . The result suggests that one of the functions of eIF1A is to prevent formation of 40S dimers . Mutant forms of eIF1A lacking either the positively charged N-terminal region or the negatively charged C-terminal region were constructed and tested for their ability to confer cell growth as the sole source of eIF1A . Either deletion supports cell growth, albeit at a slower rate, and causes a reduction in polysomes, although eIF1A lacking the N-terminal region is more deleterious . Therefore the charged terminal regions contribute to, but are not absolutely essential for, eIF1A function.

Biotechnol Bioeng, 2001 Sep, 76(2), 99 - 107
Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae; Morawski B et al.; Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower K(m) for peroxide, and higher thermostability . Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50 degrees C . A single mutation (N175S) in the HRP active site was found to improve thermal stability . Introducing this mutation into an HRP variant evolved for higher activity yielded HRP 13A7-N175S, whose half-life at 60 degrees C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St . Louis, MO) . The variant is also more stable in the presence of H(2)O(2), SDS, salts (NaCl and urea), and at different pH values . Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications . Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide .

Nucleic Acids Res, 2001 Aug 15, 29(16), 3297 - 303
Repression of rRNA synthesis due to a secretory defect requires the C-terminal silencing domain of Rap1p in Saccharomyces cerevisiae; Miyoshi K et al.; A secretory defect causes specific transcriptional repression of both ribosomal protein and ribosomal RNA genes, suggesting the coupling of plasma membrane and ribosome syntheses . We previously reported that the rap1-17 allele, which produced C-terminally truncated Rap1p, derepressed transcription of ribosomal protein genes when the secretory pathway was blocked . In this paper, we demonstrate that the rap1-17 mutation also leads to significant attenuation of transcriptional repression of rRNA genes due to a secretory defect . In contrast, the rap1-2 temperature-sensitive allele containing a unique missense mutation in the middle of the coding sequence has only a weak effect on repression . These results suggest that the C-terminal silencing domain of Rap1p is required for transcriptional repression of rDNA in response to a secretory defect . We also demonstrated that transcriptional regulation of ribosomal protein genes in response to nitrogen limitation was not affected by the rap1-17 allele, suggesting that the mechanism of nitrogen response is distinct from that of the secretory response.

J Biol Chem, 2001 Nov 2, 276(44), 41455 - 64 Epub 2001 Aug 14.
The CWH8 gene encodes a dolichyl pyrophosphate phosphatase with a luminally oriented active site in the endoplasmic reticulum of Saccharomyces cerevisiae; Fernandez F et al.; Mutations in the CWH8 gene, which encodes an ER transmembrane protein with a phosphate binding pocket in Saccharomyces cerevisiae, result in a deficiency in dolichyl pyrophosphate (Dol-P-P)-linked oligosaccharide intermediate synthesis and protein N-glycosylation (van Berkel, M . A., Rieger, M., te Heesen, S., Ram, A . F., van den Ende, H., Aebi, M., and Klis, F . M . (1999) Glycobiology 9, 243-253) . Genetic, enzymological, and topological approaches were taken to investigate the potential role of Cwh8p in Dol-P-P/Dol-P metabolism . Overexpression of Cwh8p in the yeast double mutant strain, lacking LPP1/DPP1, resulted in an impressive increase in Dol-P-P phosphatase activity, a relatively small increase in Dol-P phosphatase activity, but no change in phosphatidate (PA) phosphatase activity in microsomal fractions . The Dol-P-P phosphatase encoded by CWH8 is optimally active in the presence of 0.5% octyl glucoside and relatively unstable in Triton X-100, distinguishing this activity from the lipid phosphatases encoded by LPP1 and DPP1 . Stoichiometric amounts of P(i) and Dol-P are formed during the enzymatic reaction indicating that Cwh8p cleaves the anhydride linkage in Dol-P-P . Membrane fractions from Sf-9 cells expressing Cwh8p contained a 30-fold higher level of Dol-P-P phosphatase activity, a slight increase in Dol-P phosphatase activity, but no increase in PA phosphatase relative to controls . This is the first report of a lipid phosphatase that hydrolyzes Dol-P-P/Dol-P but not PA . In accord with this enzymatic function, Dol-P-P accumulated in cells lacking the Dol-P-P phosphatase . Topological studies using different approaches indicate that Cwh8p is a transmembrane protein with a luminally oriented active site . The specificity, subcellular location, and topological orientation of this novel enzyme are consistent with a role in the re-utilization of the glycosyl carrier lipid for additional rounds of lipid intermediate biosynthesis after its release during protein N-glycosylation reactions.

Appl Microbiol Biotechnol, 2001 Jul, 56(1-2), 249 - 54
The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures; Laroche C et al.; Membrane fluidity in whole cells of Saccharomyces cerevisiae W303-1A was estimated from fluorescence polarization measurements using the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, over a wide range of temperatures (6-35 degrees C) and at seven levels of osmotic pressure between 1.38 MPa and 133.1 MPa . An increase in phase transition temperatures was observed with increasing osmotic pressure . At 1.38 MPa, a phase transition temperature of 12 +/- 2 degrees C was observed, which increased to 17 +/- 4 degrees C at 43.7 MPa, 21+/- 7 degrees C at 61.8 MPa, and 24 +/- 9 degrees C at an osmotic pressure of 133.1 MPa . From these results we infer that, with increases in osmotic pressure, the change in phospholipid conformation occurs over a larger temperature range . These results allow the representation of membrane fluidity as a function of temperature and osmotic pressure . Osmotic shocks were applied at two levels of osmotic pressure and at nine temperatures, in order to relate membrane conformation to cell viability.

Biochem Soc Trans, 2001 Aug, 29(Pt 4), 436 - 41
Structure and function of Pet100p, a molecular chaperone required for the assembly of cytochrome c oxidase in Saccharomyces cerevisiae; Forsha D et al.; The assembly of cytochrome c oxidase in the inner mitochondrial membranes of eukaryotic cells requires the protein products of a large number of nuclear genes . In yeast, some of these act globally and affect the assembly of several respiratory-chain protein complexes, whereas others act in a cytochrome c oxidase-specific fashion . Many of these yeast proteins have human counterparts, which when mutated lead to energy-related diseases . One of these proteins, Pet100p, is a novel molecular chaperone that functions to incorporate a subcomplex containing cytochrome c oxidase subunits VII, VIIa and VIII into holo-(cytochrome c oxidase) . Here we report the topological disposition of Pet100p in the inner mitochondrial membrane and show that its C-terminal domain is essential for its function as a cytochrome c oxidase-specific 'assembly facilitator'.

RNA, 2001 Aug, 7(8), 1084 - 96
Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae; Meskauskas A et al.; Our previous demonstration that mutants of 5S rRNA called mof9 can specifically alter efficiencies of programmed ribosomal frameshifting (PRF) suggested a role for this ubiquitous molecule in the maintenance of translational reading frame, though the repetitive nature of the 5S rDNA gene (>100 copies/cell) inhibited more detailed analyses . However, given the known interactions between 5S rRNA and ribosomal protein L5 (previously called L1 or YL3) encoded by an essential, single-copy gene, we monitored the effects of a series of well-defined rpl5 mutants on PRF and virus propagation . Consistent with the mof9 results, we find that the rpl5 mutants promoted increased frameshifting efficiencies in both the -1 and +1 directions, and conferred defects in the ability of cells to propagate two endogenous viruses . Biochemical analyses demonstrated that mutant ribosomes had decreased affinities for peptidyl-tRNA . Pharmacological studies showed that sparsomycin, a peptidyltransferase inhibitor that specifically increases the binding of peptidyl-tRNA with ribosomes, was antagonistic to the frameshifting defects of the most severe mutant, and the extent of sparsomycin resistance correlated with the severity of the frameshifting defects in all of the mutants . These results provide biochemical and physiological evidence that one function of L5 is to anchor peptidyl-tRNA to the P-site . A model is presented describing how decreased affinity of ribosomes for peptidyl-tRNA can affect both -1 and +1 frameshifting, and for the effects of sparsomycin.

Microbiology, 2001 Aug, 147(Pt 8), 2037 - 44
Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae; Walther K et al.; Glucose-repressible alcohol dehydrogenase II, encoded by the ADH2 gene of the yeast Saccharomyces cerevisiae, is transcriptionally controlled by the activator Adr1, binding UAS1 of the control region . However, even in an adr1 null mutant, a substantial level of gene derepression can be detected, arguing for the existence of a further mechanism of activation . Here it is shown that the previously identified UAS2 contains a distantly related variant of the carbon source-responsive element (CSRE) initially found upstream of gluconeogenic genes . In a mutant defective for the CSRE-binding factor Cat8, derepression of an ADH2-lacZ fusion was reduced to about 12% of the wild-type level . Gene expression in a cat8 adr1 double mutant decreased almost to the basal level of the glucose-repressed promoter . CSRE(ADH2) present in a single copy turned out to be a weak UAS element, while a significant synergism of gene activation was found in the presence of at least two copies . Its importance for regulated gene activation was confirmed by site-directed mutagenesis of the CSRE in the natural ADH2 control region . Direct binding of Cat8 to CSRE(ADH2) could be shown by electrophoretic retardation of the corresponding protein/DNA complex in the presence of a specific antibody . In contrast to what was shown previously for CSRE sequence variants, no significant influence of the isofunctional activator Sip4 on CSRE(ADH2) was detected . In conclusion, these results show a derepression of ADH2 by synergistically acting regulators Adr1 (interacting with UAS1) and Cat8, binding to UAS2 (=CSRE(ADH2)).

J Cell Sci, 2001 Jun, 114(Pt 12), 2231 - 9
Three dimensional configuration of the secretory pathway and segregation of secretion granules in the yeast Saccharomyces cerevisiae; Rambourg A et al.; The structural elements of the secretory pathway in the budding yeast Saccharomyces cerevisiae were analyzed by 3D stereo-electron microscopy using relatively thick sections in which membranes were selectively impregnated . In a wild-type strain, tubular networks of various sizes and staining properties were distributed throughout the cytoplasm . As a rule, wide-meshed, lightly stained polygonal networks were connected to more or less fenestrated sheets of endoplasmic reticulum (ER) . Some of these networks were continuous with more intensely stained networks and narrower meshes that displayed at their intersections nodular dilations that progressively increased in size and staining properties to reach those of secretion granules . Such networks presumably corresponded to Golgi elements . Indeed, stacked cisternae typical of the mammalian Golgi apparatus are rarely found in wild-type cells . However, if it is assumed that the Golgi apparatus plays a key role in the segregation and maturation of secretion granules, then tubular networks with nodular dilations should be equivalent to parts of this organelle . In correlation with the increase in size and density of the nodules there was a decrease in diameter and staining intensity of the interconnecting tubules . These results parallel observations on the formation of secretory granules in mammalian cells and suggest that the segregation of secretory material is concomitant with the progressive perforation and tubulization of previously unperforated sheets . When the sec21-3 thermosensitive mutant was examined at the nonpermissive temperature (37 degrees C), the secretory pathway was blocked at exit from the ER, which started to accumulate as clusters of narrow, anastomosed, unperforated ribbon-like elements . When the block was released by shifting down to permissive temperature (24 degrees C), tubular networks of various sizes and caliber, presumably Golgi in nature, formed as soon as 5 minutes after release of the block . At later time intervals, granules of various sizes and densities appeared to be released by rupture of these tubular networks or even to form at the edges of ER fenestrae . These observations support a dynamic maturation process in which the formation of secretion granules occurs by means of an oriented series of membrane transformations starting at the ER and culminating with the liberation of secretion granules from Golgi networks.

J Bacteriol, 2001 Sep, 183(17), 4985 - 93
The Saccharomyces cerevisiae Isw2p-Itc1p complex represses INO1 expression and maintains cell morphology; Sugiyama M et al.; In the yeast Saccharomyces cerevisiae, IRE1 encodes a bifunctional protein with transmembrane kinase and endoribonuclease activities . HAC1 encodes a transcription factor which has a basic leucine zipper domain . Both gene products play a crucial role in the unfolded protein response . Mutants in which one of these genes is defective also show the inositol-auxotrophic (Ino(-)) phenotype, but the reason for this has not been clear . To investigate the mechanism underlying the Ino(-) phenotype, we screened a multicopy suppressor gene which can suppress the Ino(-) phenotype of the Delta hac1 strain . We obtained a truncated form of the ITC1 gene that has a defect in its 3' region . Although the truncated form of ITC1 clearly suppressed the Ino(-) phenotype of the Delta hac1 strain, the full-length ITC1 had a moderate effect . The gene products of ITC1 and ISW2 are known to constitute a chromatin-remodeling complex (T . Tsukiyama, J . Palmer, C . C . Landel, J . Shiloach, and C . Wu, Genes Dev . 13:686--697, 1999) . Surprisingly, the deletion of either ITC1 or ISW2 in the Delta hac1 strain circumvented the inositol requirement and caused derepression of INO1 even under repression conditions, i.e., in inositol-containing medium . These data indicate that the Isw2p-Itc1p complex usually represses INO1 expression and that overexpression of the truncated form of ITC1 functions in a dominant negative manner in INO1 repression . It is conceivable that the repressor function of this complex is regulated by the C-terminal region of Itc1p.

J Bacteriol, 2001 Sep, 183(17), 4950 - 7
Transcriptional regulation of the two sterol esterification genes in the yeast Saccharomyces cerevisiae; Jensen-Pergakes K et al.; Saccharomyces cerevisiae transcribes two genes, ARE1 and ARE2, that contribute disproportionately to the esterification of sterols . Are2p is the major enzyme isoform in a wild-type cell growing aerobically . This likely results from a combination of differential transcription initiation and transcript stability . By using ARE1 and ARE2 promoter fusions to lacZ reporters, we demonstrated that transcriptional initiation from the ARE1 promoter is significantly reduced compared to that from the ARE2 promoter . Furthermore, the half-life of the ARE2 mRNA is approximately 12 times as long as that of the ARE1 transcript . We present evidence that the primary role of the minor sterol esterification isoform encoded by ARE1 is to esterify sterol intermediates, whereas the role of the ARE2 enzyme is to esterify ergosterol, the end product of the pathway . Accordingly, the ARE1 promoter is upregulated in strains that accumulate ergosterol precursors . Furthermore, ARE1 and ARE2 are oppositely regulated by heme . Under heme-deficient growth conditions, ARE1 was upregulated fivefold while ARE2 was down-regulated . ARE2 requires the HAP1 transcription factor for optimal expression, and both ARE genes are derepressed in a rox1 (repressor of oxygen) mutant genetic background . We further report that the ARE genes are not subject to end product inhibition; neither ARE1 nor ARE2 transcription is altered in an are mutant background, nor does overexpression of either ARE gene alter the response of the ARE-lacZ reporter constructs . Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate . Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.

Mol Microbiol, 2001 Jul, 41(2), 489 - 502
Genetic analysis of the signalling pathway activated by external amino acids in Saccharomyces cerevisiae; Bernard F et al.; The permease-like amino acid sensor Ssy1p of Saccharomyces cerevisiae is required for transcriptional induction, in response to external amino acids, of several genes encoding peptide and amino acid permeases . Among them is AGP1 encoding a low-affinity, broad-specificity amino acid permease important for the utilization of amino acids as a nitrogen source . We report here data from experiments aimed at identifying components of the signalling pathway activated by Ssy1p . Overproduction of the large amino-terminal tail of Ssy1p interferes negatively with the induction of AGP1 in wild-type cells . Furthermore, overproduction of this domain can relieve growth defects of a ssy1 null strain, indicating that the N-terminal tail of Ssy1p is an important functional element of the pathway . Consistent with a role for Ssy1p in the recognition of amino acids, a mutant form of the protein with a Thr to Ile substitution in the eighth predicted transmembrane domain is competent for the induction of AGP1 by leucine but not by other amino acids . In a screen for other mutants defective in the Ssy1p pathway, we confirmed that PTR3 and SSY5 encode additional factors essential for AGP1 expression in response to multiple amino acids . Data obtained by overproducing Ptr3p and Ssy5p in ssy1Delta, ptr3Delta and ssy5Delta mutants suggest that Ptr3p acts downstream from Ssy1p and Ssy5p downstream from Ptr3p in the transduction pathway . Furthermore, two-hybrid experiments indicated that Ptr3p interacts with Ssy5p and that Ptr3p can self-associate . Finally, the Cys-6-Zn2 transcription factor Uga35p/Dal81p required for the induction of AGP1 is also essential for the expression of two other genes under Ssy1p-Ptr3p-Ssy5p control, namely BAP2 and PTR2, suggesting that the protein is yet another component of the amino acid signalling pathway.

Arch Biochem Biophys, 2001 Aug 15, 392(2), 326 - 32
Requirements of Cyc2p and the porin, Por1p, for ionic stability and mitochondrial integrity in Saccharomyces cerevisiae; Sanchez NS et al.; It was previously demonstrated that Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein; that the cyc2-Delta2 deletion lacking the entire gene causes a diminution to only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo-cytochrome c; that the deletion causes a defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources; and that this defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a nonionic compound . We report that por1-Delta strains lacking the mitochondrial porin, Por1p, but not por2-Delta strains lacking the related porin, share some phenotypes similar to the cyc2-Delta2 strain, including hypersensitivity to KCl in glycerol medium . Moreover, spontaneous swelling in the presence of ATP was detected in mitochondria from the cyc2-Delta2 strain, while swelling could be detected in mitochondria from the other strains only after the addition of KCl . Thus, highly unspecific membrane permeation may be triggered by ATP in the cyc2-Delta2 strain . We suggest that Por1p and Cyc2p, in addition to their own unique functions, serve to maintain the osmotic stability of mitochondria, but by different mechanisms .

Mol Cell Biol, 2001 Sep, 21(17), 5790 - 6
Interaction of the Srb10 kinase with Sip4, a transcriptional activator of gluconeogenic genes in Saccharomyces cerevisiae; Vincent O et al.; Sip4 is a Zn(2)Cys(6) transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae . The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4 . Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptional repression but also positively regulates Gal4 . We show that Srb10 is required for phosphorylation of Sip4 during growth in nonfermentable carbon sources and that the catalytic activity of Srb10 stimulates the ability of LexA-Sip4 to activate transcription of a reporter . Srb10 and Sip4 coimmunoprecipitate from cell extracts and interact in two-hybrid assays, suggesting that Srb10 regulates Sip4 directly . We also present evidence that the Srb10 and Snf1 kinases interact with different regions of Sip4 . These findings support the view that the Srb10 kinase not only plays negative roles in transcriptional control but also has broad positive roles during growth in carbon sources other than glucose.

Mol Cell Biol, 2001 Sep, 21(17), 5710 - 22
Inhibition of acetyl coenzyme A carboxylase activity restores expression of the INO1 gene in a snf1 mutant strain of Saccharomyces cerevisiae; Shirra MK et al.; Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis . To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1Delta strains . We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the beta subunit of fatty acid synthase . Acc1 was shown previously to be phosphorylated and inactivated by Snf1 . Here we show that snf1Delta strains with increased Acc1 activity exhibit decreased INO1 transcription . Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A . Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1Delta strains . Together, these findings indicate that the inositol auxotrophy of snf1Delta strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains . These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.

