Yeasts

Prostate, 1999 Oct 1, 41(2), 134 - 42
Use of a yeast assay to detect functional alterations in p53 in prostate cancer: review and future directions; deVere White RW et al.; BACKGROUND: While many studies have suggested that p53 mutations are common in human cancers, the functional activity of these mutant alleles has not yet been fully addressed . We believe that information about the functional status of individual p53 mutants will prove to be important for a better understanding of the role of p53 in tumor development and progression . Ultimately, this information could also influence treatment decisions for individual cancer patients . METHODS: A recently developed yeast functional assay can be used to assess the transactivational activity of p53 mutants . Furthermore, this assay is more sensitive than single strand conformational polymorphism (SSCP) for detection of p53 mutations . In this review, we summarize the mechanism of this new technique and describe its applications in cancer research, with an emphasis on prostate cancer . RESULTS: The use of the yeast functional assay provides a simple, sensitive, and reproducible method for detecting p53 mutations and for determining the transactivational activity and dominant-negative role of individual p53 mutants . CONCLUSIONS: This method may be adapted to analyze other transcriptional factors, including the human androgen receptor .

Mutat Res, 1999 Jul 21, 444(1), 117 - 22
Protective effect of the yeast glucomannan against cyclophosphamide-induced mutagenicity; Chorvatovicova D et al.; Glucomannan (GM) isolated from Candida utilis with molecular weight 30 kDa was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei was evaluated in polychromatic erythrocytes of mouse bone marrow . GM administration by either route decreased significantly (p<0.002) the clastogenic effect of CP . The protective effect was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b . wt . (body weight) . The fact that GM was effective also at oral administration is indicative of the passage of GM molecules through the wall of the gastrointestinal tract . The important characteristics of GM isolated from C . utilis, such as good water solubility, relatively small molecular weight (30 kDa), and antimutagenic effect exerted also at oral administration, appear to be promising features for its prospective use as a natural protective agent.

Nucleosides Nucleotides, 1999 Jun-Jul, 18(6-7), 1459 - 61
Interaction of complementary oligonucleotides with the 3'-end of yeast tRNA(Phe); Petyuk V et al.; Interaction of yeast tRNA(Phe) with oligodeoxyribonucleotides (ONs), complementary to the nucleotides 62-76 was investigated . Results of gel-mobility shift assay and RNase A probing evidence that the ONs containing the sequence complementary to the tRNA ACCA end can easily invade the hairpin structure under physiological conditions . The limiting step of association process is the tRNA unfolding.

Nat Biotechnol, 1999 Sep, 17(9), 878 - 83
Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling; Cismowski MJ et al.; We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways . Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen) . Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified . One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha . A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.

Mol Biol Cell, 1999 Sep, 10(9), 2987 - 3001
Functional dissection and hierarchy of tubulin-folding cofactor homologues in fission yeast; Radcliffe PA et al.; We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules . Alp11(B) contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21(E) does not . Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats . Immunoprecipitation analysis shows that Alp11(B) interacts with both alpha-tubulin and Alp21(E), but not with the cofactor D homologue Alp1, whereas Alp21(E) also interacts with Alp1(D) . The cellular amount of alpha-tubulin is decreased in both alp1 and alp11 mutants . Overproduction of Alp11(B) results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of alpha-tubulin . Both full-length Alp11(B) and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to alpha-tubulin . Deletion of alp11 is suppressed by multicopy plasmids containing either alp21(+) or alp1(+), whereas alp21 deletion is rescued by overexpression of alp1(+) but not alp11(+) . Finally, the alp1 mutant is not complemented by either alp11(+) or alp21(+) . The results suggest that cofactors operate in a linear pathway (Alp11(B)-Alp21(E)-Alp1(D)), each with distinct roles.

Mol Biol Cell, 1999 Sep, 10(9), 2879 - 89
Import into and degradation of cytosolic proteins by isolated yeast vacuoles; Horst M et al.; In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins . The physiological condition of the cell often determines the degradation pathway of a specific protein . In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles . After starvation of the cells, protein uptake increases . Uptake and degradation are temperature dependent and show biphasic kinetics . Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles . Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase . We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway . This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells.

J Biol Chem, 1999 Sep 10, 274(37), 26057 - 64
Substrate- and inhibitor-induced conformational changes in the yeast V-ATPase provide evidence for communication between the catalytic and proton-translocating sectors; Landolt-Marticorena C et al.; The vacuolar-type H(+)-ATPases (V-ATPases) are composed of two distinct sectors, a catalytic complex (V(1)) involved in ATP hydrolysis and a membrane-associated complex (V(0)) mediating proton translocation across a lipid bilayer . To date, little is known about the mechanism by which these two functions are coupled . We sought to examine the impact of nucleotide and cation binding on the structure of the core components of the catalytic complex and to determine whether conformational changes within the catalytic complex impact subunits of the membrane-associated complex . Nucleotide- and cation- induced changes in the catalytic core of the V-ATPase were investigated by monitoring changes in the rate and pattern of tryptic digests . ATP.Mg-induced changes were detected in both the catalytic (Vma1p or 69 kDa) and the regulatory subunits (Vma2p or 60 kDa) of the V(1) sector . ATP alone increased the rate of trypsinization of the regulatory subunit, but did not have any effect on Vma1p . Surprisingly, ATP also had an impact on the 95-kDa subunit, a component of the V(0) sector of the V-ATPase . Although the presence of divalent cations had no impact on the V(1) sector, the rate of trypsinization of the 95-kDa subunit was greatly enhanced . The effect of divalent cations on the structure of the 95-kDa subunit was abrogated when trypsinization was performed in the absence of the catalytic sector . Addition of bafilomycin A(1), a V-ATPase inhibitor that putatively binds to the 95-kDa subunit, increased the rate of trypsinization of the catalytic subunit . These data suggest that structural alterations within the V(1) sector result in alterations within the V(0) sector and vice versa . Clearly, a structural link must exist to couple the two sectors . The 95-kDa subunit is ideally suited to fulfill this role . Hydropathy analysis suggests a bipartite structure, with the NH(2)-terminal portion predicted to lie in an aqueous environment and the C-terminal portion predicted to contain 6 transmembrane segments . Tryptic digests of sealed vacuolar vesicles and immunofluorescence studies revealed that the large hydrophilic NH(2)-terminal domain of the 95-kDa subunit is localized toward the cytosol . This region therefore is ideally positioned to interact with components of the V(1) complex, potentially functioning as the elusive link between the two sectors of the V-ATPase.

Plant Foods Hum Nutr, 1999, 53(2), 175 - 81
The relationship between soil copper content and copper resistance in yeast of an ultisol in midwestern Nigeria; Ekundayo EO et al.; Yeast isolates were obtained from the brewer's stock of the Guinness brewery in Benin City, midwestern Nigeria, and from local sources such as mango and sugar cane . The fruits were exposed to the immediate soil environment in eleven selected sites in the Ugbowo and Ikpoba Hill areas of Benin City, Edo State of midwestern Nigeria . A total of twenty-seven yeast isolates were obtained . Fourteen isolates were identified as Saccharomyces cerevisiae and 13 as S . carlsbergensis . Analysis of resistance to copper sulphate revealed that the two brewer's yeast isolates were resistant to 3.5 mM and 4.2 mM CuSO4, respectively, while the local yeasts were resistant to higher concentrations ranging from 6.5 to 16.5 mM . The percentage copper sulphate resistance of yeast isolates obtained from Mangifera inidica ranged from 52.4 to 73.8%, while the percentage copper sulphate resistance of yeast isolates obtained from Saccharum officinarum ranged from 51.8 to 62.6% . The two brewer's yeast isolates (BY2 and BY4) had a percentage CuSO4 resistance of 32.4 and 38.5%, respectively . Comparison of soil copper concentration and copper resistance levels in selected yeast isolates indicated that the soil copper concentrations generally influenced the ability of yeasts to resist copper.

Neoplasma, 1999, 46(2), 80 - 8
FASAY: a simple functional assay in yeast for identification of p53 mutation in tumors; Smardova J; Alteration of the p53 tumor suppressor gene is the most common genetic defect known to occur in human tumors . Germ-line p53 mutations significantly increase the risk of developing diverse malignancies . FASAY is a simple functional assay for germ-line and somatic mutations in the p53 gene altering the transactivation capability of the p53 protein . The method was successfully used for mutation analysis of p53 in various cell lines, somatic tumor cells and blood cells . In addition, FASAY was also found effective as a tool for basic research of binding of mutant p53 proteins to promoters of different p53 target genes.

Genes Dev, 1999 Aug 15, 13(16), 2148 - 58
The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases; Allmang C et al.; We previously identified a complex of 3' --> 5' exoribonucleases, designated the exosome, that is expected to play a major role in diverse RNA processing and degradation pathways . Further biochemical and genetic analyses have revealed six novel components of the complex . Therefore, the complex contains 11 components, 10 of which are predicted to be 3' --> 5' exoribonucleases on the basis of sequence homology . Human homologs were identified for 9 of the 11 yeast exosome components, three of which complement mutations in the respective yeast genes . Two of the newly identified exosome components are homologous to known components of the PM-Scl particle, a multisubunit complex recognized by autoimmune sera of patients suffering from polymyositis-scleroderma overlap syndrome . We demonstrate that the homolog of the Rrp4p exosome subunit is also a component of the PM-Scl complex, thereby providing compelling evidence that the yeast exosome and human PM-Scl complexes are functionally equivalent . The two complexes are similar in size, and biochemical fractionation and indirect immunofluorescence experiments show that, in both yeast and humans, nuclear and cytoplasmic forms of the complex exist that differ only by the presence of the Rrp6p/PM-Scl100 subunit exclusively in the nuclear complex.

J Biol Chem, 1999 Sep 3, 274(36), 25921 - 6
Regulated degradation of yeast ornithine decarboxylase; Toth C et al.; Ornithine decarboxylase (ODC) declines in cells that accumulate an excess of polyamines, the downstream products of the enzyme . Superfluous production of polyamines is thus prevented . In animal cells, polyamines reduce ODC activity by accelerating its degradation . Similar down-regulation of ODC activity has been observed in the budding yeast Saccharomyces cerevisiae, but induced degradation has not been documented . Here we show using pulse-chase analysis that the loss of enzyme activity is the result of increased degradation of ODC . Polyamines reduce the half-life of the newly synthesized protein from 3 h to approximately 10 min . Degradation of bulk ODC pools is also accelerated by polyamines, but the absolute rate of turnover is slower, with a half-life of 5 h in untreated and 1 h in treated cells . Newly synthesized ODC polypeptide thus undergoes a process of maturation that renders it relatively resistant to both basal and polyamine-induced degradation . Proteasome mutants have a blunted or absent regulatory response, implicating both the core protease and the regulatory cap of the proteasome in induced degradation of yeast ODC.

J Biol Chem, 1999 Sep 3, 274(36), 25682 - 90
Stable association of 70-kDa heat shock protein induces latent multisite specificity of a unisite-specific endonuclease in yeast mitochondria; Mizumura H et al.; The multisite-specific endonuclease Endo.SceI of yeast mitochondria is unique among endonucleases because its 50-kDa subunit forms a stable dimer with the mitochondrial 70-kDa heat shock protein (mtHSP70), which otherwise fulfills a chaperone function by binding transiently to unfolded proteins . Here we show that the mtHSP70 subunit confers broader sequence specificity, greater stability, and higher activity on the 50-kDa subunit . The 50-kDa subunit alone displayed weaker activity and highly sequence-specific endonuclease activity . The 50-kDa protein exists as a heterodimer with mtHSP70 in vivo, allowing Endo.SceI to cleave specifically at multiple sites on mitochondrial DNA . Endo.SceI may have evolved from a highly specific endonuclease that gained broader sequence specificity after becoming a stable partner of mtHSP70.

J Pept Sci, 1999 Jun, 5(6), 245 - 50
Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8; Goetz M et al.; Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem . Strong interchain associations within the peptide-resin matrix have to be overcome . A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures . A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis . In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described . The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

DNA Cell Biol, 1999 Aug, 18(8), 653 - 61
The product of the primary response gene BRF1 inhibits the interaction between 14-3-3 proteins and cRaf-1 in the yeast trihybrid system; Bustin SA et al.; The 14-3-3 proteins are small abundant cytosolic eukaryotic proteins that associate with and modulate the activity of numerous other proteins . The 14-3-3 beta isoform has been shown to bind to the product of the protooncogene cRaf-1 and to facilitate its activation by Ras . Using the yeast two-hybrid system, we have demonstrated that 14-3-3 beta and another isoform, 14-3-3 tau, bind to the product of the primary response gene BRF1 and that the interaction between each isoform and BRF1 is significantly stronger than that with cRaf-1 . We further demonstrated that the charge of residue 187 in 14-3-3 beta regulates its affinity for both BRF1 and cRaf-1 . The interaction of either isoform with BRF1 requires both proteins to be fully intact . When all three proteins are coexpressed in a yeast trihybrid system, BRF1 interferes significantly with the binding of 14-3-3 to full-length cRaf-1 as well as to its regulatory and kinase domains . Using quantitative reverse transcription-polymerase chain reaction, 14-3-3 beta and BRF1 were found to be coexpressed in four different human tissues, suggesting a biologic role for their interaction in the regulation of cRaf-1-mediated signal transduction processes.

J Anim Sci, 1999 Aug, 77(8), 2172 - 9
Effects of dietary levels of selenium-enriched yeast and sodium selenite as selenium sources fed to growing-finishing pigs on performance, tissue selenium, serum glutathione peroxidase activity, carcass characteristics, and loin quality; Mahan DC et al.; This research evaluated the efficacy of inorganic and organic Se sources for growing-finishing pigs, as measured by performance and various tissue, serum, carcass, and loin quality traits . A total of 351 crossbred pigs were allotted at an average BW of 20.4 kg to six replicates of a 2x4 factorial experiment in a randomized complete block design . Pigs were fed diets containing Se-enriched yeast (organic) or sodium selenite (inorganic), each at .05, .10, .20, or .30 mg Se/kg diet . A non-Se-fortified basal diet was a ninth treatment group . Five pigs per pen were bled initially and at 30-d intervals with serum analyzed for Se and glutathione peroxidase (GSH-Px) activity . At 55 kg BW, one pig per pen from each of three replicates was killed, and tissues were collected for Se analysis . At 105 kg BW, the remaining pigs in the three replicates were killed, carcass measurements were collected, tissues were analyzed for Se, and loin quality was evaluated for pH, drip loss, and lightness . No performance or carcass measurement benefit resulted from either Se source or dietary Se levels . Pigs had a lower serum Se concentration and GSH-Px activity when the basal diet was fed, but both increased as dietary Se level increased (P<.01) . Serum GSH-Px activities were increased by pig age and reached a plateau when the diet contained approximately .10 mg Se/kg (P<.01) at d 30, and 60 of the trial, and at .05 mg Se/kg diet at d 90 of the trial . The organic Se group fed .05 and .10 mg Se/kg had serum GSH-Px activities that tended to be lower than those of pigs fed the inorganic Se source, but GSH-Px activities in both groups were similar at higher Se levels . Tissue Se contents increased linearly as the dietary Se level increased, but the increase was markedly higher when organic Se was fed, resulting in an interaction (P<.01) response . Loin drip loss, pH, and lightness were unaffected (P>.15) by organic Se source or level, but there was a trend for a higher drip loss (P = .11) and a linear (P<.01) increase in loin paleness when the inorganic Se level increased . These results indicate that neither Se source nor Se level had an effect on pig performance or carcass measurements, but organic Se source increased tissue Se concentrations . Inorganic Se may, however, have a detrimental effect on loin quality, as reflected by higher drip loss and a paler color . Using serum GSH-Px activity as the measurement criterion, the supplemental dietary Se requirement did not seem to exceed .10 and .05 mg Se/kg diet for the growing and finishing phases, respectively, when added to a basal diet containing .06 mg Se/kg.

Trends Genet, 1999 Sep, 15(9), 337 - 40
Multi-organellar disorders of pigmentation: intracellular traffic jams in mammals, flies and yeast; Spritz RA; Several different mutant genes in humans, mice and Drosophila, most of which were identified initially on the basis of reduced pigmentation, have been associated with defects of multiple cytoplasmic organelles - melanosomes, lysosomes and granules . Recent discoveries show that several of these mutations directly affect components in the pathway of organelle-specific protein trafficking, and provide new insights into the relationships of these pathways in mammals, flies and yeast.

J Cell Biol, 1999 Aug 23, 146(4), 741 - 54
Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane; Schneiter R et al.; Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells . Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed . Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%) . The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20% . Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane . Most notably, the plasma membrane is enriched in saturated species of PS and PE . The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further . The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species . This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.

Curr Opin Microbiol, 1999 Aug, 2(4), 348 - 52
Cell wall dynamics in yeast; Smits GJ et al.; The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level . It is a dynamic and highly regulated structure . This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

Cryobiology, 1999 Aug, 39(1), 80 - 7
A dual role for intracellular trehalose in the resistance of yeast cells to water stress; Sano F et al.; It is well known that yeast cells survive environmental stresses such as desiccation and freezing and there is evidence that these phenomena may be related to the presence of trehalose in the cells . However, the molecular mechanism by which trehalose might exert an influence on cell functions remains unknown . In this report, thermogravimetry and differential thermal analysis were used to estimate the amount of bound water in yeast cells . It is shown that when the trehalose content is greater than 2-3% of the cell dry weight, the amount of bound water is drastically decreased and the viability of the dried cells is increased . This implies that a major portion of the bound water is replaced by trehalose . In addition, measurements of the NMR spin-lattice relaxation time of the intracellular water protons show that trehalose acts as a water-structuring agent in hydrated yeast cells . This dual role is essential for high resistance to water stress in yeast cells .

Biotechnol Bioeng, 1999 Oct 20, 65(2), 176 - 81
Slow heat rate increases yeast thermotolerance by maintaining plasma membrane integrity; Martinez de Maranon I et al.; Thermal resistance of Saccharomyces cerevisiae was found to be drastically dependent on the kinetics of heat perturbation . Yeasts were found to be more resistant to a plateau of 1 h at 50 degrees C after a slope of temperature increase (slow and linear temperature increments) than after a shock (sudden temperature change) . Thermotolerance was mainly acquired between 40-50 degrees C during a heat slope, i.e., above the maximal temperature of growth . The death of the yeasts subjected to a heat shock might be related to the loss of membrane integrity: intracellular contents extrusion, i.e., membrane permeabilization, was found to precede cell death . However, the permeabilization did not precede cell death during a heat slope and, therefore, membrane permeabilization was a consequence rather than a cause of cell death . During a slow temperature increase, yeasts which remain viable may have time to adapt their plasma membrane and thus maintain membrane integrity .

Int J Biol Macromol, 1999 Aug, 25(4), 345 - 51
Identification of simian cyclophilin A as a calreticulin-binding protein in yeast two-hybrid screen and demonstration of cyclophilin A interaction with calreticulin; Reddy PA et al.; Cyclophilin A (CyPA) was identified as one of the calreticulin (CR)-binding proteins in a yeast two-hybrid screen utilizing simian cDNA expression-library . The simian CyPA protein had 96% identity with that of human, differing only at eight amino acid residues . We further established CyPA-CR interaction by incubation of glutathione transferase-fused CyPA (GST-CyPA) and CR proteins with CV-1 cyto-lysates, followed by CR and CyPA-specific immuno-blot analysis . The immunosuppressive drug cyclosporin A, a CyPA ligand, did not inhibit CyPA-CR interaction . Our results established a new property of CyPA binding activity to CR . Since CR is a Ca2+-binding protein, CR-CyPA interactions may be important in signaling pathways for induction of Ca2+-dependent cellular processes.

FEBS Lett, 1999 Aug 6, 456(2), 281 - 4
Low iron concentration and aconitase deficiency in a yeast frataxin homologue deficient strain; Foury F; Deletion of the yeast frataxin homologue, YFH1, elicits accumulation of iron in mitochondria and mitochondrial defects . We report here that in the presence of an iron chelator in the culture medium, the concentration of iron in mitochondria is the same in wild-type and YFH1 deletant strains . Under these conditions, the activity of the respiratory complexes is restored . However, the activity of the mitochondrial aconitase, a 4Fe-4S cluster-containing protein, remains low . The frataxin family bears homology to a bacterial protein family which confers resistance to tellurium, a metal closely related to sulfur . Yfh1p might control the synthesis of iron-sulfur clusters in mitochondria.

Methods, 1999 Jul, 18(3), 368 - 76, 323
In vitro DNA replication in yeast nuclear extracts; Pasero P et al.; Two assays have been developed for studying DNA replication in vitro based on nuclear extracts isolated from budding yeast cells synchronized in S phase . In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G(1) . In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented with nucleotides and an energy-regenerating system . Semiconservative DNA replication is monitored by substitution of newly synthesized DNA with bromodeoxyuridine 5'-triphosphate (BrdUTP) and density gradient analysis . In addition, neutral-neutral two-dimensional gel analyses and, in the case of nuclei, detection of newly synthesized DNA in replication foci by DIG-dUTP incorporation can be used to monitor replication .

Methods, 1999 Jul, 18(3), 356 - 67
Genetic methods for characterizing the cis-acting components of yeast DNA replication origins; Huberman JA; Small circular plasmids containing replication origins and, in some cases, centromeres, can replicate autonomously in the nuclei of all tested yeast species . Because this autonomous replication is dependent on the replication origin within the plasmid, measurements of the efficiency of autonomous replication (by the methods summarized here) permit evaluation of the effects of mutations on origin function . Although alternative methods are available for genetic characterization of replication origins in other organisms, the simplicity of the autonomous replication assay in yeasts has permitted development of the deepest understanding to date of eukaryotic replication origin structure . This information has come primarily from studies with Saccharomyces cerevisiae . However, there are many other yeast species, each with its own variety of replication origins . Use of the methods summarized here to characterize origins in other yeast species is likely to provide additional insights into eukaryotic replication origin structure .

Nucleic Acids Res, 1999 Aug 1, 27(15), 3001 - 8
New yeast genes important for chromosome integrity and segregation identified by dosage effects on genome stability; Ouspenski II et al.; Phenotypes produced by gene overexpression may provide important clues to gene function . Here, we have performed a search for genes that affect chromo-some stability when overexpressed in the budding yeast Saccharomyces cerevisiae . We have obtained clones encompassing 30 different genes . Twenty-four of these genes have been previously characterized . Most of them are involved in chromatin dynamics, cell cycle control, DNA replication or mitotic chromosome segregation . Six novel genes obtained in this screen were named CST (chromosome stability) . Based on the pattern of genomic instability, inter-action with checkpoint mutations and sensitivity to chromosome replication or segregation inhibitors, we conclude that overexpression of CST4 specifically interferes with mitotic chromosome segregation, and CST6 affects some aspect of DNA metabolism . The other CST genes had complex pleiotropic phenotypes . We have created deletions of five genes obtained in this screen, CST9, CST13, NAT1, SBA1 and FUN30 . None of these genes is essential for viability, and deletions of NAT1 and SBA1 cause chromosome instability, a phenotype not previously associated with these genes . This work shows that analysis of dosage effects is complementary to mutational analysis of chromosome transmission fidelity, as it allows the identification of chromosome stability genes that have not been detected in mutational screens.

Mol Cell Biol, 1999 Sep, 19(9), 6110 - 9
The yeast trimeric guanine nucleotide-binding protein alpha subunit, Gpa2p, controls the meiosis-specific kinase Ime2p activity in response to nutrients; Donzeau M et al.; Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development . Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation . Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro . Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen . In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function . Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium . These observations are consistent with the following model . In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels . Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium . The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted . This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision.

Mol Cell Biol, 1999 Sep, 19(9), 6098 - 109
Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast; Vujcic M et al.; In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity . Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302) . Another, ARS303, resides between HML and the CHA1 gene, and its function is not known . Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320 . Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III . However, at their native locations, where the two ARSs are in a cluster with ARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), or cis-acting mutations that delete the E and I silencers containing ARS elements . These results suggest that, for the HML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis- and trans-acting components . Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301, the E silencer . Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome . The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

Mol Cell Biol, 1999 Sep, 19(9), 6029 - 40
Functional analysis of the yeast Glc7-binding protein Reg1 identifies a protein phosphatase type 1-binding motif as essential for repression of ADH2 expression; Dombek KM et al.; In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose . Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2 . Constitutive ADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2 expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells . To further elucidate the functional role of Reg1 in repressing ADH2 expression, deletions scanning the entire length of the protein were analyzed . Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression . Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional . Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses . Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex . Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source . When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression . These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression . This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm.

Mol Cell Biol, 1999 Sep, 19(9), 5943 - 51
SMG-2 is a phosphorylated protein required for mRNA surveillance in Caenorhabditis elegans and related to Upf1p of yeast; Page MF et al.; mRNAs that contain premature stop codons are selectively degraded in all eukaryotes tested, a phenomenon termed "nonsense-mediated mRNA decay" (NMD) or "mRNA surveillance." NMD may function to eliminate aberrant mRNAs so that they are not translated, because such mRNAs might encode deleterious polypeptide fragments . In both yeasts and nematodes, NMD is a nonessential system . Mutations affecting three yeast UPF genes or seven nematode smg genes eliminate NMD . We report here the molecular analysis of smg-2 of Caenorhabditis elegans . smg-2 is homologous to UPF1 of yeast and to RENT1 (also called HUPF1), a human gene likely involved in NMD . The striking conservation of SMG-2, Upf1p, and RENT1/HUPF1 in both sequence and function suggests that NMD is an ancient system, predating the divergence of most eukaryotes . Despite similarities in the sequences of SMG-2 and Upf1p, expression of Upf1p in C . elegans does not rescue smg-2 mutants . We have prepared anti-SMG-2 polyclonal antibodies and identified SMG-2 on Western blots . SMG-2 is phosphorylated, and mutations of the six other smg genes influence the state of SMG-2 phosphorylation . In smg-1, smg-3, and smg-4 mutants, phosphorylation of SMG-2 was not detected . In smg-5, smg-6, and smg-7 mutants, a phosphorylated isoform of SMG-2 accumulated to abnormally high levels . In smg-2(r866) and smg-2(r895) mutants, which harbor single amino acid substitutions of the SMG-2 nucleotide binding site, phosphorylated SMG-2 accumulated to abnormally high levels, similar to those observed in smg-5, smg-6, and smg-7 mutants . We discuss these results with regard to the in vivo functions of SMG-2 and NMD.

Acta Biochim Pol, 1999, 46(1), 211 - 5
Phosphorylation of yeast ribosomal proteins by CKI and CKII in the presence of heparin; Wojda I et al.; We have found that heparin has a different effect on Trichosporon cutaneum ribosomal protein phosphorylation by CKI and by CKII . In the presence of heparin, modification of 13 kDa, 19 kDa and 38 kDa proteins catalyzed by CKII was inhibited, while in the case of CKI, in addition to protein of 15 kDa, phosphorylation of 20 kDa and 35 kDa proteins was detected . It was also found that, in the presence of heparin, phosphorylation of P proteins (13 kDa and 38 kDa) by ribosome-bound protein kinases was inhibited . Moreover at the same conditions modification of 40 kDa protein was observed in all four yeast species tested.

Acta Biochim Pol, 1999, 46(1), 163 - 72
Thermodynamic contribution of nucleoside modifications to yeast tRNA(Phe) anticodon stem loop analogs; Agris PF et al.; The determination of the structural and functional contributions of natural modified nucleosides to tRNA has been limited by lack of an approach that can systematically incorporate the modified units . We have produced a number of oligonucleotide analogs, of the anticodon of yeast tRNA(Phe) by, combining standard automated synthesis for the major nucleosides with specialty chemistries for the modified nucleosides . In this study, both naturally occurring and unnatural modified nucleotides were placed in native contexts . Each oligonucleotide was purified and the nucleoside composition determined to validate the chemistry . The RNAs were denatured and analyzed to determine the van't Hoff thermodynamic parameters . Here, we report the individual thermodynamic contributions for Cm, Gm, m1G, m5C, psi . In addition m5m6U, m1psi, and m3psi, were introduced to gain additional understanding of the physicochemical contribution of psi and m5C at an atomic level . These oligonucleotides demonstrate that modifications have measurable thermodynamic contributions and that loop modifications have global contributions.

Hum Genet, 1999 Jun, 104(6), 443 - 8
Hereditary spastic paraplegia: mitochondrial metalloproteases of yeast; Pearce DA; Hereditary spastic paraplegia (HSP) is a genetically heterogenous group of inherited neurodegenerative disorders . Recently, an autosomal recessive form of HSP was mapped to 16q24.3, and subsequently the defective gene associated to HSP was identified and designated SPG7 . The SPG7 gene product was predicted to encode a protein of 795 amino acids, and is called paraplegin . Paraplegin is highly homologous to a class of well studied yeast ATP-dependent zinc metalloproteases, which show 55%, 55% and 52% identity, respectively, to Afg3p, Rca1p and Yme1p . Mutation of either Afg3p, Rca1p or Yme1p in yeast results in pleiotropic effects with regard to growth, respiration and, particularly, in the assembly and/or degradation of more than one mitochondrial protein complex . Taking into account the homology of paraplegin to these yeast ATP-dependent zinc metalloproteases and what is known about their function, allows us to speculate as to what function paraplegin plays in normal individuals.

Can J Microbiol, 1999 Jun, 45(6), 472 - 9
Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat shock proteins and trehalose; Swan TM et al.; The response of a yeast unsaturated fatty acid auxotroph, defective in delta 9-desaturase activity, to heat and ethanol stresses was examined . The most heat- and ethanol-tolerant cells had membranes enriched with oleic acid (C18:1), followed in order by cells enriched with linoleic (C18:2) and linolenic (C18:3) acids . Cells subjected to a heat shock (25-37 degrees C for 30 min) accumulated trehalose and synthesized typical heat shock proteins . Although there were no obvious differences in protein profiles attributable to lipid supplementation of the mutant, relative protein synthesis as determined by densitometric analysis of autoradiograms suggested that hsp expression was different . However, there was no consistent relationship between the synthesis of heat shock proteins and the acquisition of thermotolerance in the lipid supplemented auxotroph or related wild type . Furthermore, trehalose accumulation was also not closely related to stress tolerance . On the other hand, the data presented indicated a more consistent role for membrane lipid composition in stress tolerance than trehalose, heat shock proteins, or ergosterol . We suggest that the sensitivity of C18:3-enriched cells to heat and ethanol may be attributable to membrane damage associated with increases in membrane fluidity and oxygen-derived free radical attack of membrane lipids.