BMC Microbiol . 2001;1(1):11 . Epub 2001 Jul 16.
Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae; Pereira MD et al.; BACKGROUND: Living cells constantly sense and adapt to redox shifts by the induction of genes whose products act to maintain the cellular redox environment . In the eukaryote Saccharomyces cerevisiae, while stationary cells possess a degree of constitutive resistance towards oxidants, treatment of exponential phase cultures with sub-lethal stresses can lead to the transient induction of protection against subsequent lethal oxidant conditions . The sensors of oxidative stress and the corresponding transcription factors that activate gene expression under these conditions have not yet been completely identified . RESULTS: We report the role of SOD1, SOD2 and TPS1 genes (which encode the cytoplasmic Cu/Zn-superoxide dismutase, the mitochondrial Mn-isoform and trehalose-6-phosphate synthase, respectively) in the development of resistance to oxidative stress . In all experimental conditions, the cultures were divided into two parts, one was immediately submitted to severe stress (namely: exposure to H2O2, heat shock or ethanol stress) while the other was initially adapted to 40 degrees C for 60 min . The deficiency in trehalose synthesis did not impair the acquisition of tolerance to H2O2, but this disaccharide played an essential role in tolerance against heat and ethanol stresses . We also verified that the presence of only one Sodp isoform was sufficient to improve cellular resistance to 5 mM H2O2 . On the other hand, while the lack of Sod2p caused high cell sensitivity to ethanol and heat shock, the absence of Sod1p seemed to be beneficial to the process of acquisition of tolerance to these adverse conditions . The increase in oxidation-dependent fluorescence of crude extracts of sod1 mutant cells upon incubation at 40 degrees C was approximately 2-fold higher than in sod2 and control strain extracts . Furthermore, in Western blots, we observed that sod mutants showed a different pattern of Hsp104p and Hsp26p expression also different from that in their control strain . CONCLUSIONS: Trehalose seemed not to be essential in the acquisition of tolerance to H2O2 stress, but its absence was strongly felt under water stress conditions such as heat and alcoholic stresses . On the other hand, Sod1p could be involved in the control of ROS production; these reactive molecules could signal the induction of genes implicated within cell tolerance to heat and ethanol . The effects of this deletion needs further investigation.

Yeast, 2001 Aug, 18(11), 1003 - 14
Multicopy suppressors of the sly1 temperature-sensitive mutation in the ER-Golgi vesicular transport in Saccharomyces cerevisiae; Kosodo Y et al.; Saccharomyces cerevisiae Sly1 protein is a member of the Sec1/Munc18-family proteins, which are essential for vesicular trafficking, but their exact biological roles are yet to be determined . A temperature-sensitive sly1 mutant arrests the vesicular transport from the ER to Golgi compartments at 37 degrees C . We screened for multicopy suppressor genes that restore the colony formation of the sly1(ts) mutant to discover functionally interacting components . The suppressor genes obtained were classified as: (1) those that encode a multifunctional suppressor, SSD1; (2) heat shock proteins, SSB1 and SSB2; (3) cell surface proteins, WSC1, WSC2 and MID2; (4) ER-Golgi transport proteins, USO1 and BET1; and (5) an as-yet-uncharacterized protein, HSD1 (high-copy suppressor of SLY1 defect 1) . By epitope tagging of the gene product, we found that Hsd1 protein is an ER-resident membrane protein . Its overproduction induced enlargement of ER-like membrane structures .

Proc Natl Acad Sci U S A, 2001 Aug 14, 98(17), 9760 - 5 Epub 2001 Jul 31.
Interactions of Exo1p with components of MutLalpha in Saccharomyces cerevisiae; Tran PT et al.; Previously, we reported evidence suggesting that Saccharomyces cerevisiae MutLalpha, composed of Mlh1p and Pms1p, was a functional member of the gyrase b/Hsp90/MutL (GHL) dimeric ATPase superfamily characterized by highly conserved ATPase domains . Similar to other GHL ATPases, these putative ATPase domains of MutLalpha may be important for the recruitment and/or activation of downstream effectors . One downstream effector candidate is Exo1p, a 5'-3' double stranded DNA exonuclease that has previously been implicated in DNA mismatch repair (MMR) . Here we report yeast two-hybrid results suggesting that Exo1p can interact physically with MutLalpha through the Mlh1p subunit . We also report epistasis analysis involving MutLalpha ATPase mutations combined with exo1Delta . One interpretation of our genetic results is that MutLalpha ATPase domains function to direct Exo1p and other functionally redundant exonucleases during MMR . Finally, our results show that much of the increase in spontaneous mutation observed in an exo1Delta strain is REV3-dependent, in turn suggesting that Exo1p is also involved in one or more MMR-independent mutation avoidance pathways.

Arch Microbiol, 2001 Jul, 176(1-2), 69 - 78
The role of amino acids in the regulation of hydrogen sulfide production during ultradian respiratory oscillation of Saccharomyces cerevisiae; Sohn H et al.; We previously demonstrated that periodic H2S production during aerobic continuous culture of Saccharomyces cerevisiae resulted in ultradian respiratory oscillation, and that H2S production was dependent on the activity of sulfate uptake and the level of sulfite . To investigate the mechanism of regulation of the sulfate assimilation pathway and of respiratory oscillation, several amino acids were pulse-injected into cultures during respiratory oscillation . Injection of sulfur amino acids or their derivatives perturbed respiratory oscillation, with changes in the H2S production profile . Four major regulators of H2S production in the sulfate assimilation pathway and respiratory oscillation were identified: (1) O-acetylhomoserine, not O-acetylserine, as a sulfide acceptor, (2) homoserine/threonine as a regulator of O-acetylhomoserine supply, (3) methionine/S-adenosyl methionine as a negative regulator of sulfate assimilation, and (4) cysteine (or its derivatives) as an essential regulator . The results obtained after the addition of DL-propargylglycine (5 microM and 100 microM) and cystathionine (50 microM) suggested that the intracellular cysteine level and cystathionine gamma-lyase, rather than methionine/S-adenosylmethionine, play an essential role in the regulation of sulfate assimilation and respiratory oscillation . Based on these results and those of our previous reports, we propose that periodic depletion of cysteine (or its derivatives), which is involved in the detoxification of toxic materials originating from respiration, causes periodic H2S production.

J Biol Chem, 2001 Oct 19, 276(42), 38808 - 13 Epub 2001 Jul 30.
Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene; Bai Y et al.; The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4 . In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene . Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis . In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive . Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain . Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme . Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium . The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

J Biol Chem, 2001 Oct 26, 276(43), 39645 - 52 Epub 2001 Jul 30.
Overexpression of eIF4E in Saccharomyces cerevisiae causes slow growth and decreased alpha-factor response through alterations in CLN3 expression; Anthony C et al.; The association of G(1) cyclins and Cdc28/cyclin-dependent protein kinase catalyzes the cell cycle entry (Start) in budding yeast . Activation of Start is presumed to be triggered by a post-transcriptional increase in Cln3 during early G(1) . Cells arrested by mating pheromone show a loss of cyclin-dependent protein kinase activity caused by transcriptional shutoff of cyclins and/or inhibition by Far1 . We report that overexpression of eIF4E (Cdc33), a rate-limiting translation initiation factor, causes an increase in CLN3 mRNA translation, which results in increased expression of CLN2 and in slow growth and decreased alpha-factor response . This phenotype was abrogated in a Deltacln3 or Deltacln2 background . We isolated the transcription factor MBP1 as a multicopy suppressor of the growth and alpha-factor response defects . Furthermore, elevated MBP1, a transcriptional regulator of cyclins, altered the transcriptional start site in CLN3 mRNA, shifting it 45 nucleotides upstream of the normal . This lengthened 5'-untranslated region likely reduces translation efficiency and down-regulates CLN3 protein synthesis, thereby correcting for the excess translation promoted by elevated Cdc33 . In addition, the CLN2 mRNA level returned to normal . We propose that regulation of translation initiation by Cdc33 plays a pivotal role in the activation of Start and cell cycle progression in budding yeast.

Biochemistry, 2001 Aug 7, 40(31), 9177 - 86
Pre-steady-state kinetic studies of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase mutants identify residues involved in catalysis; Farazi TA et al.; MyristoylCoA:protein N-myristoyltransferase (Nmt, EC 2.3.1.97), a member of the GCN5 acetyltransferase (GNAT) superfamily, is an essential eukaryotic enzyme that catalyzes covalent attachment of myristate (C14:0) to the N-terminal Gly of proteins involved in myriad cellular functions . The 2.5 A resolution structure of a ternary complex of Saccharomyces cerevisiae Nmt1p with a bound substrate peptide (GLYASKLA) and nonhydrolyzable myristoylCoA analogue {Farazi, T . A., et al . (2001) Biochemistry 40, 6335} was used as the basis for a series of mutagenesis experiments designed to define the enzyme's catalytic mechanism . The kinetic properties of an F170A/L171A Nmt mutant are consistent with the proposal that their main chain amides, located in a beta-bulge structure conserved among GNATs, function as an oxyanion hole to polarize the thioester carbonyl of bound myristoylCoA prior to subsequent nucleophilic attack . Removal of the two C-terminal residues (M454 and L455) produces a 300--400-fold reduction in the chemical transformation rate and converts the rate-limiting step from a step after the transformation to the transformation event itself . This finding is consistent with the main chain C-terminal carboxylate of L455 functioning as a catalytic base that abstracts a proton from the N-terminal Gly ammonium of the bound peptide to generate the nucleophilic amine . Mutating N169 and T205 in concert reduces the rate of the chemical transformation, supporting their role as components of an H-bonding network that facilitates attack of the Gly1 amine and stabilizes the tetrahedral intermediate.

Biochem Biophys Res Commun, 2001 Aug 3, 285(5), 1186 - 91
Ntg2 of Saccharomyces cerevisiae repairs the oxidation products of 8-hydroxyguanine; Kim JE et al.; In Escherichia coli, endonuclease III (endo III) repairs the oxidation products of 8-OHGua . However, the corresponding repair enzymes in eukaryotes have not been identified . Here we report that 8-hydroxyguanine (8-OHGua) is highly sensitive to further oxidation . We also show that Ntg2, a functional homolog of endo III in Saccharomyces cerevisiae, is capable of nicking the irradiated duplex DNA containing 8-OHGua . Moreover, Ntg2 formed a stable complex with the DNA upon incubation with NaBH(4) . In contrast, Ntg1, another functional homolog of endo III, showed no such activities . These findings indicate that Ntg2 is, at least in part, responsible for repairing the oxidation products of 8-OHGua in eukaryotic cells .

J Biol Chem, 2001 Oct 5, 276(40), 37051 - 9 Epub 2001 Jul 27.
The Acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import, activation, and intracellular Utilization; Faergeman NJ et al.; Exogenous long-chain fatty acids are activated to coenzyme A derivatives prior to metabolic utilization . In the yeast Saccharomyces cerevisiae, the activation of these compounds prior to metabolic utilization proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p . Faa1p or Faa4p are essential for long-chain fatty acid import, suggesting that one or both of these enzymes are components of the fatty acid transport system, which also includes Fat1p . By monitoring the intracellular accumulation of the fluorescent long-chain fatty acid analogue 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, long-chain fatty acid transport was shown to be severely restricted in a faa1 Delta faa4 Delta strain . These data established for the first time a mechanistic linkage between the import and activation of exogenous fatty acids in yeast . To investigate this linkage further, oleoyl CoA levels were defined following incubation of wild type and mutant cells with limiting concentrations of exogenous oleate . These studies demonstrated oleoyl CoA levels were reduced to less than 10% wild-type levels in faa1 Delta and faa1 Delta faa4 Delta strains . Defects in metabolic utilization and intracellular trafficking were also found in the fatty acyl-CoA synthetase-deficient strains . The faa1 Delta faa4 Delta strain had a marked reduction in endogenous acyl-CoA pools, suggesting these enzymes play a role in maintenance of endogenous acyl-CoA pools, metabolism and trafficking . In addition, this strain had levels of in vivo beta-oxidation of exogenous oleate reduced 3-fold when compared with the isogenic parent . Northern analyses demonstrated an additional defect in fatty acid trafficking as FAA1 or FAA4 were required for the transcriptional regulation of the genes encoding the peroxisomal enzymes acyl-CoA oxidase (POX1) and medium-chain acyl-CoA synthetase (FAA2) . These data support the hypothesis that fatty acyl-CoA synthetase (Faa1p or Faa4p) functions as a component of the fatty acid import system by linking import and activation of exogenous fatty acids to intracellular utilization and signaling.

J Appl Microbiol, 2001 Aug, 91(2), 306 - 11
The influence of dissolved CO(2) concentration on the death kinetics of Saccharomyces cerevisiae; Shimoda M et al.; AIMS: The effects of temperature and concentration of dissolved CO(2) on the inactivation of Saccharomyces cerevisiae were investigated using a plug-flow system . METHODS AND RESULTS: Several combinations of pressure (4, 6, 8, 10 mega-Pa (MPa)) and temperature (30, 34, 36, 38 degrees C) were used . The D-values obtained were 0.14 min at 8 MPa and 38 degrees C, and 0.15 min at 10 MPa and 36 degrees C . The log D-values were related linearly to the treatment temperature and to the dissolved CO(2) concentration . The thermal resistance constant (zCO(2)(T)) was 9.5 degrees C in the media, including significant levels of CO(2), and the CO(2) resistance constant was z(temp.)(gamma)=7.2 gamma . CONCLUSION: This work has shown that inactivation followed first-order death kinetics, and the effects of temperature and CO(2) concentration were consistent through the critical temperature and pressure of CO(2) . Therefore, it is feasible to estimate D-values at any temperature and any CO(2) concentration . SIGNIFICANCE AND IMPACT OF THE STUDY: Non-thermal inactivation of micro-organisms in acidic beverages could be realized by the present technique.

J Cell Biol, 2001 Jul 23, 154(2), 331 - 44
Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome; Kornitzer D et al.; Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed mammalian cells . In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle . Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species . E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants . Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest . The E4orf4-PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation . Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.

Gene, 2001 Jul 11, 272(1-2), 93 - 101
Gene dosage affects the expression of the duplicated NHP6 genes of Saccharomyces cerevisiae; Kolodrubetz D et al.; Nhp6Ap and Nhp6Bp, which are 87% identical in sequence, are moderately abundant, chromosome-associated proteins from Saccharomyces cerevisiae . In wild type cells Nhp6Ap is present at three times the level of Nhp6Bp . The effects of altering NHP6A or NHP6B gene number on the expression of its partner has been examined using Northern blots and reporter genes . Deletion of NHP6A led to a three-fold increase in NHP6B synthesis while an extra copy of NHP6A reduced NHP6B expression two-fold . Changes in the NHP6B gene copy number caused more moderate changes in NHP6A synthesis . The regulation of one NHP6 gene by the other uses a mechanism that detects the level of Nhp6 protein (or RNA) rather than gene number, since overexpression of Nhp6B protein from a single gene led to a dramatic decrease in NHP6A synthesis . Deletion analysis showed that the regulatory element involved in gene dosage compensation maps to a 190 bp segment in the NHP6B promoter . The simplest model, that each Nhp6 protein can act as a transcriptional repressor at the other NHP6 gene, is not true since purified Nhp6A protein does not bind specifically to the NHP6B promoter region . Instead, Nhp6p appears to interact with or through another protein in regulating transcription from the NHP6 genes.

Biochim Biophys Acta, 2001 Jun 29, 1532(3), 234 - 47
A genetic screen for ethanolamine auxotrophs in Saccharomyces cerevisiae identifies a novel mutation in Mcd4p, a protein implicated in glycosylphosphatidylinositol anchor synthesis; Storey MK et al.; A genetic screen for ethanolamine auxotrophs has identified a novel mutant allele of the morphogenesis checkpoint dependent (MCD)-4 gene, designated mcd4-P301L . In the presence of a null allele for the phosphatidylserine (PtdSer) decarboxylase 1 gene (psd1 Delta), the mcd4-P301L mutation confers temperature sensitivity for growth on minimal medium . This growth defect is reversed by either ethanolamine or choline supplementation . Incubation of mutant cells with {(3)H}serine followed by analysis of the aminoglycerophospholipids demonstrated a 60% decrease in phosphatidylethanolamine (PtdEtn) formation compared to parental cells . Chemical analysis of phospholipid content after culture under non-permissive conditions also demonstrated a 60% decrease in the PtdEtn pool compared to the parental strain . Although the morphogenesis checkpoint dependent (MCD)-4 gene and its homologues have been shown to play a role in glycosylphosphatidylinositol (GPI) anchor synthesis, the mcd4-P301L strain displayed normal incorporation of {(3)H}inositol into both proteins and lipids . Thus, a defect in GPI anchor synthesis does not explain either the ethanolamine auxotrophy or biochemical phenotype of this mutant . We also examined the growth characteristics and PtdSer metabolism of a previously described mcd4-174 mutant strain, with defects in GPI anchor synthesis, protein modification and cell wall maintenance . The mcd4-174, psd1 Delta strain is a temperature sensitive ethanolamine auxotroph that requires osmotic support for growth, and displays normal PtdEtn formation compared to parental cells . These results reveal important genetic interactions between PSD1 and MCD4 genes, and provide evidence that Mcd4p can modulate aminoglycerophospholipid metabolism, in a way independent of its role in GPI anchor synthesis.

J Biol Chem, 2001 Sep 21, 276(38), 35614 - 21 Epub 2001 Jul 23.
Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae; Chung N et al.; In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids in the heat stress response through inhibition of nutrient import (Chung, N., Jenkins, G . M., Hannun, Y . A., Heitman, J., and Obeid, L . M . (2000) J . Biol . Chem . 275, 17229-17232) . In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition . When cells were treated with exogenous phytosphingosine (PHS, 20 microm) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, dihydrosphingosine, C(2)-phytoceramide (PHC), and stearylamine, only PHS inhibited growth . Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine . Again this effect was specific to PHS . Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself . By using mutant yeast strains defective in various steps in sphingolipid metabolism, we further determined the specificity of PHS . The elo2Delta strain, which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of ceramides and to be hypersensitive to PHS (5 microm), suggesting that PHS does not need to be converted to PHC . The lcb4Delta lcb5Delta strain is defective in the conversion of PHS to PHS 1-phosphate, and it was as sensitive to PHS as the wild-type strain . The syr2Delta mutant strain was defective in the conversion of DHS to PHS . Interestingly, this strain was resistant to high concentrations of DHS (40 microm) that inhibited the growth of an isogenic wild-type strain, demonstrating that DHS needs to be converted to PHS to inhibit growth . Together, these data demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely related and yet unidentified metabolite.

Biochemistry, 2001 Jul 31, 40(30), 9049 - 58
Temperature-induced denaturation and renaturation of triosephosphate isomerase from Saccharomyces cerevisiae: evidence of dimerization coupled to refolding of the thermally unfolded protein; Benitez-Cardoza CG et al.; The thermal denaturation of the dimeric enzyme triosephosphate isomerase (TIM) from Saccharomyces cerevisiae was studied by spectroscopic and calorimetric methods . At low protein concentration the structural transition proved to be reversible in thermal scannings conducted at a rate greater than 1.0 degrees C min(-1) . Under these conditions, however, the denaturation-renaturation cycle exhibited marked hysteresis . The use of lower scanning rates lead to pronounced irreversibility . Kinetic studies indicated that denaturation of the enzyme likely consists of an initial first-order reaction that forms thermally unfolded (U) TIM, followed by irreversibility-inducing reactions which are probably linked to aggregation of the unfolded protein . As judged from CD measurements, U possesses residual secondary structure but lacks most of the tertiary interactions present in native TIM . Furthermore, the large increment in heat capacity upon denaturation suggests that extensive exposure of surface area occurs when U is formed . Above 63 degrees C, reactions leading to irreversibility were much slower than the unfolding process; as a result, U was sufficiently long-lived as to allow an investigation of its refolding kinetics . We found that U transforms into nativelike TIM through a second-order reaction in which association is coupled to the regain of secondary structure . The rate constants for unfolding and refolding of TIM displayed temperature dependences resembling those reported for monomeric proteins but with considerably larger activation enthalpies . Such large temperature dependences seem to be determinant for the occurrence of kinetically controlled transitions and thus constitute a simple explanation for the hysteresis observed in thermal scannings.