J Mol Biol, 1999 Aug 27, 291(4), 761 - 73
Yeast aspartyl-tRNA synthetase residues interacting with tRNA(Asp) identity bases connectively contribute to tRNA(Asp) binding in the ground and transition-state complex and discriminate against non-cognate tRNAs; Eriani G et al.; Crystallographic studies of the aspartyl-tRNA synthetase-tRNA(Asp)complex from yeast identified on the enzyme a number of residues potentially able to interact with tRNA(Asp) . Alanine replacement of these residues (thought to disrupt the interactions) was used in the present study to evaluate their importance in tRNA(Asp)recognition and acylation . The results showed that contacts with the acceptor A of tRNA(Asp)by amino acid residues interacting through their side-chain occur only in the acylation transition state, whereas those located near the G73 discriminator base occur also during initial binding of tRNA(Asp) . Interactions with the anticodon bases provide the largest free energy contribution to stability of the enzyme-tRNA complex in its ground state . These contacts also favour catalysis, by acting connectively with each other and with those of G73, as shown by multiple mutant analysis . This implies structural communication transmitting the anticodon recognition signal to the distally located acylation site . This signal might be conveyed via tRNA(Asp)as suggested by the observed conformational change of this molecule upon interaction with AspRS . From binding free energy values corresponding to the different AspRS-tRNA(Asp)interaction domains, it might be concluded that upon complex formation, the anticodon interacts first . Finally, acylation efficiencies of AspRS mutants in the presence of pure tRNA(Asp)and non-fractionated tRNAs indicate that residues involved in the binding of identity bases also discriminate against non-cognate tRNAs .

Cytogenet Cell Genet, 1999, 85(3-4), 217 - 20
A novel human gene, encoding a potential membrane protein conserved from yeast to man, is strongly expressed in testis and cancer cell lines; Veitia RA et al.; We have characterized a novel human gene (C14orf1) which codes for a polypeptide homologous to the yeast protein Yer044c . Both the human and yeast proteins are predicted to be highly basic and to present several potential, evolutionarily conserved, transmembrane domains . C14orf1 mRNA was found to be particularly abundant in the adult testis and in several cancer cell lines . The gene maps to chromosome band 14q24 . Further investigations should be performed to understand the role of C14orf1 in the testis and the significance of its strong expression in the cell lines studied here.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9533 - 8
Studying interactions of four proteins in the yeast two-hybrid system: structural resemblance of the pVHL/elongin BC/hCUL-2 complex with the ubiquitin ligase complex SKP1/cullin/F-box protein; Pause A et al.; The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners . However, the system fails to detect protein-protein interactions that require the presence of additional components of a multisubunit complex . Here we demonstrate that the vector YIpDCE1 can be used to express elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel-Lindau tumor-suppressor gene product (pVHL) . Only when pVHL and elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/elongin BC/hCUL-2 complex . This system was then used to map the binding region of hCUL-2 for the VBC complex . The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex . The elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with elongins B and C . These results reveal a striking structural resemblance of pVHL/elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions . Thus, it seems possible that pVHL/elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome.

Nature, 1999 Aug 5, 400(6744), 573 - 6
Oligopeptide-repeat expansions modulate 'protein-only' inheritance in yeast; Liu JJ et al.; The yeast {PSI+} element represents a new type of genetic inheritance, in which changes in phenotype are transmitted by a 'protein only' mechanism reminiscent of the 'protein-only' transmission of mammalian prion diseases . The underlying molecular mechanisms for both are poorly understood and it is not clear how similar they might be . Sup35, the {PSI+} protein determinant, and PrP, the mammalian prion determinant, have different functions, different cellular locations and no sequence similarity; however, each contains five imperfect oligopeptide repeats-PQGGYQQYN in Sup35 and PHGGGWGQ in PrP . Repeat expansions in PrP produce spontaneous prion diseases . Here we show that replacing the wild-type SUP35 gene with a repeat-expansion mutation induces new {PSI+} elements, the first mutation of its type among these newly described elements of inheritance . In vitro, fully denatured repeat-expansion peptides can adopt conformations rich in beta-sheets and form higher-order structures much more rapidly than wild-type peptides . Our results provide insight into the nature of the conformational changes underlying protein-based mechanisms of inheritance and suggest a link between this process and those producing neurodegenerative prion diseases in mammals.

EMBO J, 1999 Aug 16, 18(16), 4485 - 97
Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast; Neecke H et al.; We studied the response of nucleotide excision repair (NER)-defective rad14Delta cells to UV irradiation in G(1) followed by release into the cell cycle . Only a subset of checkpoint proteins appears to mediate cell cycle arrest and regulate the timely activation of replication origins in the presence of unrepaired UV-induced lesions . In fact, Mec1 and Rad53, but not Rad9 and the Rad24 group of checkpoint proteins, are required to delay cell cycle progression in rad14Delta cells after UV damage in G(1) . Consistently, Mec1-dependent Rad53 phosphorylation after UV irradiation takes place in rad14Delta cells also in the absence of Rad9, Rad17, Rad24, Mec3 and Ddc1, and correlates with entry into S phase . Two-dimensional gel analysis indicates that late replication origins are not fired in rad14Delta cells UV-irradiated in G(1) and released into the cell cycle, which instead initiate DNA replication from early origins and accumulate replication and recombination intermediates . Progression through S phase of UV-treated NER-deficient mec1 and rad53 mutants correlates with late origin firing, suggesting that unregulated DNA replication in the presence of irreparable UV-induced lesions might result from a failure to prevent initiation at late origins.

EMBO J, 1999 Aug 16, 18(16), 4383 - 93
Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis; Wendland B et al.; The mammalian protein epsin is required for endocytosis . In this study, we have characterized two homologous yeast proteins, Ent1p and Ent2p, which are similar to mammalian epsin . An essential function for the highly conserved N-terminal epsin N-terminal homology (ENTH) domain was revealed using deletions and randomly generated temperature-sensitive ent1 alleles . Changes in conserved ENTH domain residues in ent1(ts) cells revealed defects in endocytosis and actin cytoskeleton structure . The Ent1 protein was localized to peripheral and internal punctate structures, and biochemical fractionation studies found the protein associated with a large, Triton X-100-insoluble pellet . Finally, an Ent1p clathrin-binding domain was mapped to the final eight amino acids (RGYTLIDL*) in the Ent1 protein sequence . Based on these and other data, we propose that the yeast epsin-like proteins are essential components of an endocytic complex that may act at multiple stages in the endocytic pathway.

J Bioenerg Biomembr, 1999 Apr, 31(2), 105 - 17
Activation and deactivation of F0F1-ATPase in yeast mitochondria; Schouppe C et al.; The regulation of membrane-bound proton F0F1 ATPase by the protonmotive force and nucleotides was studied in yeast mitochondria . Activation occurred in whole mitochondria and the ATPase activity was measured just after disrupting the membranes with Triton X-100 . Deactivation occurred either in whole mitochondria uncoupled with FCCP, or in disrupted membranes . No effect of Triton X-100 on the ATPase was observed, except a slow reactivation observed only in the absence of MgADP . Both AMPPNP and ATP increased the ATPase deactivation rate, thus indicating that occupancy of nucleotidic sites by ATP is more decisive than catalytic turnover for this process . ADP was found to stimulate the energy-dependent ATPase activation . ATPase deactivated at the same rate in uncoupled and disrupted mitochondria This suggests that deactivation is not controlled by rebinding of some soluble factor, like IF1, but rather by the conversion of the F1.IF1 complex into an inactive form.

J Bioenerg Biomembr, 1999 Apr, 31(2), 85 - 94
Subunit f of the yeast mitochondrial ATP synthase: topological and functional studies; Roudeau S et al.; Modified versions of subunit f were produced by mutagenesis of the ATP17 gene of Saccharomyces cerevisiae . A version of subunit f devoid of the last 28 amino acid residues including the unique transmembranous domain complemented the oxidative phosphorylation of the null mutant . However, a two-fold decrease in the specific ATP synthase activity was measured and attributed to a decrease in the stability of the mutant ATP synthase complex as shown by the low oligomycin-sensitive ATPase activity at alkaline pH . The modification or not by nonpermeant maleimide reagents of cysteine residues introduced at the N and C termini of subunit f indicated a Nin-Cout orientation . From the C terminus of subunit f it was possible to cross-link subunit 4 (also called subunit b), which is another component of the F0 sector and which also displays a short hydrophilic segment exposed to the intermembrane space.

Can J Microbiol, 1999 May, 45(5), 413 - 7
Clues to the origin of high external invertase activity in immobilized growing yeast: prolonged SUC2 transcription and less susceptibility of the enzyme to endogenous proteolysis; de Alteriis E et al.; Expression of the SUC2 gene encoding invertase was studied using free and gelatin-immobilized yeast cells to try to explain the high activity of this enzyme exhibited by immobilized cells when allowed to grow in a nutrient medium . The results indicated that at least two factors are probably responsible for the accumulation of invertase in immobilized cells . First, the expression of the SUC2 gene was maintained throughout growth in immobilized cells, whereas its expression was only transient in free cells . Second, invertase of immobilized cells was shown to be less susceptible to endogenous proteolytic attack than that of the corresponding free cells . These results have been interpreted, respectively, in terms of diffusional limitations and changes in the pattern of invertase glycosylation due to growth of yeast in an immobilized state.

Nucleic Acids Res, 1999 Sep 1, 27(17), 3610 - 20
Nucleotide excision repair in a constitutive and inducible gene of a yeast minichromosome in intact cells; Li S et al.; Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in a yeast minichromosome, having a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene and varied regions of chromatin structure . Transcription of GAL1:URA3 increased >150-fold, while HIS3 expression decreased <2-fold when cells were switched from glucose to galactose medium . Following galactose induction, four nucleosomes were displaced or rearranged in the GAL3-GAL10 region . However, no change in nucleosome arrangement was observed in other regions of the minichromosome following induction, indicating that only a few plasmid molecules actively transcribe at any one time . Repair at 269 cis-syn CPD sites revealed moderate preferential repair of the transcribed strand of GAL1:URA3 in galactose, consistent with transcription-coupled repair in a fraction of these genes . Many sites upstream of the transcription start site in the transcribed strand were also repaired faster upon induction . There is remarkable repair heterogeneity in the HIS3 gene and preferential repair is seen only in a short sequence immediately downstream of the transcription start site . Finally, a mild correlation of repair heterogeneity with nucleosome positions was observed in the transcribed strand of the inactive GAL1:URA3 gene and this correlation was abolished upon galactose induction.

Nucleic Acids Res, 1999 Sep 1, 27(17), 3503 - 9
Origin and properties of non-coding ORFs in the yeast genome; Mackiewicz P et al.; In a recent paper we have estimated the total number of protein coding open reading frames (ORFs) in the Saccharomyces cerevisiae genome, based on their properties, at about 4800 . This number is much smaller than the 5800-6000 which is widely accepted . In this paper we analyse differences between the set of ORFs with known phenotypes annotated in the Munich Information Centre for Protein Sequences (MIPS) database and ORFs for which the probability of coding, counted by us, is very low . We have found that many of the latter ORFs have properties of antisense sequences of coding ORFs, which suggests that they could have been generated by duplication of coding sequences . Since coding sequences generate ORFs inside themselves, with especially high frequency in the antisense sequences, we have looked for homology between known proteins and hypothetical polypeptides generated by ORFs under consideration in all the six phases . For many ORFs we have found paralogues and orthologues in phases different than the phase which had been assumed in the MIPS database as coding.

J Biol Chem, 1999 Aug 20, 274(34), 24431 - 7
Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p); Komano H et al.; The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase . Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain . Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation . Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg . No cleavage was seen when both Lys and Arg were absent . No significant enhancement was seen with multiple basic residues . However, cleavage always occurred carboxyl to the most COOH-terminal basic residue . V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely . These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)' . The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0 . However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast.

J Biol Chem, 1999 Aug 20, 274(34), 24257 - 62
Synergistic interaction between yeast nucleotide excision repair factors NEF2 and NEF4 in the binding of ultraviolet-damaged DNA; Guzder SN et al.; Saccharomyces cerevisiae RAD4, RAD7, RAD16, and RAD23 genes function in the nucleotide excision repair (NER) of ultraviolet light (UV)-damaged DNA . Previous biochemical studies have shown that the Rad4 and Rad23 proteins are associated in a stoichiometric complex named NEF2, and the Rad7 and Rad16 proteins form another stoichiometric complex named NEF4 . While NEF2 is indispensable for the incision of UV-damaged DNA in the in vitro reconstituted system, NEF4 stimulates the incision reaction . Both NEF2 and NEF4 bind UV-damaged DNA, which raises the intriguing possibility that these two complexes cooperate to achieve the high degree of specificity for DNA damage demarcation required for nucleotide excision repair in vivo . Consistent with this hypothesis, we find that NEF2 and NEF4 bind in a synergistic fashion to UV-damaged DNA in a reaction that is dependent on ATP . We also purify the Rad7 protein and show that it binds DNA but has no preference for UV-damaged DNA . Rad7 physically interacts with NEF2, suggesting a role for Rad7 in linking NEF2 with NEF4.

J Biol Chem, 1999 Aug 20, 274(34), 24220 - 31
Recovery of the yeast cell cycle from heat shock-induced G(1) arrest involves a positive regulation of G(1) cyclin expression by the S phase cyclin Clb5; Li X et al.; In the yeast Saccharomyces cerevisiae, heat shock stress induces a variety of cellular responses including a transient cell cycle arrest before G(1)/S transition . Previous studies have suggested that this G(1) delay is probably attributable to a reduced level of the G(1) cyclin gene (CLN1 and CLN2) transcripts . Here we report our finding that the G(1) cyclin Cln3 and the S cyclin Clb5 are the key factors required for recovery from heat shock-induced G(1) arrest . Heat shock treatment of G(1) cells lacking either CLN3 or CLB5/CLB6 functions leads to prolonged cell cycle arrest before the initiation of DNA synthesis, concomitant with a severe deficiency in bud formation . The inability of the clb5 clb6 mutant to resume normal budding after heat shock treatment is unanticipated, since the S phase cyclins are generally thought to be required mainly for initiation of DNA synthesis and have no significant roles in bud formation in the presence of functional G(1) cyclins . Further studies reveal that the accumulation of G(1) cyclin transcripts is markedly delayed in the clb5 clb6 mutant following heat shock treatment, indicating that the CLN gene expression may require Clb5/Clb6 to attain a threshold level for driving the cell cycle through G(1)/S transition . Consistent with this assumption, overproduction of Clb5 greatly enhances the transcription of at least two G(1) cyclin genes (CLN1 and CLN2) in heat-shocked G(1) cells . These results suggest that Clb5 may positively regulate the expression of G(1) cyclins during cellular recovery from heat shock-induced G(1) arrest . Additional evidence is presented to support a role for Clb5 in maintaining the synchrony between budding and DNA synthesis during normal cell division as well.

J Biol Chem, 1999 Aug 20, 274(34), 24038 - 46
Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms; Evans DR et al.; The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes . This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer . Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function . However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation . Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p . Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells . The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.

J Biol Chem, 1999 Aug 20, 274(34), 23752 - 60
Functional analysis of a homopolymeric (dA-dT) element that provides nucleosomal access to yeast and mammalian transcription factors; Koch KA et al.; Eukaryotic organisms ranging from yeast to humans maintain a large amount of genetic information in the highly compact folds of chromatin, which poses a large DNA accessibility barrier to rapid changes in gene expression . The ability of the yeast Candida glabrata to survive copper insult requires rapid transcriptional autoactivation of the AMT1 copper-metalloregulatory transcription factor gene . The kinetics of AMT1 autoactivation is greatly enhanced by homopolymeric (dA-dT) element (A16)-mediated nucleosomal accessibility for Amt1p to a metal response element in this promoter . Analysis of the nucleosomal positional requirements for the A16 element reveal an impaired ability of the A16 element to stimulate AMT1 autoregulation when positioned downstream of the metal response element within the nucleosome, implicating an inherent asymmetry to the nucleosome positioned within the AMT1 promoter . Importantly, we demonstrate that the A16 element functions to enhance nucleosomal access and hormone-stimulated transcriptional activation for the mammalian glucocorticoid receptor, in a rotational phase-dependent manner . These data provide compelling evidence that nucleosomal homopolymeric (dA-dT) elements provide enhanced DNA access to diverse classes of transcription factors and suggest that these elements may function in this manner to elicit rapid transcriptional responses in higher eukaryotic organisms.

RNA, 1999 Aug, 5(8), 1055 - 70
Overexpression of truncated Nmd3p inhibits protein synthesis in yeast; Belk JP et al.; The yeast NMD3 gene was identified in a two-hybrid screen using the nonsense-mediated mRNA decay factor, Upf1p, as bait . NMD3 was shown to encode an essential, highly conserved protein that associated principally with free 60S ribosomal subunits . Overexpression of a truncated form of Nmd3p, lacking 100 C-terminal amino acids and most of its Upf1p-interacting domain, had dominant-negative effects on both cell growth and protein synthesis and promoted the formation of polyribosome half-mers . These effects were eliminated by truncation of an additional 100 amino acids from Nmd3p . Overexpression of the nmd3delta100 allele also led to increased synthesis and destabilization of some ribosomal protein mRNAs, and increased synthesis and altered processing of 35S pre-rRNA . Our data suggest that Nmd3p has a role in the formation, function, or maintenance of the 60S ribosomal subunit and may provide a link for Upf1p to 80S monosomes.

RNA, 1999 Aug, 5(8), 1042 - 54
Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition; Chung S et al.; Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects . The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain . Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development . We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor . CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame . Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage . Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association . Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p . Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1) . Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.

Mol Cell, 1999 Jul, 4(1), 75 - 83
A role for transcriptional repressors in targeting the yeast Swi/Snf complex; Dimova D et al.; Genetic and biochemical studies indicate that the evolutionarily conserved Swi/Snf complex acts at a subset of genes to help transcriptional activators function on chromatin templates . The mechanism by which this complex is targeted to specific chromosomal loci remains unknown . We show that Swi/Snf is required for expression of the yeast histone HTA1-HTB1 locus because of the role of Hir1p and Hir2p corepressors in negatively regulating transcription . Snf5p, Snf2p/Swi2p, and Swi3p, three components of the yeast Swi/Snf complex, coimmunoprecipitate with each Hir protein, and Snf5p is maximally associated with the HTA1-HTB1 promoter when the Hir-based repression system is intact and the Swi/Snf complex is functional . The data support a role for the Hir repressors in the gene-specific targeting of Swi/Snf.

Mol Cell, 1999 Jul, 4(1), 21 - 33
SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex; Kitagawa K et al.; We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function . Sgt1p physically associates with Skp1p in vivo and in vitro . SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content . Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function . Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p . Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase . sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro . Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution.

Genes Dev, 1999 Aug 1, 13(15), 1950 - 9
Pds1p of budding yeast has dual roles: inhibition of anaphase initiation and regulation of mitotic exit; Cohen-Fix O et al.; Progression through mitosis is controlled by protein degradation that is mediated by the anaphase-promoting complex/cyclosome (APC/C) and its associated specificity factors . In budding yeast, APC/C(Cdc20) promotes the degradation of the Pds1p anaphase inhibitor at the metaphase-to-anaphase transition, whereas APC/C(Cdh1) promotes the degradation of the mitotic cyclins at the exit from mitosis . Here we show that Pds1p has a novel activity as an inhibitor of mitotic cyclin destruction, apparently by preventing the activation of APC/C(Cdh1) . This activity of Pds1p is independent of its activity as an anaphase inhibitor . We propose that the dual role of Pds1p as an inhibitor of anaphase and of cyclin degradation allows the cell to couple the exit from mitosis to the prior completion of anaphase . Finally, these observations provide a novel regulatory paradigm in which the sequential degradation of two substrates is determined by the substrates themselves, such that an early substrate inhibits the degradation of a later one.

Genes Dev, 1999 Aug 1, 13(15), 1912 - 7
Localization of bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site; Park HO et al.; Yeast cells of different cell type exhibit distinct budding patterns that reflect the organization of the actin cytoskeleton . Bud1p (Rsr1p), a Ras-like GTPase, and Bud2p, a GTPase-activating protein for Bud1p, are essential for proper budding pattern . We show that Bud2p is localized at the presumptive bud site in G(1) cells in all cell types and that this localization is independent of Bud1p . Bud2p subsequently localizes to the mother-bud neck after bud emergence; this localization depends on the integrity of the septins . These observations indicate that Bud2p becomes positioned in G(1) cells by recognizing cell type-specific landmarks at the presumptive bud site.

Plant Physiol, 1999 Aug, 120(4), 1193 - 204
Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system; Moon YH et al.; A MADS family gene, OsMADS6, was isolated from a rice (Oryza sativa L.) young flower cDNA library using OsAMDS1 as a probe . With this clone, various MADS box genes that encode for protein-to-protein interaction partners of the OsMADS6 protein were isolated by the yeast two-hybrid screening method . On the basis of sequence homology, OsMADS6 and the selected partners can be classified in the APETALA1/AGAMOUS-LIKE9 (AP1/AGL9) family . One of the interaction partners, OsMADS14, was selected for further study . Both genes began expression at early stages of flower development, and their expression was extended into the later stages . In mature flowers the OsMADS6 transcript was detectable in lodicules and also weakly in sterile lemmas and carpels, whereas the OsMADS14 transcript was detectable in sterile lemmas, paleas/lemmas, stamens, and carpels . Using the yeast two-hybrid system, we demonstrated that the region containing of the 109th to 137th amino acid residues of OsMADS6 is indispensable in the interaction with OsMADS14 . Site-directed mutation analysis revealed that the four periodical leucine residues within the region are essential for this interaction . Furthermore, it was shown that the 14 amino acid residues located immediately downstream of the K domain enhance the interaction, and that the two leucine residues within this region play an important role in that enhancement.

Curr Microbiol, 1999 Sep, 39(3), 168 - 72
Stochastic nucleosome positioning in a yeast chromatin region is not dependent on histone H1; Puig S et al.; To gain a better understanding of the function of the yeast histone H1, its role in nucleosome positioning was studied . With this objective in mind, we analyzed a chromatin region of the yeast Chromosome (Chr) IX, in which there are two closely packed open reading frames (ORFs), POT1 and YIL161w . This locus shows a regular ladder of 13 stochastically positioned nucleosomes, which is unaffected by the absence of the HHO1 gene . This suggests that histone H1 has no effect on nucleosome positioning in yeast.

Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 734 - 9
Activator-specific requirement for the general transcription factor IIE in yeast; Sakurai H et al.; The general transcription factor (TF) IIE is required for mRNA synthesis of many, but not all, genes in yeast . In the transcription process, TFIIE regulates TFIIH kinase activity that phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II . The CTD and the CTD kinase Kin28, a subunit of TFIIH, have been shown to be dispensable for activation of several heat shock genes and the copper metallothionein gene CUP1 . Here we analyzed requirement of TFIIE for transcription of these genes and found that TFIIE is necessary for activation of the heat shock genes by heat shock transcription factor Hsf1 . By contrast, transcription of CUP1 mediated by both Hsf1 and copper-activated transcription factor Ace1 was inducible after inactivating TFIIE . These results show that both TFIIE and the CTD/the CTD kinase exhibit "gene specificities" which are overlapping, but not identical to each other, and thereby suggest that TFIIE functions with or without involvement of the CTD/the CTD kinase depending on the gene to be transcribed .

Arch Biochem Biophys, 1999 Aug 15, 368(2), 338 - 46
Mechanism of inhibition of yeast squalene synthase by substrate analog inhibitors; Kalinowski SS et al.; Squalene synthase catalyzes the reductive condensation of two identical substrate molecules, farnesyl diphosphate, to the hydrocarbon squalene via an obligatory intermediate, presqualene pyrophosphate . Since the kinetic mechanism of the transformation is sequential, two substrate binding pockets that recognize the same molecule must exist in the enzyme active site . This raises the possibility of a choice of binding pockets for inhibitors that are designed as substrate or reaction intermediate analogs and thus may provide some information on the mechanism of differentiation of the two identical molecules . In this report, we have investigated the mechanism of inhibition of a series of farnesyl diphosphate analog inhibitors . The inhibitors fall into two categories . One class of compounds binds to free enzyme as well as the enzyme substrate complex, and the binding is refractory to the concentration of the substrate . The second class binds only to the free enzyme, and its binding is significantly modulated by the substrate concentration . Very modest structural changes in the compounds appear to dictate which class of inhibitor any compound may fall into . The significance of these observations with respect to the mechanism of the enzyme are discussed .

J Med Virol, 1999 Sep, 59(1), 78 - 83
Ability of yeast Ty-VLPs (virus-like particles) containing varicella-zoster virus (VZV)gE and assembly protein fragments to induce in vitro proliferation of human lymphocytes from VZV immune patients; Welsh MD et al.; Yeast Ty virus-like particles (VLPs) containing viral protein inserts have previously been shown to be potent immunogens, inducing both humoral and cell mediated immunity (CMI) . The antigenicity of hybrid VLPs containing fragments of the varicella-zoster virus (VZV) gE protein or the assembly protein (AP) was assessed by lymphocyte proliferation . Peripheral blood mononuclear cells (PBMCs) from patients with a recent natural VZV infection were stimulated in vitro with VZV-VLPs together with control antigens . PBMC samples from both varicella (85%) and zoster (75%) patients proliferated in responses to at least one of the gE VZV-VLPs . As reported for the first time, VZV specific lymphocyte responses were also identified towards the VZV AP in two varicella and two zoster patient samples . The results demonstrate specific CMI recognition of the VZV gE fragments tested and the VZV AP delivered in the form of recombinant Ty-VLPs, and highlights their potential use as a recombinant antigen delivery system for vaccination.

J Biol Chem, 1999 Aug 13, 274(33), 23599 - 609
Efficient processing of DNA ends during yeast nonhomologous end joining . Evidence for a DNA polymerase beta (Pol4)-dependent pathway; Wilson TE et al.; Repair of DNA double strand breaks by nonhomologous end joining (NHEJ) requires enzymatic processing beyond simple ligation when the terminal bases are damaged or not fully compatible . We transformed yeast with a series of linearized plasmids to examine the role of Pol4 (Pol IV, DNA polymerase beta) in repair at a variety of end configurations . Mutation of POL4 did not impair DNA polymerase-independent religation of fully compatible ends and led to at most a 2-fold reduction in the frequency of joins that require only DNA polymerization . In contrast, the frequency of joins that also required removal of a 5'- or 3'-terminal mismatch was markedly reduced in pol4 (but not rev3, exo1, apn1, or rad1) yeast . In a chromosomal double strand break assay, pol4 mutation conferred a marked increase in sensitivity to HO endonuclease in a rad52 background, due primarily to loss of an NHEJ event that anneals with a 3'-terminal mismatch . The NHEJ activity of Pol4 was dependent on its nucleotidyl transferase function, as well as its unique amino terminus . Paradoxically, in vitro analyses with oligonucleotide substrates demonstrated that although Pol4 fills gaps with displacement of mismatched but not matched 5' termini, it lacks both 5'- and 3'-terminal nuclease activities . Pol4 is thus specifically recruited to perform gap-filling in an NHEJ pathway that must also involve as yet unidentified nucleases.

J Biol Chem, 1999 Aug 13, 274(33), 23203 - 9
An interaction between the N-terminal region and the core domain of yeast TFIIB promotes the formation of TATA-binding protein-TFIIB-DNA complexes; Bangur CS et al.; The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by eukaryotic RNA polymerase II . We previously identified a yeast TFIIB mutant (R64E) that exhibited increased activity in the formation of stable TATA-binding protein-TFIIB-DNA (DB) complexes in vitro . We report here that the homologous human TFIIB mutant (R53E) also displayed increased activity in DB complex formation in vitro . Biochemical analyses revealed that the increased activity of the R64E mutant in DB complex formation was associated with an altered protease sensitivity of the protein and an enhanced interaction between the N-terminal region and the C-terminal core domain . These results suggest that the intramolecular interaction in yeast TFIIB stabilizes a productive conformation of the protein for the association with promoter-bound TATA-binding protein.

Mol Biol Cell, 1999 Aug, 10(8), 2745 - 57
Fission yeast Pob1p, which is homologous to budding yeast Boi proteins and exhibits subcellular localization close to actin patches, is essential for cell elongation and separation; Toya M et al.; The fission yeast pob1 gene encodes a protein of 871 amino acids carrying an SH3 domain, a SAM domain, and a PH domain . Gene disruption and construction of a temperature-sensitive pob1 mutant indicated that pob1 is essential for cell growth . Loss of its function leads to quick cessation of cellular elongation . Pob1p is homologous to two functionally redundant Saccharomyces cerevisiae proteins, Boi1p and Boi2p, which are necessary for cell growth and relevant to bud formation . Overexpression of pob1 inhibits cell growth, causing the host cells to become round and swollen . In growing cells, Pob1p locates at cell tips during interphase and translocates near the division plane at cytokinesis . Thus, this protein exhibits intracellular dynamics similar to F-actin patches . However, Pob1p constitutes a layer, rather than patches, at growing cell tips . It generates two split discs flanking the septum at cytokinesis . The pob1-defective cells no longer elongate but swell gradually at the middle, eventually assuming a lemon-like morphology . Analysis using the pob1-ts allele revealed that Pob1p is also essential for cell separation . We speculate that Pob1p is located on growing plasma membrane, possibly through the function of actin patches, and may recruit proteins required for the synthesis of cell wall.

Mol Biol Cell, 1999 Aug, 10(8), 2607 - 18
The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins; Chen RH et al.; The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle . When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability . We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle . Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p . This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins . In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE . Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p.

Mol Biol Cell, 1999 Aug, 10(8), 2583 - 94
The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast; Swaminathan S et al.; Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation . In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome . In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked . We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome . Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome . Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well . In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase . Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells . This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin . Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells . Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis . We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.

Mol Biol Cell, 1999 Aug, 10(8), 2559 - 72
Multiple sex pheromones and receptors of a mushroom-producing fungus elicit mating in yeast; Fowler TJ et al.; The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors . Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis . A complex set of pheromone-receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone . The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein . Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor . The specificity of interaction among pheromone-receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone-receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms.

Mol Biol Cell, 1999 Aug, 10(8), 2531 - 46
Protein kinase activity and identification of a toxic effector domain of the target of rapamycin TOR proteins in yeast; Alarcon CM et al.; In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1 . The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton . A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified . We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin . We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast . Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect . Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation . Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains . In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Analyst, 1998 Dec, 123(12), 2585 - 8
Determination of oestrogen concentrations in bovine plasma by a recombinant oestrogen receptor-reporter gene yeast bioassay; Burdge GC et al.; A recombinant cell yeast bioassay (RCBA) was applied to the generic measurement of bovine plasma oestrogen concentration . Samples were prepared by diethyl ether extraction of plasma following addition of {3H}17 beta-oestradiol as internal standard; organic and aqueous phases were separated by freezing (recovery 97.1 +/- 0.7%) and dried extract reconstituted in culture medium (recovery 31.4 +/- 4.5%) . Plasma oestrogen concentrations were measured by incubation of extracts with yeast containing a stable human oestrogen receptor (hER) and a reporter construct comprising an hER response element regulating beta-galactosidase expression . The linearity of response for the analysis of spiked plasma samples using the RCBA, following corrections, is described by y = 0.8994x - 0.111 (r2 = 0.9776, P < 0.0001) . Inter-assay variation for endogenous oestrogen was 11.5% for > 1 pg ml-1 . Plasma oestrogen concentrations for intact (n = 5) and castrated (n = 3) males were < 0.5 pg ml-1, and 3.7 +/- 2.6 pg ml-1 for luteal phase females (n = 10) . Analysis by RCBA of sequential samples from heifers during the reproductive cycle failed to detect the pre-ovulatory increase in plasma 17 beta-oestradiol as determined by radioimmunoassay (RIA) (maximal concentrations 2.09 +/- 2.1 pg ml-1 and 32.6 +/- 14.6 pg ml-1, respectively) . Interestingly, when samples were hydrolysed using Helix pomatia glucuronidase the RCBA gave concentrations (29.5 +/- 8.9 pg ml-1) not significantly different to those obtained by RIA . These preliminary findings suggest that a substantial proportion of plasma oestrogen during the pre-ovulatory period may be conjugated . These data indicate the potential of the RCBA to measure biologically active and physiological levels of plasma oestrogens in cattle . One potentially valuable application of this generic oestrogen assay could be in surveillance programmes to detect illegal use of anabolic oestrogens in live-stock where the identity of the analyte may be unknown.

Mutat Res, 1999 Aug 11, 429(1), 13 - 26
Cell division transforms mutagenic lesions into deletion-recombinagenic lesions in yeast cells; Galli A et al.; Cell proliferation has been recognized as an important factor in human and experimental carcinogenesis . Point mutations as well as larger chromosomal rearrangements are involved in the initiation of cancer . In this paper we compared the relative potencies of radiation and chemical carcinogens for inducing point mutations vs . deletions in cell cycle arrested with dividing cells of Saccharomyces cerevisiae . Point mutation substrates and deletion (DEL) recombination substrates were constructed with the genes CDC28 and TUB2 that are required for cell cycle progression through G1 and G2, respectively . The carcinogens ionizing radiation, UV, MMS, EMS and 4-NQO induced point mutations in G1 and in G2 arrested as well as in dividing cells . UV, MMS, EMS and 4-NQO caused very weak if any increases in DEL recombination in G1 or G2 arrested cells, but large increases in dividing cells . When cells treated with carcinogen either in G1 or G2 were allowed to progress through the cell cycle, a time-dependent increase in DEL recombination was seen . Ionizing radiation and the site-specific endonuclease I-SceI, which both directly create double-strand breaks, induced DEL recombination in G1 as well as in G2 arrested cells . In conclusion, UV-, MMS-, EMS- and 4-NQO-induced DNA damage was converted during DNA replication to a lesion capable of inducing DEL recombination which is probably a DNA strand break . Thus, cell proliferation is not necessary to turn DNA alkylation or UV damage into a mutagenic lesion but to convert the damage into a lesion that induces DNA deletions . These results are discussed with respect to mechanisms of carcinogenesis .

Gene, 1999 Aug 5, 236(1), 53 - 61
Insertion of expanded CAG trinucleotide repeat motifs into a yeast artificial chromosome containing the human Machado-Joseph disease gene; Cemal CK et al.; Machado-Joseph disease or spinocerebellar ataxia 3 (SCA3) is a progressive neurodegenerative disorder caused by pathological expansion of a trinucleotide repeat motif present within exon 4 of the MJD1 gene . Previous attempts to create a transgenic animal model have failed to produce a neurological deficit truly representative of the disease phenotype . This appears to be the result of inappropriate expression of the mutant protein in neuronal populations generally spared in the disease state . Introduction of a human disease gene in the context of a yeast artificial chromosome clone containing endogenous regulatory elements would enhance the potential for correct tissue/cell-specific expression at physiological levels . We report the introduction of expanded CAG repeat motifs into a 250kb yeast artificial chromosome clone spanning the MJD1 locus using two rounds of homologous recombination . Transformants exhibited both expansions and contractions of the motif with alleles ranging in size from 48 to 84 repeat units . The availability of these clones for modelling of the disease in transgenic animals should allow elucidation of the role of repeat length in the phenotypic spectrum of the disease.

Gene, 1999 Aug 5, 236(1), 33 - 42
Monitoring dynamics of gene expression in yeast during stationary phase; Paz I et al.; The commonly used genetic approaches in yeast are designed to identify defects in cell/colony growth . In order to identify genes which control molecular mechanisms during quiescence ('stationary phase'), different tactics are required . We describe the development of a new genetic approach based on the previous observations that gene expression in quiescent Saccharomyces cerevisiae cells is largely repressed . For studying the mechanism controlling the repression of gene expression in stationary phase, we use UBI4-lacZ as a reporter gene . The product of this fusion gene was shown previously to encode an unstable protein in dividing cells . We show here that it is also unstable in stationary cells . We demonstrate that the relatively short half-life of this reporter protein can be utilized to monitor the dynamics of the repression of gene expression during stationary phase in liquid culture, using ACT1 or SSA3 promoters as the model promoters . By adapting a colony color test, we show that the reporter gene can also be used to monitor gene expression in quiescent colonies, thus serving as a tool to screen for defects in the regulation of this process during growth arrest . The utility of the approach was demonstrated by confirming the defects of top1Delta and bcy1Delta cells to appropriately express the ACT1p-UBI4-lacZ in stationary phase . The mutant colonies were easily discernible from wild-type colonies by our color test . Finally, using SSA3p-UBI4-lacZ as the reporter gene, we found that the 5'-untranslated region of SSA3 mRNA is sufficient to repress translation of the reporter mRNA after entry of the cells into stationary phase . The possibility that the short length of the SSA3 5'-untranslated region is a major determinant of the inefficient translation of SSA3p-UBI4-lacZ in stationary phase is discussed.

Biochemistry, 1999 Aug 3, 38(31), 10004 - 12
Effects of substitution of tryptophan 412 in the substrate activation pathway of yeast pyruvate decarboxylase; Li H et al.; Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at W412, located on the putative substrate activation pathway and linking E91 on the alpha domain with W412 on the gamma domain of the enzyme . While C221 on the beta domain is the residue at which substrate activation is triggered {Baburina, I., et al . (1994) Biochemistry 33, 5630-5635; Baburina, I., et al . (1996) Biochemistry 35, 10249-10255}, that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 {Baburina, I., et al . (1998) Biochemistry 37, 1235-1244; Baburina, I., et al . (1998) Biochemistry 37, 1245-1255}, thence to E91 on the alpha domain {Li, H., and Jordan, F . (1999) Biochemistry 38, 9992-10003}, and then on to W412 on the gamma domain and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains {Arjunan, D., et al . (1996) J . Mol . Biol . 256, 590-600} . Substitution at W412 with F and A was carried out, resulting in active enzymes with specific activities about 4- and 10-fold lower than that of the wild-type enzyme . Even though W412 interacts with E91 and H115 via a main chain hydrogen bond donor and acceptor, respectively, there is clear evidence for the importance of the indole side chain of W412 from a variety of experiments: thermostability, fluorescence quenching, and the binding constants of the thiamin diphosphate, and circular dichroism spectroscopy, in addition to conventional steady-state kinetic measurements . While the substrate activation is still prominent in the W412F variant, its level is very much reduced in the W412A variant, signaling that the size of the side chain is also important in positioning the amino acids surrounding the active center to achieve substrate activation . The fluorescence studies demonstrate that W412 is a relatively minor contributor to the well-documented fluorescence of apopyruvate decarboxylase in its native state . The information about the W412 variants provides strong additional support for the putative substrate activation pathway from C221 --> H92 --> E91 --> W412 --> G413 --> thiamin diphosphate . The accumulating evidence for the central role of the beta domain in stabilizing the overall structure is summarized.

Biochemistry, 1999 Aug 3, 38(31), 9992 - 10003
Role of glutamate 91 in information transfer during substrate activation of yeast pyruvate decarboxylase; Li H et al.; Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at E91, located on the putative substrate activation pathway and linking the alpha and gamma domains of the enzyme . While C221 on the beta domain is the residue at which substrate activation is triggered {Baburina, I., et al . (1994) Biochemistry 33, 5630-5635; Baburina, I., et al . (1996) Biochemistry 35, 10249-10255}, that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 {Baburina, I . , et al . (1998) Biochemistry 37, 1235-1244}, thence to E91 on the alpha domain, and then on to W412 on the gamma domain {Li, H., and Jordan, F . (1999) Biochemistry 38, 10004-10012} and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains {Arjunan, D., et al . (1996) J . Mol . Biol . 256, 590-600} . Substitution at E91 with Q, D, or A led to modest reductions in the specific activity (4-, 5-, and 30-fold), as well as in both the turnover number and the catalytic efficiency, in that order . Interestingly, the Hill coefficient was only slightly reduced for the E91D variant, but cooperativity was virtually abolished for the E91Q and E91A variants . The thermal stability of the variants was reduced in the following order: wild type > E91Q > E91D > E91A; circular dichroism and fluorescence experiments also demonstrated that the tertiary structure of the enzyme was affected by these substitutions . The variants could be purified as apoenzymes, demonstrating their impaired ability to bind thiamin diphosphate . Apparently, the charge at residue 91 is quite important for maintaining optimal cooperativity . To maintain strong domain-domain interactions, the length of the side chain at position 91 with hydrogen bonding potential to W412 is sufficient.

J Appl Microbiol, 1999 Jul, 87(1), 1 - 7
Yeast cell mortality related to a high-pressure shift: occurrence of cell membrane permeabilization; Perrier-Cornet JM et al.; The shrinkage of yeast cells caused by high-pressure treatment (250 MPa, 15 min) was investigated using direct microscopic observation . A viable staining method after treatment allowed the volume variation of two populations to be distinguished: an irreversible volume decrease (about 35% of the initial volume) of pressure-inactivated cells during pressure holding time, and viable cells, which were less affected . A mass transfer was then induced during high-pressure treatment . Causes of this transfer seem to be related to a pressure-induced membrane permeabilization, allowing a subsequent leakage of internal solutes, where three ions (Na+, K+ and Ca2+), plus endogenous glycerol, were verified . This glycerol leakage was found to occur after yeast pressurization in a medium having low water activity, although the yeast was not inactivated . All these observations lead to the hypothesis that pressure-induced cell permeabilization could be the cause of yeast inactivation under pressure.

FEBS Lett, 1999 Jul 9, 454(3), 201 - 6
Assessment of uncoupling activity of the human uncoupling protein 3 short form and three mutants of the uncoupling protein gene using a yeast heterologous expression system; Hagen T et al.; The human uncoupling protein 3 gene generates two mRNA transcripts, uncoupling protein 3L and uncoupling protein 3S, which are predicted to encode long and short forms of the uncoupling protein 3 protein, respectively . While uncoupling protein 3L is similar in length to the other known uncoupling proteins, uncoupling protein 3S lacks the last 37 C-terminal residues . A splice site mutation in the human uncoupling protein 3 gene, resulting in the exclusive expression of uncoupling protein 3S, and a number of point mutations in the uncoupling protein 3 gene have been described . This study compares the biochemical activity of uncoupling protein 3S as well as three mutants of the uncoupling protein 3 gene (V9M, V102I, R282C) with that of uncoupling protein 3L utilizing a yeast expression system . All proteins were expressed at similar levels and had qualitatively similar effects on parameters related to the uncoupling function . Both uncoupling protein 3S and uncoupling protein 3L decreased the yeast growth rate by 35 and 52%, increased the whole yeast basal O2 consumption by 26 and 48%, respectively, and decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodide . In isolated mitochondria, uncoupling protein 3S and uncoupling protein 3L caused a similar (33 and 35%, respectively) increase in state 4 respiration, which was relatively small compared to uncoupling protein 1 (102% increase) . A truncated version of uncoupling protein 3S, lacking the last three C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif that is highly conserved among all mitochondrial carriers, had a greatly reduced uncoupling activity . The two naturally occurring uncoupling protein 3 mutants, V9M and V102I, were similar to uncoupling protein 3L with respect to effects on the yeast growth and whole yeast O2 consumption . The R282C mutant had a reduced effect compared to uncoupling protein 3L . In summary, uncoupling protein 3S and the three mutants of uncoupling protein 3 appear to be functional proteins with biochemical activities similar to uncoupling protein 3L, although uncoupling protein 3S and the R282C mutant have a modestly reduced function.

FEBS Lett, 1999 Jul 9, 454(3), 181 - 6
Induction of vacuolar Ca2+-ATPase and H+/Ca2+ exchange activity in yeast mutants lacking Pmr1, the Golgi Ca2+-ATPase; Marchi V et al.; We have analyzed Ca2+ transport activity in defined subcellular fractions of an isogenic set of wild-type and mutant yeast . The results, together with measurements of polypeptide expression levels and promoter::reporter gene activity, show that the Golgi Ca2+-ATPase, Pmr1, is the major Ca2+ pump under normal growth conditions . In the absence of Pmr1, we show a massive, calcineurin-dependent compensatory induction of the vacuolar Ca2+-ATPase, Pmc1 . In addition, H+/Ca2+ exchange activity, that may be distinct from the vacuolar exchanger Vcx1, is also increased.

Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9033 - 8
Binding of elongin A or a von Hippel-Lindau peptide stabilizes the structure of yeast elongin C; Botuyan MV et al.; Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals . Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis . Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL . To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL . Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region . Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers . Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer . Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins . These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL.

Genetics, 1999 Aug, 152(4), 1543 - 56
Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers; Jones S et al.; Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport . While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment . Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well . Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation . Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases . Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function . Sec7p itself has an important role in the yeast secretory pathway . However, its mechanism of action is not yet understood . We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes . Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases . The Sec7-domain of Sec7p is sufficient for this activity . Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA . These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast . The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway.

Eur J Biochem, 1999 Jul, 263(1), 118 - 27
ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa; Beauvoit B et al.; The role of the nuclear-encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria . As the subunit VIa-null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP-regulating sites were characterized with respect to their location and their kinetic effect: (a) intra-mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of ATP on the wild-type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated by subunit VIa . However, at physiological ionic strength (i.e . approximately 200 mM), activation by ATP was absent but inhibition was not impaired; (b) in ethanol-respiring mitochondria, when the electron flux was modulated using a protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild-type) or inhibition (null-mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of cytochrome oxidase on respiratory flux, decreased (wild-type) or increased (null-mutant) in the presence of ATP; (d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild-type but not for the mutant subunit . Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration . This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP.

EMBO J, 1999 Aug 2, 18(15), 4321 - 31
Characterization of Sm-like proteins in yeast and their association with U6 snRNA; Mayes AE et al.; Seven Sm proteins associate with U1, U2, U4 and U5 spliceosomal snRNAs and influence snRNP biogenesis . Here we describe a novel set of Sm-like (Lsm) proteins in Saccharomyces cerevisiae that interact with each other and with U6 snRNA . Seven Lsm proteins co-immunoprecipitate with the previously characterized Lsm4p (Uss1p) and interact with each other in two-hybrid analyses . Free U6 and U4/U6 duplexed RNAs co-immunoprecipitate with seven of the Lsm proteins that are essential for the stable accumulation of U6 snRNA . Analyses of U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs in Lsm-depleted strains suggest that Lsm proteins may play a role in facilitating conformational rearrangements of the U6 snRNP in the association-dissociation cycle of spliceosome complexes . Thus, Lsm proteins form a complex that differs from the canonical Sm complex in its RNA association(s) and function . We discuss the possible existence and functions of alternative Lsm complexes, including the likelihood that they are involved in processes other than pre-mRNA splicing.

EMBO J, 1999 Aug 2, 18(15), 4180 - 95
Interaction of the yeast gamma-tubulin complex-binding protein Spc72p with Kar1p is essential for microtubule function during karyogamy; Pereira G et al.; The spindle pole body component Kar1p has a function in nuclear fusion during conjugation, a process known as karyogamy . The molecular role of Kar1p during this process is poorly understood . Here we show that the yeast gamma-tubulin complex-binding protein Spc72p interacts directly with the N-terminal domain of Kar1p, thereby targeting the gamma-tubulin complex to the half bridge, a substructure of the spindle pole body, where it organizes microtubules . This binding of Spc72p to Kar1p has only a minor role during vegetative growth, whereas it becomes essential for karyogamy in mating cells, explaining the important role of Kar1p in this process . We also show that the localization of Spc72p within the spindle pole body changes throughout the cell cycle and even more strongly in response to mating pheromone . Taken together, these observations suggest that the relocalization of Spc72p within the spindle pole body is the 'landmark' event in the pheromone-induced reorganization of the cytoplasmic microtubules.

J Biol Chem, 1999 Aug 6, 274(32), 22770 - 4
The yeast heme-responsive transcriptional activator Hap1 is a preexisting dimer in the absence of heme; Hon T et al.; In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC . Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription . Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones . Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids . Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1 . We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme . By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC . Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities . Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding . Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer.

J Biol Chem, 1999 Aug 6, 274(32), 22763 - 9
Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase; Branda SS et al.; Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease . Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity . Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form . A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M . (1998) Hum . Mol . Gen . 7, 1485-1489), suggesting that a different peptidase might be required for production of mature size frataxin . However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin . MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein . In this way, MPP could influence frataxin function and indirectly affect mitochondrial iron homeostasis.

J Biol Chem, 1999 Aug 6, 274(32), 22668 - 78
A conserved domain of yeast RNA triphosphatase flanking the catalytic core regulates self-association and interaction with the guanylyltransferase component of the mRNA capping apparatus; Lehman K et al.; The 549-amino acid yeast RNA triphosphatase Cet1p catalyzes the first step in mRNA cap formation . Cet1p consists of three domains as follows: (i) a 230-amino acid N-terminal segment that is dispensable for catalysis in vitro and for Cet1p function in vivo; (ii) a protease-sensitive segment from residues 230 to 275 that is dispensable for catalysis but essential for Cet1p function in vivo; and (iii) a catalytic domain from residues 275 to 539 . Sedimentation analysis indicates that purified Cet1(231-549)p is a homodimer . Cet1(231-549)p binds in vitro to the yeast RNA guanylyltransferase Ceg1p to form a 7.1 S complex that we surmise to be a trimer consisting of two molecules of Cet1(231-549)p and one molecule of Ceg1p . The more extensively truncated protein Cet1(276-549)p, which cannot support cell growth, sediments as a monomer and does not interact with Ceg1p . An intermediate deletion protein Cet1(246-549)p, which supports cell growth only when overexpressed, sediments principally as a discrete salt-stable 11.5 S homo-oligomeric complex . These data implicate the segment of Ceg1p from residues 230 to 275 in regulating self-association and in binding to Ceg1p . Genetic data support the existence of a Ceg1p-binding domain flanking the catalytic domain of Cet1p, to wit: (i) the ts growth phenotype of 2mu CET1(246-549) is suppressed by overexpression of Ceg1p; (ii) a ts alanine cluster mutation CET1(201-549)/K250A-W251A is suppressed by overexpression of Ceg1p; and (iii) 15 other cet-ts alleles with missense changes mapping elsewhere in the protein are not suppressed by Ceg1p overexpression . Finally, we show that the in vivo function of Cet1(275-549)p is completely restored by fusion to the guanylyltransferase domain of the mouse capping enzyme . We hypothesize that the need for Ceg1p binding by yeast RNA triphosphatase can by bypassed when the triphosphatase catalytic domain is delivered to the RNA polymerase II elongation complex by linkage in cis to the mammalian guanylyltransferase.

Cell, 1999 Jul 23, 98(2), 249 - 59
Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region; Blat Y et al.; Sister chromatid cohesion is mediated by evolutionary conserved chromosomal proteins, termed "cohesins." Using an extension of chromatin immunoprecipitation, we have analyzed the distribution of cohesins Mcd1/ Sccl and Smc1 along yeast chromosome III . Both proteins occur preferentially at the same approximately 23 positions . Sites in a approximately 50 kb region around the centromere give especially intense signals . Prominent centric region binding appears to emerge from a more even distribution, probably by differential loss of cohesins along the chromosome arms . Cohesin binding peaks correspond closely to peaks of high local AT composition, a base composition periodicity of approximately 15 kb that is distinct from the approximately 50 kb periodicity of base composition isochores, consistent with axis association of cohesins . The methodology described can be used to analyze the distribution of any DNA-binding protein and, via microchips, along entire genomes.

Antonie Van Leeuwenhoek, 1999 Apr, 75(3), 245 - 51
Trehalose accumulation by tropical yeast strains submitted to stress conditions; Ribeiro MJ et al.; Trehalose, a non-reducing disaccharide that accumulates in Saccharomyces cerevisiae, has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate which may be mobilized during stress . However, most of these studies have been done with strains isolated from European or Asian habitats of temperate climate . In this study, yeasts living in tropical environments, isolated from different microhabitats in Southeastern Brazil, were used to evaluate whether trehalose contributes to survival under osmotic, ethanol and heat stress . The survival under severe stress was compared to a well-characterized laboratorial wild-type strain (D273-10B) . Most of the Saccharomyces cerevisiae strains isolated from Drosophila in Tropical Rain Forest were able to accumulate trehalose after a preconditioning treatment at 40 degrees C for 1 h . The amount of intracellular trehalose levels was better correlated with survival during a challenging heat shock at 50.5 degrees C for 8 min . Saccharomyces cerevisiae and Candida guilliermondii were observed to be thermotolerant as well as osmotolerant . No clear correlation between intracellular trehalose levels and survival could be derived during ethanol stress . In some cases, the amount of trehalose accumulated before the ethanol stress seemed to play an important role for the survival of these strains.

J Cell Biol, 1999 Jul 26, 146(2), 453 - 64
DAip1, a Dictyostelium homologue of the yeast actin-interacting protein 1, is involved in endocytosis, cytokinesis, and motility; Konzok A et al.; The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes.We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia . In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions.Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1 . DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates . Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate . On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin . Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.

J Cell Biol, 1999 Jul 26, 146(2), 373 - 87
The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell; Drgonova J et al.; Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase . Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization . Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C . In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature . Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature . This was also the case for rho1(E45I) when placed in a different genetic background . Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I) . Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly . The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone . The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1 . Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds . Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.

J Cell Biol, 1999 Jul 26, 146(2), 273 - 84
Expression of the 180-kD ribosome receptor induces membrane proliferation and increased secretory activity in yeast; Becker F et al.; Expression of the canine 180-kD ribosome receptor (p180) in yeast cells resulted in a marked proliferation of intracellular membranes . The type of membranes observed varied with the expression of specific portions of p180 . Rough membranes predominated when the ribosome binding domain of p180 was present, whereas expression constructs lacking this region resulted in smooth membranes . Northern analysis indicated that expression of the NH(2)-terminal 767 amino acids (DeltaCT), which include the ribosome binding domain, upregulated the transcription and translation of genes involved in exocytosis . The membranes that were proliferated were functional as these cells overcame a temperature-sensitive translocation defect . Most significantly, cells that overexpressed DeltaCT and proliferated rough endoplasmic reticulum exhibited severalfold higher levels of secretion of an ectopically expressed secretory protein . We conclude that p180 expression triggers a cascade of events leading to an increase in secretory potential akin to the terminal differentiation of mammalian secretory cells and tissues.

Appl Environ Microbiol, 1999 Aug, 65(8), 3594 - 8
Transport and utilization of hexoses and pentoses in the halotolerant yeast Debaryomyces hansenii; Nobre A et al.; Debaryomyces hansenii is a yeast species that is known for its halotolerance . This organism has seldom been mentioned as a pentose consumer . In the present work, a strain of this species was investigated with respect to the utilization of pentoses and hexoses in mixtures and as single carbon sources . Growth parameters were calculated for batch aerobic cultures containing pentoses, hexoses, and mixtures of both types of sugars . Growth on pentoses was slower than growth on hexoses, but the values obtained for biomass yields were very similar with the two types of sugars . Furthermore, when mixtures of two sugars were used, a preference for one carbon source did not inhibit consumption of the other . Glucose and xylose were transported by cells grown on glucose via a specific low-affinity facilitated diffusion system . Cells derepressed by growth on xylose had two distinct high-affinity transport systems for glucose and xylose . The sensitivity of labeled glucose and xylose transport to dissipation of the transmembrane proton gradient by the protonophore carbonyl cyanide m-chlorophenylhydrazone allowed us to consider these transport systems as proton symports, although the cells displayed sugar-associated proton uptake exclusively in the presence of NaCl or KCl . When the V(max) values of transport systems for glucose and xylose were compared with glucose- and xylose-specific consumption rates during growth on either sugar, it appeared that transport did not limit the growth rate.

Appl Environ Microbiol, 1999 Aug, 65(8), 3325 - 7
The spheroplast lysis assay for yeast in microtiter plate format; Ovalle R et al.; A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time . The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time . Clonal differences were determined to be the major source of variation . Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes.

Am J Clin Nutr, 1999 Aug, 70(2), 208 - 12
Plasma lipid changes after supplementation with beta-glucan fiber from yeast; Nicolosi R et al.; BACKGROUND: Dietary fiber has been shown to improve blood lipids . OBJECTIVE: The purpose of this study was to evaluate the effect on serum lipids of a yeast-derived beta-glucan fiber in 15 free-living, obese, hypercholesterolemic men . DESIGN: After a 3-wk period in which subjects ate their usual diet, 15 g fiber/d was added to the diet for 8 wk and then stopped for 4 wk . Plasma lipids were measured weekly during baseline and at week 7 and 8 of fiber consumption, and again at week 12 . RESULTS: Compared with baseline, fiber consumption significantly reduced plasma total cholesterol (by 8% at week 7 and 6% at week 8; P < 0.05 using Bonferroni correction); week 12 values did not differ from baseline . No significant differences were noted between baseline LDL cholesterol and values at weeks 7, 8, or 12 when comparing individual groups by using Bonferroni correction, even though the overall one-way analysis of variance with repeated measures was highly significant (P < 0.001) . LDL-cholesterol concentrations did decline by 8% at week 8 compared with baseline . There was a significant effect of diet on plasma HDL-cholesterol concentrations (P < 0.005 by one-way ANOVA with repeated measures) . However, a group difference was observed only between baseline and week 12 (16% increase; P < 0.05 by Bonferroni correction) . Triacylglycerol concentrations did not change . CONCLUSIONS: The yeast-derived beta-glucan fiber significantly lowered total cholesterol concentrations and was well tolerated; HDL-cholesterol concentrations rose, but only 4 wk after the fiber was stopped.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1301 - 5
Mastigobasidium, a new teleomorphic genus for the perfect state of ballistosporous yeast Bensingtonia intermedia; Golubev WI; A new genus, Mastigobasidium, is proposed for teliospore-forming, xylose-lacking, ballistosporogenous, glucuronate-positive yeasts . The distinguishing features of the genus are: germination of the teliospore by several long aseptate hyphae; curved phragmometabasidia development on the apices of these hyphae; and production of basidiospores on a peg in clusters . The type strain of heterothallic, nitrate-negative species Mastigobasidium intermedium is VKM Y-2720T (Bullera intermedia type strain) and the allotype strain is VKM Y-2727AL (Sporobolomyces weijmanii type strain).

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1287 - 94
Genetic diversity in the yeast species Malassezia pachydermatis analysed by multilocus enzyme electrophoresis; Midreuil F et al.; Fifty-two strains of the yeast species Malassezia pachydermatis were analysed by multilocus enzyme electrophoresis . M . pachydermatis appeared to be genetically heterogeneous . A total of 27 electrophoretic types were identified that could be divided into five distinct groups with different host specificities . The diversity revealed by this electrophoretic method matched remarkably well the reported genetic variability obtained by comparing large subunit rRNA sequences . This study also suggests that genetic exchanges can occur in the anamorphic species M . pachydermatis.

Biochim Biophys Acta, 1999 Jul 30, 1439(2), 167 - 74
Regulation and function of PLDs in yeast; Rudge SA et al.; While yeast contain multiple phospholipase D activities, only one, encoded by SPO14, appears to be a member of the phosphatidylcholine-specific phospholipase D gene family . Genetic analyses have revealed a role for this enzyme in regulated membrane trafficking events.

Biochemistry, 1999 Jul 27, 38(30), 9693 - 703
Nucleotide sequences surrounding the nonanucleotide promoter motif influence the activity of yeast mitochondrial promoter; Biswas TK; The highly conserved nonanucleotide (5'-TATAAGTAA{+2}) promoter sequence dictates initiation of gene-specific transcription by the mitochondrial (mt) RNA polymerase in yeast mitochondria . However, transcriptional efficiency of the nonanucleotide promoter in different mt genes varies severalfold . To explore the regulatory role of the promoter-proximal template sequence in mt transcription, different deletion, nucleotide (nt) substitution, and tandem promoter constructs were analyzed under in vitro transcription reaction conditions . It has been found that the conserved nonanucleotide promoter plus more than 9 nt of nonconserved sequence 3' to the promoter were absolutely essential for mt gene-specific transcription . In addition, approximately 300 nt of nonspecific DNA sequence 5' to the promoter was also important for efficient transcription . Interestingly, introduction of consecutive T residues in the early transcribed sequence of the template strongly inhibited mt transcription at low nt concentrations (i.e., 5 microM UTP) . In contrast, neither other nt clusters nor a bacterial terminator-like sequences at that location inhibited mt transcription . Under the nonproductive reaction conditions, the full-length transcript from the mt polyT template was drastically reduced with the formation of several short abortive oligoribonucleotides . These results suggest that the transcriptional efficacy of the yeast mt promoter is influenced by sequence 3' to the promoter.

Genes Cells, 1999 May, 4(5), 291 - 7
Involvement of CRM1, a nuclear export receptor, in mRNA export in mammalian cells and fission yeast; Watanabe M et al.; BACKGROUND: CRM1, an evolutionarily conserved protein, was shown to be a receptor for leucine-rich nuclear export signal (NES)-dependent protein transport . In lower eukaryotes CRM1 is reported to be required for the export of mRNA, however, involvement of the NES-dependent transport pathway in mRNA export in higher eukaryotes has not been established . RESULTS: We have found that treatment of mammalian cells with leptomycin B (LMB), a specific inhibitor of CRM1, induces the nuclear accumulation of endogenous mRNA, probably due to the inhibition of its export . In fission yeasts, the nuclear accumulation of mRNA also occurred in cells treated with LMB or in a temperature-sensitive crm1 mutant at a restrictive temperature . A synthetic mRNA that was injected into the nucleus of mammalian cultured cells was exported from the nucleus within 5 h . This export was inhibited by both wheat germ agglutinin and a temperature of 4 degrees C . Importantly, this mRNA export was inhibited by LMB or by an excess amount of the NES peptide-conjugates . LMB treatment, on the other hand, rapidly induced the nuclear entry of RanBP1, a factor involved in the active nucleocytoplasmic transport, although the treatment did not interfere with a nuclear localization signal-dependent transport system within 7 h . CONCLUSION: These results suggest that CRM1 is involved in mRNA export in eukaryotic cells.