J Bacteriol, 2001 Aug, 183(16), 4910 - 3
YLR209c encodes Saccharomyces cerevisiae purine nucleoside phosphorylase; Lecoq K et al.; The yeast YLR209c (PNP1) gene encodes a protein highly similar to purine nucleoside phosphorylases . This protein specifically metabolized inosine and guanosine . Disruption of PNP1 led to inosine and guanosine excretion in the medium, thus showing that PNP1 plays an important role in the metabolism of these purine nucleosides in vivo.

Mol Cell Biol, 2001 Aug, 21(16), 5374 - 88
The Sgs1 helicase of Saccharomyces cerevisiae inhibits retrotransposition of Ty1 multimeric arrays; Bryk M et al.; Ty1 retrotransposons in the yeast Saccharomyces cerevisiae are maintained in a genetically competent but transpositionally dormant state . When located in the ribosomal DNA (rDNA) locus, Ty1 elements are transcriptionally silenced by the specialized heterochromatin that inhibits rDNA repeat recombination . In addition, transposition of all Ty1 elements is repressed at multiple posttranscriptional levels . Here, we demonstrate that Sgs1, a RecQ helicase required for genome stability, inhibits the mobility of Ty1 elements by a posttranslational mechanism . Using an assay for the mobility of Ty1 cDNA via integration or homologous recombination, we found that the mobility of both euchromatic and rDNA-Ty1 elements was increased 32- to 79-fold in sgs1Delta mutants . Increased Ty1 mobility was not due to derepression of silent rDNA-Ty1 elements, since deletion of SGS1 reduced the mitotic stability of rDNA-Ty1 elements but did not stimulate their transcription . Furthermore, deletion of SGS1 did not significantly increase the levels of total Ty1 RNA, protein, or cDNA and did not alter the level or specificity of Ty1 integration . Instead, Ty1 cDNA molecules recombined at a high frequency in sgs1Delta mutants, resulting in transposition of heterogeneous Ty1 multimers . Formation of Ty1 multimers required the homologous recombination protein Rad52 but did not involve recombination between Ty1 cDNA and genomic Ty1 elements . Therefore, Ty1 multimers that transpose at a high frequency in sgs1Delta mutants are formed by intermolecular recombination between extrachromosomal Ty1 cDNA molecules before or during integration . Our data provide the first evidence that the host cell promotes retrotransposition of monomeric Ty1 elements by repressing cDNA recombination.

J Biol Chem, 2001 Sep 14, 276(37), 34738 - 42 Epub 2001 Jul 18.
The phosphatase C(X)5R motif is required for catalytic activity of the Saccharomyces cerevisiae Acr2p arsenate reductase; Mukhopadhyay R et al.; Acr2p detoxifies arsenate by reduction to arsenite in Saccharomyces cerevisiae . This reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism . Acr2p has a HC(X)(5)R motif, the signature sequence of the phosphate binding loop of the dual-specific and protein-tyrosine phosphatase family . In Acr2p these are residues His-75, Cys-76, and Arg-82, respectively . Acr2p has another sequence, (118)HCR, that is absent in phosphatases . Acr2p also has a third cysteine residue at position 106 . Each of these cysteine residues was changed individually to serine residues, whereas the histidine and arginine residues were altered to alanines . Cells of Escherichia coli heterologously expressing the majority of the mutant ACR2 genes retained wild type resistance to arsenate, and the purified altered Acr2p proteins exhibited normal enzymatic properties . In contrast, cells expressing either the C76S or R82A mutations lost resistance to arsenate, and the purified proteins were inactive . These results suggest that Acr2p utilizes a phosphatase-like Cys(X)(5)Arg motif as the catalytic center to reduce arsenate to arsenite.

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8263 - 9
Links between replication and recombination in Saccharomyces cerevisiae: a hypersensitive requirement for homologous recombination in the absence of Rad27 activity; Debrauwere H et al.; The RAD27 gene of Saccharomyces cerevisiae encodes a 5'-3' flap exo/endonuclease, which plays an important role during DNA replication for Okazaki fragment maturation . Genetic studies have shown that RAD27 is not essential for growth, although rad27 Delta mutants are temperature sensitive . Moreover, they exhibit increased sensitivity to alkylating agents, enhanced spontaneous recombination, and repetitive DNA instability . The conditional lethality conferred by the rad27 Delta mutation indicates that other nuclease(s) can compensate for the absence of Rad27 . Indeed, biochemical and genetical analyses indicate that Okazaki fragment processing can be assured by other enzymatic activities or by alternative pathways such as homologous recombination . Here we present the results of a screen that makes use of a synthetic lethality assay to identify functions required for the survival of rad27 Delta strains . Altogether, we confirm that all genes of the Rad52 recombinational repair pathway are required for the survival of rad27 Delta strains at both permissive (23 degrees C) and semipermissive (30 degrees C) temperatures for growth . We also find that several point mutations that confer weaker phenotypes in mitotic than in meiotic cells (rad50S, mre11s) and additional gene deletions (com1/sae2, srs2) exhibit synthetic lethality with rad27 Delta and that rad59 Delta exhibits synergistic effects with rad27 Delta . This and previous studies indicate that homologous recombination is the primary, but not only, pathway that functions to bypass the replication defects that arise in the absence of the Rad27 protein.

Mol Genet Genomics, 2001 Jun, 265(4), 622 - 35
Characterization of Gac1p, a regulatory subunit of protein phosphatase type I involved in glycogen accumulation in Saccharomyces cerevisiae; Wu X et al.; GAC1 and GLC7 encode regulatory and catalytic subunits, respectively, of a type 1 phosphatase (PP1) in Saccharomyces cerevisiae that controls glycogen synthesis by regulating the phosphorylation state of glycogen synthase (Gsy2p) . To investigate the role of Gac1p in this process, a set of GAC1 deletions were tested for their ability to complement a gac1 null mutation and to associate with Glc7p and with Gsy2p . The N-terminal 93 amino acids of Gaclp are necessary and sufficient for the interaction with Glc7p, whereas a region spanning residues 130-502 is required for Gsy2p binding . Both domains are required for full activity in vivo, although the Glc7p-binding domain retains some residual activity and can alter the phosphorylase a phosphatase activity of Glc7p in vitro . Further mutational analysis showed that Val71 and Phe73 of Gaclp are necessary for binding to Glc7p, while Asn356 and Tyr357 of Gaclp are necessary for binding to Gsy2p . These results suggest that Gac1p targets PPI to its substrate Gsy2p and that Gac1p may alter the catalytic activity of PP . Our data also show that overexpression of Gac1p affects glucose repression and ion homeostasis, two additional targets of GLC7, suggesting that multiple regulatory subunits compete for Glc7p binding in vivo.

Mol Genet Genomics, 2001 Jun, 265(4), 585 - 95
A mutation in SPC42, which encodes a component of the spindle pole body, results in production of two-spored asci in Saccharomyces cerevisiae; Ishihara S et al.; In the yeast Saccharomyces cerevisiae, SPC42 is an essential gene, which encodes one of the major components of the spindle pole body (SPB) . We report on a mutation in the SPC42 gene (spc42-102) that results in a sporulation-specific defect . Mitotic growth of haploid and diploid spc42-102 strains is normal and both exhibit the same growth rates as the isogenic wild-type strains . Many diploid spc42-102/spc42-102 cells undergo normal meiotic nuclear divisions, producing four haploid nuclei . However, a significant fraction of meiotic spc42-102/spc42-102 cells contain two immature SPBs and aberrant nuclei that are not surrounded by a prospore membrane . Some 40% of the resultant asci contain only two spores, while wild-type diploid cells almost always produce four-spored asci . Segregation of auxotrophic markers that are tightly linked to the centromere reveals that two-spore asci formed from spc42-102/spc42-102 diploid cells exclusively contain nonsister haploid spores . Western analysis and measurements of the fluorescent signal from an Spc42p-GFP (green fluorescent protein) fusion reveal that the mutant strain fails to accumulate Spc42p at meiosis . Thus, our results suggest that insufficiency of Spc42p during meiosis results in a pair of immature nonsister SPBs that are not enclosed by prospore membrane.

J Biol Chem, 2001 Sep 14, 276(37), 34832 - 9 Epub 2001 Jul 16.
Modulation of myosin function by isoform-specific properties of Saccharomyces cerevisiae and muscle tropomyosins; Strand J et al.; Tropomyosin is an extended coiled-coil protein that influences actin function by binding longitudinally along thin filaments . The present work compares cardiac tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and TPM2, that are much shorter than vertebrate tropomyosins . Unlike cardiac tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is discontinuous; it is interrupted by a 4-residue deletion . TPM1 has two such deletions, which flank the 38-residue partial gene duplication that causes TPM1 to span five actins instead of the four of TPM2 . Each of the three tropomyosin isoforms modulates actin-myosin interactions, with isoform-specific effects on cooperativity and strength of myosin binding . These different properties can be explained by a model that combines opposite effects, steric hindrance between myosin and tropomyosin when the latter is bound to a subset of its sites on actin, and also indirect, favorable interactions between tropomyosin and myosin, mediated by mutually promoted changes in actin . Both of these effects are influenced by which tropomyosin isoform is present . Finally, the tropomyosins have isoform-specific effects on in vitro sliding speed and on the myosin concentration dependence of this movement, suggesting that non-muscle tropomyosin isoforms exist, at least in part, to modulate myosin function.

J Biol Chem, 2001 Sep 14, 276(37), 34441 - 4 Epub 2001 Jul 16.
The GATA transcription factors GLN3 and GAT1 link TOR to salt stress in Saccharomyces cerevisiae; Crespo JL et al.; One of the most recent functions assigned to the TOR signaling pathway in yeast is the coordination of the transcription of genes involved in nutrient utilization . Here we show that transcription of ENA1, a gene encoding a lithium and sodium ion transporter essential for salt tolerance in yeast, is controlled by the TOR signaling pathway . First, ENA1 expression is strongly induced under TOR-inactivating conditions . Second, the absence of the TOR-controlled GATA transcription factors GLN3 and GAT1 results in reduced basal and salt-induced expression of ENA1 . Third, a gln3 gat1 mutant displays a pronounced sensitivity to high concentrations of lithium and sodium . Fourth, TOR1, similar to ENA1, is required for growth under saline stress conditions . In summary, our results suggest that TOR plays a role in the general response to saline stress by regulating the transcription of ENA1 via GLN3 and GAT1.

J Biol Chem, 2001 Sep 21, 276(38), 35458 - 64 Epub 2001 Jul 13.
DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex; Trujillo KM et al.; Saccharomyces cerevisiae RAD50 and MRE11 genes are required for the nucleolytic processing of DNA double-strand breaks . We have overexpressed Rad50 and Mre11 in yeast cells and purified them to near homogeneity . Consistent with the genetic data, we show that the purified Rad50 and Mre11 proteins form a stable complex . In the Rad50.Mre11 complex, the protein components exist in equimolar amounts . Mre11 has a 3' to 5' exonuclease activity that results in the release of mononucleotides . The addition of Rad50 does not significantly alter the exonucleolytic function of Mre11 . Using homopolymeric oligonucleotide-based substrates, we show that the exonuclease activity of Mre11 and Rad50.Mre11 is enhanced for substrates with duplex DNA ends . We have examined the endonucleolytic function of Mre11 on defined, radiolabeled hairpin structures that also contain 3' and 5' single-stranded DNA overhangs . Mre11 is capable of cleaving hairpins and the 3' single-stranded DNA tail . These endonuclease activities of Mre11 are enhanced markedly by Rad50 but only in the presence of ATP . Based on these results, we speculate that the Mre11 nuclease complex may mediate the nucleolytic digestion of the 5' strand at secondary structures formed upon DNA strand separation.

Genetics, 2001 Jul, 158(3), 989 - 97
The defect in transcription-coupled repair displayed by a Saccharomyces cerevisiae rad26 mutant is dependent on carbon source and is not associated with a lack of transcription; Bucheli M et al.; Nucleotide excision repair (NER) is an evolutionarily conserved pathway that removes DNA damage induced by ultraviolet irradiation and various chemical agents that cause bulky adducts . Two subpathways within NER remove damage from the genome overall or the transcribed strands of transcribing genes (TCR) . TCR is a faster repair process than overall genomic repair and has been thought to require the RAD26 gene in Saccharomyces cerevisiae . Rad26 is a member of the SWI/SNF family of proteins that either disrupt chromatin or facilitate interactions between the RNA Pol II and transcription activators . SWI/SNF proteins are required for the expression or repression of a diverse set of genes, many of which are differentially transcribed in response to particular carbon sources . The remodeling of chromatin by Rad26 could affect transcription and/or TCR following formation of DNA damage and other stress-inducing conditions . We speculate that another factor(s) can substitute for Rad26 under particular growth conditions . We therefore measured the level of repair and transcription in two different carbon sources and found that the defect in the rad26 mutant for TCR was dependent on the type of carbon source . Furthermore, TCR did not correlate with transcription rate, suggesting that disruption of RAD26 leads to a specific defect in DNA repair and not transcription.

Genetics, 2001 Jul, 158(3), 973 - 88
Suppressors of ssy1 and ptr3 null mutations define novel amino acid sensor-independent genes in Saccharomyces cerevisiae; Forsberg H et al.; Ssy1p and Ptr3p are components of the yeast plasma membrane SPS amino acid sensor . In response to extracellular amino acids this sensor initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and amino acid permeases (AAPs) . As a result of diminished leucine uptake capabilities, ssy1Delta leu2 and ptr3Delta leu2 mutant strains are unable to grow on synthetic complete medium (SC) . Genes affecting the functional expression of AAPs were identified by selecting spontaneous suppressing mutations in amino acid sensor-independent (ASI) genes that restore growth on SC . The suppressors define 11 recessive (asi) complementation groups and 5 dominant (ASI) linkage groups . Strains with mutations in genes assigned to these 16 groups fall into two phenotypic classes . Mutations in the class I genes (ASI1, ASI2, ASI3, TUP1, SSN6, ASI13) derepress the transcription of AAP genes . ASI1, ASI2, and ASI3 encode novel membrane proteins, and Asi1p and Asi3p are homologous proteins that have conserved ubiquitin ligase-like RING domains at their extreme C termini . Several of the class II genes (DOA4, UBA1, BRO1, BUL1, RSP5, VPS20, VPS36) encode proteins implicated in controlling aspects of post-Golgi endosomal-vacuolar protein sorting . The results from genetic and phenotypic analysis indicate that SPS sensor-initiated signals function positively to facilitate amino acid uptake and that two independent ubiquitin-mediated processes negatively modulate amino acid uptake.

Eur J Biochem, 2001 Jul, 268(14), 3930 - 6
In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae; Martins AM et al.; The kinetics of glyoxalase I {(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5} and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis . Initial rate analysis was performed by hyperbolic regression of initial rates using the program HYPERFIT . Glyoxalase I exhibited saturation kinetics on 0.05-2.5 mM hemithioacetal concentration range, with kinetic parameters Km 0.53 +/- 0.07 mM and V (3.18 +/- 0.16) x 10(-2) mM.min(-1) . Glyoxalase II also showed saturation kinetics in the SD-lactoylglutathione concentration range of 0.15-3 mM and Km 0.32 +/- 0.13 mM and V (1.03 +/- 0.10) x 10(-3) mM.min(-1) were obtained . The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program GEPASI . Several optimization methods were used to minimize the sum of squares of residuals . The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis-Menten model . The kinetic parameters obtained, Km 0.62 +/- 0.18 mM and V (2.86 +/- 0.01) x 10(-2) mM.min(-1), were in agreement with those obtained by initial rate analysis . The results obtained for glyoxalase II, using either the irreversible Michaelis-Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with Km . The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.

Nucleic Acids Res, 2001 Jul 15, 29(14), 3069 - 79
Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease; Bae SH et al.; In order to gain insights into the structural basis of the multifunctional Dna2 enzyme involved in Okazaki fragment processing, we performed biochemical, biophysical and genetic studies to dissect the domain structure of Dna2 . Proteolytic digestion of Dna2 using subtilisin produced a 127 kDa polypeptide that lacked the 45 kDa N-terminal region of Dna2 . Further digestion generated two subtilisin-resistant core fragments of approximately equal size, 58 and 60 kDa . Surprisingly, digestion resulted in a significant (3- to 8-fold) increase in both ATPase and endonuclease activities compared to the intact enzyme . However, cells with a mutant DNA2 allele lacking the corresponding N-terminal region were severely impaired in growth, being unable to grow at 37 degrees C, indicating that the N-terminal region contains a domain critical for a cellular function(s) of Dna2 . Analyses of the hydrodynamic properties of and in vivo complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa domain revealed that Dna2 is active as the monomer and thus the defect in the mutant Dna2 protein is not due to its inability to multimerize . In addition, we found that the N-terminal 45 kDa domain interacts physically with a central region located between the two catalytic domains . Our results suggest that the N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the enzymatic activities of Dna2 by serving as a site for intra- and intermolecular interactions essential for optimal function of Dna2 in Okazaki fragment processing . The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing.

Nucleic Acids Res, 2001 Jul 15, 29(14), 2927 - 37
Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae; Hong B et al.; Using molecular genetic techniques, we have generated and characterized six temperature sensitive (ts) alleles of nop2 . All failed to support growth at 37 degrees C and one was also formamide sensitive (fs) and failed to grow on media containing 3% formamide . Conditional lethality is not due to rapid turnover of mutant Nop2p proteins at 37 degrees C . Each allele contains between seven and 14 amino acid substitutions and one possesses a nonsense mutation near the C-terminus . Mapping experiments with one allele, nop2-4, revealed that a subset of the amino acid substitutions conferred the ts phenotype and that these mutations have an additive effect . All six mutants exhibited dramatic reductions in levels of 60S ribosome subunits under non-permissive conditions as well as some reduction at permissive temperature . Processing of 27S pre-rRNA to mature 25S rRNA was defective in all six mutants grown under non-permissive conditions . Levels of the 40S ribosomal subunit and 18S rRNA were not significantly affected . Amino acid substitutions in nop2 conditional alleles are discussed in the context of the hypothesis that Nop2p functions both as an RNA methyltransferase and a trans-acting factor in rRNA processing and large ribosomal subunit biogenesis.

Mol Biol Cell, 2001 Jul, 12(7), 2147 - 70
A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae; Ni L et al.; A genome-wide screen of 4168 homozygous diploid yeast deletion strains has been performed to identify nonessential genes that participate in the bipolar budding pattern . By examining bud scar patterns representing the sites of previous cell divisions, 127 mutants representing three different phenotypes were found: unipolar, axial-like, and random . From this screen, 11 functional classes of known genes were identified, including those involved in actin-cytoskeleton organization, general bud site selection, cell polarity, vesicular transport, cell wall synthesis, protein modification, transcription, nuclear function, translation, and other functions . Four characterized genes that were not known previously to participate in bud site selection were also found to be important for the haploid axial budding pattern . In addition to known genes, we found 22 novel genes (20 are designated BUD13-BUD32) important for bud site selection . Deletion of one resulted in unipolar budding exclusively from the proximal pole, suggesting that this gene plays an important role in diploid distal budding . Mutations in 20 other novel BUD genes produced a random budding phenotype and one produced an axial-like budding defect . Several of the novel Bud proteins were fused to green fluorescence protein; two proteins were found to localize to sites of polarized cell growth (i.e., the bud tip in small budded cells and the neck in cells undergoing cytokinesis), similar to that postulated for the bipolar signals and proteins that target cell division site tags to their proper location in the cell . Four others localized to the nucleus, suggesting that they play a role in gene expression . The bipolar distal marker Bud8 was localized in a number of mutants; many showed an altered Bud8-green fluorescence protein localization pattern . Through the genome-wide identification and analysis of different mutants involved in bipolar bud site selection, an integrated pathway for this process is presented in which proximal and distal bud site selection tags are synthesized and localized at their appropriate poles, thereby directing growth at those sites . Genome-wide screens of defined collections of mutants hold significant promise for dissecting many biological processes in yeast.

Mol Biol Cell, 2001 Jul, 12(7), 1957 - 71
Topological and mutational analysis of Saccharomyces cerevisiae Ste14p, founding member of the isoprenylcysteine carboxyl methyltransferase family; Romano JD et al.; Eukaryotic proteins that terminate in a CaaX motif undergo three processing events: isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation . In Saccharomyces cerevisiae, the latter step is mediated by Ste14p, an integral endoplasmic reticulum membrane protein . Ste14p is the founding member of the isoprenylcysteine carboxyl methyltransferase (ICMT) family, whose members share significant sequence homology . Because the physiological substrates of Ste14p, such as Ras and the yeast a-factor precursor, are isoprenylated and reside on the cytosolic side of membranes, the Ste14p residues involved in enzymatic activity are predicted to be cytosolically disposed . In this study, we have investigated the topology of Ste14p by analyzing the protease protection of epitope-tagged versions of Ste14p and the glycosylation status of Ste14p-Suc2p fusions . Our data lead to a topology model in which Ste14p contains six membrane spans, two of which form a helical hairpin . According to this model most of the Ste14p hydrophilic regions are located in the cytosol . We have also generated ste14 mutants by random and site-directed mutagenesis to identify residues of Ste14p that are important for activity . Notably, four of the five loss-of-function mutations arising from random mutagenesis alter residues that are highly conserved among the ICMT family . Finally, we have identified a novel tripartite consensus motif in the C-terminal region of Ste14p . This region is similar among all ICMT family members, two phospholipid methyltransferases, several ergosterol biosynthetic enzymes, and a group of bacterial open reading frames of unknown function . Site-directed and random mutations demonstrate that residues in this region play a critical role in the function of Ste14p.

Curr Biol, 2001 Jun 26, 11(12), 991 - 5
Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae; Sjogren C et al.; The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process . Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair . This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation . The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair . We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast . Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair . Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.

Yeast, 2001 Jul, 18(10), 923 - 30
Hexokinase PII: structural analysis and glucose signalling in the yeast Saccharomyces cerevisiae; Mayordomo I et al.; Hexokinase PII (Hxk2) is a yeast glucose phosphorylating enzyme that, besides its role in glycolysis, seems to have an additional role in glucose signalling . To study the domains in Hxk2 that may participate in this latter process, we have constructed 11 mutant alleles using site-directed mutagenesis . Six of them were clustered charged-to-alanine mutants in which clusters of charged residues were changed to alanine residues . Two of them contained substitutions in Ser15 to either alanine or glutamic acid and three of them had deletions at either the N-terminus or the C-terminus of the protein . In most of them, the catalytic activity correlated directly with their functionality in glucose signalling . However, we found two mutants (Delta1-15 and Delta476-486) that, having low catalytic activity, were still fully functional in glucose signalling . This may indicate that other factors and not just the catalytic activity of the enzyme may be important for the functionality of the protein in glucose signalling .

Proc Natl Acad Sci U S A, 2001 Jul 17, 98(15), 8542 - 7 Epub 2001 Jul 10.
The endopolyphosphatase gene: essential in Saccharomyces cerevisiae; Sethuraman A et al.; Endopolyphosphatases (Ppn1) from yeast and animal cells hydrolyze inorganic polyphosphate (poly P) chains of many hundreds of phosphate residues into shorter lengths . The limit digest consists predominantly of chains of 60 (P(60)) and 3 (P(3)) P(i) residues . Ppn1 of Saccharomyces cerevisiae, a homodimer of 35-kDa subunits (about 352-aa) is of vacuolar origin and requires the protease activation of a 75-kDa (674-aa) precursor polypeptide . The Ppn1 gene (PPN1) now has been cloned, sequenced, overexpressed, and deleted . That PPN1 encodes Ppn1 was verified by a 25-fold increase in Ppn1 when overexpressed under a GAL promoter and also by several peptide sequences that match exactly with sequences in a yeast genome ORF, the mutation of which abolishes Ppn1 activity . Null mutants in Ppn1 accumulate long-chain poly P and are defective in growth in minimal media . A double mutant of PPN1 and PPX1 (the gene encoding a potent exopolyphosphatase) loses viability rapidly in stationary phase . Whether this loss is a result of the excess of long-chain poly P or to the lack of shorter chains (i.e., poly P(60) and P(3)) is unknown . Overexpression of the processed form of Ppn1 should provide a unique and powerful reagent to analyze poly P when the chain termini are unavailable to the actions of polyPase and poly P kinase.

Cell Motil Cytoskeleton, 2001 Jun, 49(2), 67 - 77
Mutagenesis of beta-tubulin cysteine residues in Saccharomyces cerevisiae: mutation of cysteine 354 results in cold-stable microtubules; Gupta ML Jr et al.; Cysteine residues play important roles in the control of tubulin function . To determine which of the six cysteine residues in beta-tubulin are critical to tubulin function, we mutated the cysteines in Saccharomyces cerevisiae beta-tubulin individually to alanine and serine residues . Of the twelve mutations, only three produced significant effects: C12S, C354A, and C354S . The C12S mutation was lethal in the haploid, but the C12A mutation had no observable phenotype . Based on interactive views of the electron crystallographic structure of tubulin, we suggest that substitution of serine for cysteine at this position has a destabilizing effect on the interaction of tubulin with the exchangeable GTP . The two C354 mutations, although not lethal, produced dramatic effects on microtubules and cellular processes that require microtubules . The C354 mutant cells had decreased growth rates, a slowed mitosis, increased resistance to benomyl, and impaired nuclear migration and spindle assembly . The C354A mutation produced a more severe phenotype than the C354S mutation: the haploid cells had chromosome segregation defects, only 50% of cells in a culture were viable, and a significant percentage of the cells were misshapened . Cytoplasmic microtubules in the C354S and C354A cells were longer than in the control strain and spindle structures appeared shorter and thicker . Both cytoplasmic and spindle microtubules in the two C354 mutants were extremely stable to cold temperature . After 24 h at 4 degrees C, the microtubules were still present and, in fact, very long and thick tubulin polymers had formed . Evidence exists to indicate that the C354 residue in mammalian tubulin is near the colchicine binding site and the electron crystal structure of tubulin places the residue at the interface between the alpha- and beta-subunits . The sulfhydryl group is situated in a polar environment, which may explain why the alanine mutation is more severe than the serine mutation . When the C12S and the two C354 mutations were made in a diploid strain, the mutated tubulin was incorporated into microtubules and the resulting heterozygotes had phenotypes that were intermediate between those of the mutated haploids and the wild-type strains . The results suggest that the C12 and C354 residues play important roles in the structure and function of tubulin.

J Bacteriol, 2001 Aug, 183(15), 4580 - 7
Cytotoxic and genotoxic consequences of heat stress are dependent on the presence of oxygen in Saccharomyces cerevisiae; Davidson JF et al.; Lethal heat stress generates oxidative stress in Saccharomyces cerevisiae, and anaerobic cells are several orders of magnitude more resistant than aerobic cells to a 50 degrees C heat shock . Here we characterize the oxidative effects of this heat stress . The thermoprotective effect in anaerobic cells was not due to expression of HSP104 or any other heat shock gene, raising the possibility that the toxicity of lethal heat shock is due mainly to oxidative stress . Aerobic but not anaerobic heat stress caused elevated frequencies of forward mutations and interchromosomal DNA recombination . Oxidative DNA repair glycosylase-deficient strains under aerobic conditions showed a powerful induction of forward mutation frequencies compared to wild-type cells, which was completely abolished under anaerobiosis . We also investigated potential causes for this oxygen-dependent heat shock-induced genetic instability . Levels of sulfhydryl groups, dominated mainly by the high levels of the antioxidant glutathione (reduced form) and levels of vitamin E, decreased after aerobic heat stress but not after anaerobic heat stress . Aerobic heat stress also led to an increase in mitochondrial membrane disruption of several hundredfold, which was 100-fold reduced under anaerobic conditions.

Mol Microbiol, 2001 Jun, 40(6), 1391 - 401
The glycerol channel Fps1p mediates the uptake of arsenite and antimonite in Saccharomyces cerevisiae; Wysocki R et al.; The Saccharomyces cerevisiae FPS1 gene encodes a glycerol channel protein involved in osmoregulation . We present evidence that Fps1p mediates influx of the trivalent metalloids arsenite and antimonite in yeast . Deletion of FPS1 improves tolerance to arsenite and potassium antimonyl tartrate . Under high osmolarity conditions, when the Fps1p channel is closed, wild-type cells show the same degree of As(III) and Sb(III) tolerance as the fps1Delta mutant . Additional deletion of FPS1 in mutants defective in arsenite and antimonite detoxification partially suppresses their hypersensitivity to metalloid salts . Cells expressing a constitutively open form of the Fps1p channel are highly sensitive to both arsenite and antimonite . We also show by direct transport assays that arsenite uptake is mediated by Fps1p . Yeast cells appear to control the Fps1p-mediated pathway of metalloid uptake, as expression of the FPS1 gene is repressed upon As(III) and Sb(III) addition . To our knowledge, this is the first report describing a eukaryotic uptake mechanism for arsenite and antimonite and its involvement in metalloid tolerance.

Genes Cells, 2001 Jun, 6(6), 495 - 506
Yeast Saccharomyces cerevisiae has two cis-prenyltransferases with different properties and localizations . Implication for their distinct physiological roles in dolichol synthesis; Sato M et al.; BACKGROUND: Dolichol is a family of long-chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER) . We have identified a key enzyme of the dolichol synthesis, cis-prenyltransferase, as Rer2p from Saccharomyces cerevisiae . We have also isolated a multicopy suppressor of an rer2 mutant and named it SRT1 . It encodes a protein similar to Rer2p but its function has not been established . RESULTS: The cis-prenyltransferase activity of Srt1p has been proved biochemically in the lysate of yeast cells lacking Rer2p . The polyprenol product of Srt1p is longer in chain length than that of Rer2p and is not sufficiently converted to dolichol and dolichyl phosphate, unlike that of Rer2p . The subcellular localization of these two isozymes has been examined by immunofluorescence microscopy and by the use of GFP fusion proteins . Whereas GFP-Rer2p is localized to the continuous ER and some dots associated with the ER, GFP-Srt1p shows only punctate localization patterns . Immunofluorescence double staining with Erg6p, a marker of lipid particles in yeast, indicates that Srt1p is mainly localized to lipid particles (lipid bodies) . RER2 is mainly expressed in the early logarithmic phase, while the expression of SRT1 is induced in the stationary phase . CONCLUSIONS: We have shown that yeast has two active cis-prenyltransferases with different properties . This result implies that the two isozymes have different physiological roles during the life cycle of the yeast.

Plant J, 2001 May, 26(3), 329 - 38
Arabidopsis UVH3 gene is a homolog of the Saccharomyces cerevisiae RAD2 and human XPG DNA repair genes; Liu Z et al.; To identify mechanisms of DNA repair in Arabidopsis thaliana, we have analyzed a mutant (uvh3) which exhibits increased sensitivity to ultraviolet (UV) light, H2O2 and ionizing radiation and displays a premature senescence phenotype . The uvh3 locus was mapped within chromosome III to the GL1 locus . A cosmid contig of the GL1 region was constructed, and individual cosmids were used to transform uvh3 mutant plants . Cosmid N9 was found to confer UV-resistance, H2O2-resistance and a normal senescence phenotype following transformation, indicating that the UVH3 gene is located on this cosmid and that all three phenotypes are due to the same mutation . Analysis of cosmid N9 sequences identified a gene showing strong similarity to two homologous repair genes, RAD2 (Saccharomyces cerevisiae) and XPG (human), which encode an endonuclease required for nucleotide excision repair of UV-damage . The uvh3 mutant was shown to carry a nonsense mutation in the coding region of the AtRAD2/XPG gene, thus revealing that the UVH3 gene encodes the AtRAD2/XPG gene product . In humans, the homologous XPG protein is also involved in removal of oxygen-damaged nucleotides by base excision repair . We discuss the possibility that the increased sensitivity of the uvh3 mutant to H2O2 and the premature senescence phenotype might result from failure to repair oxygen damage in plant tissues . Finally, we show that the AtRAD2/XPG gene is expressed at moderate levels in all plant tissues.

Mol Cell Biol, 2001 Aug, 21(15), 5031 - 40
Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae; Pluta K et al.; Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood . Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III . We show here that tRNA levels are elevated in maf1 mutant cells . In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1 cell extracts . Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect . Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III . These results indicate that Maf1p acts as a negative effector of Pol III synthesis . This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

Genetika, 2001 May, 37(5), 602 - 9
{Suppression of frameshift mutation as a result of partial inactivation of translation termination factors in Saccharomyces cerevisiae yeast}; Kulikov VN et al.; Special search for frameshift mutations, which are suppressed by the cytoplasmic {PSI} factor and by omnipotent nonsense suppressors (recessive mutations in the SUP35 and SUP45 genes), partially inactivating a translation termination complex, was initiated in the LYS2 gene in the yeast Saccharomyces cerevisiae . Mutations were obtained after exposure to UV light and treatment with a mixture consisting of 1.6- and 1.8-dinitropyrene (DNP) . This mixture was shown to induce mutations of the frameshift type with a high frequency . The majority of these mutations were insertions of one A or T, which is in good agreement with the data obtained in studies of DNP-induced mutagenesis in other eukaryotes . Frameshift suppression in yeast was first shown on the example of the mutation obtained in this work (lys2-90), which carried the insertion of an extra T in the sequence of five T . This frameshift suppression was shown to occur in the presence of the {PSI} factor (i.e., due to the prion form of the translation release factor eRF3) and as a result of mutations in genes SUP35 or SUP45, which partially inactivate translation termination factors eRF3 and eRF1, respectively . Alternative mechanisms of programmed translational frameshifting in the course of translation and the possibility of enhancing the effectiveness of such frameshifting in the presence of the {PSI} factor are considered.

FEBS Lett, 2001 Jun 29, 500(1-2), 12 - 6
An interplay between the TOM complex and porin isoforms in the yeast Saccharomyces cerevisiae mitochondria; Antos N et al.; The outer mitochondrial membrane of Saccharomyces cerevisiae contains two isoforms of mitochondrial porin, known also as the voltage-dependent anion channel . The isoform termed here porin1 displays channel-forming activity enabling metabolite transport whereas the second one, termed here porin2, does not form a channel and its function is still not clear . We have shown recently that in the absence of porin1, the channel within the protein import machinery (the TOM complex) is essential for metabolite transport across the outer membrane {Kmita and Budzinska, Biochim . Biophys . Acta 1509 (2000) 6044-6050} . Here, we report that the TOM complex channel may also serve as a supplementary pathway for metabolites in the presence of porin1 when the permeability of the latter is limited and the role of the TOM complex seems to increase when porin2 is depleted.

Genes Genet Syst, 2001 Apr, 76(2), 141 - 7
Regulation of the localization of Dbf2 and mob1 during cell division of saccharomyces cerevisiae; Yoshida S et al.; The mitotic exit network (MEN) governs Cdk inactivation . In budding yeast, MEN consists of the protein phosphatase Cdc14, the ras-like GTPase Tem1, protein kinases Cdc15, Cdc5, Dbf2 and Dbf2-binding protein Mob1 . Tem1, Dbf2, Cdc5 and Cdc15 have been reported to be localized at the spindle pole body (SPB) . Here we report changes of the localization of Dbf2 and Mob1 during cell division . Dbf2 and Mob1 localize to the SPBs in anaphase and then moves to the bud neck, just prior to actin ring assembly, consistent with their role in cytokinesis . The neck localization, but not SPB localization, of Dbf2 was inhibited by the Bub2 spindle checkpoint . Cdc14 is the downstream target of Dbf2 in Cdk inactivation, but we found that the neck localization of DbP2 and Mob1 was dependent on the Cdc14 activity, suggesting that Dbf2 and Mob1 function in cytokinesis at the end of the mitotic signaling cascade.

J Biol Chem, 2001 Aug 24, 276(34), 31825 - 30 Epub 2001 Jun 28.
Saccharomyces cerevisiae Adr1p governs fatty acid beta-oxidation and peroxisome proliferation by regulating POX1 and PEX11; Gurvitz A et al.; Saccharomyces cerevisiae Adr1p is essential for fatty acid degradation and peroxisome proliferation . Here, the role of Adr1p was examined with respect to the transcriptional regulation of the Pip2p-Oaf1p dependent genes POX1 and PEX11 . POX1 encodes the rate-limiting enzyme of peroxisomal beta-oxidation, acyl-CoA oxidase . The POX1 promoter was shown to contain a canonical Adr1p element (UAS1), within which the oleate response element (ORE) was nested . PEX11 codes for a peroxin that is critical for normal peroxisome proliferation, and its promoter was shown similarly to contain a UAS1-like element overlapping the ORE . Northern analysis demonstrated that transcriptional up-regulation of both POX1 and PEX11 was abolished in adr1 Delta mutant cells, and immunoblotting confirmed that the abundance of their gene products was dramatically reduced . Studies of an overlapping ORE/UAS1 arrangement in the CTA1 promoter revealed synergy between these elements . We conclude that overlapping ORE and UAS1 elements in conjunction with their binding factors Pip2p-Oaf1p and Adr1p coordinate the carbon flux through beta-oxidation with peroxisome proliferation.

J Biol Chem, 2001 Sep 21, 276(38), 35396 - 404 Epub 2001 Jun 28.
Protein oxidation in G0 cells of Saccharomyces cerevisiae depends on the state rather than rate of respiration and is enhanced in pos9 but not yap1 mutants; Aguilaniu H et al.; Immunodetection of protein carbonyl groups demonstrates that growth arrest elicited by carbon or nitrogen starvation causes an increased oxidation of proteins in Saccharomyces cerevisiae . Mutant analysis suggests that the response regulator Pos9p is involved in mitigating self-inflicted oxidative damages in G(0) cells, whereas Yap1p is primarily required in growing cells . The data also suggest that oxidation of target proteins is not a priori an effect of arrest of cell division or nutrient depletion and cannot be explained by the respiratory activity alone nor a high ratio of catabolic/anabolic activity in G(0) cells . Instead, we observed that starvation elicits a transition in the respiratory state (from phosphorylating to nonphosphorylating respiration) and that this transition is associated with a stepwise increase in protein oxidation . During carbon starvation, this transition and increase in oxidation occurs immediately as the carbon source is depleted, growth is arrested, and the respiratory rate falls drastically . In contrast, during nitrogen starvation and excess carbon the respiratory state transition and stepwise increase in protein oxidation are markedly delayed and occur long after the nitrogen source has been depleted and division and growth-arrested . Oxidation in G(0) cells could be enhanced by treating cells with low concentrations of antimycin A and attenuated with myxothiazol, indicating that protein oxidation is intimately linked to reactive oxygen species generated by semiquinones of the Q-cycle . Thus, the work presented suggests that the degree of coupling in the mitochondrial respiratory apparatus rather then the overall rate of respiration affects the degree of protein oxidation in nondividing yeast cells.

Mol Cell, 2001 Jun, 7(6), 1191 - 200
Regulated ARE-mediated mRNA decay in Saccharomyces cerevisiae; Vasudevan S et al.; The stability of several oncogene, cytokine, and growth factor transcripts is tightly regulated by signaling pathways through an ARE (AU-rich element) present in their 3'-UTRs . We have identified a yeast transcript, TIF51A, whose stability is regulated through its AU-rich 3'-UTR . We demonstrate that the mammalian TNFalpha and c-fos AREs regulate turnover of a reporter yeast transcript in a similar manner . AREs stabilize the transcript in glucose media and function as destabilizing elements in media lacking glucose or when the Hog1p/p38 MAP kinase pathway is inhibited . Significantly, both yeast and mammalian AREs promote deadenylation-dependent decapping in the yeast system . Furthermore, the yeast ELAV homolog, Pub1p, regulates the stability mediated by the TNFalpha ARE . These results demonstrate that yeast possess a regulatable mechanism for ARE-mediated decay and suggest conservation of this turnover process from yeast to humans.