Genes Dev, 1999 Jul 15, 13(14), 1871 - 83
Collisions between yeast chromosomal loci in vivo are governed by three layers of organization; Burgess SM et al.; The relative probabilities that different pairs of chromosomal loci will collide with one another in vegetatively growing diploid yeast cells have been assessed using a genetic assay for Cre/loxP site-specific recombination . Recombination rates have been determined for 18 different pairs of loxP sites representing diverse pairs of positions within the genome . Overall, relative collision probabilities vary over an eightfold range . Within this range, a hierarchy comprising three levels of organization can be discerned . First, collisions between loci on nonhomologous chromosomes are governed by nonspecific centromere clustering . Second, a sequence is closer to allelic or nearby sequences on its homolog than to sequences on nonhomologous chromosomes, an effect most simply attributed to homolog pairing . Third, a sequence can be closer to other sequences nearby on the same chromosome than to sequences on other chromosomes . These findings provide a framework for assessing the role of chromosome disposition in cellular processes such as DNA repair and gene expression . Also the possibility is raised that genome-wide coalignment of homologs is not the fundamental raison d'etre of the somatic pairing process . We suggest instead that pairing may exist to promote juxtaposition of homologous regions within irregular genome complements.

Curr Biol, 1999 Jul 15, 9(14), 767 - 70
Role of yeast SIR genes and mating type in directing DNA double-strand breaks to homologous and non-homologous repair paths; Lee SE et al.; Eukaryotes have acquired many mechanisms to repair DNA double-strand breaks (DSBs) {1} . In the yeast Saccharomyces cerevisiae, this damage can be repaired either by homologous recombination, which depends on the Rad52 protein, or by non-homologous end-joining (NHEJ), which depends on the proteins yKu70 and yKu80 {2} {3} . How do cells choose which repair pathway to use? Deletions of the SIR2, SIR3 and SIR4 genes - which are involved in transcriptional silencing at telomeres and HM mating-type loci (HMLalpha and HMRa) in yeast {4} - have been reported to reduce NHEJ as severely as deletions of genes encoding Ku proteins {5} . Here, we report that the effect of deleting SIR genes is largely attributable to derepression of silent mating-type genes, although Sir proteins do play a minor role in end-joining . When DSBs were made on chromosomes in haploid cells that retain their mating type, sir Delta mutants reduced the frequency of NHEJ by twofold or threefold, although plasmid end-joining was not affected . In diploid cells, sir mutants showed a twofold reduction in the frequency of NHEJ in two assays . Mating type also regulated the efficiency of DSB-induced homologous recombination . In MATa/MATalpha diploid cells, a DSB induced by HO endonuclease was repaired 98% of the time by gene conversion with the homologous chromosome, whereas in diploid cells with an alpha mating type (matDelta/MATalpha) repair succeeded only 82% of the time . Mating-type regulation of genes specific to haploid or diploid cells plays a key role in determining which pathways are used to repair DSBs.

J Biol Chem, 1999 Jul 30, 274(31), 21943 - 52
Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast; Russell SJ et al.; The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system . Yeast has been an important model organism for understanding eukaryotic proteasome structure and function . Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits . The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence . In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle . We have also determined the cellular abundance and stoichiometry of these proteasome subunits . Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus . Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast . This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.

J Biol Chem, 1999 Jul 30, 274(31), 21688 - 94
Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II; Olland S et al.; Catalysis of ATP hydrolysis by two NH(2)-terminal fragments of yeast DNA topoisomerase II was studied in the absence and presence of DNA, and in the absence and presence of inhibitor ICRF-193 . The results indicate that purified Top2-(1-409), a fragment containing the NH(2)-terminal 409 amino acids of the yeast enzyme, is predominantly monomeric, with a low level of ATPase owing to weak association of two monomers to form a catalytically active dimer . The ATPase activity of Top2-(1-409) is independent of DNA in a buffer containing 100 mM NaCl, in which intact yeast DNA topoisomerase II exhibits robust DNA-dependent ATPase and DNA transport activities . Purified Top2-(1-660), a fragment containing the NH(2)-terminal 660 amino acid of the yeast enzyme, appears to be dimeric in the absence or presence of DNA, and the ATPase activity of the protein is significantly stimulated by DNA . These results are consistent with a model in which binding of an intact DNA topoisomerase II to DNA places the various subfragments of the enzyme in a way that makes the intramolecular dimerization of the ATPase domains more favorable . We believe that this alignment of subfragments is mainly achieved through the binding of the enzyme to the DNA segment within which the enzyme makes transient breaks . The ATPase activity of Top2-(1-409) is inhibited by ICRF-193, suggesting that the bisdioxopiperazine class of DNA topoisomerase II inhibitors directly interacts with the paired ATPase domains of the enzyme.

J Biol Chem, 1999 Jul 30, 274(31), 21589 - 97
PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides . Effect of insulin; Sbrissa D et al.; One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation . Two such species, PtdIns 5-P and PtdIns 3,5-P(2), have now been identified in mammalian cells, but their biosynthesis remains unclear . We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D., and Ikonomov, O . (1999) Mol . Cell . Biol . 19, 623-634) . Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P(2) by HPLC analyses . Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P(2) but to a substantially lesser extent than PtdIns isolated from natural sources . Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine . By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity . HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells . However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235 . In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P(2) generation, implying PIKfyve has a key role in their biosynthesis.

Acta Vet Scand, 1999, 40(1), 23 - 34
The influence of supplements of selenite, selenate and selenium yeast on the selenium status of dairy heifers; Ortman K et al.; The aim of the study was to define possible differences between selenite, selenate and selenium yeast on various aspects of selenium status in growing cattle . Twenty-four Swedish Red and White dairy heifers were fed no supplementary selenium for 6 months . The basic diet contained 0.026 mg selenium/kg feed dry matter (DM) . After the depletion period the animals were divided into 4 groups; group I-III received 2 mg additional selenium daily as sodium selenite, sodium selenate, and a selenium yeast product, respectively . Group IV, the control group, received no additional selenium . The total dietary selenium content for groups I-III during the supplementation period was 0.25 mg/kg DM . After the depletion period the mean concentration of selenium in blood (640 nmol/l) and plasma (299 nmol/l) and the activity of GSH-Px in erythrocytes (610 mukat/l) were marginal, but after 3 months of supplementation they were adequate in all 3 groups . The concentration of selenium in blood and plasma was significantly higher in group III than in groups I and II, but there was no significant difference between groups I and II . The activity of GSH-Px in erythrocytes did not differ between any of the supplemented groups . The animals in the control group had significantly lower concentrations of selenium in blood and plasma and lower activities of GSH-Px in erythrocytes than those in the supplemented groups . The activity of GSH-Px in platelets was also increased by the increased selenium intake . There was no difference in the concentration of triiodothyronine (T3) between any of the groups, but the concentration of thyroxine (T4) was significantly higher in the unsupplemented control group.

FEMS Microbiol Lett, 1999 Jul 1, 176(1), 205 - 12
Development of a yeast-based assay system for monitoring microsatellite instability; Larkin K et al.; Simple sequence repeats (microsatellites) are found in all eukaryotic genomes . Instabilities within these sequences have been associated with several human disorders including Huntington's chorea and myotonic dystrophy . Further studies have identified links between microsatellite instability, faulty mismatch repair and certain human cancers, in particular a form of hereditary colorectal cancer . The assay system described here consists of a congenic set of yeast strains mutated in DNA replication and mismatch repair genes and assay plasmids with which it is possible to measure differences in microsatellite stability in the range of 5-850-fold . The development of this technology will allow monitoring of environmental and dietary influences on the genomic stability in the context of human disease.

Science, 1999 Jul 23, 285(5427), 578 - 82
An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells; Cormack BP et al.; Candida glabrata is an important fungal pathogen of humans that is responsible for about 15 percent of mucosal and systemic candidiasis . Candida glabrata adhered avidly to human epithelial cells in culture . By means of a genetic approach and a strategy allowing parallel screening of mutants, it was possible to clone a lectin from a Candida species . Deletion of this adhesin reduced adherence of C . glabrata to human epithelial cells by 95 percent . The adhesin, encoded by the EPA1 gene, is likely a glucan-cross-linked cell-wall protein and binds to host-cell carbohydrate, specifically recognizing asialo-lactosyl-containing carbohydrates.

Biochem J, 1999 Aug 1, 341 ( Pt 3), 477 - 82
Functional divergence between the half-sites of the DNA-binding sequence for the yeast transcriptional regulator Rap1p; Idrissi FZ et al.; The yeast transcriptional regulator Rap1p binds to the DNA consensus sequence ACACCCAYACAYYY . We have previously shown that DNA-binding sites in which all four Y (Y=T or C) positions were Ts (UASrpg sequences) synergized more efficiently to activate transcription than sequences in which all Ys were Cs (telomere sequences) {F.-Z . Idrissi, J . Fernandez-Larrea and B . Pina (1998) J . Mol . Biol . 284, 925-935} . Here we provide evidence that the DNA consensus sequence for Rap1p behaves as a combination of two ACAYYY half-sites with different functionality, the presence of Ts in the second half-site being the determinant for the transcriptional behaviour of the UASrpg sequences . DNA structure in the different complexes with Rap1p varied from being relatively uniform to appear rather distorted, this also being dependent on the presence of Ts in the second half-site . These distortions did not cause sharp bends or kinks in the DNA molecule . Computer analysis suggests that high-affinity binding of Rap1p to UASrpg sequences requires a rearrangement of the C-terminal Myb domain of the protein . We propose that the structural alterations in Rap1p-DNA complexes, both in the DNA and in the protein, affect the transcription potential of the complex in an allosteric manner . We also propose that the dimeric nature of the Rap1 DNA-binding domain is a key structural feature that explains the disparate functions of its DNA-binding sites in vivo.

Biochemistry, 1999 Jul 20, 38(29), 9465 - 70
Dynamic mobility of genetically expressed fusion protein between cytochrome P4501A1 and NADPH-cytochrome P450 reductase in yeast microsomes; Yamada M et al.; A fusion protein of rat liver CYP1A1 with NADPH-cytochrome P450 reductase was expressed genetically in yeast microsomal membranes . This flavo-cytochrome is active in 6-hydroxylation of zoxazolamine . Rotational diffusion of the fusion protein was examined by observing the flash-induced absorption anisotropy r(t) of the P450.CO complex . Theoretical analysis of r(t) was performed based on a "rotation-about-membrane normal" model . The absorption anisotropy decayed within 2 ms to a time-independent value r(3) . Forty percent of the fusion protein rotated with a rotational relaxation time phi of 1.35 ms . Treatment with high salt increased the mobile population of the fusion protein to 62% with phi = 0.96 ms . The mobile population of the fusion protein is close to that of CYP1A1 coexpressed with the P450 reductase and greater than that of CYP1A1 alone {Iwase et al . (1991) Biochemistry 30, 8347-8351} . The large mobile population of the fusion protein provides evidence that CYP1A1 is mobilized by forming associations with P450 reductase in microsomal membranes.

Biochemistry, 1999 Jul 20, 38(29), 9242 - 53
Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation; Rajesh S et al.; The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied by heteronuclear multidimensional NMR spectroscopy . Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)C(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitin binding interface of YUH1 . This interface mapped on the secondary structure of YUH1 suggests a wide area of contact for ubiquitin, encompassing the N-terminus, alpha1, alpha4, beta2, beta3, and beta6, coincident with the high specificity of YUH1 for ubiquitin . The presence of several hydrophobic clusters in the ubiquitin binding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactions in contacting ubiquitin . The residues in the binding interface exhibit a high percentage of homology among the members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of the ubiquitin binding interface reported in this study . The secondary structure of YUH1, from our NMR studies, was similar to the recently determined structure of its human homologue ubiquitin C-terminal hydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3 . This region in YUH1 (helix H3 of UCH-L3) was least perturbed upon ubiquitin binding . Therefore, the binding interface was mapped onto the corresponding residues in the UCH-L3 crystal structure . A model for ubiquitin binding to YUH1 is proposed, in which a good correlation was observed for the lateral binding of ubiquitin to UCH-L3 (YUH1), stabilized by the electrostatic and hydrophobic interactions.

Biochemistry, 1999 Jul 13, 38(28), 9137 - 45
Role of lysine 240 in the mechanism of yeast pyruvate kinase catalysis; Bollenbach TJ et al.; Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kinase (Lys 269 in muscle PK) to Met . K240M has an absolute requirement for FBP for catalysis . K240M is 100- and 1000-fold less active than wild-type YPK in the presence of Mn(2+) and Mg(2+), respectively . Steady-state fluorescence titration data suggest that the substrate PEP binds to K240M with the same affinity as it does to wild-type YPK . The rate of phosphoryl transfer in K240M has been decreased >1000-fold compared to wild-type YPK . The detritiation of 3-{(3)H}pyruvate catalyzed by YPK occurs at a rate significantly greater than the spontaneous rate . Detritiation of pyruvate by wild-type YPK occurs as a divalent metal- and FBP-dependent process requiring ATP . There is no detectable detritiation of pyruvate catalyzed by K240M . The solvent deuterium isotope effect on k(cat) is 2.7 +/- 0.2 and 1.6 +/- 0.1 for the wild type and for K240M YPK, respectively . This suggests that the isotope sensitive step in the PK reaction does not involve Lys 240 and that the enolpyruvate intermediate is still protonated by K240M . Isotope trapping was used to characterize enolpyruvate protonation by K240M . While there was enrichment of the methyl protons of pyruvate from labeled solvent formed by catalysis with muscle PK and wild-type YPK, only background levels of tritium were trapped with K240M . In K240M, the proton donor exchanges protons with the solvent at a higher rate relative to turnover than does the proton donor in wild-type YPK . The pH-rate profile of K240M exhibits the loss of a pK(a) value of 8 . 8 observed with wild-type YPK . The above data and recent crystal structure data suggest that Lys 240 interacts with the phosphoryl group of phosphoenolpyruvate and helps to stabilize the pentavalent phosphate transition state during phosphoryl transfer . Phosphoryl transfer is highly coupled to proton transfer, or Lys 240 also affects enolate protonation.

FEBS Lett, 1999 Jul 2, 454(1-2), 11 - 5
Mutational analysis of subunit i beta2 (MECL-1) demonstrates conservation of cleavage specificity between yeast and mammalian proteasomes; Salzmann U et al.; Proteasomes are the major protein-degrading complexes in the cytosol and regulate many cellular processes . To examine the functional importance of the MC14/MECL-1 proteasome active site subunits, cell lines expressing a catalytically inactive form of MECL-1 were established . Whereas mutant MECL-1 was readily incorporated into cytosolic proteasomes, replacing the constitutive MC14 subunit, removal of the prosequence was incomplete indicating that its processing required autocatalytic cleavage . Functional analyses showed that the absence of the MC14/MECL-1 active sites abrogated proteasomal trypsin-like activity, but did not affect other catalytic activities . Our data demonstrate a conservation of cleavage specificity between mammalian and yeast proteasomes.

Cell, 1999 Jul 9, 98(1), 91 - 103
A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis; Klein F et al.; A multisubunit complex, called cohesin, containing Smc1p, Smc3p, Scc1p, and Scc3p, is required for sister chromatid cohesion in mitotic cells . We show here that Smc3p and a meiotic version of Scc1p called Rec8p are required for cohesion between sister chromatids, for formation of axial elements, for reciprocal recombination, and for preventing hyperresection of double-strand breaks during meiosis . Both Rec8p and Smc3p colocalize with chromosome cores independently of synapsis during prophase I and largely disappear from chromosome arms after pachytene but persist in the neighborhood of centromeres until the onset of anaphase II . The eukaryotic cell's cohesion apparatus is required both for the repair of recombinogenic lesions and for chromosome segregation and therefore appears to lie at the heart of the meiotic process.

Mol Microbiol, 1999 Jul, 33(2), 274 - 83
The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons; Boy-Marcotte E et al.; The heat shock transcription factor Hsf1p and the stress-responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae . Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild-type strains using {35S}-methionine labelling and two-dimensional gel electrophoresis . Among 52 proteins induced by a shift from 25 degrees C to 38 degrees C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p . The two sets of proteins overlapped only slightly . Three proteins were induced independently of these transcription factors, suggesting the involvement of other transcription factor(s) . The Ras/cAMP/PKA signalling pathway cAMP had a negative effect on the induction of the Msn2p/Msn4p regulon, but did not affect the Hsf1p regulon . Thus, the two types of transcription factor are regulated differently and control two sets of functionally distinct proteins, suggesting two different physiological roles in the heat shock cellular response.

Mol Microbiol, 1999 Jul, 33(1), 52 - 62
The prepropeptide of vacuolar aminopeptidase I is necessary and sufficient to target the fluorescent reporter protein GFP to the vacuole of yeast by the Ccvt pathway; Martinez E et al.; We have studied the capacity of the prepro amino extension of vacuolar protease leucine aminopeptidase I (API) to target the fluorescent reporter protein GFP to the vacuole of yeast . The preproGFP chimera constructed by extending the amino end of GFP with the prepro-part of API is rapidly degraded in both wild-type WCG cells and WCG 11/21a cells deficient in the proteasome . In contrast, the chimera expressed in WCG-PP cells deficient in both proteasome activity and vacuolar proteinase A accumulates in the vacuole, where it remains stable . Replacement of Gly by Ile-7, a substitution that prevents folding of the pre-part into an amphipathic helix and inhibits the targeting of the API precursor to the vacuole, inhibits the targeting of preproGFP to the vacuole . The separated pre- and pro-parts of the API precursor do not target GFP to the vacuole . Targeting of preproGFP to the vacuole is independent of its levels of expression, as the fluorescent protein localizes to the vacuole in cells expressing the protein under the control of both the GAL 1/10 or the API promoter . The preproGFP expressed under both promoters is recovered as monomers from cytosolic cell extracts . PreproGFP expressed under the API promoter is packed into cytoplasmic bodies that penetrate into the vacuolar lumen to release the protein . Altogether our results show that the prepro-part of the API precursor is necessary and sufficient to target the green fluorescent reporter protein to the vacuole.

RNA, 1999 Jul, 5(7), 959 - 71
The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex; Kim DH et al.; The yeast PRP44 gene, alternatively named as BRR2, SLT22, RSS1, or SNU246, encodes a 246-kDa protein with putative RNA helicase function during pre-mRNA splicing . The protein is a typical DEAD/H family member, but unlike most other members of this family, it contains two putative RNA helicase domains, each with a highly conserved ATPase motif . Prior to this study little was known about functional roles for these two domains . We present genetic and biochemical evidence that ATPase motifs of only the first helicase domain are required for cell viability and pre-mRNA splicing . Overexpression of mutations in the first domain results in a dominant negative phenotype, and extracts from these mutant strains inhibit in vitro pre-mRNA splicing . In vitro analyses of affinity purified proteins revealed that only the first helicase domain possesses poly (U)-dependent ATPase activity . Overexpression of a dominant negative protein in vivo reduces the relative abundance of free U4 and U6 snRNA with a concomitant accumulation of the U4/U6 duplex . Accumulation of the U4/U6 duplex was relieved by overexpression of wild-type Prp44p . Three DEAD/H box proteins, Prp16p, Prp22p and Prp44p, have previously been shown to affect U4/U6 unwinding activity in vitro . The possible role of these proteins in mediating this reaction in vivo was explored following induced expression of ATPase domain mutants in each of these . Although overexpression of the mutant form of either Prp16p, Prp22p, or Prp44p was lethal, only expression of the mutant Prp44p resulted in accumulation of the U4/U6 helix . Our results, when combined with previously published in vitro results, support a direct role for Prp44p in unwinding of the U4/U6 helix.

RNA, 1999 Jul, 5(7), 909 - 17
Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS; Kufel J et al.; We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis . Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed . In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA . The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210) . We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0 . The sites of in vivo cleavage in the 3' ETS were mapped by primer extension . Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49 . These are in good agreement with the consensus Rnt1p cleavage site . Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.

Mol Cell Biol, 1999 Aug, 19(8), 5535 - 47
A fission yeast gene, him1(+)/dfp1(+), encoding a regulatory subunit for Hsk1 kinase, plays essential roles in S-phase initiation as well as in S-phase checkpoint control and recovery from DNA damage; Takeda T et al.; Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G(1)/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans . Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins . We have identified him1(+) from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1 . Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro . him1(+) is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G(1) . The protein level is low at START in G(1), increases at the G(1)/S boundary, and is maintained at a high level throughout S phase . Him1 protein is hyperphosphorylated at G(1)/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation . Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment . The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate . In keeping with this result, a database search indicates that him1(+) is identical to rad35(+) . Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

Mol Cell Biol, 1999 Aug, 19(8), 5405 - 16
Ssy1p and Ptr3p are plasma membrane components of a yeast system that senses extracellular amino acids; Klasson H et al.; Mutations in SSY1 and PTR3 were identified in a genetic selection for components required for the proper uptake and compartmentalization of histidine in Saccharomyces cerevisiae . Ssy1p is a unique member of the amino acid permease gene family, and Ptr3p is predicted to be a hydrophilic protein that lacks known functional homologs . Both Ssy1p and Ptr3p have previously been implicated in relaying signals regarding the presence of extracellular amino acids . We have found that ssy1 and ptr3 mutants belong to the same epistasis group; single and ssy1 ptr3 double-mutant strains exhibit indistinguishable phenotypes . Mutations in these genes cause the nitrogen-regulated general amino acid permease gene (GAP1) to be abnormally expressed and block the nonspecific induction of arginase (CAR1) and the peptide transporter (PTR2) . ssy1 and ptr3 mutations manifest identical differential effects on the functional expression of multiple specific amino acid transporters . ssy1 and ptr3 mutants have increased vacuolar pools of histidine and arginine and exhibit altered cell growth morphologies accompanied by exaggerated invasive growth . Subcellular fractionation experiments reveal that both Ssy1p and Ptr3p are localized to the plasma membrane (PM) . Ssy1p requires the endoplasmic reticulum protein Shr3p, the amino acid permease-specific packaging chaperonin, to reach the PM, whereas Ptr3p does not . These findings suggest that Ssy1p and Ptr3p function in the PM as components of a sensor of extracellular amino acids.

Yeast, 1999 Jul, 15(10B), 1001 - 8
A large-scale sonication assay for cell wall mutant analysis in yeast; Ruiz C et al.; The complete yeast genome contains a large number of genes of unknown biological function . Simple, rapid and reliable specific screens are valuable tools in exploring gene function via systematic phenotypic analysis of large mutant collections . This report provides a new approach for monitoring changes in cell wall strength, based on the deleterious effects caused by ultrasound on the yeast cell surface . Sonication can thus be used for the screening of mutants affected in the architecture or stability of the cell wall, since such mutants are expected to have an altered sensitivity to this treatment compared to that of a wild-type . The experimental procedure, consisting in the quantification of damaged cells after a mild sonication treatment, by means of flow cytometry, can be applied on a large scale . The usefulness of the sonication assay as a primary screen for cell wall-related mutants is evaluated on the collection of calcofluor white-hypersensitive and -resistant mutants obtained by Lussier et al . (1997) . A further phenotypic characterization of the sonication-hypersensitive mutants within the calcofluor white collection is also presented .

Yeast, 1999 Jul, 15(10B), 987 - 1000
Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals; Kucharczyk R et al.; A PCR-based method for targeted gene deletion by kanMX4 module was used to construct complete deletion mutants of six individual open reading frames from chromosome II: YBR128c, YBR131w, YBR133c, YBR137w, YBR138c and YBR142w . The ORFs were deleted in two diploid strains, FY1679 and W303 . Sporulation and tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD-box RNA helicase, is an essential gene . A systematic phenotypic analysis of the deleted mutants was carried out . Homozygous diploids ybr128cDelta/ybr128cDelta and ybr131wDelta/ybr131wDelta did not sporulate . The ybr131cDelta mutant whether haploid or homozygous diploid, in addition displayed an increased sensitivity to Caffeine, Calcium and Zinc, and to emphasize this phenotype we named the gene CCZ1 . ORF YBR133c was independently reported by others as Histone Synthetic Lethal (HSL7) (Ma et al., 1996) . We found that the aberrant morphology characteristic for ybr133cDelta (hsl7Delta) cells was observed in W303 but not in FY1679 genetic background . Furthermore, we observed that deletion of YBR133c had a pleiotropic effect under a wide range of conditions, including increased sensitivity to calcium, caffeine, calcofluor white, vanadate and verapamil . The effects of the deletion were reinforced in W303 background . We found no phenotypic effects of the two remaining deletions, ybr137wDelta and ybr138cDelta .

Yeast, 1999 Jul, 15(10B), 973 - 86
Chemotyping of yeast mutants using robotics; Rieger KJ et al.; By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed . Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories . Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547-1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics . The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors . Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae . In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype . The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper-pleiotropic phenotype with sensitivities to a dozen or more compounds . Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in-depth functional analysis and a more precise definition of molecular targets .

Yeast, 1999 Jul, 15(10B), 963 - 72
Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines; Knop M et al.; Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used . In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR-based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994) . To further employ the power of this strategy, a variety of novel tags was constructed . These tags were combined with different selectable marker genes, resulting in PCR amplificable modules . Only one set of primers is required for the amplification of any module . Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope-tagged proteins .

EMBO J, 1999 Jul 15, 18(14), 3897 - 908
Clathrin functions in the absence of heterotetrameric adaptors and AP180-related proteins in yeast; Huang KM et al.; The major coat proteins of clathrin-coated vesicles are the clathrin triskelion and heterotetrameric associated protein (AP) complexes . The APs are thought to be involved in cargo capture and recruitment of clathrin to the membrane during endocytosis and sorting in the trans-Golgi network/endosomal system . AP180 is an abundant coat protein in brain clathrin-coated vesicles, and it has potent clathrin assembly activity . In Saccharomyces cerevisiae, there are 13 genes encoding homologs of heterotetrameric AP subunits and two genes encoding AP180-related proteins . To test the model that clathrin function is dependent on the heterotetrameric APs and/or AP180 homologs, yeast strains containing multiple disruptions in AP subunit genes, as well as in the two YAP180 genes, were constructed . Surprisingly, the AP deletion strains did not display the phenotypes associated with clathrin deficiency, including slowed growth and endocytosis, defective late Golgi protein retention and impaired cytosol to vacuole/autophagy function . Clathrin-coated vesicles isolated from multiple AP deletion mutants were morphologically indistinguishable from those from wild-type cells . These results indicate that clathrin function and recruitment onto membranes are not dependent upon heterotetrameric adaptors or AP180 homologs in yeast . Therefore, alternative mechanisms for clathrin assembly and coated vesicle formation, as well as the role of AP complexes and AP180-related proteins in these processes, must be considered.

EMBO J, 1999 Jul 15, 18(14), 3888 - 96
Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway; Mizushima N et al.; Autophagy is an intracellular bulk degradation system that is ubiquitous for eukaryotic cells . In this process, cytoplasmic components are enclosed in autophagosomes and delivered to lysosomes/vacuoles . We recently found that a protein conjugation system, in which Apg12p is covalently attached to Apg5p, is indispensable for autophagy in yeast . Here, we describe a novel coiled-coil protein, Apg16p, essential for autophagy . Apg16p interacts with Apg12p-conjugated Apg5p and less preferentially with unconjugated Apg5p . Moreover, the coiled-coil domain of Apg16p mediates self-multimerization that leads to cross-linking of Apg5p molecules and formation of a stable protein complex . Apg16p is not essential for the Apg12p-Apg5p conjugation reaction . These results suggest that the Apg12p-Apg5p conjugate requires Apg16p to accomplish its role in the autophagy pathway, and Apg16p is a key molecule as a linker to form the Apg12p-Apg5p-Apg16p multimer.

Anal Biochem, 1999 Jul 15, 272(1), 71 - 9
Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy; Teleman A et al.; (31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast . The yeast cells have been extracted using chloroform into a neutral aqueous phase . The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra . Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances . In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified . Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium . No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium . Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts .

FEBS Lett, 1999 Jun 25, 453(3), 369 - 74
Identification of human GC-box-binding zinc finger protein, a new Krüppel-like zinc finger protein, by the yeast one-hybrid screening with a GC-rich target sequence; Lisowsky T et al.; A new human zinc finger DNA-binding protein was identified by using a yeast one-hybrid selection system . Two versions of the cDNA, encoding the same protein, were detected that differ for a 584 bp extension at the 5' region . Sequence analysis showed that the longer clone is a full length version containing part of the 5' untranslated region . The smaller version was fused in frame with the yeast GAL4 activation domain whereas the 5' region of the longer clone displayed a stop codon interrupting the fusion with the GAL4 domain . Nevertheless, this clone activated the yeast HIS3 reporter gene with the same efficiency as the smaller version . Sequence comparison of the derived protein with the database showed that it belongs to a family of zinc finger DNA-binding proteins which regulate the expression of genes involved in cell proliferation . Expression of the protein in an in vitro system, DNA-binding studies and genetic experiments identify this factor as a new zinc finger DNA-binding protein which binds GC-rich sequences and contains a domain probably functioning as a transcriptional activator . The new human protein identified in this study was therefore named GC-box-binding zinc finger protein).

Comput Chem, 1999 Jun 15, 23(3-4), 233 - 50
Promoter analysis of co-regulated genes in the yeast genome; Zhang MQ; The use of high density DNA arrays to monitor gene expression at a genome-wide scale constitutes a fundamental advance in biology . In particular, the expression pattern of all genes in Saccharomyces cerevisiae can be interrogated using microarray analysis where cDNAs are hybridized to an array of more than 6000 genes in the yeast genome . In an effort to build a comprehensive Yeast Promoter Database and to develop new computational methods for mapping upstream regulatory elements, we started recently in an on going collaboration with experimental biologists on analysis of large-scale expression data . It is well known that complex gene expression patterns result from dynamic interacting networks of genes in the genetic regulatory circuitry . Hierarchical and modular organization of regulatory DNA sequence elements are important information for our understanding of combinatorial control of gene expression . As a bioinformatics attempt in this new direction, we have done some computational exploration of various initial experimental data . We will use cell-cycle regulated gene expression as a specific example to demonstrate how one may extract promoter information computationally from such genome-wide screening . Full report of the experiments and of the complete analysis will be published elsewhere when all the experiments are to be finished later in this year (Spellman, P.T., et al . 1998 . Mol . Biol . Cell 9, 3273-3297).

Biochem Biophys Res Commun, 1999 Jul 14, 260(3), 799 - 805
Oligomerized Ced-4 kills budding yeast through a caspase-independent mechanism; Tao W et al.; In Caenorhabdtis elegans, Ced-3, Ced-4, and Ced-9 are components of a cell suicide program . Ced-4 facilitates the proteolytic activation of the caspase, Ced-3, while Ced-9 opposes Ced-3/Ced-4 killing . To examine the interactions among these proteins they were expressed in Saccharomyces cerevisiae . Ced-3 and Ced-4 were lethal when expressed alone, revealing an intrinsic Ced-4 killing activity . Coexpression of Ced-9 blocked Ced-3- and Ced-4-induced killing, showing Ced-9 can independently antagonize the action of both proteins . Ced-3- but not Ced-4-toxicity was attenuated by coexpression of the caspase inhibitors, CrmA and p35 . Thus, besides its Ced-3- and Ced-9-dependent action in C . elegans, Ced-4 has an additional Ced-9-dependent, Ced-3-independent killing mechanism in yeast . Two-hybrid analysis confirmed that Ced-4 formed heteromers with Ced-9 . In addition, Ced-4 formed homomers and mutation of its nucleoside triphosphate binding motif eliminated both homomerization and cell killing . We suggest the caspase-independent lethality of Ced-4 in yeast is mediated by a Ced-4 homomer .