Nature, 2001 Jun 28, 411(6841), 1073 - 6
Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae; Myung K et al.; Gross chromosome rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions and inversions, are often observed in cancer cells . Spontaneous GCRs are rare in Saccharomyces cerevisiae; however, the existence of mutator mutants with increased genome instability suggests that GCRs are actively suppressed . Here we show by genetic analysis that these genome rearrangements probably result from DNA replication errors and are suppressed by at least three interacting pathways or groups of proteins: S-phase checkpoint functions, recombination proteins and proteins that prevent de novo addition of telomeres at double-strand breaks (DSBs) . Mutations that inactivate these pathways cause high rates of GCRs and show synergistic interactions, indicating that the pathways that suppress GCRs all compete for the same DNA substrates.

Yeast, 2001 Jun 30, 18(9), 781 - 7
Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene; Guerreiro P et al.; Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement . Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene . The other five genes are non-essential, and no phenotypes could be associated to their inactivation . Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the beta-subunit of the succinyl-CoA ligase . For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416 .

Proc Natl Acad Sci U S A, 2001 Jul 3, 98(14), 7801 - 5 Epub 2001 Jun 26.
A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca(2+)-permeable channel in the yeast vacuolar membrane; Palmer CP et al.; The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels . We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae . Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family . By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance . In physiological conditions, tens of micromolar cytoplasmic Ca(2+) activates the YVC1 current carried by cations including Ca(2+) across the vacuolar membrane . Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes . Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene . This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

Acta Biol Hung, 2001, 52(2-3), 325 - 33
Cytoskeleton in regenerating protoplasts and restoration of cell polarity in the yeast Saccharomyces cerevisiae; Svoboda A et al.; The actin cables and microtubules were disrupted during protoplasting in Saccharomyces cerevisiae cells . In the process of protoplast regeneration, the cytoplasmic microtubules were the first to be restored; the actin patches remained regularly distributed under the surface of the growing protoplast . After the cell wall had been regenerated in a gelatine medium, the actin patches aggregated into clusters, which marked the site of bud development . An incomplete cell wall was the apparent cause for uncoupling between karyokinesis and cytokinesis . The presence of several nuclei in the cell gave rise to several buds emerging simultaneously and was probably related to their random positions . In haploids, however, the axial type of budding was also obvious in the reverting protoplasts . These observations suggest that actin is a structure involved in the restoration of polar growth during protoplast regeneration, and that the cell wall plays an important role in this process: in its absence, actin fails to polarise.

Mutat Res, 2001 Jul 12, 486(2), 137 - 46
Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae; Friedl AA et al.; The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in post-replicational repair of DNA damage . We have investigated the genetic interactions between mutations in these genes with respect to cell survival and ectopic gene conversion following treatment of logarithmic and early stationary cells with UV- and gamma-rays . We find that the genetic interaction between the rad5 and rad18 mutations depends on DNA damage type and position in the cell cycle at the time of treatment . Inactivation of SRS2 suppresses damage sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic phase . When irradiated in stationary phase, the srs2 mutation enhances the sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants . Irrespective of the growth phase, the srs2 mutation reduces the frequency of damage-induced ectopic gene conversion in rad5 and rad18 mutants . In addition, we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for SCR . We propose a model in which the Srs2 protein has pro-recombinogenic or anti-recombinogenic activity, depending on the context of the DNA damage.

FEMS Microbiol Lett, 2001 Jun 25, 200(2), 181 - 4
Molecular cloning and identification of UTR1 of a yeast Saccharomyces cerevisiae as a gene encoding an NAD kinase; Kawai S et al.; UTR1 of the yeast Saccharomyces cerevisiae was cloned from the genomic DNA by polymerase chain reaction and expressed in Escherichia coli . Characterization of the purified UTR1p revealed that UTR1p is a NAD kinase consisting of six identical subunits with a molecular mass of 60 kDa . UTR1p specifically phosphorylated NAD in the presence of ATP, dATP, or CTP as phosphoryl donors, and was most active at pH 8.0, 30 degrees C . Km values of UTR1p for NAD and ATP were determined to be 0.50 mM and 0.60 mM, respectively.

FEBS Lett, 2001 Jun 22, 499(3), 230 - 4
Mathematical analysis of a mechanism for autonomous metabolic oscillations in continuous culture of Saccharomyces cerevisiae; Wolf J et al.; Autonomous metabolic oscillations were observed in aerobic continuous culture of Saccharomyces cerevisiae . Experimental investigation of the underlying mechanism revealed that several pathways and regulatory couplings are involved . Here a hypothetical mechanism including the sulfate assimilation pathway, ethanol degradation and respiration is transformed into a mathematical model . Simulations confirm the ability of the model to produce limit cycle oscillations which reproduce most of the characteristic features of the system.

Traffic, 2001 Jul, 2(7), 449 - 64
Nuclear localization of the Saccharomyces cerevisiae HMG protein NHP6A occurs by a Ran-independent nonclassical pathway; Yen YM et al.; The Saccharomyces cerevisiae non-histone protein 6-A (NHP6A) is a member of the high-mobility group 1/2 protein family that bind and bend DNA of mixed sequence . NHP6A has only one high-mobility group 1/2 DNA binding domain and also requires a 16-amino-acid basic tail at its N-terminus for DNA binding . We show in this report that nuclear accumulation of NHP6A is strictly correlated with its DNA binding properties since only nonhistone protein 6 A-green fluorescent protein chimeras that were competent for DNA binding were localized to the nucleus . Despite the requirement for basic residues within the N-terminal segment for DNA binding and nuclear accumulation, this region does not appear to contain a nuclear localization signal . Moreover, NHP6A does not bind to the yeast nuclear localization signal receptor SRP1 and nuclear targeting of NHP6A does not require the function of the 14 different importins . Unlike histone H2B1 which contains a classical nuclear localization signal, entry of NHP6A into the nucleus was found to be independent of Ran as judged by coexpression of Ran GTPase mutants and was shown to occur at 0 degrees C after a 15-min induction . These unusual properties lead us to suggest that NHP6A entry into the nucleus proceeds by a nonclassical Ran-independent pathway.

Eur J Biochem, 2001 Jun, 268(12), 3416 - 22
The ABC transporter Pdr5p mediates the efflux of nonsteroidal ecdysone agonists in Saccharomyces cerevisiae; Hu W et al.; We have previously shown that the synthetic nonsteroidal ecdysone agonist tebufenozide (RH-5992) is actively excluded by resistant cells of insects . To identify the transporter that could be involved in the efflux of RH-5992, the role of three ATP binding cassette transporters, Pdr5p, Snq2p and Ycf1p, has been studied using transporter-deletion mutants of yeast Saccharomyces cerevisiae . PDR5 (pleiotropic drug resistance 5) deletion mutants (Deltapdr5 and Deltapdr5Deltasnq2) retained significantly higher levels of 14C-radiolabeled RH-5992 within the cells when compared to wild-type strain or single deletion mutants of SNQ2 (Deltasnq2) and YCF1 (Deltaycf1) . Introduction of an expression vector containing the PDR5 gene into the PDR5 single deletion mutant reversed the effect, resulting in the active exclusion of {14C}RH-5992 from these cells as efficiently as the wild-type cells . These results demonstrated that the ABC transporter Pdr5p but not Snq2p or Ycf1p was responsible for the active exclusion of {14C}RH-5992 in yeast . This exclusion was temperature-dependent and was blocked by the ATPase inhibitors oligomycin and vanadate, indicating that the efflux was an active process . The mutants with the PDR5 deletion can also selectively accumulate {14C}RH-0345 and {14C}RH-2485, but not {14C}RH-5849, indicating that these three compounds share the same transporter Pdr5p for efflux.

RNA, 2001 Jun, 7(6), 896 - 903
Identification of cis-acting elements involved in 3'-end formation of Saccharomyces cerevisiae 18S rRNA; van Beekvelt CA et al.; In yeast, the 3' end of mature 18S rRNA is generated by endonucleolytic cleavage of the 20S precursor at site D . Available data indicate that the major cis-acting elements required for this processing step are located in relatively close proximity to the cleavage site . To identify these elements, we have studied the effect of mutations in the mature 18S and ITS1 sequences neighboring site D on pre-rRNA processing in vivo . Using clustered point mutations, we found that alterations in the sequence spanning site D from position -5 in 18S rRNA to +6 in ITS1 reduced the efficiency of processing at this site to different extents as demonstrated by the lower level of the mature 18S rRNA and the increase in 20S pre-rRNA in cells expressing only mutant rDNA units . More detailed analysis revealed an important role for the residue located 2 nt upstream from site D (position -2), whereas sequence changes at position -1, +1, and +2 relative to site D had no effect . The data further demonstrate that the proposed base pairing between the 3' end of 18S rRNA and the 5' end of ITS1 is not important for efficient and accurate processing at site D, nor for the formation of functional 40S ribosomal subunits . These results were confirmed by analyzing the accumulation of the D-A2 fragment derived from the mutant 20S pre-rRNA in cells that lack the Xrn1p exonuclease responsible for its degradation . The latter results also showed that the accuracy of cleavage was affected by altering the spacer sequence directly downstream of site D but not by mutations in the 18S rRNA sequence preceding this site.

J Bacteriol, 2001 Jul, 183(14), 4386 - 8
New plasmid system to select for Saccharomyces cerevisiae purine-cytosine permease affinity mutants; Wagner R et al.; The FCY2 gene of Saccharomyces cerevisiae encodes a purine-cytosine permease (PCP) that mediates the active transport of purines and cytosine . A structure-function model for this PCP has been recently proposed . In this study, we developed a plasmid-based system that generated a number of affinity-mutated alleles, enabling us to define new amino acids critical for permease function.

Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7134 - 9
Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae; Sagermann M et al.; In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments . We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme . This is the first structure of any component of a V-type ATPase . The H-subunit is not required for assembly but plays an essential regulatory role . Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins . A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself . The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins . A cleft between the amino- and carboxyl-terminal domains also suggests another possible site of interaction with other factors.

Curr Genet, 2001 May, 39(3), 127 - 36
A novel putative reductase (Cpd1p) and the multidrug exporter Snq2p are involved in resistance to cercosporin and other singlet oxygen-generating photosensitizers in Saccharomyces cerevisiae; Ververidis P et al.; Phytopathogenic Cercospora species produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers which absorb light energy and produce extremely cytotoxic, reactive oxygen species . In this work, we used Saccharomyces cerevisiae as a model system for the identification and cloning of genes whose products mediate cercosporin detoxification . Two genesexpressed in high-copy number vectors conferred cercosporin resistance to an otherwise sensitive strain . One gene codes for Snq2p, a well-characterized multidrug, ABC-type, efflux protein . The other, designated CPD1 (Cercosporin Photosensitizer Detoxification), encodes a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases . We showed that over-expression of either of these proteins can also mediate resistance to other singlet oxygen-generating compounds . The involvement of Snq2p and Cpd1p in photosensitizer detoxification reinforces previous observations which suggested that singlet oxygen acts on membrane lipids and that cellular resistance to cercosporin is mediated by a mechanism involving toxin efflux and/or toxin reduction.

FEMS Microbiol Lett, 2001 Jun 12, 200(1), 53 - 8
Interaction of Knr4 protein, a protein involved in cell wall synthesis, with tyrosine tRNA synthetase encoded by TYS1 in Saccharomyces cerevisiae; Dagkessamanskaia A et al.; The Knr4 protein, known to be involved in the regulation of cell wall assembly in Saccharomyces cerevisiae, strongly interacts with the tyrosine tRNA synthetase protein encoded by TYS1 as demonstrated by the genetic two-hybrid system and a biochemical pull-down experiment using GST--Tys1p fusion . Data reported here raise the possibility that this physical interaction between these proteins is required for dityrosine formation during the sporulation process . In addition, it is shown that the efficiency of spores formation was drastically reduced in diploid cells homozygous for the disruption of KNR4 or for a temperature-sensitive mutation of TYS1, although this effect could be independent of their protein interaction . Altogether, these data provide novel functions of Knr4p and Tys1p to those that were known before.

Mutat Res, 2001 Jul 1, 478(1-2), 169 - 76
Mitotic recombination and inactivation in Saccharomyces cerevisiae induced by UV-radiation (254 nm) and hyperthermia depend on UV fluence rate; Petin VG et al.; In experiments with wild-type diploid yeast cells of Saccharomyces cerevisiae, the synergistic interaction of ultraviolet (UV) light (wavelength, 254 nm) and heat (45--60 degrees C) was studied both for mutagenic and inactivation effects . Simultaneous hyperthermia and UV light treatments increase the frequency of UV-induced mitotic intergenic recombination (crossing-over) and cell inactivation . The enhancing effect was a function of UV light fluence rate . It is concluded that the effect of hyperthermia on low fluence UV or high fluence UV irradiation results in comparable effects on survival and mitotic recombination suggesting similar modulation by hyperthermia of the effects induced by UV at different fluence rates . The interpretation of the data obtained was carried out within the widely accepted point of view considering the synergistic effects as a result of repair ability damage.

Mol Genet Genomics, 2001 May, 265(3), 560 - 8
A role for Sds3p, a component of the Rpd3p/Sin3p deacetylase complex, in maintaining cellular integrity in Saccharomyces cerevisiae; Vannier D et al.; The SDS3 gene was identified in a suppressor screen for mutations that enhance position-effect silencing in yeast . Cells that are defective in SDS3 have pleiotropic phenotypes, similar to those seen in the absence of the histone deacetylase components Rpd3p and Sin3p, including meiotic defects and improper regulation of the HO gene . To gain further insight into SDS3 function we undertook an epistasis analysis with other SDS genes . We found that sds3 is synthetically lethal in combination with a deletion of the SWI6 (SDS11) gene, which encodes a cell-cycle regulator . sds3 swi6 double mutants do not display a specific cell-cycle arrest phenotype, but instead die due to cell lysis . Constitutive expression of the G1 cyclin gene CLN2 restores viability to an sds3 swi6 strain, as does overexpression of SKT5/ CHS4, which encodes a regulatory subunit of chitin synthase III, and SSD1, a gene previously implicated in ensuring cell-cycle progression and cellular integrity . Significantly, growth in the presence of 1 M sorbitol or overexpression of PKC1 also partially suppresses the lethal phenotype of the sds3 swi6 strain . This lethality in the absence of SWI6 function most probably reflects an important or essential role for Sds3p in the Rpd3p/Sin3p histone deacetylase complex, since RPD3 and SIN3 mutations are also synthetically lethal in combination with swi6 and these phenotypes are also rescued by elevated dosage of SKT5/CHS4, SSD1, or PCK1 . Taken together, these data indicate that the transcription factor Swi6p and the Rpd3p-based deacetylase complex act in parallel pathways to activate genes required for cell wall biosynthesis.

Mol Genet Genomics, 2001 May, 265(3), 461 - 8
Marker structure and recombination substrate environment influence conversion preference of broken and unbroken alleles in Saccharomyces cerevisiae; Weng Y et al.; Double-strand break (DSB)-induced gene conversion was investigated using plasmid x chromosome (P x C) and chromosomal direct-repeat recombination substrates with markers arranged such that functional (selected) products could not arise by longpatch mismatch repair initiated from the DSB . As seen previously with analogous substrates, these substrates yield products with discontinuous conversion tracts, albeit at low frequency . Most conversion tracts were of minimum length, suggesting that heteroduplex DNA (hDNA) is limiting, or that co-repair imposes selective pressure against products with more extensive hDNA . When functional products can arise by long-patch mismatch repair, the broken allele is converted in nearly all products . In contrast, in the absence of long-patch mismatch repair, unbroken alleles are frequently converted, and we show that such conversion depends on both marker structure (i.e., long palindromic vs . nonpalindromic insertions) and the chromosomal environment of the recombination substrate . We propose that conversion of unbroken alleles is largely a consequence of the segregation of unrepaired markers, and that differences in mismatch repair efficiency underlie the observed effects of marker structure and chromosome environment on allele conversion preference.

Genetics, 2001 Jun, 158(2), 563 - 72
Interaction of the repressors Nrg1 and Nrg2 with the Snf1 protein kinase in Saccharomyces cerevisiae; Vyas VK et al.; The Snf1 protein kinase is essential for the transcription of glucose-repressed genes in Saccharomyces cerevisiae . We identified Nrg2 as a protein that interacts with Snf1 in the two-hybrid system . Nrg2 is a C(2)H(2) zinc-finger protein that is homologous to Nrg1, a repressor of the glucose- and Snf1-regulated STA1 (glucoamylase) gene . Snf1 also interacts with Nrg1 in the two-hybrid system and co-immunoprecipitates with both Nrg1 and Nrg2 from cell extracts . A LexA fusion to Nrg2 represses transcription from a promoter containing LexA binding sites, indicating that Nrg2 also functions as a repressor . An Nrg1 fusion to green fluorescent protein is localized to the nucleus, and this localization is not regulated by carbon source . Finally, we show that VP16 fusions to Nrg1 and Nrg2 allow low-level expression of SUC2 in glucose-grown cells, and we present evidence that Nrg1 and Nrg2 contribute to glucose repression of the DOG2 gene . These results suggest that Nrg1 and Nrg2 are direct or indirect targets of the Snf1 kinase and function in glucose repression of a subset of Snf1-regulated genes.

Genetics, 2001 Jun, 158(2), 549 - 62
A role for the Swe1 checkpoint kinase during filamentous growth of Saccharomyces cerevisiae; La Valle R et al.; In this study we show that inactivation of Hsl1 or Hsl7, negative regulators of the Swe1 kinase, enhances the invasive behavior of haploid and diploid cells . The enhancement of filamentous growth caused by inactivation of both genes is mediated via the Swe1 protein kinase . Whereas Swe1 contributes noticeably to the effectiveness of haploid invasive growth under all conditions tested, its contribution to pseudohyphal growth is limited to the morphological response under standard assay conditions . However, Swe1 is essential for pseudohyphal differentiation under a number of nonstandard assay conditions including altered temperature and increased nitrogen . Swe1 is also required for pseudohyphal growth in the absence of Tec1 and for the induction of filamentation by butanol, a related phenomenon . Although inactivation of Hsl1 is sufficient to suppress the defect in filamentous growth caused by inactivation of Tec1 or Flo8, it is insufficient to promote filamentous growth in the absence of both factors . Moreover, inactivation of Hsl1 will not bypass the requirement for nitrogen starvation or growth on solid medium for pseudohyphal differentiation . We conclude that the Swe1 kinase modulates filamentous development under a broad spectrum of conditions and that its role is partially redundant with the Tec1 and Flo8 transcription factors.

Mol Biol Cell, 2001 Jun, 12(6), 1801 - 10
Identification of a Saccharomyces cerevisiae gene that is required for G1 arrest in response to the lipid oxidation product linoleic acid hydroperoxide; Alic N et al.; Reactive oxygen species cause damage to all of the major cellular constituents, including peroxidation of lipids . Previous studies have revealed that oxidative stress, including exposure to oxidation products, affects the progression of cells through the cell division cycle . This study examined the effect of linoleic acid hydroperoxide, a lipid peroxidation product, on the yeast cell cycle . Treatment with this peroxide led to accumulation of unbudded cells in asynchronous populations, together with a budding and replication delay in synchronous ones . This observed modulation of G1 progression could be distinguished from the lethal effects of the treatment and may have been due to a checkpoint mechanism, analogous to that known to be involved in effecting cell cycle arrest in response to DNA damage . By examining several mutants sensitive to linoleic acid hydroperoxide, the YNL099c open reading frame was found to be required for the arrest . This gene (designated OCA1) encodes a putative protein tyrosine phosphatase of previously unknown function . Cells lacking OCA1 did not accumulate in G1 on treatment with linoleic acid hydroperoxide, nor did they show a budding, replication, or Start delay in synchronous cultures . Although not essential for adaptation or immediate cellular survival, OCA1 was required for growth in the presence of linoleic acid hydroperoxide, thus indicating that it may function in linking growth, stress responses, and the cell cycle . Identification of OCA1 establishes cell cycle arrest as an actively regulated response to oxidative stress and will enable further elucidation of oxidative stress-responsive signaling pathways in yeast.

Mol Biol Cell, 2001 Jun, 12(6), 1645 - 69
Dynamic localization of the Swe1 regulator Hsl7 during the Saccharomyces cerevisiae cell cycle; Cid VJ et al.; In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form . Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase . Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase . In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly . We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins . With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1 . A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1 . When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1 . GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot . Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle . Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB) . These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay . Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.

J Biol Chem, 2001 Aug 24, 276(34), 32136 - 44 Epub 2001 Jun 14.
Gln3p nuclear localization and interaction with Ure2p in Saccharomyces cerevisiae; Kulkarni AA et al.; Gln3p is one of two well characterized GATA family transcriptional activation factors whose function is regulated by the nitrogen supply of the cell . When nitrogen is limiting, Gln3p and Gat1p are concentrated in the nucleus where they bind GATA sequences upstream of nitrogen catabolite repression (NCR)-sensitive genes and activate their transcription . Conversely, in excess nitrogen, these GATA sequences are unoccupied by Gln3p and Gat1p because these transcription activators are excluded from the nucleus . Ure2p binds to Gln3p and Gat1p and is required for NCR-sensitive transcription to be repressed and for nuclear exclusion of these transcription factors . Here we show the following . (i) Gln3p residues 344-365 are required for nuclear localization . (ii) Replacing Ser-344, Ser-347, and Ser-355 with alanines has minimal effects on GFP-Gln3p localization . However, replacing Gln3p Ser-344, Ser-347, and Ser-355 with aspartates results in significant loss of its ability to be concentrated in the nucleus . (iii) N and C termini of the Gln3p region required for it to complex with Ure2p and be excluded from the nucleus are between residues 1-103 and 301-365, respectively . (iv) N and C termini of the Ure2p region required for it to interact with Gln3p are situated between residues 101-151 and 330-346, respectively . (v) Loss of Ure2p residues participating in either dimer or prion formation diminishes its ability to carry out NCR-sensitive regulation of Gln3p activity.

J Cell Biol, 2001 Jun 11, 153(6), 1327 - 36
Prion filament networks in {URE3} cells of Saccharomyces cerevisiae; Speransky VV et al.; The {URE3} prion (infectious protein) of yeast is a self-propagating, altered form of Ure2p that cannot carry out its normal function in nitrogen regulation . Previous data have shown that Ure2p can form protease-resistant amyloid filaments in vitro, and that it is aggregated in cells carrying the {URE3} prion . Here we show by electron microscopy that {URE3} cells overexpressing Ure2p contain distinctive, filamentous networks in their cytoplasm, and demonstrate by immunolabeling that these networks contain Ure2p . In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates . However, these distributions do not resemble the single, large networks seen in {URE3} cells, nor do the control cells exhibit cytoplasmic filaments . In {URE3} cell extracts, Ure2p is present in aggregates that are only partially solubilized by boiling in SDS and urea . In these aggregates, the NH(2)-terminal prion domain is inaccessible to antibodies, whereas the COOH-terminal nitrogen regulation domain is accessible . This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains . These observations support and further specify the concept of the {URE3} prion as a self-propagating amyloid.

Am J Physiol Cell Physiol, 2001 Jul, 281(1), C33 - 45
Expression and characterization of the anion transporter homologue YNL275w in Saccharomyces cerevisiae; Zhao R et al.; A search of the yeast Saccharomyces cerevisiae genome has revealed an open reading frame, YNL275w, which encodes a 576-amino acid protein that shows sequence similarity to the family of mammalian Cl-/HCO3- anion exchangers and Na+/HCO3- cotransporters . This yeast protein also has a very similar hydropathy profile to the mammalian HCO3- transporters, indicating a similar membrane topology and structure . A V5 epitope and His6-tagged version of Ynl275wp was expressed in yeast and was localized to the plasma membrane by subcellular fractionation and immunofluorescence labeling . The protein was purified by nickel affinity chromatography and was found not to be N-glycosylated . The protein's mobility on SDS-PAGE gels was not altered by treatment with N-glycanase F, alpha-mannosidase, or by mutation of each of the five consensus N-glycosylation sites . The protein did not bind to concanavalin A by lectin blotting or lectin affinity chromatography . The expressed protein bound specifically to a stilbene disulfonate inhibitor resin (SITS-Affi-Gel), and this binding could be competed by certain anions (HCO3-, Cl-, NO3-, and I-) but not by others (SO4(2-) and PO4(3-)) . These results suggest that the yeast gene YNL275w encodes a nonglycosylated anion transport protein, localized to the plasma membrane.

Mol Microbiol, 2001 Jun, 40(5), 1085 - 96
SUT1p interaction with Cyc8p(Ssn6p) relieves hypoxic genes from Cyc8p-Tup1p repression in Saccharomyces cerevisiae; Regnacq M et al.; SUT1 is a hypoxic gene encoding a nuclear protein that belongs to the Zn{II}2Cys-6 family . It has been shown that constitutive expression of SUT1 induces exogenous sterol uptake in aerobically growing Saccharomyces cerevisiae cells . A differential display approach was used to identify genes whose transcription is modified upon SUT1 induction . Within the promoter sequence of one of these genes, DAN1, we identified the region responsive to SUT1 and showed that it has a strong repressive activity when cloned in the vicinity of distinct promoters . Upon SUT1 constitutive expression in aerobiosis, the repression is released, allowing enhanced transcription of the reporter gene . We provide evidence that the repression is promoted by the Cyc8p(Ssn6p)-Tup1p co-repressor and that release of repression is the result of a physical interaction between Sut1p and Cyc8p . Moreover, genetic data suggest that complete derepression of the reporter gene requires a functional Cyc8p . In addition, we show that Sut1p is involved in the induction of hypoxic gene transcription when the cells are shifted from aerobiosis to anaerobiosis.

Mol Microbiol, 2001 Jun, 40(5), 1067 - 83
The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage; Rep M et al.; A major part of the transcriptional response of yeast cells to osmotic shock is controlled by the HOG pathway and several downstream transcription factors . Sko1p is a repressor that mediates HOG pathway-dependent regulation by binding to CRE sites in target promoters . Here, we report five target genes of Hog1p-Sko1p: GRE2, AHP1, SFA1, GLR1 and YML131w . The two CREs in the GRE2 promoter function as activating sequences and, hence, bind (an) activator protein(s) . However, the two other yeast CRE-binding proteins, Aca1p and Aca2p, are not involved in regulation of the GRE2 promoter under osmotic stress . In the absence of the co-repressor complex Tup1p-Ssn6p/Cyc8p, which is recruited by Sko1p, stimulation by osmotic stress is still observed . These data indicate that Sko1p is not only required for repression, but also involved in induction upon osmotic shock . All five Sko1p targets encode oxidoreductases with demonstrated or predicted roles in repair of oxidative damage . Altered basal expression levels of these genes in hog1Delta and sko1Delta mutants may explain the oxidative stress phenotypes of these mutants . All five Sko1p target genes are induced by oxidative stress, and induction involves Yap1p . Although Sko1p and Yap1p appear to mediate osmotic and oxidative stress responses independently, Sko1p may affect Yap1p promoter access or activity . The five Sko1p target genes described here are suitable models for studying the interplay between osmotic and oxidative responses at the molecular and physiological levels.

Mol Microbiol, 2001 Jun, 40(5), 1059 - 66
Transcriptional control of the GAL/MEL regulon of yeast Saccharomyces cerevisiae: mechanism of galactose-mediated signal transduction; Bhat PJ et al.; In the yeast Saccharomyces cerevisiae, the interplay between Gal3p, Gal80p and Gal4p determines the transcriptional status of the genes needed for galactose utilization . The interaction between Gal80p and Gal4p has been studied in great detail; however, our understanding of the mechanism of Gal3p in transducing the signal from galactose to Gal4p has only begun to emerge recently . Historically, Gal3p was believed to be an enzyme (catalytic model) that converts galactose to an inducer or co-inducer, which was thought to interact with GAL80p, the repressor of the system . However, recent genetic analyses indicate an alternative 'protein-protein interaction model' . According to this model, Gal3p is activated by galactose, which leads to its interaction with Gal80p . Biochemical and genetic experiments that support this model provided new insights into how Gal3p interacts with the Gal80p-Gal4p complex, alleviates the repression of Gal80p and thus allows Gal4p to activate transcription . Recently, a galactose-independent signal was suggested to co-ordinate the induction of GAL genes with the energy status of the cell.

Mol Microbiol, 2001 May, 40(4), 1020 - 6
Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae; Sugajska E et al.; The trehalose-degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen-starved glucose-repressed yeast (Saccharomyces cerevisiae) cells . Trehalase activation is mediated by phosphorylation . Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles . Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro . To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22 . Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source . Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive . After the addition of glucose or of a nitrogen source to starved cells, Pph22-overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress-regulated element-controlled genes . Deletion of the SCH9 gene encoding a protein kinase involved in nutrient-induced signal transduction restored glucose-induced trehalase activation in Pph22-overexpressing cells . Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient-induced signal transduction pathways in the absence of nutrients.

Mol Microbiol, 2001 May, 40(4), 890 - 9
Conformational changes play a role in regulating the activity of the proline utilization pathway-specific regulator in Saccharomyces cerevisiae; Des Etages SA et al.; In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources . Changes in target gene expression occur through an alteration in activity of the DNA-bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins . Here, we report that the 'on' conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non-inducibility of the pathway . In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease . These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.

Appl Microbiol Biotechnol, 2001 May, 55(4), 471 - 5
Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses; Shibasaki S et al.; The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP) . The emission from EGFP on the cell surface was affected by changes in pH . The amount of EGFP on the cell surface, displayed as alpha-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0 . The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 10(4)-10(5) molecules of alpha-agglutinin-fused molecules per cell were expressed . Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.

Appl Microbiol Biotechnol, 2001 May, 55(4), 454 - 62
Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae; Takahashi S et al.; The role of the prosequence of Rhizopus oryzae lipase (ROL) with a preprosequence was analyzed by an expression system using Saccharomyces cerevisiae . When the mature portion of ROL (mROL) fused to the pre-alpha-factor leader sequence was expressed, secretion of active mROL was not observed . However, when mROL was synthesized together with the prosequence in trans (individually and coincidentally), secretion of active mROL was observed . The results indicate that the prosequence of ROL helped correct folding of mROL and its subsequent secretion from the yeast cells, and that physical linkage (cis) of the prosequence to the mature region was not prerequisite . From the expression of the ROL mutants with deletions at the N-terminal end of the prosequence together with mROL in trans, the residues from 20 to 37 in the prosequence were essential for the secretion, and those from 38 to 57 were essential for the formation of the active ROL and might play a role as an intramolecular chaperone . The results using the fragment of the prosequence confirmed that these residues (20-57) were significant for in vivo folding and secretion of active mROL.

J Biol Chem, 2001 Aug 24, 276(34), 31561 - 6 Epub 2001 Jun 07.
Identification of a Delta 4 fatty acid desaturase from Thraustochytrium sp . involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea; Qiu X et al.; The existence of Delta 4 fatty acid desaturation in the biosynthesis of docosahexanoic acid (DHA) has been questioned over the years . In this report we describe the identification from Thraustochytrium sp . of two cDNAs, Fad4 and Fad5, coding for Delta 4 and Delta 5 fatty acid desaturases, respectively . The Delta 4 desaturase, when expressed in Saccharomyces cerevisiae, introduced a double bond at position 4 of 22:5(n-3) and 22:4(n-6) resulting in the production of DHA and docosapentanoic acid . The enzyme, when expressed in Brassica juncea under the control of a constitutive promoter, desaturated the exogenously supplied substrate 22:5(n-3), resulting in the production of DHA in vegetative tissues . These results support the notion that DHA can be synthesized via Delta 4 desaturation and suggest the possibility that DHA can be produced in oilseed crops on a large scale.

Arch Biochem Biophys, 2001 Jun 15, 390(2), 195 - 205
Expression and degradation of the cystic fibrosis transmembrane conductance regulator in Saccharomyces cerevisiae; Kiser GL et al.; Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein . Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins . To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p . Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells . Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker . Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin beta-lactone . Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment . Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p . Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation . These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae .

Mol Cell Biol, 2001 Jul, 21(13), 4321 - 9
Identification of a peroxisomal ATP carrier required for medium-chain fatty acid beta-oxidation and normal peroxisome proliferation in Saccharomyces cerevisiae; van Roermund CW et al.; We have characterized the role of YPR128cp, the orthologue of human PMP34, in fatty acid metabolism and peroxisomal proliferation in Saccharomyces cerevisiae . YPR128cp belongs to the mitochondrial carrier family (MCF) of solute transporters and is localized in the peroxisomal membrane . Disruption of the YPR128c gene results in impaired growth of the yeast with the medium-chain fatty acid (MCFA) laurate as a single carbon source, whereas normal growth was observed with the long-chain fatty acid (LCFA) oleate . MCFA but not LCFA beta-oxidation activity was markedly reduced in intact ypr128cDelta mutant cells compared to intact wild-type cells, but comparable activities were found in the corresponding lysates . These results imply that a transport step specific for MCFA beta-oxidation is impaired in ypr128cDelta cells . Since MCFA beta-oxidation in peroxisomes requires both ATP and CoASH for activation of the MCFAs into their corresponding coenzyme A esters, we studied whether YPR128cp is an ATP carrier . For this purpose we have used firefly luciferase targeted to peroxisomes to measure ATP consumption inside peroxisomes . We show that peroxisomal luciferase activity was strongly reduced in intact ypr128cDelta mutant cells compared to wild-type cells but comparable in lysates of both cell strains . We conclude that YPR128cp most likely mediates the transport of ATP across the peroxisomal membrane.

J Biol Chem, 2001 Aug 24, 276(34), 31551 - 60 Epub 2001 Jun 04.
Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae; Greenberg RB et al.; Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways . In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs) . In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points . We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation . Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic . Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs . Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions . The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination . A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.

FEBS Lett, 2001 Jun 1, 498(1), 98 - 103
Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae; Alexandre H et al.; DNA microarrays were used to investigate the expression profile of yeast genes in response to ethanol . Up to 3.1% of the genes encoded in the yeast genome were up-regulated by at least a factor of three after 30 min ethanol stress (7% v/v) . Concomitantly, 3.2% of the genes were down-regulated by a factor of three . Of the genes up-regulated in response to ethanol 49.4% belong to the environmental stress response and 14.2% belong to the stress gene family . Our data show that in addition to the previously identified ethanol-induced genes, a very large number of genes involved in ionic homeostasis, heat protection, trehalose synthesis and antioxidant defence also respond to ethanol stress . It appears that a large number of the up-regulated genes are involved in energy metabolism . Thus, 'management' of the energy pool (especially ATP) seems to constitute an ethanol stress response and to involve different mechanisms.

J Am Chem Soc, 2001 Jun 13, 123(23), 5507 - 17
Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: correlation of structure with reactivity in the multicopper oxidases; Machonkin TE et al.; Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomyces cerevisiae . Fet3p is functionally homologous to ceruloplasmin (Cp) in that both are ferroxidases . However, by sequence homology Fet3p is more similar to fungal laccase, and both contain a type 1 Cu site that lacks the axial methionine ligand present in the functional type 1 sites of Cp . To determine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circular dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-depleted mutants . The spectroscopic features of the type 1 Cu site of Fet3p are nearly identical to those of fungal laccase, indicating a very similar three-coordinate geometry . We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics . From poised potential redox titrations, the E degrees of the type 1 Cu site is 427 mV, which is low for a three-coordinate type 1 Cu site . The kinetics of reduction of the type 1 Cu sites of four different multicopper oxidases with two different substrates were compared . The type 1 site of a plant laccase (Rhus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky organic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.029 and 0.013 s(-)(1), respectively) . On the other hand, the type 1 site of a fungal laccase (Coprinus cinereus) is reduced very rapidly by both substrates (k(obs) > 23 s(-)(1)) . In contrast, both Fet3p and Cp are rapidly reduced by Fe(II) (k(obs) > 23 s(-)(1)), but only very slowly by 1,4-hydroquinone (10- and 100-fold more slowly than plant laccase, respectively) . Semiclassical theory is used to analyze the origin of these differences in reactivity in terms of type 1 Cu site accessibility to specific substrates.

Biosci Biotechnol Biochem, 2001 Apr, 65(4), 993 - 5
Expression of marY1, a gypsy-type LTR-retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake, in the budding yeast Saccharomyces cerevisiae; Murata H et al.; marY1 is a gypsy-type LTR-retroelement present in the genome of the ectomycorrhizal homobasidiomycete Tricholoma matsutake . We document here that a marY1-lacZ gene fusion was expressed in the budding yeast Saccharomyces cerevisiae . The finding strongly suggests that marY1 is activated by trans-regulatory factors common to higher fungi, and may be useful for the development of new recombinant systems in ectomycorrhizal fungi and homobasidiomycetes.

J Biol Chem, 2001 Aug 3, 276(31), 28694 - 9 Epub 2001 May 31.
Mutation in PMR1, a Ca(2+)-ATPase in Golgi, confers salt tolerance in Saccharomyces cerevisiae by inducing expression of PMR2, an Na(+)-ATPase in plasma membrane; Park SY et al.; Sodium tolerance in yeast is enhanced by continuous activation of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase that is required for modulation of the Na(+) efflux mechanism . We isolated several salt-tolerant mutations with the treatment of ethylmethane sulfonate under high salt stress . One of the mutations was mapped in the PMR1 gene . Pmr1p, the P-type Ca(2+)-ATPase in the Golgi apparatus, regulates a cytosolic Ca(2+) level in various responses . Cytosolic Ca(2+) concentration in the pmr1 mutant is highly maintained, and thus calcineurin is activated continuously . The treatment of FK506, a specific inhibitor of calcineurin, abolishes the salt-tolerant phenotype of the pmr1 mutant . Activated calcineurin induces the expression of PMR2, encoding the P-type Na(+)-ATPase, through the specific transcription factor, Tcn1p/Crz1p . Also, expression of the PMR2::lacZ reporter gene in the pmr1 mutant was higher than that in wild type . We propose that the pmr1 mutation confers salt tolerance through continuous activation of calcineurin and that Pmr1p might act as a major Ca(2+)-ATPase under high salt stress.

EMBO J, 2001 Jun 1, 20(11), 2655 - 65
C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p; Guillas I et al.; Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum (ER) . When both genes are deleted, cells cannot transport glycosylphosphatidylinositol (GPI)-anchored proteins from the ER to the Golgi at a normal rate . Here we show that microsomes or detergent extracts from lag1lac1 double mutants lack an activity transferring C26 fatty acids from C26-coenzyme A onto dihydrosphingosine or phytosphingosine . As a consequence, in intact cells, the normal ceramides and inositolphosphorylceramides are drastically reduced . lag1lac1 cells compensate for the lack of normal sphingolipids by making increased amounts of C26 fatty acids, which become incorporated into glycerophospholipids . They also contain 20- to 25-fold more free long chain bases than wild type and accumulate very large amounts of abnormally polar ceramides . They make small amounts of abnormal mild base-resistant inositolphospholipids . The lipid remodelling of GPI-anchored proteins is severely compromised in lag1lac1 double mutants since only few and mostly abnormal ceramides are incorporated into the GPI anchors . The participation of Lag1p and Lac1p in ceramide synthesis may explain their role in determining longevity.