J Cell Biol, 1999 Jul 12, 146(1), 125 - 40
Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9; Lehman K et al.; We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p . These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn . SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis . We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane . In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion . This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton . Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex . Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase . Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

J Cell Biol, 1999 Jul 12, 146(1), 85 - 98
A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome; Vida T et al.; We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY) . Yeast spheroplasts are lysed by extrusion through polycarbonate filters . After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes . A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes . When these membranes are incubated together with ATP and cytosolic extracts, approximately 50% of the radiolabeled proCPY is processed to mature CPY . Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive . Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer . Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport . Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90% . Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays . This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.

Bioorg Med Chem, 1999 May, 7(5), 887 - 94
The linkage of catalysis and regulation in enzyme action: oxidative diversion in the hysteretically regulated yeast pyruvate decarboxylase; Hajipour G et al.; The reaction catalyzed by the thiamin-diphosphate-dependent yeast pyruvate decarboxylase, which is hysteretically regulated by pyruvate, undergoes paracatalytic oxidative diversion by 2,6-dichlorophenolindophenol, which traps a carbanionic intermediate and diverts the product from acetaldehyde to acetate (Christen, P . Meth . Enzymol . 1977, 46, 48) . This reaction is now shown to exhibit an oxidant on-rate constant somewhat faster than that for pyruvate in the normal catalytic cycle and a product off-rate constant about 60-fold smaller than that for acetaldehyde . Both on-rates and off-rates exhibit an inverse solvent isotope effect of 1.5-2, observed in normal catalysis as a signal of sulfhydryl addition to the keto group of pyruvate at the allosteric regulatory site . The findings are consistent with a model for regulation in which the sulfhydryl-addition process mediates access to a fully catalytically competent active site, the oxidative-diversion reaction being forced to make use of the normal entry exit machinery.

Chromosoma, 1999 Jul, 108(3), 146 - 61
The ZDS1 and ZDS2 proteins require the Sir3p component of yeast silent chromatin to enhance the stability of short linear centromeric plasmids; Roy N et al.; Yeast artificial chromosome (YAC) clones of Saccharomyces cerevisiae containing a centromere, origin of replication, two telomeres and a >50 kb insert of DNA are maintained as normal yeast chromosomes . However, short linear centromeric plasmids of 10-15 kb in size (short YACs) are missegregated at a much higher frequency than long YACs or 10-15 kb circular centromeric plasmids . A search for genes that stabilized short linear centromeric plasmids when present in multiple copies per cell uncovered ZDS1, which reduced the rate at which cells lost the short YAC, increased the fraction of cells that maintained the short YAC and decreased the number of short YACs per cell . Multiple copies of ZDS2, a homolog of ZDS1, had similar effects . Genes near yeast telomeres are transcriptionally silenced by the recruitment of proteins encoded by the SIR2, SIR3 and SIR4 genes (Sir2p, Sir3p and Sir4p) . Multiple copies of ZDS1 and ZDS2 caused an increase in telomeric silencing . In addition, ZDS1 and ZDS2 both required the open reading frame encoding the N-terminal 174 amino acids of Sir3p to stabilize short YACs . Thus, the short YAC stability assay revealed a silencing-independent function for the Sir3p N-terminus . Two-hybrid analysis indicated that Zds1p and Zds2p interact with Sir2p, Sir3p, Sir4p or the yeast telomere binding protein Rap1p . Deletion of both ZDS1 and ZDS2 made short YACs, but not a 100 kb YAC, extremely unstable and also caused a 70 bp increase in the length of the telomeric TG1-3 repeats . These data indicate that short YACs can be stabilized by trans-acting factors and suggest that the proteins encoded by ZDS1 and ZDS2 alter short YAC stability by interacting with proteins that function at the telomere.

Mol Biol Cell, 1999 Jul, 10(7), 2377 - 91
Yeast Dam1p is required to maintain spindle integrity during mitosis and interacts with the Mps1p kinase; Jones MH et al.; We have identified a mutant allele of the DAM1 gene in a screen for mutations that are lethal in combination with the mps1-1 mutation . MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint . Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel . The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p . DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex . We report here that cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature . Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function . We have observed that a Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis.

Mol Biol Cell, 1999 Jul, 10(7), 2265 - 83
Sla2p is associated with the yeast cortical actin cytoskeleton via redundant localization signals; Yang S et al.; Sla2p, also known as End4p and Mop2p, is the founding member of a widely conserved family of actin-binding proteins, a distinguishing feature of which is a C-terminal region homologous to the C terminus of talin . These proteins may function in actin cytoskeleton-mediated plasma membrane remodeling . A human homologue of Sla2p binds to huntingtin, the protein whose mutation results in Huntington's disease . Here we establish by immunolocalization that Sla2p is a component of the yeast cortical actin cytoskeleton . Deletion analysis showed that Sla2p contains two separable regions, which can mediate association with the cortical actin cytoskeleton, and which can provide Sla2p function . One localization signal is actin based, whereas the other signal is independent of filamentous actin . Biochemical analysis showed that Sla2p exists as a dimer in vivo . Two-hybrid analysis revealed two intramolecular interactions mediated by coiled-coil domains . One of these interactions appears to underlie dimer formation . The other appears to contribute to the regulation of Sla2p distribution between the cytoplasm and plasma membrane . The data presented are used to develop a model for Sla2p regulation and interactions.

Mol Biol Cell, 1999 Jul, 10(7), 2235 - 50
Pleiotropic alterations in lipid metabolism in yeast sac1 mutants: relationship to "bypass Sec14p" and inositol auxotrophy; Rivas MP et al.; SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes . This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy . Elucidation of how sac1 mutants effect "bypass Sec14p" will provide insights into Sec14p function in vivo . We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway . This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e . phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase . Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p . These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p . A significant fate for this DAG is consumption by the CDP-choline pathway . Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene.

Bioorg Med Chem Lett, 1999 Jun 21, 9(12), 1683 - 6
Binding and catalysis by yeast aldose reductase: a substrate-analog approach with new aldose derivatives; Hadwiger P et al.; 5-Deoxy-D-xylofuranose derivatives and a range of new 5,6-dideoxy analogs of D-glucofuranose bearing azido or fluoro substituents were synthesised and employed as substrates of the NADH-dependent aldehyde reduction catalysed by yeast aldose reductase . In terms of catalytic efficiencies, these products proved to be superior to the parent compounds.

Acta Biochim Pol, 1998, 45(4), 935 - 40
Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: an indication of a functional interaction between 3' and 5' ends of mitochondrial mRNAs; Wegierski T et al.; Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA . Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs . We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions . Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.

Cancer Res, 1999 Jul 1, 59(13), 3175 - 9
The human p53 negative regulatory domain mediates inhibition of reporter gene transactivation in yeast lacking thioredoxin reductase; Merrill GF et al.; Stimulation of target gene transcription by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase . LexA/p53 fusion proteins were used to study the basis for thioredoxin reductase dependence . A fusion protein containing all 393 of the residues of p53 efficiently and specifically stimulated transcription of a LexOP-LacZ reporter gene in wild-type yeast but was several-fold less effective in delta trr1 yeast lacking the thioredoxin reductase gene . Thus, even when p53 was tethered to a reporter gene by a heterologous DNA-binding domain, reporter gene transactivation remained dependent on thioredoxin reductase . A fusion protein containing only the activation domain of p53 stimulated reporter gene transcription equally in wild-type and delta trr1 cells, suggesting that p53 residues downstream from the activation domain created the requirement for thioredoxin reductase . Experiments using additional LexA/p53 truncation mutations indicated that the p53 negative regulatory domain, rather than the DNA-binding or oligomerization domains, created the requirement for thioredoxin reductase . The fusion protein results suggested that, under oxidative conditions, the negative regulatory domain inhibited the ability of DNA-bound p53 to stimulate transcription . However, deletion of the negative regulatory domain did not alleviate the requirement of non-LexA-containing p53 for thioredoxin reductase . The results, thus, suggest that oxidative conditions inhibit both DNA binding and transactivation by p53, and that inhibition of the latter requires the negative regulatory domain.

Immunol Lett, 1999 May 3, 68(1), 155 - 9
Demonstration of the interaction of thioredoxin with p40phox, a phagocyte oxidase component, using a yeast two-hybrid system; Nishiyama A et al.; Thioredoxin (TRX) has disulfide reducing activity and is reported to be involved in various cellular functions including the promotion of cell growth and apoptosis . To help understand the molecular mechanism through which TRX is involved in immunological systems, we screened a cDNA library derived from a B-cell population of Epstein-Barr virus-transformed human peripheral blood lymhocyte for TRX binding proteins by use of a yeast two-hybrid system . Among plasmids from positive clones, a plasmid contained an insert which has homology with human p40phox, a cytosolic component of phagocyte oxidase . This insert sequence extended from the base + 181 to the stop codon of p40phox . The entire coding region of p40phox was shown to interact with TRX both in assays of histidine prototrophy and beta-galactosidase activity; in contrast, no interaction was observed with substituted mutant TRX (C32S/C35S), which lacks reducing activity . These results showed that p40phox interacts with TRX and indicated the possibility of TRX-dependent regulation of phagocyte oxidase activity.

Gene, 1999 Jul 8, 234(2), 285 - 95
Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures; Utsugi T et al.; A tom1-1 mutant was isolated from Saccharomyces cerevisiae . At high temperatures, 60% of the cells were arrested as dumbbell forms with a single large nucleus containing duplicated DNA and a short spindle . Electron-microscopy showed electron-dense structures scattered within the nucleus . Indirect immunofluorescent microscopy revealed these structures to be fragmented nucleoli since the dotted structures were stained with anti-Nop1(fibrillarin) antibody in large regions of the nuclei . Fluorescent in situ hybridization analysis using oligo(dT) revealed nuclear accumulation of poly(A)+RNA . We cloned TOM1 which encodes a large protein (380kDa) with a hect (homologous to E6-AP C terminus)-domain at its C terminus . Deletions of either this hect-region or the entire gene made cellular growth temperature-sensitive . Site-directed mutagenesis of the conserved cysteine residue (tom1C3235A) in the hect-domain, supposed to be necessary for thioester-bond formation with ubiquitin, abolished the gene function . When a functional glutathione S-transferase (GST)-tagged hect protein was overproduced, it facilitated the protein conjugation with a myc-tagged ubiquitinRA, while this was not seen when GST-hectC3235A was overproduced . The protein conjugation with a hemagglutinin-tagged Smt3 was not affected by the overproduction of GST-hect . Taken together, we suggest that Tom1 is a ubiquitin ligase . As a multi-copy suppressor of tom1, we isolated STM3/NPI46/FPR3 which encodes a nucleolar nucleolin-like protein . We discuss possible functions of Tom1 with respect to the pleiotropic defects of nuclear division, maintenance of nuclear structure, and nucleocytoplasmic transport.

Arch Biochem Biophys, 1999 Jul 15, 367(2), 240 - 9
Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes; Fan F et al.; Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently . Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP . Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold . The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers . In the consensus nucleotide-binding betaalphabeta fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers . Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers . NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2'-phosphate of NADP . The Gly203Arg change in SceADH does not significantly affect the kinetics . The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme . SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+ . The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions .

Plant Mol Biol, 1999 May, 40(1), 167 - 77
Identification of a rice APETALA3 homologue by yeast two-hybrid screening; Moon YH et al.; A cDNA clone OsMADS16 was isolated from the rice young inflorescence cDNA expression library by the yeast two-hybrid screening method with OsMADS4 as bait . We have previously shown that the OsMADS4 gene is a member of the PI family and that the MADS-box gene is involved in controlling development of the second and third whorls of rice flowers . The sequence comparison indicated that OsMADS16 belongs to the AP3 family . The OsMADS16 protein contains a PI-derived motif, FAFRVVPSQPNLH, that is a conserved sequence in AP3 family genes at the C-terminal region . In addition, OsMADS16 contains a paleoAP3 motif, YGGNHDLRLG, downstream of the PI-derived motif . The paleoAP3 motif is a consensus sequence in the C-terminal region of the AP3 family genes of lower eudicot and magnolid dicot species . RNA blot analysis showed that the OsMADS16 gene was expressed in the second and third whorls, whereas the OsMADS4 transcripts were present in the second, third, and fourth whorls . These expression patterns of the OsMADS16 and OsMADS4 genes are very similar to those of AP3 and PI, respectively . In the yeast two-hybrid system, OsMADS4 interacted only with OsMADS16 among several rice MADS genes investigated, suggesting that OsMADS4 and OsMADS16 function as a heterodimer in specifying sepal and petal identities . The OsMADS16 protein displayed transcription activation ability in yeast, whereas AP3 did not . It was also shown in yeast that OsMADS16 interacted with PI whereas OsMADS4 did not interact with AP3 . These differences between OsMADS16 and AP3 indicate that the functions of the AP3 family genes of monocots and dicots diverged during molecular evolution processes of the B function genes . Deletion analysis showed that the 155-200 amino acid region of the OsMADS16 protein plays an important role in the transcription activation ability.

Mol Gen Genet, 1999 Jun, 261(4-5), 788 - 95
Genetic evidence for interactions between yeast importin alpha (Srp1p) and its nuclear export receptor, Cse1p; Schroeder AJ et al.; The yeast Srp1p protein functions as an import receptor for proteins bearing basic nuclear localization signals . Cse1p, the yeast homolog of mammalian CAS, recycles Srp1p back to the cytoplasm after import substrates have been released into the nucleoplasm . In this report we describe genetic interactions between SRP1 and CSE1 . Results from genetic suppression and synthetic lethality studies demonstrate that these gene products interact to ensure accurate chromosome segregation . We also describe new mutant alleles of CSE1 and analyze a new temperature-sensitive allele of CSE1, cse1-2 . This allele causes high levels of chromosome missegregation and cell cycle arrest during mitosis at the nonpermissive temperature.

Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7815 - 20
A protein-protein interaction map of yeast RNA polymerase III; Flores A et al.; The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain . This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme . In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC . Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits . These regions overlapped with highly conserved domains . C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37) . Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

J Cell Sci, 1999 Aug, 112 ( Pt 15), 2529 - 37
Rvs167p, the budding yeast homolog of amphiphysin, colocalizes with actin patches; Balguerie A et al.; In this report, we have shown that the yeast amphiphysin-like protein Rvs167p was localized mainly in small cortical patches throughout the cell in unbudding cells . During budding, the patches were polarized at bud emergence site . During mating, Rvs167p was concentrated at the tip of the shmoo . Rvs167p colocalized with actin patches during yeast vegetative growth and mating . Complete disruption of the actin cytoskeleton using Latrunculin-A did not affect Rvs167p localization in patches throughout the cell . In rvs167 mutant cells, actin patches are mislocalized and in rvs161 or abp1 mutant cells, Rvs167p localization is not affected . These observations suggest that Rvs167p may localize the actin cortical complex properly . Finally, the amphiphysin-conserved N-terminal domain of Rvs167p, called the BAR domain, was required but not sufficient for the correct localization of the protein.

Yeast, 1999 Jun 15, 15(8), 715 - 20
In vivo construction of cDNA libraries for use in the yeast two-hybrid system; Fusco C et al.; We describe a simple and efficient one-step method to make cDNA libraries using homologous recombination in yeast . cDNA from any source, together with a linear vector, is used to transform yeast . Through homologous recombination and gap repair, the cDNA is unidirectionally incorporated into the yeast expression vector in vivo . The cDNA-encoded proteins can then be screened for potential protein-protein interactions with a bait already present in the yeast . This method allows rapid construction and screening of cDNA libraries, even from extremely small amounts of mRNA, and can replace the use of conventional cDNA libraries.

Biochemistry, 1999 Jul 6, 38(27), 8647 - 56
A distinctive RNA fold: the solution structure of an analogue of the yeast tRNAPhe T Psi C domain; Koshlap KM et al.; The structure of an analogue of the yeast tRNAPhe T Psi C stem-loop has been determined by NMR spectroscopy and restrained molecular dynamics . The molecule contained the highly conserved modification ribothymidine at its naturally occurring position . The ribothymidine-modified T Psi C stem-loop is the product of the m5U54-tRNA methyltransferase, but is not a substrate for the m1A58-tRNA methyltransferase . Site-specific substitutions and 15N labels were used to confirm the assignment of NOESY cross-peaks critical in defining the global fold of the molecule . The structure is unusual in that the loop folds far over into the major groove of the curved stem . This conformation is stabilized by both stacking interactions and hydrogen bond formation . Furthermore, this conformation appears to be unique among RNA hairpins of similar size . There is, however, a considerable resemblance to the analogous domain in the crystal structure of the full-length yeast tRNAPhe . We believe, therefore, that the structure we have determined may represent an intermediate in the folding pathway during the maturation of tRNA.

Gene, 1999 Jun 24, 234(1), 149 - 59
Cloning, characterisation, and functional expression of the Mus musculus SKD1 gene in yeast demonstrates that the mouse SKD1 and the yeast VPS4 genes are orthologues and involved in intracellular protein trafficking; Scheuring S et al.; The mouse SKD1 protein displays a high degree of sequence identity (62%) to the yeast Vps4 protein, which is involved in the transport of proteins out of a prevacuolar/endosomal compartment . We isolated the mouse SKD1 locus and found that the SKD1 gene is split into 11 exons covering a region of 29kb of the genome . Interestingly, the exon/intron structure reflects to a certain degree the proposed domain structure of the protein, since the 5' located coiled-coil region and the AAA domain are flanked by introns . Analysis of the promoter region, which revealed features common for 'housekeeping genes', is consistent with previous results of a mouse multi-tissue Northern blot, confirming that SKD1 is a ubiquitously expressed gene . Expression of the full-length SKD1 cDNA in a vps4 disrupted yeast strain suppressed the temperature-sensitive growth defect of the vps4 mutant strain . Overexpression of wild type and expression of mutant Vps4 and SKD1 proteins, harbouring single amino acid exchanges in their AAA domains, induced a dominant-negative vacuolar protein sorting defect in wild type yeast cells, indicating that mouse SKD1 protein and yeast Vps4p fulfil similar functions.

EMBO J, 1999 Jul 1, 18(13), 3820 - 33
DDP1, a single-stranded nucleic acid-binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy; Cortes A et al.; The centromeric dodeca-satellite of Drosophila forms altered DNA structures in vitro in which its purine-rich strand (G-strand) forms stable fold-back structures, while the complementary C-strand remains unstructured . In this paper, the purification and characterization of DDP1, a single-stranded DNA-binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca-satellite C-strand, is presented . In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca-satellite sequences . DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts . During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes . In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co-localizing with the heterochromatin protein HP1 . DDP1 is a multi-KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy . Expression of DDP1 complements a Deltascp160 deletion in yeast . These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin.

Biochem J, 1999 Jul 15, 341 ( Pt 2), 445 - 52
Endocytic delivery of intramolecularly quenched substrates and inhibitors to the intracellular yeast Kex2 protease1; Henkel MK et al.; Kex2 in the yeast Saccharomyces cerevisiae is a transmembrane, Ca2+-dependent serine protease of the subtilisin-like pro-protein convertase (SPC) family with specificity for cleavage after paired basic amino acids . At steady state, Kex2 is predominantly localized in late Golgi compartments and initiates the proteolytic maturation of pro-protein precursors that transit the distal secretory pathway . However, Kex2 localization is not static, and its itinerary apparently involves transiting out of the late Golgi and cycling back from post-Golgi endosomal compartments during its lifetime . We tested whether the endocytic pathway could deliver small molecules to Kex2 from the extracellular medium . Here we report that intramolecularly quenched fluorogenic substrates taken up into intact yeast revealed fluorescence due to specific cleavage by Kex2 protease in endosomal compartments . Furthermore, the endocytic delivery of protease inhibitors interfered with Kex2 activity for precursor protein processing . These observations reveal that the endocytic pathway does intersect with the cycling itinerary of active Kex2 protease . This strategy of endocytic drug delivery has implications for modulating SPC protease activity needed for hormone, toxin and viral glycoprotein precursor processing in human cells.

Chemosphere, 1999 Jun, 38(14), 3303 - 12
Applicability of a yeast oestrogen screen for the detection of oestrogen-like activities in environmental samples; Rehmann K et al.; A (xeno)oestrogen bioassay was introduced, using a genetically modified yeast strain which produces a fusion protein encompassing the human oestrogen receptor hormone binding domain and the yeast GAL4-DNA binding domain . Upon binding of appropriate substances this fusion protein recognises the respective DNA sequence thereby enhancing the transcription of a beta-galactosidase reporter gene . The bioassay procedure was evaluated by screening 30 compounds, including some known or suspected (xeno)oestrogens and determining EC50-values for 17 beta-oestradiol, 1.5 nM, 4-tert.-octylphenol, 6.7 microM and bisphenol A, 104 microM . Toluene extracts from different environmental matrices were tested for their oestrogenic activity . The positive test results obtained with a sewage sludge extract indicated the applicability of this bioassay for environmental monitoring.

Genetics, 1999 Jul, 152(3), 953 - 63
The effect of DNA replication mutations on CAG tract stability in yeast; Schweitzer JK et al.; CAG repeat tracts are unstable in yeast, leading to frequent contractions and infrequent expansions in repeat tract length . To compare CAG repeats to other simple repeats and palindromic sequences, we examined the effect of DNA replication mutations, including alleles of pol alpha, pol delta, pol epsilon, and PCNA (proliferating cell nuclear antigen), on tract stability . Among the polymerase mutations, the pol delta mutation (pol3-14) destabilizes tracts with either CAG or CTG as the lagging strand template . One pol alpha mutation, pol1-1, destabilizes the orientation with CAG as the lagging strand template, but it has little effect on the CTG orientation . In contrast, the pol1-17 mutation has no effect on either orientation . Similarly, mutations in the proofreading functions of pol delta and pol epsilon, as well as a temperature-sensitive pol epsilon mutation, pol2-18, have no effect on tract stability . Three PCNA mutations, pol30-52, pol30-79, and pol30-90, all have drastic effects on tract stability . Of the three, pol30-52 is unique in yielding small tract changes that are indicative of an impairment in mismatch repair . These results show that while CAG repeats are destabilized by many of the same mutations that destabilize other simple repeats, they also have some behaviors that are suggestive of their potential to form hairpin structures.

Genetics, 1999 Jul, 152(3), 881 - 93
In vivo analysis of the domains of yeast Rvs167p suggests Rvs167p function is mediated through multiple protein interactions; Colwill K et al.; Morphological changes during cell division in the yeast Saccharomyces cerevisiae are controlled by cell-cycle regulators . The Pcl-Pho85p kinase complex has been implicated in the regulation of the actin cytoskeleton at least in part through Rvs167p . Rvs167p consists of three domains called BAR, GPA, and SH3 . Using a two-hybrid assay, we demonstrated that each region of Rvs167p participates in protein-protein interactions: the BAR domain bound the BAR domain of another Rvs167p protein and that of Rvs161p, the GPA region bound Pcl2p, and the SH3 domain bound Abp1p . We identified Rvs167p as a Las17p/Bee1p-interacting protein in a two-hybrid screen and showed that Las17p/Bee1p bound the SH3 domain of Rvs167p . We tested the extent to which the Rvs167p protein domains rescued phenotypes associated with deletion of RVS167: salt sensitivity, random budding, and endocytosis and sporulation defects . The BAR domain was sufficient for full or partial rescue of all rvs167 mutant phenotypes tested but not required for the sporulation defect for which the SH3 domain was also sufficient . Overexpression of Rvs167p inhibits cell growth . The BAR domain was essential for this inhibition and the SH3 domain had only a minor effect . Rvs167p may link the cell cycle regulator Pcl-Pho85p kinase and the actin cytoskeleton . We propose that Rvs167p is activated by phosphorylation in its GPA region by the Pcl-Pho85p kinase . Upon activation, Rvs167p enters a multiprotein complex, making critical contacts in its BAR domain and redundant or minor contacts with its SH3 domain.

Protein Sci, 1999 Jun, 8(6), 1250 - 6
The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid; Erskine PT et al.; The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 A resolution . The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263 . It is known that this lysine forms a Schiff base link with substrate bound at the enzyme's so-called P-site . The carboxyl group of laevulinic acid makes hydrogen bonds with the side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain >NH group of Ser290 . The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site . Our analysis strongly suggests that the same interactions will be made by P-side substrate and also indicates that the substrate that binds at the enzyme's A-site will interact with the enzyme's zinc ion bound by three cysteines (133, 135, and 143) . Inhibitor binding caused a substantial ordering of the active site flap (residues 217-235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.

Genes Dev, 1999 Jun 15, 13(12), 1627 - 41
Somatic pairing of homologs in budding yeast: existence and modulation; Burgess SM et al.; FISH analysis of well-spread chromosomes reveals that homologs are paired in vegetatively growing budding yeast diploid cells, via multiple interstitial interactions, and independent of recA homologs and mating type heterozygosity . Pairing is present during G1 and G2, and in cells arrested at G1 by mating pheromone, but is disrupted during S phase . Thus, somatic pairing is qualitatively analogous to premeiotic and early meiotic pairing . S-phase pairing disruption occurs by a complex intranuclear program involving regional, nucleus-wide, and temporal determinants . Pairing is also disrupted in two G2-arrest conditions (cdc13ts and nocodazole) . Together these findings suggest that cell cycle signals may provoke pairing disruption by modulating underlying chromosome and/or chromatin structure . Whether the cell chooses to disrupt pairing contacts or not (e.g., S phase and G2 arrest, but not G1 arrest or normal G1 or G2), could be dictated by functional considerations involving homolog/sister discrimination.

Eur J Cell Biol, 1999 May, 78(5), 349 - 56
Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization; Hofhaus G et al.; The yeast scERV1 gene product is involved in the biogenesis of mitochondria and is indispensable for viability and regulation of the cell cycle . Recently the general importance of this gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue . The homologous mammalian ALR (Augmenter of Liver Regeneration) genes from man, mouse and rat are involved in the phenomenon of liver regeneration . A low expression rate of the genes is found in all investigated cells and mammalian tissues but it is specifically induced after damage of liver organs and is especially high during spermatogenesis . The alignment of the different proteins identifies a highly conserved carboxy terminus with more than 40% identical amino acids between yeast and mammals . The conserved carboxy terminus is functionally interchangeable between distantly related species like yeast and man . In contrast, the amino terminal parts of the proteins display a high degree of variability and significant differences even among closely related species . This finding leads to the problem whether the amino termini have comparable or divergent functions in different species . In this study we demonstrate by heterologous complementation experiments in yeast that the complete human ALR protein with its own amino terminus is not able to substitute for the yeast scERV1 protein . Fusion proteins of Alrp and scErv1p with the green fluorescence protein were created to investigate the respective subcellular localizations of these homologous proteins in yeast and human cells . In yeast cells human Alrp accumulates in the cytoplasm in contrast to yeast scErv1p that is preferentially associated with yeast mitochondria . Comparable studies with human cells clearly show that the homologous human Alrp is located in the cytosol of these cells . Fractionation experiments and antibody tests with yeast and human mitochondria and cellular extracts verify these findings.

Biol Chem, 1999 May, 380(5), 525 - 30
Activation of DNA replication in yeast by recruitment of the RNA polymerase II transcription complex; Stagljar I et al.; Activators of transcription are known to also play an important and direct role in activating DNA replication . However, the mechanism whereby they stimulate replication has remained elusive . One model suggests that, in the context of replication origins, transcriptional activators work by interacting with replication factors . We show that a defined, single interaction between a DNA-bound derivative of the activator Gal4 and Gal11P, a mutant form of the RNA polymerase II holoenzyme component Gal11, suffices for stimulating DNA replication as it does for transcription . Moreover, recruitment of TBP, which can activate transcription from a gene promoter, also stimulates DNA replication from an origin site . These results strongly argue that transcriptional activators may not necessarily need to contact DNA replication factors directly, but can stimulate replication by recruiting the RNA polymerase II transcription complex to DNA.

Mar Biotechnol (NY), 1999 May, 1(3), 230 - 238
Osmoregulation and the Genetic Induction of Glycerol-3-phosphate Dehydrogenase by NaCl in the Euryhaline Yeast Debaryomyces hansenii; Thome PE et al.; : In the yeasts Saccharomyces cerevisiae and Debaryomyces hansenii, glycerol-3-phosphate dehydrogenase (NAD+-GPD) is believed to be the enzyme controlling the synthesis of glycerol in osmotically challenged cells . Consistent with this concept, immunoblot analyses demonstrated quantitative increases in NAD+-GPD 30 minutes after the addition of NaCl to actively growing D . hansenii cells; Northern blot analyses showed increases in GPD-messenger RNA within 15 minutes after the addition of salt . There appears to be an upper limit of salt concentration above which the cells appear to be less responsive . The results we describe support the concept of transcriptional regulation of NAD+-GPD in D . hansenii.

J Bacteriol, 1999 Jul, 181(13), 3886 - 9
Amino acid residues in the omega-minus region participate in cellular localization of yeast glycosylphosphatidylinositol-attached proteins; Hamada K et al.; The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall . Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the omega site) and Y or N at the site 2 amino acids upstream of the omega site for cell wall localization and (ii) dibasic residues in the region upstream of the omega site (the omega-minus region) for plasma membrane localization . The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins . The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the omega-minus region . With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate . These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.

J Biol Chem, 1999 Jul 2, 274(27), 19017 - 24
Yeast transcriptional regulator Leu3p . Self-masking, specificity of masking, and evidence for regulation by the intracellular level of Leu3p; Wang D et al.; Recent work suggests that the masking of the activation domain (AD) of yeast transactivator Leu3p, observed in the absence of the metabolic signal alpha-isopropylmalate, is an intramolecular event . Much of the evidence came from the construction and analysis of a mutant form of Leu3p (Leu3-dd) whose AD is permanently masked (Wang, D., Hu, Y., Zheng, F., Zhou, K., and Kohlhaw, G . B . (1997) J . Biol . Chem . 272, 19383-19392) . In a modified two-hybrid experiment, the ADs of both wild type Leu3p and Leu3-dd were shown to interact with the remainder of the Leu3 protein, in an alpha-isopropylmalate-dependent manner . The finding that masking and unmasking proceed apparently normally when full-length Leu3p is expressed in mammalian cells is also consistent with the notion of intramolecular masking . Here we report on the identification of nine missense mutations (all of them suppressors of the Leu3-dd phenotype) that cause permanent unmasking of Leu3p . The nine mutations map to three short segments located within a 140-residue-long region of the C-terminal part of the middle region of Leu3p . These segments may be part of a spatial trap for the AD . We also performed "domain swaps" between Leu3p and Cha4p, a serine/threonine-responsive activator that, like Leu3p, belongs to the family of Zn(II)2Cys6 proteins . We show that AD masking and response to the appropriate metabolic signal only occur when a given AD remains attached to its own middle region; middle region swapping results in constitutively active proteins . Finally, we show that the extent to which Leu3p regulates reporter gene expression depends on the intracellular concentration of Leu3p . The possible physiological significance of this observation is discussed in light of the known regulation of Leu3p by Gcn4p.

J Biol Chem, 1999 Jul 2, 274(27), 18989 - 96
Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria; Lange H et al.; The import of metals, iron in particular, into mitochondria is poorly understood . Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins . We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria . By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import . Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP . Only reduced iron is functional in generating heme . Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane . Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin . The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron . Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria . Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.

Biochem Biophys Res Commun, 1999 Jun 24, 260(1), 216 - 21
Identification of cDNAs for Sox-4, an HMG-Box protein, and a novel human homolog of yeast splicing factor SSF-1 differentially regulated during apoptosis induced by prostaglandin A2/delta12-PGJ2 in Hep3B cells; Ahn SG et al.; We have examined specific genes whose expression is altered during apoptosis induced by prostaglandin (PG)A2 and Delta12-PGJ2 in human hepatocellular carcinoma Hep3B cells . Using mRNA differential display, we have identified two genes: one is specifically up-regulated and encodes for human Sox-4 (Sry-HMG box gene) and the other is significantly down-regulated and is the human homolog of yeast Ssf-1, a novel splicing factor . Northern blot analysis confirmed their differential expressions . Interestingly, Sox-4 was highly expressed in subcutaneous tumors grown in nude mice as a xenograft from Hep3B cells . These results suggest that the expression of Sox-4 may be related to the apoptosis pathway leading to cell death as well as to tumorigenesis, and that Ssf-1 gene may serve as a negative regulator of PGA2/Delta12-PGJ2-mediated Hep3B cell apoptosis .

Plant Mol Biol, 1999 Apr, 39(6), 1079 - 90
Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast; Nedelkina S et al.; cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells . These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5'-untranslated region and a 140 bp 3'-untranslated region . The open reading frame, determined by sequence similarity, codes for a protein with a predicted Mr of 59229 and a pI of 8.8 . It contains the conserved cysteine haem-binding site found in all cytochrome P450s . The protein encoded by this cDNA diverges however from other CYP73As in its N- and C-terminus and in four domains internally, so that overall sequence similarity is in the range 58-66% . Many clones contained an identical intron, which may be associated with a novel regulatory mechanism . Sequence similarity is sufficient for it to be classified as CYP73A15, although it is the least similar member of this family classified so far . The cDNA was expressed in yeast . Successful expression of cinnamate 4-hydroxylase activity required removal of the intron . High-level expression also required modification of the N-terminus to that of CYP73A1 . Yeast did not process the intron at all and the leader sequence for A15 was not as compatible as that of A1 . The mRNA for CYP73A15 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean but induction was more transient than that of phenylalanine ammonia-lyase (PAL) . In contrast, induction in cells undergoing xylogenesis was much more coordinate with PAL . The cloned cDNA may represent a cinnamate 4-hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the majority of CYP73As cloned so far are involved.

FEMS Immunol Med Microbiol, 1999 Jun, 24(2), 131 - 9
Antigen-specific response of murine immune system toward a yeast beta-glucan preparation, zymosan; Miura T et al.; Zymosan, a particulate beta-glucan preparation from Saccharomyces cerevisiae, shows various biological activities, including anti-tumor activity . We have previously shown that soluble beta-glucan initiated anti-tumor activity was long-lived and was effective even by prophylactic treatment at 1 month prior to tumor challenge . However, the activity by zymosan was relatively short-lived . Antigen-specific responses of mice to zymosan might be a causative mechanism . In this paper, mice were immunized with zymosan and antibody production and antigen-specific responses of lymphocytes to zymosan were analyzed . Sera of zymosan immune mice contained zymosan-specific IgG assessed by enzyme-linked immunosorbent assay and FACS . Spleen and bone marrow cells of zymosan-immune mice showed higher cytokine production in response to zymosan . Specificity of zymosan-specific responses were also analyzed using various derivatives prepared from zymosan . These facts strongly suggested that mice recognize zymosan as antigen in addition to non-specific immune stimulant.

Proc Natl Acad Sci U S A, 1999 Jun 22, 96(13), 7288 - 93
Genetic dissection of the budding yeast Arp2/3 complex: a comparison of the in vivo and structural roles of individual subunits; Winter DC et al.; In previous work, we identified the yeast Arp2/3 complex, which localizes to cortical actin patches and is required for their motility and integrity in vivo . This complex contains proteins homologous to each subunit of the Acanthamoeba and human Arp2/3 complex except for a 40-kDa subunit (p40), which was missing from the purified yeast complex . Here, we demonstrate by using immunoprecipitation and gel-filtration analysis that Arc40p, the homolog of p40 identified from the yeast genome database, associates with the yeast Arp2/3 complex . We have carried out gene disruptions of each subunit of the yeast Arp2/3 complex to study each subunit's role in the function of the complex . Surprisingly, we find that only ARC40 is fully essential for cell viability . Strains lacking each of the other subunits exhibit varying degrees of defects in cell growth and viability and in assembly and polarization of cortical actin patches . We have also examined each subunit's role in maintaining the structural integrity of the Arp2/3 complex . Arp2p, Arp3p, and Arc40p fall into the monomer pool in Deltaarc19 and Deltaarc35 cells, suggesting that Arc19p and Arc35p are the central scaffolding components of the complex . Arp2p and Arp3p do not have major roles in maintaining complex integrity, and Arc15p is required for association of Arp2p and Arc40p, but not other subunits, with the complex . These results provide evidence that each subunit contributes differently to the assembly and function of the Arp2/3 complex.

Proc Natl Acad Sci U S A, 1999 Jun 22, 96(13), 7226 - 31
Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins; Stevens SW et al.; The yeast U4/U6.U5 pre-mRNA splicing small nuclear ribonucleoprotein (snRNP) is a 25S small nuclear ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells . The yeast U4/U6.U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation . We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities . In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6.U5 snRNP proteins, and four novel proteins . Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs . The purification of the low abundance U4/U6.U5 snRNP from yeast and the powerful sequencing methodologies using small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.

RNA, 1999 Jun, 5(6), 779 - 93
The roles of Rrp5p in the synthesis of yeast 18S and 5.8S rRNA can be functionally and physically separated; Eppens NA et al.; The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8Ss) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions . To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence . Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function . Biochemical analysis clearly demonstrated the presence of two distinct functional domains . Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8Ss rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA . The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region . The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis . They can even be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.

FEBS Lett, 1999 Jun 4, 452(1-2), 77 - 81
Nuclear RNA export in yeast; Strasser K et al.; Eukaryotic cells massively exchange macromolecules (proteins and RNAs) between the nucleus and cytoplasm through the nuclear pore complexes . Whereas a mechanistic picture emerges of how proteins are imported into and exported from the nucleus, less is known about nuclear exit of the different classes of RNAs . However, the yeast Saccharomyces cerevisiae offers an experimental system to study nuclear RNA export in vivo and thus to genetically dissect the different RNA export machineries . In this review, we summarize our current knowledge and recent progress in identifying components involved in nuclear RNA export in yeast.

FEBS Lett, 1999 Jun 4, 452(1-2), 57 - 60
Mitochondrial assembly in yeast; Grivell LA et al.; The yeast Saccharomyces cerevisiae is likely to be the first organism for which a complete inventory of mitochondrial proteins and their functions can be drawn up . A survey of the 340 or so proteins currently known to be localised in yeast mitochondria reveals the considerable investment required to maintain the organelle's own genetic system, which itself contributes seven key components of the electron transport chain . Translation and respiratory complex assembly are particularly expensive processes, together requiring around 150 of the proteins so far known . Recent developments in both areas are reviewed and approaches to the identification of novel mitochondrial proteins are discussed.

Biotechniques, 1999 Jun, 26(6), 1102 - 6
Differential sensitivity to 5-fluoro-orotic acid as a screen for bait RNA-independent false positives in a yeast three-hybrid system; Park YW et al.; The yeast three-hybrid system presents a valuable tool for detecting and analyzing RNA-protein interactions in vivo . A major drawback of the use of such a transcriptional reporter-based assay in a library screen is the frequent occurrence of false-positive results due to bait RNA-independent activation of the reporter gene . To minimize the isolation of false positives in three-hybrid library screens, we incorporated a rapid and simple procedure based on differential sensitivity to 5-fluoro-orotic acid . The technique effectively eliminates bait RNA-independent false positives and thus greatly enhances the efficiency of the yeast three-hybrid system.

Curr Opin Plant Biol, 1999 Jun, 2(3), 244 - 9
Zeroing in on zinc uptake in yeast and plants; Guerinot ML et al.; Zinc is an essential micronutrient . Genes responsible for zinc uptake have now been identified from yeast and plants . These genes belong to an extended family of cation transporters called the ZIP gene family . Zinc efflux genes that belong to another transporter family, the CDF family, have also been identified in yeast and Arabidopsis . It is clear that studies in yeast can greatly aid our understanding of zinc metabolism in plants.

Nucleic Acids Res, 1999 Jul 1, 27(13), 2618 - 26
Srp2, an SR protein family member of fission yeast: in vivo characterization of its modular domains; Lutzelberger M et al.; We isolated srp2, a gene encoding a protein composed of two RNA binding domains (RBDs) at the N-terminus followed by an arginine-rich region that is flanked by two short SR (serine/arginine) elements . The RBDs contain the signatures RDADDA and SWQDLKD found in RBD1 and RBD2 of all typical metazoan SR proteins . srp2 is essential for growth . We have analyzed in vivo the role of the modular domains of Srp2 by testing specific mutations in a conditional strain for complementation . We found that RBD2 is essential for function and determines the specificity of RBD1 in Srp2 . Replacement of the first RBD with RBD1 of Srp1 of fission yeast does not change this specificity . The two SR elements in the C-terminus of Srp2 are also essential for function in vivo . Cellular distribution analysis with green fluorescence protein fused to portions of Srp2 revealed that the SR elements are necessary to target Srp2 to the nucleus . Furthermore, overexpression of modular domains of Srp2 and Srp1 show different effects on pre-mRNA splicing activity of the tfIId gene . Taken together, these findings are consistent with the notion that the RBDs of these proteins may be involved in pre-mRNA recognition.

Mol Cell Biol, 1999 Jul, 19(7), 5218 - 34
Spatial organization of the core region of yeast TFIIIB-DNA complexes; Persinger J et al.; The interaction of yeast TFIIIB with the region upstream of the SUP4 tRNATyr gene was extensively probed by use of photoreactive phosphodiesters, deoxyuridines, and deoxycytidines that are site specifically incorporated into DNA . The TATA binding protein (TBP) was found to be in close proximity to the minor groove of a TATA-like DNA sequence that starts 30 nucleotides upstream of the start site of transcription . TBP was cross-linked to the phosphate backbone of DNA from bp -30 to -20 in the nontranscribed strand and from bp -28 to -24 in the transcribed strand (+1 denotes the start site of transcription) . Most of the major groove of DNA in this region was shown not to be in close proximity to TBP, thus resembling the binding of TBP to the TATA box, with one notable exception . TBP was shown to interact with the major groove of DNA primarily at bp -23 and to a lesser degree at bp -25 in the transcribed strand . The stable interaction of TBP with the major groove at bp -23 was shown to require the B" subunit of TFIIIB . The S4 helix and flanking region of TBP were shown to be proximal to the major groove of DNA by peptide mapping of the region of TBP cross-linked at bp -23 . Thus, TBP in the TFIIIB-SUP4 gene promoter region is bound in the same direction as TBP bound to the TATA box with respect to the transcription start site . The B" and TFIIB-related factor (BRF) subunits of TFIIIB are positioned on opposite sides of the TBP-DNA core of the TFIIIB complex, as indicated by correlation of cross-linking data to the crystal structure of the TBP-TATA box complex . Evidence is given for BRF binding near the C-terminal stirrup of TBP, similar to that of TFIIB near the TBP-TATA box complex . The protein clamp formed around the TBP-DNA complex by BRF and B" would help explain the long half-life of the TFIIIB-DNA complex and its resistance to polyanions and high salt . The path of DNA traversing the surface of TBP at the 3' end of the TATA-like element in the SUP4 tRNA gene is not the same as that of TBP bound to a TATA box element, as shown by the cross-linking of TBP at bp -23.

Mol Cell Biol, 1999 Jul, 19(7), 5155 - 65
Defects in components of the proteasome enhance transcriptional silencing at fission yeast centromeres and impair chromosome segregation; Javerzat JP et al.; Fission yeast centromeres are transcriptionally silent and form a heterochromatin-like structure essential for normal centromere function; this appears analogous to heterochromatin and position effect variegation in other eukaryotes . Conditional mutations in three genes designated cep (centromere enhancer of position effect) were found to enhance transcriptional silencing within centromeres . Cloning of the cep1(+) and cep2(+) genes by functional complementation revealed that they are identical to the previously described genes pad1(+) and mts2(+), respectively, which both encode subunits of the proteasome 19S cap . Like Mts2 and Mts4, epitope-tagged Cep1/Pad1 localizes to or near the nuclear envelope throughout the cell cycle . The cep mutants display a range of phenotypes depending on the temperature . Silencing within the central domain of centromeres is increased at 36 degrees C . This suggests that the proteasome is involved in regulating silencing and thus centromeric chromatin architecture, possibly by lowering the level of some chromatin-associated protein by ubiquitin-dependent degradation . This is the first report of defective proteasome function affecting heterochromatin-mediated transcriptional silencing . At 36 and 32 degrees C, the cep mutants lose chromosomes at an elevated rate, and at 18 degrees C, the mutants are cryosensitive for growth . Cytological analysis at 18 degrees C revealed a defect in sister chromatid separation while other mitotic events occurred normally, indicating that cep mutations might interfere specifically with the degradation of inhibitor(s) of sister chromatid separation . These observations suggest that 19S subunits confer a level of substrate specificity on the proteasome and raise the possibility of a link between components involved in centromere architecture and sister chromatid cohesion.

Mol Cell Biol, 1999 Jul, 19(7), 4774 - 87
Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p; Kimmelman J et al.; Cyclin-dependent kinase (CDK)-activating kinases (CAKs) carry out essential activating phosphorylations of CDKs such as Cdc2 and Cdk2 . The catalytic subunit of mammalian CAK, MO15/Cdk7, also functions as a subunit of the general transcription factor TFIIH . However, these functions are split in budding yeast, where Kin28p functions as the kinase subunit of TFIIH and Cak1p functions as a CAK . We show that Kin28p, which is itself a CDK, also contains a site of activating phosphorylation on Thr-162 . The kinase activity of a T162A mutant of Kin28p is reduced by approximately 75 to 80% compared to that of wild-type Kin28p . Moreover, cells containing kin28(T162A) and a conditional allele of TFB3 (the ortholog of the mammalian MAT1 protein, an assembly factor for MO15 and cyclin H) are severely compromised and display a significant further reduction in Kin28p activity . This finding provides in vivo support for the previous biochemical observation that MO15-cyclin H complexes can be activated either by activating phosphorylation of MO15 or by binding to MAT1 . Finally, we show that Kin28p is no longer phosphorylated on Thr-162 following inactivation of Cak1p in vivo, that Cak1p can phosphorylate Kin28p on Thr-162 in vitro, and that this phosphorylation stimulates the CTD kinase activity of Kin28p . Thus, Kin28p joins Cdc28p, the major cell cycle Cdk in budding yeast, as a physiological Cak1p substrate . These findings indicate that although MO15 and Cak1p constitute different forms of CAK, both control the cell cycle and the phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II by TFIIH.

J Dent Res, 1999 Jun, 78(6), 1270 - 6
Refinement of the dentinogenesis imperfecta type II locus to an interval of less than 2 centiMorgans at chromosome 4q21 and the creation of a yeast artificial chromosome contig of the critical region; Aplin HM et al.; Dentinogenesis imperfecta type II is an autosomal-dominant disorder of dentin formation which has been mapped to the 6.6 centiMorgan D4S2691-D4S2692 interval at human chromosome 4q21 . In the current investigation, the use of four short tandem repeat polymorphisms has allowed the critical region to be refined to an interval of less than 2 centiMorgans defined by recombination events in unrelated, affected individuals from two families both of which show independent evidence for linkage to chromosome 4q21 . The creation of a yeast artificial chromosome contig of this newly defined interval has allowed us to demonstrate that the critical region encompasses approximately 2 Mb of DNA and that the dentin-specific gene, dentin sialoprotein, maps to this interval within 300 kb of dentin matrix acidic phosphoprotein 1 and bone sialoprotein . Moreover, dentin sialoprotein shows no recombination with the dentinogenesis imperfecta type II phenotype . Dentin sialoprotein is therefore a candidate for the dentinogenesis imperfecta type II locus.

FEBS Lett, 1999 May 28, 451(3), 243 - 8
Targeting of tail-anchored proteins to yeast mitochondria in vivo; Egan B et al.; Tail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain . The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed . Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain . An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes . We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo.

EMBO J, 1999 Jun 15, 18(12), 3463 - 74
Partial purification of the yeast U2 snRNP reveals a novel yeast pre-mRNA splicing factor required for pre-spliceosome assembly; Caspary F et al.; We have partially purified the U2 snRNP of Saccharomyces cerevisiae . Identification of some proteins consistently found in the purified fractions by nanoelectrospray mass spectrometry indicated the presence of a novel splicing factor named Rse1p . The RSE1 gene is essential and codes for a 148.2 kDa protein . We demonstrated that Rse1p associates specifically with U2 snRNA at low salt concentrations . In addition, we showed that Rse1p is a component of the pre-spliceosome . Depletion of Rse1p and analysis of a conditional mutant indicated that Rse1p was required for efficient splicing in vivo . In vitro Rse1p is required for the formation of pre-spliceosomes . Database searches revealed that Rse1p is conserved in humans and that it belongs to a large protein family that includes polyadenylation factors and DNA repair proteins . The characteristics of Rse1p suggest that its human homologue could be a subunit of the SF3 splicing factor.

Cell, 1999 May 28, 97(5), 621 - 33
Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast; Martin SG et al.; Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions . Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex . The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin . Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage . yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points . Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents . The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Trends Microbiol, 1999 Jun, 7(6), 247 - 52
Molecular mechanisms of yeast longevity; Jazwinski SM; The genetic analysis of yeast longevity has illuminated the underlying molecular mechanisms of aging that invoke the importance of metabolic regulation, genetic stability and stress resistance in determination of life span . The RAS genes have emerged as important modulators of life-maintenance processes and of life span itself.

Biochim Biophys Acta, 1999 Jun 15, 1432(1), 40 - 8
Rational design of a more stable yeast iso-1-cytochrome c; Lett CM et al.; Yeast iso-1-cytochrome c is one of the least stable mitochondrial cytochromes c . We have used a coordinated approach, combining the known functional and structural properties of cytochromes c, to engineer mutations into yeast iso-1-cytochrome c with the goal of selectively increasing the stability of the protein . The two redox forms of the native protein and six different mutant forms of yeast iso-1-cytochrome c were analyzed by differential scanning calorimetry (DSC) . The relative stability, expressed as the difference in the Gibb's free energy of denaturation at a given temperature between the native and mutant forms (DeltaDeltaG(Tref)), was determined for each of the proteins . In both oxidation states, the mutant proteins C102T, T69E/C102T, T96A/C102T, and T69E/T96A/C102T were more stable than the wild-type protein, respectively . The increased stability of the mutant proteins is proposed to be due to the removal of a rare surface cysteine and the stabilization of two distorted alpha-helices.

J Cell Biol, 1999 Jun 14, 145(6), 1233 - 49
A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast; Yamamoto A et al.; Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown . When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules . During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind . Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein . We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast . Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase . Thus, the dynein heavy chain is required for these oscillatory movements . Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements . In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination . In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant . Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant . These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.

Genomics, 1999 Jun 1, 58(2), 146 - 57
Novel proteins interacting with the leucine-rich repeat domain of human flightless-I identified by the yeast two-hybrid system; Fong KS et al.; The flightless-I gene encodes a member of the gelsolin-like family of actin-binding proteins linked to a leucine-rich repeat (LRR) domain . It is required for cellularization during early embryogenesis and normal development of the indirect flight muscles in Drosophila melanogaster . Although the association between actin and the gelsolin-like domain of the human Flightless-I homologue (FLI) has been established, its biological role is unknown . The human FLI gene is mapped within the Smith-Magenis microdeletion region of chromosome 17 . We report the identification of two related genes, LRRFIP1 and LRRFIP2, encoding proteins that interact with the LRR domain of human FLI using the yeast two-hybrid system . LRRFIP1 exhibits sequence identity with the TRIP RNA-binding protein and GCF-2 transcriptional repressor, which are also related to the murine FLAP-1 gene . LRRFIP2 is a novel gene that shares sequence homology with LRRFIP1 and FLAP-1 . LRRFIP1 and LRRFIP2 both express alternative splice variants in heart and skeletal muscle tissue . A coiled-coil domain, conserved within each encoded protein, serves as a potential interaction motif for FLI LRR . The occurrence of multiple proteins able to interact with FLI within the same tissue suggests that they may compete for the same binding site . Sequencing and PCR-directed genomic analysis indicate that LRRFIP1 and LRRFIP2 are related genes that arose from gene duplication .

Biochem Biophys Res Commun, 1999 Jun 16, 259(3), 582 - 7
Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of septin rings at the mother-bud neck in budding yeast; Takahashi Y et al.; SMT3 of Saccharomyces cerevisiae is an essential gene encoding a ubiquitin-like protein similar to mammalian SUMO-1 . When a tagged Smt3 or human SUMO-1 was expressed from GAL1 promoter, either gene rescued the lethality of the smt3 disruptant . By indirect-immunofluorescent microscopy, the HA-tagged Smt3 was detected mostly in nuclei and also at the mother-bud neck just like septin fibers . Indeed immunoprecipitation experiments revealed that Cdc3, one of septin components, was modified with Smt3 . Furthermore, the protein level of the Cdc3-Smt3 conjugate was reduced and the septin rings disappeared in a ubc9-1 mutant at a restrictive temperature, where the Smt3 conjugation system should be defective . Thus, we conclude that Smt3 was conjugated to Cdc3 in septin rings localized at the mother-bud neck . Around the time of cytokinesis the Cdc3-Smt3 conjugate disappeared . We discuss the biological significance of this Smt3 conjugation to a septin component .

J Biol Chem, 1999 Jun 18, 274(25), 17893 - 900
Human exonuclease 1 functionally complements its yeast homologues in DNA recombination, RNA primer removal, and mutation avoidance; Qiu J et al.; Yeast exonuclease 1 (Exo1) is induced during meiosis and plays an important role in DNA homologous recombination and mismatch correction pathways . The human homolog, an 803-amino acid protein, shares 55% similarity to the yeast Exo1 . In this report, we show that the enzyme functionally complements Saccharomyces cerevisiae Exo1 in recombination of direct repeat DNA fragments, UV resistance, and mutation avoidance by in vivo assays . Furthermore, the human enzyme suppresses the conditional lethality of a rad27Delta mutant, symptomatic of defective RNA primer removal . The purified recombinant enzyme not only displays 5'-3' double strand DNA exonuclease activity, but also shows an RNase H activity . This result indicates a back-up function of exonuclease 1 to flap endonuclease-1 in RNA primer removal during lagging strand DNA synthesis.

J Biol Chem, 1999 Jun 18, 274(25), 17545 - 50
Role of residues 311/312 in actin-tropomyosin interaction . In vitro motility study using yeast actin mutant e311a/r312a; Gerson JH et al.; According to the Lorenz et al . (Lorenz, M., Poole, K . J., Popp, D., Rosenbaum, G., and Holmes, K . C . (1995) J . Mol . Biol . 246, 108-119) atomic model of the actin-tropomyosin complex, actin residue Asp-311 (Glu-311 in yeast) is predicted to have a high binding energy contribution to actin-tropomyosin binding . Using the yeast actin mutant E311A/R312A in the in vitro motility assays, we have investigated the role of these residues in such interactions . Wild type (wt) yeast actin, like skeletal alpha-actin, is fully regulated when complexed with tropomyosin (Tm) and troponin (Tn) . Structure-function comparisons of the wt and E311A/R312A actins show no significant differences between them, and the unregulated F-actins slide at similar speeds in the in vitro motility assay . However, in the presence of Tm and Tn, the mutation increases both the sliding speed and the number of moving filaments at high pCa values, shifting the speed-pCa curve nearly 0.5 pCa units to the left . Tm alone (no Tn) inhibits the motilities of both actins at low heavy meromyosin densities but potentiates only the motility of the mutant actin at high heavy meromyosin densities . Actin-Tm binding measurements indicate no significant difference between wt and E311A/R312A actin in Tm binding . These results implicate allosteric effects in the regulation of actomyosin function by tropomyosin.

Genes Dev, 1999 Jun 1, 13(11), 1412 - 21
Cell cycle-regulated histone acetylation required for expression of the yeast HO gene; Krebs JE et al.; Expression of the yeast HO gene in late G1 of the cell cycle requires the SWI/SNF chromatin remodeling complex, the Gcn5p histone acetyltransferase, and two different sequence-specific transcriptional activators, Swi5p and Swi4p/Swi6p . We have used chromatin immunoprecipitation assays to investigate the role of each of these trans-acting factors in establishing a cell cycle-regulated domain of histone acetylation surrounding the HO upstream regulatory region . We detect a approximately 1-kb domain of H3 and H4 acetylation that is established in mid-G1, prior to and independent of HO transcription, which then declines with kinetics similar to inactivation of HO . This cell cycle burst of histone acetylation requires Gcn5p, SWI/SNF, and the Swi5p activator, but occurs in the absence of the Swi4p activator . We also find that inactivation of the Sin3p/Rpd3p deacetylase complex leads to a high level of acetylation at the HO locus throughout the cell cycle . We propose a sequential model for activation of HO in which the Swi5p-dependent recruitment of the Gcn5p acetyltransferase requires chromatin remodeling events by the SWI/SNF complex.

Mol Biol Rep, 1999 Apr, 26(1-2), 131 - 5
GFP-labelling of 26S proteasomes in living yeast: insight into proteasomal functions at the nuclear envelope/rough ER; Enenkel C et al.; 26S proteasomes are multisubunit protease complexes that play the central role in the ubiquitin-dependent protein degradation pathway . The proteolytically active core is formed by the 20S proteasome . Regulatory subunits, principally the 19S cap complex, confer the specificity towards ubiquitinated substrates and an ATP-dependence on proteolysis . Green fluorescence protein (GFP)-tagged versions of either an alpha-subunit of the 20S core or an ATPase subunit of the 19S cap complex were functionally incorporated into the protease complex, thus allowing to monitor the subcellular distribution of 26S proteasomes in living yeast . Our localization studies suggest that proteasomal proteolysis mainly occurs at the nuclear envelope (NE)/rough ER . Implications of proteasomal functions at the NE/rough ER are discussed in the context of published work on ER degradation and with regard to possible targeting mechanisms.

Biochem Biophys Res Commun, 1999 Jun 7, 259(2), 391 - 400
RNase treatment of yeast and mammalian cell extracts affects in vitro substrate methylation by type I protein arginine N-methyltransferases; Frankel A et al.; Type I protein arginine N-methyltransferases catalyze the formation of omega-NG-monomethylarginine and asymmetric omega-NG, NG-dimethylarginine residues using S-adenosyl-l-methionine as the methyl donor . In vitro these enzymes can modify a number of soluble methyl-accepting substrates in yeast and mammalian cell extracts including several species that interact with RNA . We treated normal and hypomethylated Saccharomyces cerevisiae and RAT1 cell extracts with RNase prior to in vitro methylation by recombinant protein N-arginine methyltransferases and found that the methylation of certain polypeptides is enhanced up to 12-fold whereas that of others is diminished . 2-D gel electrophoresis of RNase-treated yeast extracts allowed us to tentatively identify the glycine- and arginine-rich (GAR) domain-containing proteins Gar1, Nop1, Sbp1, and Npl3 as major methyl-acceptors based on their known isoelectric points and apparent molecular weights . These results suggest that the methylation and RNA-binding of GAR domain-containing proteins in vivo may regulate protein-nucleic acid or protein-protein interactions .

Mol Microbiol, 1999 May, 32(4), 753 - 64
In budding yeast, reactive oxygen species induce both RAS-dependent and RAS-independent cell cycle-specific arrest; Wanke V et al.; The role of mild oxidative stresses elicited by diethylmaleate (DEM)-induced glutathione depletion in the progression of the yeast cell cycle has been investigated . We found that different wild-type strains are sensitive to oxidative stresses induced by similar DEM doses: approximately 1 mM on YPD plates, 5-10 mM in shaken flasks . At lower doses, DEM caused a transient decrease in growth rate, largely because of a decreased G1-to-S transition . Treatment with higher DEM doses leads to complete growth arrest, with most cells found in the unbudded G1 phase of the cell cycle . DEM treatment resulted in transcriptional induction of stress-responsive element (STRE)-controlled genes and was relieved by treatment with the antioxidant N-acetyl cysteine . Reciprocal shift experiments with cdc25 and cdc28 mutants showed that the major cell cycle arrest point was located in the Start area, at or near the CDC25-mediated step, before the step mediated by the CDC28 cyclin-dependent kinase . The DEM-induced G1 arrest requires a properly regulated RAS pathway and can be bypassed by overexpressing the G1-specific cyclin CLN2 . However, cells with either a deregulated RAS pathway or overexpressing CLN2 failed to grow and arrested as budded cells, indicating that a second DEM-sensitive cell cycle step exists.

Med Mycol, 1999 Apr, 37(2), 139 - 45
Ultrastructural characterization of the agent of systemic yeast infection of owl monkeys; Miller GF et al.; Systemic infection by an unclassified yeast-like organism has been encountered sporadically in wild-caught owl monkeys (Aotus sp.) from South America . The infection is presumably acquired in the wild; the incubation period ranges from months to years . The disease is indolent and clinical signs are non-specific . The diagnosis is based on histopathologic observation of yeast-like cells in multiple internal organs . Most cells appear to be phagocytized by macrophages, however, many are apparently free in the extracellular space . Other inflammatory infiltrates, including neutrophils, lymphocytes, plasma cells, and multinucleated giant cells, are conspicuously absent . Cells are thick-walled, globose to oval, range from 5 to 8 micron in diameter, and reproduce by narrow-based budding of single daughter cells . Attempts to cultivate the organism on artificial media have failed . Yeast cell ultrastructure was studied using transmission electron microscopy . The cell wall is multilayered, and the internal structure is markedly heterogenous . In some cells, the cytoplasm is lightly electron-opaque, finely granular and lacks recognizable organelles or nuclei . In others, the cytoplasm is electron dense and contains mitochondria, ribosome-like granules, and a multilobulated nucleus . This organism differs from other recognized pathogenic yeast in its combined light microscopic appearance, organ involvement and host response . Ultrastructurally, it most closely resembles Loboa loboi.