Physiol Chem Phys Med NMR, 2000, 32(2), 137 - 44
Cystamine transport in spheroplasts of Saccharomyces cerevisiae; Ravera R et al.; This work is the first demonstration that cystamine is actively accumulated in spheroplasts of Saccharomyces cerevisiae . We have identified and quantitatively determined the transported cystamine in extracts of spheroplasts that have been incubated over different time periods and in the presence of different amounts of cystamine . The method used, already reported in literature for the identification of natural aliphatic polyamines in biological fluids, consists of a derivatization of spheroplast extracts with dabsyl-chloride and subsequent chromatographic analysis in HPLC . Our results show that cystamine accumulation is a function of time, it increases up to 2.5 min then decreases . Transport is inhibited by natural aliphatic polyamines, which, at the same concentration of cystamine (1 mM), cause a decrease in cystamine transport of about 90% for spermidine, 50% for spermine and only 15% for putrescine . Furthermore, transport is energy-dependent as demonstrated by a significant decrease observed in the presence of 2,4-dinitrophenol, ouabain and vanadate . In particular 0.2 mM ouabain causes a decrease of more than 60% in cystamine transport . Our data suggest that cystamine is transported in Saccharomyces cerevisiae spheroplasts via the same polyamine transport system(s) known to be operating in higher eukaryotic cells.

J Biol Chem, 2001 Aug 17, 276(33), 30641 - 7 Epub 2001 May 29.
Rpb4, a non-essential subunit of core RNA polymerase II of Saccharomyces cerevisiae is important for activated transcription of a subset of genes; Pillai B et al.; A major role in the regulation of eukaryotic protein-coding genes is played by the gene-specific transcriptional regulators, which recruit the RNA polymerase II holoenzyme to the specific promoter . Several components of the mediator complex within the holoenzyme also have been shown to affect activation of different subsets of genes . Only recently has it been suggested that besides the largest subunit of RNA polymerase II, smaller subunits like Rpb3 and Rpb5 may have regulatory roles in expression of specific sets of genes . We report here, the role of Rpb4, a non-essential subunit of core RNA polymerase II, in activation of a subset of genes in Saccharomyces cerevisiae . We have shown below that whereas constitutive transcription is largely unaffected, activation from various promoters tested is severely compromised in the absence of RPB4 . This activation defect can be rescued by the overexpression of cognate activators . We have localized the region of Rpb4 involved in activation to the C-terminal 24 amino acids . We have also shown here that transcriptional activation by artificial recruitment of the TATA-binding protein (TBP) to the promoter is also defective in the absence of RPB4 . Surprisingly, the overexpression of RPB7 (the interacting partner of Rpb4) does not rescue the activation defect of all the promoters tested, although it rescues the activation defect of the heat shock element-containing promoter and the temperature sensitivity associated with RPB4 deletion . Overall, our results indicate that Rpb4 and Rpb7 play independent roles in transcriptional regulation of genes.

Arch Microbiol, 2001 Apr, 175(4), 301 - 7
Mechanisms underlying the acquisition of resistance to octanoic-acid-induced-death following exposure of Saccharomyces cerevisiae to mild stress imposed by octanoic acid or ethanol; Cabral MG et al.; Acquisition of resistance to lethal concentrations of octanoic acid was induced in cells of Saccharomyces cerevisiae grown in the presence of sublethal concentrations of this lipophilic acid or following rapid exposure (1 h) of unadapted yeast cells to mild stress imposed by the same acid . Experimental evidence indicated that the referred adaptation involved de novo protein synthesis, presumably due to the rapid induction of a plasma membrane transporter which mediates the active efflux of octanoate out of the cell . Rapid exposure of cells to mild ethanol stress also led to increased resistance to lethal concentrations of octanoic acid . This cross-resistance to octanoic-acid-induced death was below the level of resistance induced by mild octanoic acid stress and did not involve induction of the active expulsion of octanoate out of the cell . However, the rapid exposure of yeast cells to octanoic acid or ethanol led to the activation of plasma membrane H+-ATPase . The physiological role of the two stress responses examined during the present study, namely, the active efflux of octanoate specifically induced by octanoic acid and the stimulation of plasma membrane H+-ATPase activity, is discussed.

Curr Microbiol, 2001 Jun, 42(6), 403 - 7
Molecular cloning of cgrA, the gene encoding the Aspergillus nidulans ortholog of Saccharomyces cerevisiae CGR1; Sun J et al.; Saccharomyces cerevisiae CGR1 encodes a 120-amino acid protein with a predominant nucleolar localization . In this study we report the identification and cloning of the ortholog, cgrA, from Aspergillus nidulans . The cgrA gene is comprised of three exons on A . nidulans Chromosome 7 . The cDNA contains a single open reading frame (ORF) that would encode a protein of 114 amino acids with 44% sequence identity to yeast Cgr1p . A plasmid expressing cgrA complemented the impaired growth phenotype of a yeast strain that can be inducibly depleted of CGR1, and a green fluorescent protein (GFP)-tagged CgrA protein had the same nucleolar localization as the corresponding yeast protein . These results identify cgrA as the A . nidulans ortholog of yeast CGR1 and suggest evolutionary conservation of nucleolar localization mechanisms used by these proteins.

J Cell Biol, 2001 May 28, 153(5), 1085 - 96
Mba1, a novel component of the mitochondrial protein export machinery of the yeast Saccharomyces cerevisiae; Preuss M et al.; The biogenesis of mitochondria requires the integration of many proteins into the inner membrane from the matrix side . The inner membrane protein Oxa1 plays an important role in this process . We identified Mba1 as a second mitochondrial component that is required for efficient protein insertion . Like Oxa1, Mba1 specifically interacts both with mitochondrial translation products and with conservatively sorted, nuclear-encoded proteins during their integration into the inner membrane . Oxa1 and Mba1 overlap in function and substrate specificity, but both can act independently of each other . We conclude that Mba1 is part of the mitochondrial protein export machinery and represents the first component of a novel Oxa1-independent insertion pathway into the mitochondrial inner membrane.

Yeast, 2001 Jun, 18(8), 759 - 73
Identification and characterization of Saccharomyces cerevisiae mutants defective in fluid-phase endocytosis; Wiederkehr A et al.; A mutant library generated by the European Functional Analysis Network (EUROFAN) was screened for strains defective in fluid-phase endocytosis . Accumulation of Lucifer yellow in the vacuole was used as a marker for efficient endocytosis . Fourteen mutants, including ede1Delta, rcy1Delta, sys1Delta and tlg2Delta, previously described to be involved in membrane trafficking, were identified in this screen . alpha-Factor uptake, endocytosis of FM4-64, carboxypeptidase Y secretion, vacuolar morphology, and a vma2 synthetic growth defect were used as criteria to characterize the endocytic defect of the mutant strains obtained . Accordingly, eight mutant strains have endocytic phenotypes in addition to their defect in Lucifer yellow accumulation . These fluid-phase endocytosis mutants are defective at different steps of the endocytic pathway . Interestingly, only two mutants were defective for internalization, two for vacuolar protein sorting and four mutants had aberrant vacuolar morphologies . Some of the mutants identified in this screen that sort carboxypeptidase Y correctly may affect endocytosis at an early post-internalization step before the intersection of the endocytic with the vacuolar protein-sorting pathway .

Yeast, 2001 Jun, 18(8), 723 - 8
Constitutive promoter modules for PCR-based gene modification in Saccharomyces cerevisiae; DeMarini DJ et al.; Initial steps in investigating gene function often include deleting and overexpressing the gene of interest and identifying the subcellular location of the gene product . To facilitate these procedures, a number of new PCR modules, which contain selectable markers and in some cases other genetic elements (e.g promoter elements, epitope tags, and reporter genes) have been developed . These modules are used as PCR substrates to create products that can be targeted to specified locations in the yeast genome, thus modifying that genomic locus . We describe here a series of plasmids that contain a truncated version of the strong ADH1 promoter with and without amino-terminal 3HA and GST tags . Because these plasmids contain the same vector sequences as the GAL1 promoter plasmids, a constitutive and an inducible promoter can now be integrated with a minimal number of primers .

Biol Cell, 2000 Dec, 92(8-9), 629 - 37
Vacuolar morphology and cell cycle distribution are modified by leucine limitation in auxotrophic Saccharomyces cerevisiae; Cakar ZP et al.; Yeast vacuoles are highly dynamic and flexible organelles . In a previous paper, we have shown that subtle, often unrecognised amino acid limitations lead to much lower final cell densities in cultures of different commonly used auxotrophic Saccharomyces cerevisiae strains (Cakar et al., Biotechnol . Lett . 21 (1999) 611) . Here, we demonstrate for two of these strains, CEN.PK 113.6B and CBS7752, that such subtle leucine limitations also affect the number and morphology of vacuoles, and that these changes are correlated with the cell cycle in batch cultures in a similar way as is known from synchronized cultures . Morphological aspects were studied by electron microscopy, using advanced high pressure freezing/freeze-substitution techniques for sample preparation that so far have been barely successful in yeast . Cells of leucine-limited cultures had single, large vacuoles with a hexagonal tonoplast pattern and were partially arrested in G1 phase . To relieve leucine-limitation, additional leucine was supplied extracellularly via the medium or intracellularly via enhanced leucine biosynthesis due to plasmid-based expression of a leucine marker gene . Such cultures reached more than two-fold higher final optical densities in stationary phase . Cells in later growth phase were characterized by fragmented vacuoles lacking any tonoplast pattern and by a smaller proportion of cells in G1 phase . These drastic effects of subtle leucine limitation on cell physiology, vacuolar morphology and cell cycle distribution present a note of caution for morphological and cell cycle studies in yeast.

J Bacteriol, 2001 Jun, 183(12), 3791 - 4
Red fluorescent protein (DsRed) as a reporter in Saccharomyces cerevisiae; Rodrigues F et al.; We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae . Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained . A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.

Biochemistry, 2001 May 29, 40(21), 6335 - 43
Structures of Saccharomyces cerevisiae N-myristoyltransferase with bound myristoylCoA and peptide provide insights about substrate recognition and catalysis; Farazi TA et al.; MyristoylCoA:protein N-myristoyltransferase (Nmt) attaches myristate to the N-terminal Gly residue of proteins involved in a variety of signal transduction cascades, and other critical cellular functions . To gain insight about the structural basis of substrate recognition and catalysis, we determined the structures of a binary complex of Saccharomyces cerevisiae Nmt1p with myristoylCoA to 2.2 A resolution and of a ternary complex of Nmt1p with a nonhydrolyzable myristoylCoA analogue {S-(2-oxo)pentadecylCoA} and an octapeptide substrate (GLYASKLA) to 2.5 A resolution . The binary complex reveals how myristoylCoA alters the conformation of the enzyme to promote binding of both myristoylCoA and peptide and identifies the backbone amides of F170 and L171 as an oxyanion hole which polarizes the reactive thioester carbonyl . The ternary complex structure reveals details of the enzyme's peptide binding specificity and illuminates its mechanism of acyl transfer . The N-terminal Gly ammonium is positioned in close proximity to the C-terminal carboxylate of the protein, where it is poised to undergo the required deprotonation to an amine . In this conformation, the nucleophile is 6.3 A away from the thioester carbonyl . A catalytic mechanism is proposed whereby, once deprotonation is initiated, the N-terminal Gly amine can approximate the thioester carbonyl by rotating along Psi . This motion is facilitated by a H-bond network and leads to reaction between the glycine nitrogen nucleophile and the carbonyl . Loss of CoA from the tetrahedral intermediate may be facilitated by intramolecular H-bonding of the sulfur to the adenylamine of CoA . This affords a compact leaving group and lends a role for the observed bends in the CoA structure . The absolute requirement for Gly at the N-terminus of substrates is explained by the requirement for flexible rotation of its amine.

Mol Genet Genomics, 2001 Mar, 265(1), 102 - 17
Mutations in the SAM domain of STE50 differentially influence the MAPK-mediated pathways for mating, filamentous growth and osmotolerance in Saccharomyces cerevisiae; Jansen G et al.; In Saccharomyces cerevisiae, the MAPKKK Ste11p is involved in three mitogen-activated protein kinase (MAPK) pathways required for mating, filamentous growth and the SHO1-dependent response to hyperosmolarity . All three pathways are also dependent on Ste50p . Ste50p and Ste11p interact constitutively via their N-terminal regions, which include putative SAM domains . Here we show that the interaction of Ste50p and Ste11p is differentially required for modulation of Ste11p function during mating, filamentous growth and the SHO1-dependent response to hyperosmolarity . Two derivatives of Ste50p with mutations in the SAM domain were isolated and characterised . The mutant Ste50 proteins showed reduced binding to Ste11p and a tendency to form homodimers in two-hybrid and in vitro binding assays . Interestingly, these two Ste50p-SAM mutants were associated with increased activation of the mating and filamentous-growth pathways, but a reduction in the SHO1-dependent growth response to hyperosmolarity, relative to the wild-type Ste50p . Moreover, when exposed to hyperosmolarity, these Ste50p-SAM mutants activate genes in the mating (FUS1) and filamentous-growth (FLO11) pathways to higher levels than does the wild type . Thus the Ste50p-Ste11p interaction may differentially modulate the flow of information through the various MAPK-mediated pathways.

Biochimie, 2001 May, 83(5), 427 - 32
A novel family of longer chain length dolichols present in oleate-induced yeast Saccharomyces cerevisiae; Szkopinska A et al.; The typical size of the yeast dolichol family ranges from 14 to 19 isoprene units D((14-19)) with dolichol(16) being the dominating species . Induction of peroxisome proliferation by growing the cells in medium containing oleate as carbon source induces the synthesis of an additional family of longer dolichols D((19-24)) with D(21) being the most prominent . This phenomenon is abolished in the peroxisome biogenesis deficient strain in which the PEX1 gene (encoding Pex1p peroxin) has been disrupted . The total amount of dolichols in pex1Delta cells is lower than in the wild-type cells, as is the amount of phosphatidylcholine . Moreover, the levels of 3-hydroxy-3-methylglutaryl CoA reductase and farnesyl diphosphate synthase, two key enzymes in dolichol biosynthesis, are decreased in the absence of a functional PEX1 gene . The presence of longer dolichols in oleate-induced Saccharomyces cerevisiae cells, the absence of this additional family in peroxisome deficient cells, and a decrease of the total amount of dolichols in these cells indicate the involvement of peroxisomes in the biosynthesis of dolichols in this organism.

Arch Biochem Biophys, 2001 Mar 1, 387(1), 11 - 9
Structure of the unliganded cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae; Mashhoon N et al.; The structure of TPK1delta, a truncated variant of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae, was determined in an unliganded state at 2.8 A resolution and refined to a crystallographic R-factor of 19.4% . Comparison of this structure to that of its fully liganded mammalian homolog revealed a highly conserved protein fold comprised of two globular lobes . Within each lobe, root mean square deviations in Calpha positions averaged approximately equals 0.9 A . In addition, a phosphothreonine residue was found in the C-terminal domain of each enzyme . Further comparison of the two structures suggests that a trio of conformational changes accompanies ligand-binding . The first consists of a 14.7 degrees rigid-body rotation of one lobe relative to the other and results in closure of the active site cleft . The second affects only the glycine-rich nucleotide binding loop, which moves approximately equals 3 A to further close the active site and traps the nucleotide substrate . The third is localized to a C-terminal segment that makes direct contact with ligands and the ligand-binding cleft . In addition to resolving the conformation of unliganded enzyme, the model shows that the salient features of the cAMP-dependent protein kinase are conserved over long evolutionary distances.

Microbios, 2001, 105(410), 29 - 41
Reduction in mitochondrial respiratory capacity in Saccharomyces cerevisiae induced by expression of hepatitis B virus surface antigen; Chien LF et al.; Transformants of Saccharomyces cerevisiae strain TL154 (MATalpha, trp1, leu2) expressing hepatitis B virus surface antigen showed reduced rates of cell growth compared with those of nontransformed cells . The rates of phosphorylative, nonphosphorylative, and uncoupled respiration in mitochondria isolated from the transformants were reduced relative to those of mitochondria derived from nontransformed cells, regardless of whether the cells were cultured in rich or minimal medium . The electrophoretic protein profiles of cell and mitochondrial extracts did not differ substantially between transformed and nontransformed cells . These results suggest that the reduced rate of mitochondrial respiration in the transformants may be due to impairment of metabolic function rather than to inhibition of the expression of components of the respiratory chain.

Arch Biochem Biophys, 2001 Jan 15, 385(2), 301 - 10
Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides; Knoche M et al.; Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP . In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation . All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate . However, the kinetic analysis revealed distinctly differences between them . Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation . Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites . No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A . Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta cross-link . Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits.

Nucleic Acids Res, 2001 May 15, 29(10), 2020 - 5
The Saccharomyces cerevisiae RAD9 cell cycle checkpoint gene is required for optimal repair of UV-induced pyrimidine dimers in both G(1) and G(2)/M phases of the cell cycle; Al-Moghrabi NM et al.; Cells respond to DNA damage by activating both cellular growth arrest and DNA repair processes . In Saccharomyces cerevesiae the RAD9 gene controls DNA damage-mediated cell cycle arrest that is known to allow efficient repair . To ascertain whether RAD9 plays a role in DNA repair per se, the removal of UV-induced photolesions was assessed in synchronized isogenic normal and rad9 cells using the high resolution primer extension technique . The results show that RAD9 is indeed involved in the removal of photolesions from both the transcribed and the non-transcribed strands of the reporter GAL10 gene, in G(1)- as well as G(2)/M-arrested cells . Interestingly, these data also reveal that in both normal and rad9 mutant, the repair strand bias towards the transcribed stand is more pronounced in G(2)/M- than in G(1)-arrested cells . These data indicate that RAD9 coordinate the cellular response to DNA damage by activating both cell cycle checkpoint and excision repair.

Mol Genet Genomics, 2001 Apr, 265(2), 362 - 6
Stationary-phase mutations in proofreading exonuclease-deficient strains of the yeast Saccharomyces cerevisiae; Babudri N et al.; In order to understand the role of yeast polymerases in spontaneous mutagenesis in non-growing cells we have studied the effects of mutations that impair the 3'--> 5' exonuclease function of polymerases delta (pol3-01) and epsilon (pol2-4) on the spontaneous reversion frequency of the frameshift mutation his7-2 in cells starved for histidine . We showed that for each exonuclease-deficient mutant the rate of reversion per viable cell per day observed in stationary-phase cells remained constant up to the 9th day of starvation (while the number of viable cells dropped), and was very similar to that observed in the same mutants during the growth phase . These data suggest that both DNA polymerases are involved in the control of mutability in non-growing cells.

Mol Genet Genomics, 2001 Apr, 265(2), 258 - 63
Increasing Saccharomyces cerevisiae stress resistance, through the overactivation of the heat shock response resulting from defects in the Hsp90 chaperone, does not extend replicative life span but can be associated with slower chronological ageing of nondividing cells; Harris N et al.; Recent studies on Drosophila and Caenorhabditis elegans indicate that increases in stress resistance result in a longer chronological life span, an effect that must operate primarily on the postmitotic tissues of the adult . Stress resistance can be increased through decreases in Hsp90 chaperone activity, since Hsp90 acts to downregulate the activity of heat shock transcription factor . This study investigated whether the increases in stress resistance associated with reduced Hsp90 chaperone activity influence ageing in the budding yeast Saccharomyces cerevisiae, ageing being measured either as the replicative (nonchronological) senescence of budding cells or as the chronological ageing of non-dividing (stationary phase) cultures . Overactivation of the heat shock response caused no slowing of replicative senescence . In some situations though it was associated with a longer chronological life span of stationary cells, the yeast equivalent of the postmitotic state . This is consistent with the idea that stress resistance exerts its life span-extending effects primarily in postmitotic cells and tissues.