Biochemistry, 1999 Jun 8, 38(23), 7470 - 5
Effects of pressure on the kinetics of capture by yeast alcohol dehydrogenase; Cho YK et al.; High pressure causes biphasic effects on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase as expressed in the kinetic parameter V/K which measures substrate capture . Moderate pressure increases the rate of capture of benzyl alcohol by activating the hydride transfer step . This means that the transition state for hydride transfer has a smaller volume than the free alcohol plus the capturing form of enzyme, with a DeltaV of -39 +/- 1 mL/mol, a value that is relatively large . This is the first physical property of an enzymatic transition state thus characterized, and it offers new possibilities for structure-activity analyses . Pressures of >1.5 kbar decrease the rate of capture of benzyl alcohol by favoring a conformation of the enzyme which binds nicotinamide adenine dinucleotide (NAD+) less tightly . This means that the ground state for tight binding, E-NAD+, has a larger volume than the collision complex, E-NAD+, with a DeltaV of 73 +/- 2 mL/mol . The equilibrium constant of the conformational change Keq is 75 +/- 13 at 1 atm . The effects of pressure on the capture of NAD+ have no activation phase because the conformational change is now being expressed kinetically instead of thermodynamically, together with but in opposition to hydride transfer, causing the effects to cancel . For yeast alcohol dehydrogenase, this conformational change had not been detected previously, but similar conformational changes have been found by spectroscopic means in other dehydrogenases, and some of them are also sensitive to pressure . The opposite signs for the volume change of tighter binding and hydride transfer run contrary to Pauling's hypothesis that substrates are bound more tightly in the transition state than in the Michaelian reactant state.

J Nutr Sci Vitaminol (Tokyo), 1999 Jan, 45(1), 119 - 28
An evaluation of the bioavailability of selenium in high-selenium yeast; Yoshida M et al.; The bioavailability of selenium (Se) in high-Se yeast (SeY) was evaluated by measuring tissue Se accumulation and glutathione peroxidase (GSHPx) activity . For 4 weeks, 4-week-old male Wistar rats were fed a Torula yeast-based Se-deficient diet (basal diet) or a diet supplemented with a graded level (0.04, 0.08, 0.16, and 0.32 microgram/g) of Se as either sodium selenite or SeY, which was obtained from two different sources . Se supplementation did not influence growth, hematological values, or serum biochemical tests . Se contents and GSHPx activities in the liver, serum, and erythrocytes increased gradually with increases of the supplemented Se . At lower Se levels (0.04 and 0.08 microgram/g), selenite produced higher Se deposition and higher GSHPx activities than SeY did, but at a higher Se level (0.32 microgram/g), SeY showed higher measures . Strong correlations were detected between the supplementary Se levels and the tissue Se contents or GSHPX activities when the regression was fitted to this equation: R-Rb = m log X + k, where R represented tissue Se content or GSHPx activity in rats fed the diet supplemented with Se at X level, Rb corresponding mean value in rats fed the basal diet, m slope, and k constant . The bioavailability of Se in SeY, as assessed by slope ratio analysis using selenite as a reference Se, was 135% to 165% in the tissue Se content and 105% to 197% in the GSHPx activities . These results indicate that Se in SeY is more bioavailable than selenite Se, and therefore it is the preferred form for supplementation.

Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6841 - 6
Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells; Heard E et al.; Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC) . The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse . Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog . To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells . The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells . Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors . Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found . This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.

Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6835 - 40
GCN5-dependent histone H3 acetylation and RPD3-dependent histone H4 deacetylation have distinct, opposing effects on IME2 transcription, during meiosis and during vegetative growth, in budding yeast; Burgess SM et al.; Diploid yeast undergo meiosis under certain conditions of nutrient limitation, which trigger a transcriptional cascade involving two key regulatory genes . IME1 is a positive activator of IME2, which activates downstream genes . We report that Gcn5, a histone H3 acetylase, plays a central role in initiation of meiosis via effects on IME2 expression . An allele, gcn5-21, was isolated as a mutant defective in spore formation . gcn5-21 fails to carry out meiotic DNA replication, recombination, or meiotic divisions . This mutant also fails to induce IME2 transcription; IME1 transcription, however, is essentially normal . Further investigation shows that during wild-type meiosis the IME2 promoter undergoes an increase in the level of bound acetylated histone H3 . This increase is contemporaneous with meiotic induction of IME2 transcription and is absent in gcn5-21 . In contrast, the RPD3 gene, which encodes a histone H4 deacetylase and is known to be required for repression of basal IME2 transcription in growing yeast cells, is not involved in induction of IME2 transcription or IME2 histone acetlyation during meiosis . These and other results suggest that Gcn5 and Rpd3 play distinct roles, modulating transcription initiation in opposite directions under two different cellular conditions . These roles are implemented via opposing effects of the two gene products on acetylation of two different histones . Finally, we find that gcn5 and rpd3 single mutants are not defective in meiosis if acetate is absent and respiration is promoted by a metabolically unrelated carbon source . Perhaps intracellular acetate levels regulate meiosis by controlling histone acetylation patterns.

Mol Biol Cell, 1999 Jun, 10(6), 1923 - 38
Erp1p and Erp2p, partners for Emp24p and Erv25p in a yeast p24 complex; Marzioch M et al.; Six new members of the yeast p24 family have been identified and characterized . These six genes, named ERP1-ERP6 (for Emp24p- and Erv25p-related proteins) are not essential, but deletion of ERP1 or ERP2 causes defects in the transport of Gas1p, in the retention of BiP, and deletion of ERP1 results in the suppression of a temperature-sensitive mutation in SEC13 encoding a COPII vesicle coat protein . These phenotypes are similar to those caused by deletion of EMP24 or ERV25, two previously identified genes that encode related p24 proteins . Genetic and biochemical studies demonstrate that Erp1p and Erp2p function in a heteromeric complex with Emp24p and Erv25p.

Mol Biol Cell, 1999 Jun, 10(6), 1859 - 72
Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources; Kal AJ et al.; We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE) . Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source . A major fraction (>20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of approximately 6300 yeast genes . Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters) . In oleate-grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal beta-oxidation enzymes required for degradation of fatty acids . The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes . We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2 and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins . Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment . We describe a statistical method for analysis of data obtained by SAGE.

Int J Food Microbiol, 1999 Mar 15, 47(3), 171 - 8
Aroma improving in microvinification processes by the use of a recombinant wine yeast strain expressing the Aspergillus nidulans xlnA gene; Ganga MA et al.; A recombinant wine yeast strain has been constructed expressing the gene coding for beta-(1,4)-endoxylanase from Aspergillus nidulans under the control of the yeast actin gene promoter . The resulting recombinant strain is able to secrete active xylanase enzyme into the culture medium . Wines obtained by microvinification with the control and the recombinant wine yeast strain did not differ in their physicochemical characteristics although an increase in fruity aroma was organoleptically detected in the wine produced by the recombinant yeast . Also, an increase in the concentration of some esters, higher alcohols and terpenes was observed in the case of the recombinant strain.

J Biol Chem, 1999 Jun 11, 274(24), 17275 - 83
Early steps in assembly of the yeast vacuolar H+-ATPase; Kane PM et al.; Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the Vo sector, attached to a complex of peripheral membrane proteins, the V1 sector . We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions . In wild-type cells, several V1 subunits and the 100-kDa Vo subunit associate within 3-5 min, followed by addition of other Vo subunits with time . Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V1 and Vo sectors that form without any apparent V1Vo subunit interaction . Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V1 subunits and the 100-kDa Vo subunit during incubation at elevated temperature . This complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed . We propose that assembly of the yeast V-ATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V1 and Vo subunits and an independent assembly pathway requiring full assembly of V1 and Vo sectors before combination of the two sectors . The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of Vo subunits during or after exit from the endoplasmic reticulum.

J Biol Chem, 1999 Jun 11, 274(24), 17103 - 8
The human WASP-interacting protein, WIP, activates the cell polarity pathway in yeast; Vaduva G et al.; WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells . The mechanism by which WIP reorganizes actin cytoskeleton is unknown . WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis . To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast . WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells . The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain . Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth . Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry . A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome.

J Biol Chem, 1999 Jun 11, 274(24), 17017 - 24
UV-responsive genes of arabidopsis revealed by similarity to the Gcn4-mediated UV response in yeast; Zimmermann S et al.; A UV response that involves the Ras proteins and AP-1 transcription factors has recently been described in mammals and yeast . To test whether an equivalent response exists in plants, we monitored the expression of Arabidopsis histidinol dehydrogenase gene (HDH), a homologue of the yeast HIS4 gene, which is strongly induced by UV light and is a target of the transcriptional activator Gcn4 . We show that HDH mRNA levels increase specifically in response to UV-B light . Only small increases were detected upon exposure to other wavelengths . To isolate plant genes involved in this UV response, a gcn4 mutant was transfected with an Arabidopsis thaliana cDNA library . A new type of nucleotide diphosphate kinase (NDPK Ia) with a significant homology to the human tumor suppressor protein Nm23 rescued the gcn4 phenotype . NDPK Ia specifically binds to the HIS4 promoter in vitro and induces HIS4 transcription in yeast . In Arabidopsis, the NDPK Ia protein is located in the nucleus and cytosol . Expression studies in seedlings revealed that the level of NDPK Ia mRNA, like that of HDH, increases in response to UV-B light . It appears that NDPK Ia and HDH are components of a novel UV-responsive pathway in A . thaliana.

Biotechnol Prog, 1999 May, 15(3), 472 - 9
Effects of added yeast on protein transmission and flux in cross-flow membrane microfiltration
Kuberkar VT, Davis RH.
Microfiltration membranes may be used to separate valuable proteins from suspensions containing cells or cell debris . Although a clean microfiltration membrane allows for complete protein transmission and high flux, both of these quantities decline in time due to membrane fouling . Using bovine serum albumin (BSA) as a model protein, flux and protein transmission during cross-flow microfiltration were studied with and without added yeast cells . Cross-flow microfiltration of BSA-only solutions results in a BSA fouling layer with low permeability forming on the membrane surface . Due to this layer, the long-term BSA transmission is typically only 25-40% . In contrast, during microfiltration of yeast-BSA mixtures, the yeast forms a cake layer on the membrane surface . The yeast cake acts as a dynamic or secondary membrane, allowing BSA monomers to pass through but preventing protein aggregates from fouling the membrane . The result is slower flux decline and higher long-term BSA transmission of typically 60-90% . For filtration of yeast-BSA mixtures at low yeast concentrations (<1 g/L), 50-100% higher BSA recovery is obtained than for BSA-only solutions with the same BSA concentration . At high yeast concentrations (>5 g/L), the protein transmission remains high, but the recovery may be lower due to reduced flux.

EMBO J, 1999 Jun 1, 18(11), 3173 - 85
RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast; Gardner R et al.; Eukaryotic checkpoint genes regulate multiple cellular responses to DNA damage . In this report, we examine the roles of budding yeast genes involved in G2/M arrest and tolerance to UV exposure . A current model posits three gene classes: those encoding proteins acting on damaged DNA (e.g . RAD9 and RAD24), those transducing a signal (MEC1, RAD53 and DUN1) or those participating more directly in arrest (PDS1) . Here, we define important features of the pathways subserved by those genes . MEC1, which we find is required for both establishment and maintenance of G2/M arrest, mediates this arrest through two parallel pathways . One pathway requires RAD53 and DUN1 (the 'RAD53 pathway'); the other pathway requires PDS1 . Each pathway independently contributes approximately 50% to G2/M arrest, effects demonstrable after cdc13-induced damage or a double-stranded break inflicted by the HO endonuclease . Similarly, both pathways contribute independently to tolerance of UV irradiation . How the parallel pathways might interact ultimately to achieve arrest is not yet understood, but we do provide evidence that neither the RAD53 nor the PDS1 pathway appears to maintain arrest by inhibiting adaptation . Instead, we think it likely that both pathways contribute to establishing and maintaining arrest.

EMBO J, 1999 Jun 1, 18(11), 3153 - 63
The yeast poly(A)-binding protein Pab1p stimulates in vitro poly(A)-dependent and cap-dependent translation by distinct mechanisms; Otero LJ et al.; Translation initiation in extracts from Saccharomyces cerevisiae involves the concerted action of the cap-binding protein eIF4E and the poly(A) tail-binding protein Pab1p . These two proteins bind to translation initiation factor eIF4G and are needed for the translation of capped or polyadenylated mRNA, respectively . Together, these proteins synergistically activate the translation of a capped and polyadenylated mRNA . We have discovered that excess Pab1p also stimulates the translation of capped mRNA in extracts, a phenomenon that we define as trans-activation . Each of the above activities of Pab1p requires its second RNA recognition motif (RRM2) . We have found that RRM2 from human PABP cannot substitute functionally for yeast RRM2 . Using the differences between human and yeast RRM2 sequences as a guide, we have mutagenized yeast RRM2 and discovered residues that are required for eIF4G binding and poly(A)-dependent translation but not for trans-activation . Similarly, other residues within RRM2 were found to be required for trans-activation but not for eIF4G binding or poly(A)-dependent translation . These data show that Pab1p has at least two biochemically distinct activities in translation extracts.

EMBO J, 1999 Jun 1, 18(11), 3139 - 52
Post-termination ribosome interactions with the 5'UTR modulate yeast mRNA stability; Vilela C et al.; A novel form of post-transcriptional control is described . The 5' untranslated region (5'UTR) of the Saccharomyces cerevisiae gene encoding the AP1-like transcription factor Yap2 contains two upstream open reading frames (uORF1 and uORF2) . The YAP2-type of uORF functions as a cis-acting element that attenuates gene expression at the level of mRNA turnover via termination-dependent decay . Release of post-termination ribosomes from the YAP2 5'UTR causes accelerated decay which is largely independent of the termination modulator gene UPF1 . Both of the YAP2 uORFs contribute to the destabilization effect . A G/C-rich stop codon context, which seems to promote ribosome release, allows an uORF to act as a transferable 5'UTR-destabilizing element . Moreover, termination-dependent destabilization is potentiated by stable secondary structure 3' of the uORF stop codon . The potentiation of uORF-mediated destabilization is eliminated if the secondary structure is located further downstream of the uORF, and is also influenced by a modulatory mechanism involving eIF2 . Destabilization is therefore linked to the kinetics of acquisition of reinitiation-competence by post-termination ribosomes in the 5'UTR . Our data explain the destabilizing properties of YAP2-type uORFs and also support a more general model for the mode of action of other known uORFs, such as those in the GCN4 mRNA.

Cell Biochem Biophys, 1999, 30(2), 193 - 212
The yeast pheromone-responsive G alpha protein stimulates recovery from chronic pheromone treatment by two mechanisms that are activated at distinct levels of stimulus; Zhou J et al.; The pheromone response of Saccharomyces cerevisiae is mediated by a receptor-coupled heterotrimeric G protein . The beta gamma subunit of the G protein stimulates a PAK/MAP kinase cascade that leads to cellular changes preparatory to mating, while the pheromone-responsive G alpha protein, Gpa1, antagonizes the G beta gamma-induced signal . In its inactive conformation, Gpa1 sequesters G beta gamma and tethers it to the receptor . In its active conformation, Gpa1 stimulates adaptive mechanisms that downregulate the mating signal, but which are independent of alpha-beta gamma binding . To elucidate these potentially novel signaling functions of G alpha in yeast, epistasis analyses were performed using N388D, a hyperadaptive mutant form of Gpa1, and null alleles of various loci that have been implicated in adaptation . The results of these experiments indicate the existence of signaling thresholds that affect the yeast mating reaction . At low pheromone concentration, the Regulator of G Protein Signaling (RGS) homologue and putative guanosine triphosphatase (GTPase) activating protein, Sst2, appears to stimulate sequestration of G beta gamma by Gpa1 . Throughout the range of pheromone concentrations sufficient to cause cell cycle arrest, Gpa1 stimulates adaptive mechanisms that are partially dependent on Msg5 and Mpt5 . Gpa1-mediated adaptation appears to be independent of Afr1, Akr1, and the carboxy-terminus of the pheromone receptor.

J Mol Biol, 1999 Jun 11, 289(3), 473 - 90
Group II intron reverse transcriptase in yeast mitochondria . Stabilization and regulation of reverse transcriptase activity by the intron RNA; Zimmerly S et al.; Group II introns encode reverse transcriptases that function in both intron mobility and RNA splicing . The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription . Here, immunoblotting and UV cross-linking experiments show that the reverse transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62) . p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion . Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing . Although the reverse transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided . Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer . Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3' exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1 . Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62 .

Biophys J, 1999 Jun, 76(6), 3345 - 8
Real-time monitoring of yeast cell division by dielectric spectroscopy; Asami K et al.; To assay cell cycle progression in synchronized culture of yeast we have applied dielectric spectroscopy to its real-time monitoring . The dielectric monitoring is based on the electromagnetic induction method, regarded as a nonelectrode method, which has resolved the problems encountered in measurements with metal electrodes, namely electrode polarization and bubble formation on electrodes . In the synchronized culture with temperature-sensitive cell division cycle mutants, the permittivity of the culture broth showed cyclic changes at frequencies below 300 kHz . The increase and decrease in the cyclic changes of the relative permittivity correspond to the increase in cell length and bud size and to the septum formation between mother and daughter cells, respectively.

Gene, 1999 May 31, 232(2), 143 - 53
Cis-regulation of inter-allelic exchanges in mutation at human minisatellite MS205 in yeast; He Q et al.; Tandemly repeated DNA is a major component of the human genome, and includes loci contributing to human disease . Minisatellites include the most variable human loci described to date, and the mechanisms by which this variation is generated in humans have been studied in detail . Integration of human minisatellites into yeast not only provides a model for further dissecting the molecular basis of length change mutation at these loci, but also more generally allows the study of complex recombinational events in yeast . We have used human minisatellite MS205 integrated into yeast to study the structural details of length change mutations . Apart from showing that mutation at this locus in yeast has features similar to those observed at some minisatellites in humans, including meiosis-specificity, and polarity, in which exchange events are localised to one extremity of the array, we here, for the first time, directly demonstrate that a flanking element in yeast regulates the mutation process . The results therefore support the hypothesis that flanking initiators are involved in minisatellite mutation in humans . Furthermore, mutant alleles showed more complex rearrangements in one orientation than the other . The data also suggest that the mutational pathway for deletions might be different from the pathway generating inter-allelic exchanges and duplications.

Nucleic Acids Res, 1999 Jun 15, 27(12), 2560 - 7
Sequence-specific and conformation-dependent binding of yeast telomerase RNA to single-stranded telomeric DNA; Lue NF; Telomerase is a ribonucleoprotein reverse trans-criptase responsible for the maintenance of one strand of telomere terminal repeats . The mechanisms whereby telomerase recognizes chromosomal ends are not fully characterized . Earlier studies showed that the yeast telomerase RNP could bind the dG-rich strand of yeast telomeres with high affinity and sequence specificity . Further analysis of telomerase-telomere complex formation in vitro as described in this report led to the following conclusions . First, telomerase binding to short DNAs is magnesium-dependent, while binding to long DNAs is magnesium-independent, consistent with the existence of more than one interaction site . Second, binding is likely to be mediated largely through the RNA subunit of telomerase (TLC1), because de-proteinated TLC1 RNA also binds telomeres with high affinity and sequence specificity, and exhibits the same length and divalent cation dependence as telomerase RNP . The crucial role of RNA in binding is further supported by the ability of TLC1 transcripts synthesized in vitro to form stable complexes with telomeric DNA . Finally, results from deletion analysis and RNase H-mediated cleavage suggest that a specific conformation(s) of the RNA is required for stable binding, and that non-template regions of the TLC1 RNA may contribute directly or indirectly to the stability of the RNA-DNA complex.

J Cell Biol, 1999 May 31, 145(5), 993 - 1007
Yeast Bim1p promotes the G1-specific dynamics of microtubules; Tirnauer JS et al.; Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro . Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells . Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1 . During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state . Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein . Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules . This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end . Our in vivo data provide evidence of a cell cycle-specific microtubule-binding protein that promotes microtubule dynamicity.

J Cell Biol, 1999 May 31, 145(5), 979 - 91
Budding yeast Bub2 is localized at spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2; Fraschini R et al.; The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle . In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest . Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins . We investigated the role of Bub2 in budding yeast mitotic checkpoint . The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions . Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

FEBS Lett, 1999 Apr 30, 450(1-2), 27 - 34
Rpn4p acts as a transcription factor by binding to PACE, a nonamer box found upstream of 26S proteasomal and other genes in yeast; Mannhaupt G et al.; We identified a new, unique upstream activating sequence (5'-GGTGGCAAA-3') in the promoters of 26 out of the 32 proteasomal yeast genes characterized to date, which we propose to call proteasome-associated control element . By using the one-hybrid method, we show that the factor binding to the proteasome-associated control element is Rpn4p, a protein containing a C2H2-type finger motif and two acidic domains . Electrophoretic mobility shift assays using proteasome-associated control element sequences from two regulatory proteasomal genes confirmed specific binding of purified Rpn4p to these sequences . The role of Rpn4p to function as a transregulator in yeast is corroborated by its ability of stimulating proteasome-associated control element-driven lacZ expression and by experiments using the RPT4 and RPT6 gene promoters coupled to the bacterial cat gene as a reporter . Additionally, we found the proteasome-associated control element to occur in a number of promoters to genes which are related to the ubiquitin-proteasome pathway in yeast.

J Biol Chem, 1999 Jun 4, 274(23), 16040 - 6
Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast; Lee J et al.; Yap1 and Skn7 are two yeast transcriptional regulators that co-operate to activate thioredoxin (TRX2) and thioredoxin reductase (TRR1) in response to redox stress signals . Although they are both important for resistance to H2O2, only Yap1 is important for cadmium resistance, whereas Skn7 has a negative effect upon this response . The respective roles of Yap1 and Skn7 in the induction of defense genes by H2O2 were analyzed by two-dimensional gel electrophoresis . Yap1 controls a large oxidative stress response regulon of at least 32 proteins . Fifteen of these proteins also require the presence of Skn7 for their induction by H2O2 . Although about half of the Yap1 target genes do not contain a consensus Yap1 recognition motif, the control of one such gene, TSA1, involves the binding of Yap1 and Skn7 to its promoter in vitro . The co-operative control of the oxidative stress response by Yap1 and Skn7 delineates two gene subsets . Remarkably, these two gene subsets separate antioxidant scavenging enzymes from the metabolic pathways regenerating the main cellular reducing power, glutathione and NADPH . Such a specialization may explain, at least in part, the dissociated function of Yap1 and Skn7 in H2O2 and cadmium resistance.

Yeast, 1999 May, 15(7), 601 - 14
Systematic analysis of yeast strains with possible defects in lipid metabolism; Daum G et al.; Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers . Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized . Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids . To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes . These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project . First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized . The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism . During these investigations it was demonstrated that different wild-type strains, among them FY1679, CEN.PK2-1C and W303, exhibit marked differences in lipid content and lipid composition . Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function . Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined . For some genes we found evidence suggesting a possible role in lipid metabolism.

Yeast, 1999 May, 15(7), 553 - 62
Effects of weak acids on cation accumulation, delta pH and delta psi in yeast; Lopez R et al.; With glucose as carbon source, as reported by other authors (Conway et al., 1950), yeast cells accumulated large amounts of CO2, carbonic acid, bicarbonate and K+ than when grown with ethanol . The addition of propionic acid to the cells produced an increase in the potassium ion accumulation when cells were incubated with ethanol as substrate, but not when incubated with glucose . In disagreement with Ryan et al . (1971) and Ryan and Ryan (1972), the internal pH of the cells with ethanol was lower than with glucose, eliminating this factor as the limiting factor for potassium accumulation . When the membrane potential difference was estimated, it was found that it was higher with glucose as substrate than with ethanol . In addition, with ethanol the addition of propionic acid produced an increase of the membrane potential, in agreement with the idea of an accumulation of the anions of monoprotic acids, which, being not diffusible, increase the negative membrane potential inside, which can drive the accumulation of larger amounts of monovalent cations . It was also found that the addition of propionic acid to cells incubated with glucose as substrate produced an efflux of CO2 from the cells, so that an exchange of the acid appears to take place.

Yeast, 1999 May, 15(7), 541 - 51
Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1; Agaphonov MO et al.; Plasmids with different selectable markers were constructed and used to transform the Hansenula polymorpha strain DL1 . It was shown that, depending on the host mutant strain, the use of these plasmids enables rapid selection of transformants with plasmids integrated in low (1-2), moderate (6-9) or high (up to 100) copy numbers . The vectors and mutant described are potentially useful for the construction of efficient producers of heterologous proteins in H . polymorpha.

J Cell Sci, 1999 Jun, 112 ( Pt 12), 1889 - 900
The nucleolar antigen Nop52, the human homologue of the yeast ribosomal RNA processing RRP1, is recruited at late stages of nucleologenesis; Savino TM et al.; We report the molecular characterization of a novel nucleolar protein, Nop52, and its subcellular distribution during the cell cycle and nucleologenesis . This protein was originally identified with human autoantibodies which were subsequently used to clone its corresponding cDNA . Transfection experiments in mammalian cells have confirmed that this cDNA encodes a nucleolar protein that accumulates in the nucleoli and at the periphery of the chromosomes . Nop52 is the putative human homologue of the yeast ribosomal RNA processing protein RRP1 which is involved in pre-rRNA processing from 27S to 25S and 5.8S . In nucleoli, Nop52 is excluded from the ribosomal RNA transcription sites, accumulates in the granular external domain and mainly colocalizes with nucleolar proteins involved in the late processing step such as hPop1 and protein B23 . During the building process of the nucleolus at the end of mitosis, a sequential order was observed in the assembly of nucleolar proteins of early and late processing mainly via the prenucleolar body pathway . The order is the following: fibrillarin, nucleolin, Nop52 together with protein B23 in the prenucleolar bodies, and simultaneously with hPop1, and finally Ki-67 . The evolutionary conservation of Nop52 and the lethal effects observed in gene disruption experiments, predict a critical role for Nop52 in the generation of 28S rRNA.

J Bioenerg Biomembr, 1999 Feb, 31(1), 57 - 65
Structure and properties of the clathrin-coated vesicle and yeast vacuolar V-ATPases; Forgac M; The V-ATPases are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells . This review focuses on the the V-ATPases from clathrin-coated vesicles and yeast vacuoles . The V-ATPase of clathrin-coated vesicles is a precursor to that found in endosomes and synaptic vesicles, which function in receptor recycling, intracellular membrane traffic, and neurotransmitter uptake . The yeast vacuolar ATPase functions to acidify the central vacuole and to drive various coupled transport processes across the vacuolar membrane . The V-ATPases are composed of two functional domains . The V1 domain is a 570-kDa peripheral complex composed of eight subunits of molecular weight 70-14 kDa (subunits A-H) that is responsible for ATP hydrolysis . The V0 domain is a 260-kDa integral complex composed of five subunits of molecular weight 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation . Using chemical modification and site-directed mutagenesis, we have begun to identify residues that play a role in ATP hydrolysis and proton transport by the V-ATPases . A central question in the V-ATPase field is the mechanism by which cells regulate vacuolar acidification . Several mechanisms are described that may play a role in controlling vacuolar acidification in vivo . One mechanism involves disulfide bond formation between cysteine residues located at the catalytic nucleotide binding site on the 70-kDa A subunit, leading to reversible inhibition of V-ATPase activity . Other mechanisms include reversible assembly and dissociation of V1 and V0 domains, changes in coupling efficiency of proton transport and ATP hydrolysis, and regulation of the activity of intracellular chloride channels required for vacuolar acidification.

J Bioenerg Biomembr, 1999 Feb, 31(1), 49 - 56
Biosynthesis and regulation of the yeast vacuolar H+-ATPase; Kane PM; The yeast V-ATPase is highly similar to V-ATPases of higher organisms and has proved to be a biochemically and genetically accessible model for many aspects of V-ATPase function . Like other V-ATPases, the yeast enzyme consists of a complex of peripheral membrane proteins, the V1 sector, attached to a complex of integral membrane subunits, the V0 sector . Multiple pathways for biosynthetic assembly of the enzyme appear to be available to cells containing a full complement of subunits and enzyme activity may be further controlled during biosynthesis by a protease activity localized to the late Golgi apparatus . Surprisingly, the assembled V-ATPase is not a static structure . Instead, fully assembled V1V0 complexes appear to exist in a dynamic equilibrium with inactive cytosolic V1 and membrane-bound V0 complexes and this equilibrium can be rapidly shifted in response to changes in carbon source . The reversible disassembly of the yeast V-ATPase may be a novel regulatory mechanism, common to V-ATPases, that works in vivo in coordination with many other regulatory mechanisms.

J Bioenerg Biomembr, 1999 Feb, 31(1), 39 - 47
Assembly of the yeast vacuolar proton-translocating ATPase; Graham LA et al.; The yeast vacuolar proton-translocating ATPase (V-ATPase) is the best characterized member of the V-ATPase family . Biochemical and genetic screens led to the identification of a large number of genes in yeast, designated VMA, encoding proteins required to assemble a functional V-ATPase . A total of thirteen genes encode subunits of the final enzyme complex . In addition to subunit-encoding genes, we have identified three genes that code for proteins that are not part of the final V-ATPase complex yet required for its assembly . We refer to these nonsubunit Vma proteins as assembly factors, since their function is dedicated to assembling the V-ATPase . The assembly factors, Vma12p, Vma21p, and Vma22p are localized to the endoplasmic reticulum (ER) and aid the assembly of newly synthesized V-ATPase subunits that are translocated into the ER membrane . At least two of these proteins, Vma12p and Vma22p, function together in an assembly complex and interact directly with nascent V-ATPase subunits.

FEMS Microbiol Lett, 1999 May 15, 174(2), 279 - 84
Electropulsation as an alternative method for protein extraction from yeast; Ganeva V et al.; The application of series of high intensity electric pulses to a yeast suspension provoked a considerable release of some cytoplasmic proteins, glutathione reductase, 3-phosphoglycerate kinase and alcohol dehydrogenase . A maximal yield was achieved 3-8 h after pulsation . The electro-induced protein efflux was accelerated by pretreatment with the reducing agent dithiothreitol and showed a strong dependence on the growth phase and the presence of monovalent ions in the post-pulse incubation medium . The results obtained for two strains of Saccharomyces cerevisiae, PV3 (diploid) and Y47 (wild haploid), showed that electropulsation can be used for the effective extraction of cytoplasmic proteins with a preserved functional activity.