Mol Genet Genomics, 2001 Apr, 265(2), 249 - 57
The IML3/MCM19 gene of Saccharomyces cerevisiae is required for a kinetochore-related process during chromosome segregation; Ghosh SK et al.; The mcm19 mutation in budding yeast affects minichromosome maintenance . In this work we have shown that this mutation leads to defects in the segregation of minichromosomes and chromosomes . The mutant cells show defective kinetochore function as judged by three criteria-- relaxation of the transcriptional block normally associated with a CEN box, stable maintenance of a dicentric plasmid in mutant cells, and mild sensitivity to the antimicrotubule drug benomyl . The MCM19 gene has been cloned and found to be the same as IML3, which codes for the ORF YBR107C . Deletion of the gene was not lethal, nor did it confer any growth defects on the mutant cells . However, the mcm19 null mutation conferred growth defects in the presence of a mutation in the TUB1 gene coding for alpha-tubulin . Two-hybrid experiments showed an interaction between Im13p/Mcm19p and the kinetochore protein Ch14, indicating that the Im13/Mcm19 protein has a role in kinetochore function.

Arch Biochem Biophys, 2001 Apr 1, 388(1), 18 - 24
The NADH diphosphatase encoded by the Saccharomyces cerevisiae NPY1 nudix hydrolase gene is located in peroxisomes; AbdelRaheim SR et al.; The NPY1 nudix hydrolase gene of Saccharomyces cerevisiae has been cloned and shown to encode a diphosphatase (pyrophosphatase) with NADH as the preferred substrate, giving NMNH and AMP as products . NADPH, diadenosine diphosphate, NAD+, NADP+, and ADP-ribose were also utilized efficiently . Km values for NADH, NAD+, and ADP-ribose were 0.17, 0.5, and 1.3 mM and kcat values 1.5, 0.6, and 0.6 s(-1), respectively . NPY1 has a potential C-terminal tripeptide PTS1 peroxisomal targeting signal (SHL) . By fusing NPY1 to the C-terminus of yeast-enhanced green fluorescent protein, the enzyme was found to be targeted to peroxisomes . Colocalization with peroxisomal thiolase was also shown by indirect immunofluorescence . Related sequences in other organisms also have potential PTS1 signals, suggesting an important peroxisomal function for this protein . This function may be the regulation of nicotinamide coenzyme concentrations independently of those in other compartments or the elimination of oxidized nucleotide derivatives from the peroxisomal environment.

Water Res, 2001 Jun, 35(9), 2191 - 200
Removal of divalent heavy metal mixtures from water by Saccharomyces cerevisiae using crossflow microfiltration; Bayhan YK et al.; The removal of heavy metal ions, Ni2+, Cu2+ and Pb2+ using yeast (Saccharomyces cerevisiae) as carriers in a crossflow microfiltration is investigated . The effects of yeast cell and electrolyte concentrations on the transient and steady-state permeate flux and metal ion rejections are established . It is found that the metal ion rejection reaches a plateau if yeast cell concentration is greater than approximately 2 g/l as a result of cell aggregation . The binding affinity of the metals to yeast cell is Pb2+ > Cu2+ > Ni2+, which is also reflected in the metal ion rejection under identical process conditions . Because of the formation of yeast cell flocks in the presence of Pb2+, permeate flux is also higher for this metal . The presence of NaCl decreases both rejection and permeate flux for Ni2+ and Cu2+ but not for Pb2+ . When binary or ternary metal mixtures are used, the rejection of the individual metals is reduced except that of Pb2+ . It is found that the pseudo-gel concentration is unaffected by the presence of metal ions.

Electrophoresis, 2001 Apr, 22(6), 1021 - 3
Detection of Saccharomyces cerevisiae carboxylesterase activity after native and sodium dodecyl sulfate electrophoresis by using fluorescein diacetate as substrate; Lomolino G et al.; A simple method for the visualisation of wine yeast esterase (carboxylesterase EC 3.1.1.1) activity on electrophoretic gels was developed, using the fluorescent substrate fluorescein diacetate . The zymogram system allows a sensitive detection of esterase bands in only 5 min of incubation of both native and sodium dodecyl sulfate gels.

J Biol Chem, 2001 Jul 13, 276(28), 26715 - 23 Epub 2001 May 16.
Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p; Zhang H et al.; Saccharomyces cerevisiae Rad17p is necessary for cell cycle checkpoint arrests in response to DNA damage . Its known interactions with the checkpoint proteins Mec3p and Ddc1p in a PCNA-like complex indicate a sensor role in damage recognition . In a novel application of the yeast two-hybrid system and by immunoprecipitation, we show here that Rad17p is capable of increased self-interaction following DNA damage introduced by 4-nitroquinoline-N-oxide, camptothecin or partial inactivation of DNA ligase I . Despite overlap of regions required for Rad17p interactions with Rad17p or Mec3p, single amino acid substitutions revealed that Rad17p x Rad17p complex formation is independent of Mec3p . E128K (rad17-1) was found to inhibit Rad17p interaction with Mec3p but not with Rad17p . On the other hand, Phe-121 is essential for Rad17p self-interaction, and its function in checkpoint arrest but not for Mec3p interaction . These differential effects indicate that Rad17p-Rad17p interaction plays a role that is independent of the Rad17p x Mec3p x Ddc1p complex, although our results are also compatible with Rad17p-mediated supercomplex formation of the Rad17p x Mec3p x Ddc1p heterotrimer in response to DNA damage.

J Biol Chem, 2001 Aug 3, 276(31), 28659 - 66 Epub 2001 May 16.
Ammonia regulates VID30 expression and Vid30p function shifts nitrogen metabolism toward glutamate formation especially when Saccharomyces cerevisiae is grown in low concentrations of ammonia; van der Merwe GK et al.; The GATA family proteins Gln3p and Gat1p mediate nitrogen catabolite repression (NCR)-sensitive transcription in Saccharomyces cerevisiae . When cells are cultured with a good nitrogen source (glutamine, ammonia), Gln3p and Gat1p are restricted to the cytoplasm, whereas with a poor nitrogen source (proline), they localize to the nucleus, bind to the GATA sequences of NCR-sensitive gene promoters, and activate transcription . The target of rapamycin-signaling cascade and Ure2p participate in regulating the cellular localization of Gln3p and Gat1p . Rapamycin, a Tor protein inhibitor, like growth with a poor nitrogen source, promotes nuclear localization of Gln3p and Gat1p . gln3 Delta and ure2 Delta mutants are partially resistant and hypersensitive to growth inhibition by rapamycin, respectively . We show that a vid30 Delta is more rapamycin-sensitive than wild type but less so than a ure2 Delta . VID30 expression is modestly NCR-sensitive, responsive to deletion of URE2, and greatly increases in low ammonia medium . Patterns of gene expression in a vid30 Delta suggest that the Vid30p function shifts the balance of nitrogen metabolism toward the production of glutamate, especially when cells are grown in low ammonia . CAN1, DAL4, DAL5, MEP2, DAL1, DAL80, and GDH3 transcription is down-regulated by Vid30p function with proline as the nitrogen source . An effect, however, that could easily be indirect.

J Biol Chem, 2001 Jul 13, 276(28), 25661 - 71 Epub 2001 May 16.
Repression of GCN4 mRNA translation by nitrogen starvation in Saccharomyces cerevisiae; Grundmann O et al.; Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation . We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism . Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes . Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4 . Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence . Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p . Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.

Mutat Res, 2001 Jun 5, 486(1), 41 - 52
The REC41 gene of Saccharomyces cerevisiae: isolation and genetic analysis; Chepurnaya OV et al.; Recombination-deficient strains have been proven useful for the understanding of the genetic control of homologous recombination . As the genetic screens used to isolate recombination-deficient (rec(-)) yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with low or elevated rates of recombination . Using this system we isolated a collection of rec(-) mutants . We report the characterization of the REC41 gene identified in this way . REC41 is required for normal levels of interplasmid recombination and gamma-ray induced mitotic interchromosomal recombination . The rec41-1 mutant failed to grow at 37 degrees C . Microscopic analysis of plated cells showed that 45-50% of them did not form visible colonies at permissive temperature . Haploid cells of the rec41 mutant show the same gamma-ray sensitivity as wild type ones . However, the diploid rec41 mutant shows gamma-ray sensitivity which is comparable with heterozygous REC41/rec41-1 diploid cells . This fact indicates semidominance of the rec41-1 mutation . Diploid strains homozygous for the rec41 rad52 mutations had the same gamma-ray sensitivity as single rad52 diploids and exhibited dramatically decreased growth rate . The expression of the HO gene does not lead to inviability of rec41 cells . The rec41 mutation has an effect on meiosis, likely meiotic recombination, even in the heterozygous state . We cloned the REC41 gene . Sequence analysis revealed that the REC41 gene is encoded by ORF YDR245w . Earlier, this ORF was attributed to MNN10, BED1, SLC2, CAX5 genes . Two multicopy plasmids with suppressers of the rec41-1 mutation (pm21 and pm32) were isolated . The deletion analysis showed that only DNA fragments with the CDC43 and HAC1 genes can partially complement the rec41-1 mutation.

FEBS Lett, 2001 May 11, 496(2-3), 81 - 5
Ubiquitin and the SCF(Grr1) ubiquitin ligase complex are involved in the signalling pathway activated by external amino acids in Saccharomyces cerevisiae; Bernard F et al.; In response to external amino acids, the permease-like sensor Ssy1 of Saccharomyces cerevisiae activates a pathway leading to transcriptional induction of several permease genes including AGP1 and PTR2 . We previously reported that AGP1 induction requires Grr1, the F-box protein part of the SCF(Grr1) ubiquitin-ligase complex . We show here that ubiquitin, other components of SCF(Grr1) and the ubiquitin-conjugating enzyme Cdc34 are essential for AGP1 and PTR2 induction . This suggests that transduction to these genes of the amino acid signal generated by Ssy1 involves an SCF(Grr1)-catalysed ubiquitination step.

Radiat Res, 2001 Jun, 155(6), 768 - 77
Persistent genomic instability in the yeast Saccharomyces cerevisiae induced by ionizing radiation and DNA-damaging agents; Brennan RJ et al.; A "hypermutable" genome is a common characteristic of cancer cells, and it may contribute to the progressive accumulation of mutations required for the development of cancer . It has been reported that mammalian cells surviving exposure to gamma radiation display several highly persistent genomic instability phenotypes which may reflect a hypermutability similar to that seen in cancer . These phenotypes include an increased mutation frequency and a decreased plating efficiency, and they continue to be observed many generations after the radiation exposure . The underlying causes of this genomic instability have not been fully determined . We show here that exposure to gamma radiation and other DNA-damaging treatments induces a similar genomic instability in the yeast Saccharomyces cerevisiae . A dose-dependent increase in intrachromosomal recombination was observed in cultures derived from cells surviving gamma irradiation as many as 50 generations after the exposure . Increased forward mutation frequencies and low colony-forming efficiencies were also observed . Persistently elevated recombination frequencies in haploid cells were dominant after these cells were mated to nonirradiated partners, and the elevated recombination phenotype was also observed after treatment with the DNA-damaging agents ultraviolet light, hydrogen peroxide, and ethyl methanesulfonate . Radiation-induced genomic instability in yeast may represent a convenient model for the hypermutability observed in cancer cells.

Fungal Genet Biol, 2001 Mar, 32(2), 105 - 11
Expression of the lactate permease gene JEN1 from the yeast Saccharomyces cerevisiae; Andrade RP et al.; In Saccharomyces cerevisiae, lactate permease induction by lactic acid took place after transcription of JEN1 . JEN1 transcripts were undetectable 10 min after the addition of a pulse of glucose to YP-lactic acid exponentially growing cells, while the permease activity ceased after 50 min . A value of 15.1 min was found for the half-life of JEN1 mRNA, showing the involvement of a glucose-induced mechanism of mRNA degradation . The rapid decline of the carrier activity upon glucose addition points to the existence of an irreversible carbon catabolite inactivation process . Isogenic strains, deleted in genes encoding enzymes involved in lactic acid metabolism, did not express JEN1, indicating an association of the intracellular metabolism of the acid to the transcription of the permease gene . The carbon sources capable of inducing JEN1 transcription were dependent on the strain . Distinct posttranslation mechanisms appeared to be involved in the lactate carrier activity .

J Electron Microsc (Tokyo), 2001, 50(2), 133 - 7
Evidence for glycogen structures associated with plasma membrane invaginations as visualized by freeze-substitution and the Thiery reaction in Saccharomyces cerevisiae; Coulary B et al.; We have used a combination of freeze-substitution electron microscopy and specific reaction for polysaccharides to re-evaluate glycogen structures in Saccharomyces cerevisiae . We also used mutant cells devoid of glycogen to confirm the glycogenic nature of the structures described . Previously described cytoplasmic aggregates were confirmed as glycogen granules . Moreover, an original structure was discovered . This is a ring of glycogen surrounding a finger- or pleat-like plasma membrane invagination . This structure could be physiologically significant in terms of channelling glucose to or from glycogen reserves.

Proc Natl Acad Sci U S A, 2001 May 22, 98(11), 6080 - 5 Epub 2001 May 08.
Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I; Gross S et al.; Cleavage and polyadenylation of mRNA 3' ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I) . Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and/or polyadenylation . Through reconstitution of active CF I from separately expressed and purified proteins, we show that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1 . These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein . Analysis of the individual protein-protein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.

J Enzyme Inhib, 2001, 16(2), 107 - 12
Inhibition of chitin synthetase from Saccharomyces cerevisiae by a new UDP-GlcNAc analogue; Behr JB et al.; The synthesis and biological evaluation of a new UDP-GlcNAc competitor (I), designed to mimic the transition state of the sugar donor in the enzymatic reaction catalysed by chitin synthetase, is described . Compound (I) was found to competitively inhibit chitin synthetase from Saccharomyces cerevisiae with respect to UDP-GlcNAc, but displayed minimal antifungal activity.

Structure (Camb), 2001 Jan 10, 9(1), R11 - 4
Selenomethionine incorporation in Saccharomyces cerevisiae RNA polymerase II; Bushnell DA et al.; A protocol for the incorporation of SeMet into yeast proteins is described . Incorporation at a level of about 50% suffices for the location of Se sites in an anomalous difference Fourier map of the 0.5 MDa yeast RNA polymerase II . This shows the utility of the approach as an aid in the model-building of large protein complexes.

Biochim Biophys Acta, 2001 Jan 26, 1517(2), 177 - 89
Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae; Kennedy MA et al.; To identify regulatory cis-elements in the proximal promoter of the yeast ERG9 squalene synthase gene, promoter deletion analysis was performed . This approach identified two regulatory elements, one an upstream repressing cis-element (URS), and the other an upstream activating cis-element (UAS) . Electromobility shift assays (EMSAs) demonstrated that distinct proteins bind each element . Genetic screens were performed to identify yeast mutants that altered expression of ERG9 promoter-reporter gene fusions . Three non-ergosterol biosynthetic pathway genes were identified . A mutation in TPO1(YLL028W) led to a 5.5-fold increase in ERG9 expression while mutations in YER064C and SLK19 (YOR195W) led to a 3.1- and 5.6-fold decrease, respectively . Deletion analysis of these genes demonstrated that TPO1 and SLK19 specifically regulated ERG9 expression when tested with several different promoter-reporter gene fusions . Additionally, EMSAs demonstrated that extracts derived from the TPO1 deletion strain was unable to shift the repressing cis-element while protein extracts from the SLK19 deletion strain had a reduced shift of the activating cis-element . Furthermore, these two mutants showed quantitative differences in sterols and antifungal drug susceptibilities consistent with their role in regulating ERG9 expression.

Biochim Biophys Acta, 2001 Jan 12, 1544(1-2), 42 - 54
Homoserine dehydrogenase from Saccharomyces cerevisiae: kinetic mechanism and stereochemistry of hydride transfer; Jacques SL et al.; Homoserine dehydrogenase (HSD), which is required for the synthesis of threonine, isoleucine and methionine in fungi, is a potential target for novel antifungal drugs . In order to design effective inhibitors, the kinetic mechanism of Saccharomyces cerevisiae HSD and the stereochemistry of hydride transfer were examined . Product inhibition experiments revealed that yeast HSD follows an ordered Bi Bi kinetic mechanism, where NAD(P)H must bind the enzyme prior to aspartate semialdehyde (ASA) and homoserine is released first followed by NAD(P)+ . H-(1,2,4-triazol-3-yl)-D,L-alanine was an uncompetitive inhibitor of HSD with respect to NADPH (K(ii)=3.04+/-0.18 mM) and a noncompetitive inhibitor with respect to ASA (K(is)=1.64+/-0.36 mM, K(ii)=3.84+/-0.46 mM), in agreement with the proposed substrate order . Both kinetic isotope and viscosity experiments provided evidence for a very rapid catalytic step and suggest nicotinamide release to be primarily rate limiting . Incubation of HSD with stereospecifically deuterated NADP{2H} and subsaturating amounts of aspartate semialdehyde revealed that the pro-S NADPH hydride is transferred to the aldehyde . The pH dependence of steady state kinetic parameters indicate that ionizable groups with basic pKs may be involved in substrate binding, consistent with the observation of Lys223 at the enzyme active site in the recently determined 3D structure {B . DeLaBarre, P.R . Thompson, G.D . Wright, A.M . Berghuis, Nat . Struct . Biol . 7 (2000) 238-244} . These findings provide the requisite foundation for future exploitation of fungal HSD in inhibitor design.

Nucleic Acids Res, 2001 May 1, 29(9), 1943 - 50
Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I; Wang X et al.; Most methods for assessment of chromatin structure involve chemical or nuclease damage to DNA followed by analysis of distribution and susceptibility of cutting sites . The agents used generally do not permeate cells, making nuclear isolation mandatory . In vivo mapping strategies might allow detection of labile constituents and/or structures that are lost when chromatin is swollen in isolated nuclei at low ionic strengths . DNase I has been the most widely used enzyme to detect chromatin sites where DNA is active in transcription, replication or recombination . We have introduced the bovine DNase I gene into yeast under control of a galactose-responsive promoter . Expression of the nuclease leads to DNA degradation and cell death . Shorter exposure to the active enzyme allows mapping of chromatin structure in whole cells without isolation of nuclei . The validity and efficacy of the strategy are demonstrated by footprinting a labile repressor bound to its operator . Investigation of the inter-nucleosome linker regions in several types of repressed domains has revealed different degrees of protection in cells, relative to isolated nuclei.

Mol Cell Biol, 2001 Jun, 21(11), 3725 - 37
Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae; Kim HS et al.; The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function . To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast . A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1) . Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2 . The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints . RFC4 and the DNA damage checkpoint gene RAD24 were found to be epistatic with respect to DNA damage sensitivity . We show that the rfc4-2 mutant is defective in the G(1)/S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G(2)/M DNA damage checkpoint . Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways . Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.

Bioassay, Molecular Microbiology, Water Purification, Bacteria, Bioengineering, Microbes, Bioreactor, Microbe, Growth Medium, Bacterium, Activated Sludge, Microorganisms, Genome, Bacteriology, o, Bioremediation, f, Microorganism, b, Amino Acid, d, Microbiology, g, Environmental Microbiology, Botulism, Listeriosis, m, Escherichia coli, e, Microorganism, s, Neisseria, b, Antibacterial, h, Enterococcus, b, Pathogenic bacterium, e, Antibiotic, s, Gram negative, s, E. coli, p, Thermophilic, e, Streptococci, c, Antibiotic, w, Bacteriophage, b, Pseudomonas putida, e, Yeasts, c, Culture medium, a, Bacterial, s, Salmonella, p, Serratia, c, Bacilli, s, Bacterium, s, C. botulinum, p, Bacillus, e, Gram positive, s, Microbial, b, Escherichia coli, n, Cholera, s, Bacterial, Beta lactamase,

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005