FEBS Lett, 1999 Apr 23, 449(2-3), 201 - 5
The cleaved presequence is not required for import of subunit 6 of the cytochrome bc1 complex into yeast mitochondria or assembly into the complex; DeLabre ML et al.; Subunit 6 of the yeast cytochrome bc1 complex contains a 25 amino acid presequence that is not present in the mature form of the protein in the bc1 complex . The presequence of subunit 6 is atypical of presequences responsible for targeting proteins to mitochondria . Whereas mitochondrial targeting sequences rarely contain acidic residues and typically contain basic residues that can potentially form an amphiphilic structure, the presequence of subunit 6 contains only one basic amino acid and is enriched in acidic amino acids . If the 25 amino acid presequence is deleted, subunit 6 is imported into mitochondria and assembled into the cytochrome bc1 complex and the activity of the bc1 complex is identical to that from a wild-type yeast strain . However, if the C-terminal 45 amino acids are truncated from the protein, subunit 6 is not present in the mitochondria and the activity of the bc1 complex is diminished by half, identical to that of the bc1 complex from a yeast strain in which the QCR6 gene is deleted . These results indicate that the presequence of subunit 6 is not required for targeting to mitochondria or assembly of the subunit into the bc1 complex and that information necessary for targeting and import into mitochondria may be present in the C-terminus of the protein.

FEBS Lett, 1999 Apr 23, 449(2-3), 129 - 34
Assessment of uncoupling activity of uncoupling protein 3 using a yeast heterologous expression system; Zhang CY et al.; Uncoupling protein 3L, uncoupling protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae . Effects on different parameters related to the energy expenditure were studied . Both uncoupling protein 3L and uncoupling protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively . In isolated mitochondria, uncoupling protein 1 increased the state 4 respiration by 1.8-fold, while uncoupling protein 3L increased the state 4 respiration by 1.2-fold . Interestingly, mutant uncoupling protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of uncoupling protein 1 when assessed using a proteoliposome system (Bienengraeber et al . (1998) Biochem . 37, 3-8), uncoupled the mitochondrial respiration to almost the same degree as wild-type uncoupling protein 1 . Thus, absence of this histidine pair in uncoupling protein 2 and uncoupling protein 3 does not by itself rule out the possibility that these carriers have an uncoupling function . The oxoglutarate carrier had no effect on any of the studied parameters . In summary, a discordance exists between the magnitude of effects of uncoupling protein 3L and uncoupling protein 1 in whole yeast versus isolated mitochondria, with uncoupling protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria . These findings suggest that uncoupling protein 3L, like uncoupling protein 1, has an uncoupling activity . However, the mechanism of action and/or regulation of the activity of uncoupling protein 3L is likely to be different.

Glycobiology, 1999 Jun, 9(6), 547 - 55
Glycosylation of the overlapping sequons in yeast external invertase: effect of amino acid variation on site selectivity in vivo and in vitro; Reddy A et al.; Yeast invertase contains 14 sequons, all of which are glycosylated to varying degrees except for sequon 5 which is marginally glycosylated, if at all . This sequon overlaps with sequon 4 in a sequence consisting of Asn92-Asn93-Thr94-Ser95(Reddy et al., 1988, J . Biol . Chem., 263, 6978-6985) . To determine whether glycosylation at Asn93is sterically hindered by the oligosaccharide on Asn92, the latter amino acid was converted to a glutamine residue by site-directed mutagenesis of the SUC2 gene in a plasmid vector which was expressed in Saccharomyces cerevisiae . A glycopeptide encompassing sequons 3 through 6 was purified from a tryptic digest of the mutagenized invertase and sequenced by Edman degradation, which revealed that Asn93 of sequon 5 contained very little, if any, carbohydrate, despite the elimination of sequon 4 . When Ser and Thr were inverted to yield Asn-Asn-Ser-Thr carbohydrate was associated primarily with the second sequon, in agreement with numerous studies indicating that Asn-X-Thr is preferred to Asn-X-Ser as an oligosaccharide acceptor . However, when the invertase overlapping sequons were converted to Asn-Asn-Ser-Ser, both sequons were clearly glycosylated, with the latter sequon predominating . These findings rule out steric hindrance as a factor involved in preventing the glycosylation of sequon 5 in invertase . Comparable results were obtained using an in vitro system with sequon-containing tri- and tetrapeptides acceptors, in addition to larger oligosaccharide acceptors.

Eur J Biochem, 1999 Jun, 262(2), 315 - 23
Identification of subunit g of yeast mitochondrial F1F0-ATP synthase, a protein required for maximal activity of cytochrome c oxidase; Boyle GM et al.; By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase . We have designated this ORF ATP20, and its product subunit g . A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function . Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal . Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex . Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate . The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide . These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.

Genes Genet Syst, 1998 Dec, 73(6), 365 - 75
Isolation and characterization of the yeast las21 mutants, which are sensitive to a local anestheticum, tetracaine; Tohe A et al.; We isolated and characterized yeast mutants whose growth is sensitive to a local anestheticum tetracaine and, at the same time, temperature sensitive . These mutants were collectively called las mutants (local anestheticum sensitive) . The las21 mutants were analyzed in this study . The wild type LAS21 gene was cloned by exploiting temperature sensitivity of the las21 mutants and we found that LAS21 encodes ORF YJL062w which has not been analyzed before . Las21p is putative membrane protein belonging to the major facilitator super family containing plural membrane spanning domains . Complete elimination of the LAS21 ORF did not kill the cells but made their growth temperature sensitive . Interestingly, the complete loss of the LAS21 gene canceled the sensitivity to tetracaine . The ability of the las21 mutants to grow at a higher temperature was recovered in the various media containing an osmotic stabilizer or salts . Furthermore, temperature sensitivity of the las21 mutants was partially suppressed by introduction of PKC1, encoding protein kinase C, on a high copy vector . We found some genetic interactions between LAS21 and Ras/cAMP cascade genes . These results suggest that LAS21 defines unknown pathway regulating the stress response of yeast.

Biochem J, 1999 Jun 1, 340 ( Pt 2), 359 - 63
Identification of centaurin-alpha1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1; Venkateswarlu K et al.; Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3 . In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1 . Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor . This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85 . Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1 . Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

J Cell Biol, 1999 May 17, 145(4), 757 - 67
Oxygen stress: a regulator of apoptosis in yeast; Madeo F et al.; Oxygen radicals are important components of metazoan apoptosis . We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2 . Cycloheximide prevents apoptotic death revealing active participation of the cell . Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax . In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis . These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.

Mol Cell Biol, 1999 Jun, 19(6), 4480 - 94
Yeast VSM1 encodes a v-SNARE binding protein that may act as a negative regulator of constitutive exocytosis; Lustgarten V et al.; We have screened for proteins that interact with v-SNAREs of the late secretory pathway in the yeast Saccharomyces cerevisiae . A novel protein, designated Vsm1, binds tightly to the Snc2 v-SNARE in the two-hybrid system and can be coimmunoprecipitated with Snc1 or Snc2 from solubilized yeast cell extracts . Disruption of the VSM1 gene results in an increase of proteins secreted into the medium but does not affect the processing or secretion of invertase . In contrast, VSM1 overexpression in cells which bear a temperature-sensitive mutation in the Sec9 t-SNARE (sec9-4 cells) results in the accumulation of non-invertase-containing low-density secretory vesicles, inhibits cell growth and the secretion of proteins into the medium, and blocks rescue of the temperature-sensitive phenotype by SNC1 overexpression . Yet, VSM1 overexpression does not affect yeast bearing a sec9-7 allele which, in contrast to sec9-4, encodes a t-SNARE protein capable of forming a stable SNARE complex in vitro at restrictive temperatures . On the basis of these results, we propose that Vsm1 is a novel v-SNARE-interacting protein that appears to act as negative regulator of constitutive exocytosis . Moreover, this regulation appears specific to one of two parallel exocytic paths which are operant in yeast cells.

Mol Cell Biol, 1999 Jun, 19(6), 4324 - 33
A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1; Hach A et al.; Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen . In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1) . Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain . To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated . Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation . All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme . More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme . Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC . These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.

Mol Cell Biol, 1999 Jun, 19(6), 4231 - 40
Active role of a human genomic insert in replication of a yeast artificial chromosome; van Brabant AJ et al.; Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA . The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not . We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus . The human insert contains multiple sites that serve as origins of replication . The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences . In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes . We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

Mol Cell Biol, 1999 Jun, 19(6), 4153 - 8
Increased instability of human CTG repeat tracts on yeast artificial chromosomes during gametogenesis; Cohen H et al.; Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases . The mechanism and timing of the expansion events are poorly understood, however . We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable . The instability is 6 to 10 times more pronounced in meiosis than during mitotic division . The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present . Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats . The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions . In our experiments, expansions were found almost exclusively during gametogenesis . Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability . These unique patterns have novel implications for possible mechanisms of repeat instability.

Mol Cell Biol, 1999 Jun, 19(6), 4143 - 52
Accumulation of single-stranded DNA and destabilization of telomeric repeats in yeast mutant strains carrying a deletion of RAD27; Parenteau J et al.; The Saccharomyces cerevisiae RAD27 gene encodes the yeast homologue of the mammalian FEN-1 nuclease, a protein that is thought to be involved in the processing of Okazaki fragments during DNA lagging-strand synthesis . One of the predicted DNA lesions occurring in rad27 strains is the presence of single-stranded DNA of the template strand for lagging-strand synthesis . We examined this prediction by analyzing the terminal DNA structures generated during telomere replication in rad27 strains . The lengths of the telomeric repeat tracts were found to be destabilized in rad27 strains, indicating that naturally occurring direct repeats are subject to tract expansions and contractions in such strains . Furthermore, abnormally high levels of single-stranded DNA of the templating strand for lagging-strand synthesis were observed in rad27 cells . Overexpression of Dna2p in wild-type cells also yielded single-stranded DNA regions on telomeric DNA and caused a cell growth arrest phenotype virtually identical to that seen for rad27 cells grown at the restrictive temperature . Furthermore, overexpression of the yeast exonuclease Exo1p alleviated the growth arrest induced by both conditions, overexpression of Dna2p and incubation of rad27 cells at 37 degrees C . However, the telomere heterogeneity and the appearance of single-stranded DNA are not prevented by the overexpression of Exo1p in these strains, suggesting that this nuclease is not simply redundant with Rad27p . Our data thus provide in vivo evidence for the types of DNA lesions predicted to occur when lagging-strand synthesis is deficient and suggest that Dna2p and Rad27p collaborate in the processing of Okazaki fragments.

J Biol Chem, 1999 May 21, 274(21), 15251 - 61
Deletion of GPI7, a yeast gene required for addition of a side chain to the glycosylphosphatidylinositol (GPI) core structure, affects GPI protein transport, remodeling, and cell wall integrity; Benachour A et al.; Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins . Gpi7 mutants are deficient in YJL062w, herein named GPI7 . GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility . Several aspects of GPI biosynthesis are disturbed in Deltagpi7 . The extent of anchor remodeling, i.e . replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed . Gpi7p is a highly glycosylated integral membrane protein with 9-11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain . The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum . GPI7 has homologues in Saccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown . Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure . Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiester-linked side chain, possibly an ethanolamine phosphate . Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the alpha1,6-linked mannose of the GPI core structure.

J Biol Chem, 1999 May 21, 274(21), 15151 - 8
A novel nuclear export signal sensitive to oxidative stress in the fission yeast transcription factor Pap1; Kudo N et al.; Pap1, a fission yeast AP-1-like transcription factor, is negatively regulated by CRM1/exportin 1, the nuclear export factor . Pap1 was localized normally in the cytoplasm but was accumulated in the nucleus when Crm1 was inactivated by a temperature-sensitive mutation or by treatment with leptomycin B, a specific export inhibitor . Deletion of the C-terminal cysteine-rich domain (CRD) resulted in nuclear accumulation of Pap1, while a glutathione S-transferase-green fluorescent protein-CRD fusion protein was localized in the cytoplasm in a Crm1-dependent manner . Deletion and mutational analyses identified several important amino acids in a 19-amino acid region in the CRD as a nuclear export signal (NES) . Strikingly, a cysteine residue (Cys-532), in addition to two leucines and an isoleucine, was important for the NES function and the presence of at least one of the two cysteine residues was essential . Unlike classical NESs such as the human immunodeficiency virus Rev NES, the Pap1 NES lost the function upon treatment with oxidants such as diethyl maleate . The oxidative stress response is conserved through evolution, as green fluorescent protein-fused proteins bearing the Pap1 NES expressed in mammalian cells responded to diethyl maleate . These results show that the hydrophobic amino acid-rich region containing two important cysteines in Pap1 serves as a novel NES, which is sensitive to oxidative stress.

J Biol Chem, 1999 May 21, 274(21), 14655 - 61
Transmembrane topography of the 100-kDa a subunit (Vph1p) of the yeast vacuolar proton-translocating ATPase; Leng XH et al.; The membrane topography of the yeast vacuolar proton-translocating ATPase a subunit (Vph1p) has been investigated using cysteine-scanning mutagenesis . A Cys-less form of Vph1p lacking the seven endogenous cysteines was constructed and shown to have 80% of wild type activity . Single cysteine residues were introduced at 13 sites within the Cys-less mutant, with 12 mutants showing greater than 70% of wild type activity . To evaluate their disposition with respect to the membrane, vacuoles were treated in the presence or absence of the impermeant sulfhydryl reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) followed by the membrane permeable sulfhydryl reagent 3-(N-maleimidylpropionyl) biocytin (MPB) . Three of the 12 active cysteine mutants were not labeled by MPB . The mutants E3C, D89C, T161C, S266C, N447C, K450C, and S703C were labeled by MPB in an AMS-protectable manner, suggesting a cytoplasmic orientation, whereas G602C and S840C showed minimal protection by AMS, suggesting a lumenal orientation . Factor Xa cleavage sites were introduced at His-499, Leu-560, and Pro-606 . Cleavage at 560 was observed in the absence of detergent, suggesting a cytoplasmic orientation for this site . Based on these results, we propose a model of the a subunit containing nine transmembrane segments, with the amino terminus facing the cytoplasm and the carboxyl terminus facing the lumen.

EMBO J, 1999 May 17, 18(10), 2836 - 44
Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC; Moreira JM et al.; In eukaryotes, DNA is packaged into chromatin, a compact structure that must be disrupted when genes are transcribed by RNA polymerase II . For transcription to take place, chromatin is remodeled via nucleosome disruption or displacement, a fundamental transcriptional regulatory mechanism in eukaryotic organisms . Here we show that the yeast chromatin-remodeling complex, RSC (remodels the structure of chromatin), isolated on the basis of homology to the SWI/SNF complex, is required for proper transcriptional regulation and nucleosome positioning in the highly inducible CHA1 promoter . In the absence of Sth1p/Nps1p (a homolog of Swi2p/Snf2p) or of Swh3p (a homolog of Swi3p), expression of CHA1 in non-induced cells is increased to a level comparable with that of fully induced cells . Furthermore, in non-induced cells depleted for Sth1p/Nps1p or Swh3p, a nucleosome positioned over the TATA box of the CHA1 promoter is disrupted, an architectural change normally only observed during transcriptional induction . In addition, deletion of the gene-specific activator Cha4p did not affect derepression of CHA1 in cells depleted for Swh3p . Thus, CHA1 constitutes a target for the RSC complex, and we propose that RSC is essential for maintaining a repressive chromatin structure at the CHA1 promoter.

EMBO J, 1999 May 17, 18(10), 2782 - 92
Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast; Jiang Y et al.; Tor proteins, homologous to DNA-dependent protein kinases, participate in a signal transduction pathway in yeast that regulates protein synthesis and cell wall expansion in response to nutrient availability . The anti-inflammatory drug rapamycin inhibits yeast cell growth by inhibiting Tor protein signaling . This leads to diminished association of a protein, Tap42, with two different protein phosphatase catalytic subunits; one encoded redundantly by PPH21 and PPH22, and one encoded by SIT4 . We show that inactivation of either Cdc55 or Tpd3, which regulate Pph21/22 activity, results in rapamycin resistance and that this resistance correlates with an increased association of Tap42 with Pph21/22 . Furthermore, we show Tor-dependent phosphorylation of Tap42 both in vivo and in vitro and that this phosphorylation is rapamycin sensitive . Inactivation of Cdc55 or Tpd3 enhances in vivo phosphorylation of Tap42 . We conclude that Tor phosphorylates Tap42 and that phosphorylated Tap42 effectively competes with Cdc55/Tpd3 for binding to the phosphatase 2A catalytic subunit . Furthermore, Cdc55 and Tpd3 promote dephosphorylation of Tap42 . Thus, Tor stimulates growth-promoting association of Tap42 with Pph21/22 and Sit4, while Cdc55 and Tpd3 inhibit this association both by direct competition and by dephosphorylation of Tap42 . These results establish Tap42 as a target of Tor and add further refinement to the Tor signaling pathway.

Virology, 1999 May 25, 258(1), 95 - 9
Potyvirus helper component-proteinase self-interaction in the yeast two-hybrid system and delineation of the interaction domain involved; Urcuqui-Inchima S et al.; Using the yeast two-hybrid system, a screen was performed for possible interactions between the proteins encoded by the 5' region of potyviral genomes {P1, helper component-proteinase (HC-Pro), and P3} . A positive self-interaction involving HC-Pro was detected with lettuce mosaic virus (LMV) and potato virus Y (PVY) . The possibility of heterologous interaction between the HC-Pro of LMV and of PVY was also demonstrated . No interaction involving either the P1 or the P3 proteins was detected . A series of ordered deletions from either the N- or C-terminal end of the LMV HC-Pro was used to map the domain involved in interaction to the 72 N-terminal amino acids of the protein, a region known to be dispensable for virus viability but necessary for aphid transmission . A similar but less detailed analysis mapped the interacting domain to the N-terminal half of the PVY HC-Pro .

Biochem Biophys Res Commun, 1999 May 19, 258(3), 679 - 84
A family of mammalian proteins homologous to yeast Sec24p; Tang BL et al.; The Sec23p/Sec24p complex is a component of yeast coat protein II (COPII), the coat protein complex responsible for vesicle budding from the endoplasmic reticulum (ER) . Database searches and molecular cloning reveal that four different mammalian Sec24p-like proteins exist, all with about 20% amino acid identity with the yeast Sec24p . Sec24A and Sec24B share about 50% amino acid identity . Sec24D is cloned by screening a human pancreas of cDNA library with an expressed sequence tag (EST) fragment that is homologous to, but distinct from, Sec24A and Sec24B . Sec24D shares about 50% amino acid identity with the gene product of KIAA0079, which we have designated as Sec24C . These mammalian Sec24s appear to form two subclasses based on homology . Sec24A/B and Sec24C/D share about 20% identity with each other and with the yeast Sec24p . The Sec24 sequences also share weak but significant homology to the mammalian Sec23A and Sec23B . Northern blot analysis revealed that Sec24C is ubiquitously expressed . Although Sec24D transcripts are detectable in all tissues examined, they are selectively enriched in certain tissues, particularly placenta and pancreas . myc-tagged Sec24C and sec24D colocalized with Sec13, another COPII component . This colocalization suggests that Sec24C and Sec24D are indeed associated with COPII structures on membranes of the ER-Golgi boundary . The existence of at least four forms of Sec24 in mammalian cells suggest that multiple forms of COPII complex may be involved in ER export .

Biochem Biophys Res Commun, 1999 May 19, 258(3), 611 - 5
A gene coding for a ribosomal protein L41 in cycloheximide-resistant ribosomes has a promoter which is upregulated under the growth-inhibitory conditions in yeast, Candida maltosa; Mutoh E et al.; We previously found by using yeast, Candida maltosa, that cycloheximide (CYH) sensitivity of ribosomes is dependent on the 56th amino acid residues of a ribosomal protein, L413 (proline in sensitive and glutamine in resistant ribosomes) . We also revealed that in this yeast, which has both L41-P type and L41-Q type genes, the expression of the latter type genes is induced by the addition of CYH in the medium to make the cells inducibly resistant to CYH . In this paper, we analyzed the promoter region of L41-Q2a, one of the CYH-inducible L41-Q type genes and found two elements required for the induction of expression: one was a GCRE (Gcn4p-responsive element of Saccharomyces cerevisiae)-like element and the other was a GT-rich element . This promoter region was also required for its expression under some other growth inhibitory conditions . Furthermore, it was suggested that Q-type ribosomes synthesized under these conditions are more resistant to these inhibitory conditions .

J Mol Biol, 1999 May 14, 288(4), 511 - 20
Mutagenesis of yeast TFIIIB70 reveals C-terminal residues critical for interaction with TBP and C34; Andrau JC et al.; The yeast TFIIIB transcription factor is composed of three components, TBP, TFIIIB90 or B", and TFIIIB70 or BRF . TFIIIB70 is a pivotal component since it interacts with TBP, TFIIIC and RNA polymerase III (pol III) . In order to better understand the role of TFIIIB70, we mutagenized extensively three evolutionary conserved motifs of its pol III-specific C-terminal extension . Conditional mutations lying in conserved regions II and III were obtained, some of which altered the interaction with the C34 subunit of pol III and were co-lethal with rpc34 mutations . Two conditional mutations in region II impaired the interaction with TBP and were suppressed by its overexpression . The pattern of suppression of the strongest mutation by overexpression of various mutant TBP, suggested a contact between TBP-R220 and TFIIIB70-D464 residues in vivo . As expected, this TFIIIB70 mutation impaired the assembly of TFIIIB . TFIIIC.DNA complexes and affected in vitro transcription of the SUP4 tRNA gene . Our results underscore the important role of region II of TFIIIB70 in pre-initiation as well as transcription complex assembly via C34 and TBP binding .

J Mol Biol, 1999 May 14, 288(4), 505 - 10
Reverse transcription of the yeast Ty1 retrotransposon: the mode of first strand transfer is either intermolecular or intramolecular; Wilhelm M et al.; Replication of the yeast Ty1 retrotransposon occurs by a mechanism similar to that of retroviruses . According to the current model of retroviral reverse transcription, two strand transfers (the so-called minus-strand and plus-strand strong-stop DNA transfers) are required to produce full-length preintegrative DNA . Because two genomic RNA molecules are packaged inside the viral particles, the strand transfers can be either intra- or intermolecular . To study the mode of transfer of minus-strand strong-stop DNA during reverse transcription of the yeast Ty1 retrotransposon, we have analyzed the cDNA products that accumulate in the cytoplasmic virus-like particles of yeast cells harboring two marked Ty1 elements . Our results indicate that Ty1 minus-strand transfer occurs in a random manner with approximately similar frequencies of intra- and intermolecular transfer . It has been observed recently that intra- and intermolecular minus-strand transfer occur at similar frequencies during replication of a complex retrovirus such as HIV-1 . These results together with the observation that genetic recombination occurs with a high frequency during minus-strand synthesis suggest that both packaged RNA molecules are needed for the synthesis of one minus-strand DNA .

J Mol Biol, 1999 Apr 30, 288(2), 231 - 42
Active site mapping of yeast aspartyl-tRNA synthetase by in vivo selection of enzyme mutations lethal for cell growth; Ador L et al.; The active site of yeast aspartyl-tRNA synthetase has been characterised by structural and functional approaches . However, residues or structural elements that indirectly contribute to the active site organisation have still to be described . They have not been assessed by simple analysis of structural data or site-directed mutagenesis analysis, since rational targetting has proven difficult . Here, we attempt to locate these functional features by using a genetic selection method to screen a randomly mutated yeast AspRS library for mutations lethal for cell growth . This approach is an efficient method to map the active site residues, since of the 23 different mutations isolated, 13 are in direct contact with the substrates . Most of the mutations are located in a 15 A radius sphere around the ATP molecule, where they affect the very conserved residues of the class-defining motifs . The results also showed the importance of the dimer interface for the enzyme activity: a single mutation of the invariant proline residue of motif 1 led to a structural defect inactivating the enzyme . From in vivo complementation studies it appeared that the enzyme activity can be recovered by reconstitution of an intact interface through the formation of heterodimers . We also show that a single mutation affecting an interaction with G34 of the tRNA can inactivate the enzyme by inducing a relaxation of the tRNA recognition specificity . Finally, several mutants whose functional importance could not be assessed from the structural data were selected, demonstrating the importance of this type of approach in the context of a structure-function relationship study .

J Mol Biol, 1999 Apr 23, 288(1), 147 - 64
The relationship between protein structure and function: a comprehensive survey with application to the yeast genome; Hegyi H et al.; For most proteins in the genome databases, function is predicted via sequence comparison . In spite of the popularity of this approach, the extent to which it can be reliably applied is unknown . We address this issue by systematically investigating the relationship between protein function and structure . We focus initially on enzymes functionally classified by the Enzyme Commission (EC) and relate these to by structurally classified domains the SCOP database . We find that the major SCOP fold classes have different propensities to carry out certain broad categories of functions . For instance, alpha/beta folds are disproportionately associated with enzymes, especially transferases and hydrolases, and all-alpha and small folds with non-enzymes, while alpha+beta folds have an equal tendency either way . These observations for the database overall are largely true for specific genomes . We focus, in particular, on yeast, analyzing it with many classifications in addition to SCOP and EC (i.e . COGs, CATH, MIPS), and find clear tendencies for fold-function association, across a broad spectrum of functions . Analysis with the COGs scheme also suggests that the functions of the most ancient proteins are more evenly distributed among different structural classes than those of more modern ones . For the database overall, we identify the most versatile functions, i.e . those that are associated with the most folds, and the most versatile folds, associated with the most functions . The two most versatile enzymatic functions (hydro-lyases and O-glycosyl glucosidases) are associated with seven folds each . The five most versatile folds (TIM-barrel, Rossmann, ferredoxin, alpha-beta hydrolase, and P-loop NTP hydrolase) are all mixed alpha-beta structures . They stand out as generic scaffolds, accommodating from six to as many as 16 functions (for the exceptional TIM-barrel) . At the conclusion of our analysis we are able to construct a graph giving the chance that a functional annotation can be reliably transferred at different degrees of sequence and structural similarity . Supplemental information is available from + .

Arch Biochem Biophys, 1999 May 15, 365(2), 279 - 88
Yeast mutants of glucose metabolism with defects in the coordinate regulation of carbon assimilation; Dennis RA et al.; The enzymes of the glyoxylate cycle and gluconeogenesis are tightly regulated by transcriptional, posttranscriptional, and posttranslational mechanisms in Saccharomyces cerevisiae . We have previously identified four genes, ACN8, ACN9, ACN17, and ACN18, whose mutant phenotype includes two- to fourfold elevated levels of enzymes of the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism . The affected enzymes are elevated on nonfermentable carbon sources but are still fully repressed by glucose . Catabolite inactivation of the cytosolic malate dehydrogenase is not affected in the mutants . Instead, the phenotype appeared to be manifested primarily at the level of transcription . The ACN8, ACN17, and ACN18 genes were isolated by functional complementation of the respective mutant's inability to utilize acetate as a carbon and energy source, and these genes were shown to encode subunits of metabolic enzymes . ACN8 was identical to FBP1, which encodes the gluconeogenic enzyme, fructose 1,6-bisphosphatase, while ACN17 and ACN18 were identical to the SDH2 and SDH4 genes, respectively, that encode subunits of the respiratory chain and tricarboxylic acid cycle enzyme, succinate dehydrogenase . Mutants defective in other glyoxylate cycle and gluconeogenic enzymes also display the elevated enzyme phenotype, indicating that the enzyme superinduction is a general property of gluconeogenic dysfunction . Glucose 6-phosphate levels were diminished in the mutants, suggesting that endogenous glucose synthesis can regulate the expression of gluconeogenic enzymes .

J, Mar . Biotechnol. . 1998 Dec, 6(4), 255 - 259
Cell yield and superoxide dismutase activity of the marine yeast Debaryomyces hansenii under different culture conditions; Ramirez Orozco M et al.; The effect of aeration, pH, stirring rate, and temperature on the biomass production and superoxide dismutase (SOD) activity of the marine yeast Debaryomyces hansenii strain C-11 was determined . The cell biomass yield was approximately 50% in a seawater-formulated medium using glucose as the carbon source . The SOD activity increased by application of a pulse of oxygen or 0.8 mM sulfate copper into the chemical reactor . The SOD enzyme had an activity of 400 units/mg of protein in a crude extract produced under such conditions, the best activity ever reported for this enzyme in a crude preparation.

J Clin Microbiol, 1999 Jun, 37(6), 2040 - 1
Simple strategy for direct identification of medically important yeast species from positive blood culture vials; Sheppard DC et al.; We compared direct inoculation of the Auxacolor yeast identification system from positive blood culture vials to standard identification with the API 20C AUX (API 20C), using 44 prospectively collected clinical specimens and 25 seeded blood culture vials . Direct inoculation of the Auxacolor system was accurate and more rapid than standard identification with the API 20C.

Genes Dev, 1999 May 1, 13(9), 1190 - 202
Cyclin-dependent kinase and Cks/Suc1 interact with the proteasome in yeast to control proteolysis of M-phase targets; Kaiser P et al.; Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes . Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome . We report here that components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome . A mutation in Cdc28 (cdc28-1N) that interferes with Cks1 binding, or inactivation of Cks1 itself, confers stabilization of Clb2, the principal mitotic B-type cyclin in budding yeast . Surprisingly, Clb2-ubiquitination in vivo and in vitro is not affected by mutations in cks1, indicating that Cks1 is not essential for cyclosome/APC activity . However, mutant Cks1 proteins no longer physically interact with the proteasome, suggesting that Cks1 is required for some aspect of proteasome function during M-phase-specific proteolysis . We further provide evidence that Cks1 function is required for degradation of the anaphase inhibitor Pds1 . Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially relieves the metaphase block in these mutants.

Genes Dev, 1999 May 1, 13(9), 1140 - 55
A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore; Ortiz J et al.; We have established a one-hybrid screen that allows the in vivo localization of proteins at a functional Saccharomyces cerevisiae centromere . Applying this screen we have identified three proteins-Ctf19, Mcm21, and the product of an unspecified open reading frame that we named Okp1-as components of the budding yeast centromere . Ctf19, Mcm21, and Okp1 most likely form a protein complex that links CBF3, a protein complex directly associated with the CDE III element of the centromere DNA, with further components of the budding yeast centromere, Cbf1, Mif2, and Cse4 . We demonstrate that the CDE III element is essential and sufficient to localize the established protein network to the centromere and propose that the interaction of the CDE II element with the CDE III localized protein complex facilitates a protein-DNA conformation that evokes the active centromere.

Curr Opin Microbiol, 1999 Apr, 2(2), 202 - 7
Glucose repression in yeast; Carlson M; The Snf1 protein kinase is a central component of the signaling pathway for glucose repression in yeast . Recent studies have addressed the regulation of Snf1 kinase activity and elucidated mechanisms by which Snf1 controls repression and activation of glucose-repressed genes . Important advances include evidence that Snf1 regulates the localization of the Mig1 repressor and that Snf1 functions at multiple points to control Cat8 and Sip4, the activators of gluconeogenic genes.

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