S. cerevisiae

Biochim Biophys Acta, 2001 Apr 23, 1538(2-3), 181 - 9
Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae; Rudoni S et al.; The CDC25 gene product is a guanine nucleotide exchange factor for Ras proteins in yeast . Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction . To test this hypothesis we measured the levels of nucleotides in yeast cells under different growth conditions and the relative amount of Ras2-GTP . The intracellular GTP/GDP ratio was found to be very sensitive to growth conditions: the ratio is high, close to that of ATP/ADP during exponential growth, but it decreases rapidly before the beginning of stationary phase, and it drops further under starvation conditions . The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio . The relative amount of Ras2-GTP changes in a parallel way suggesting that there is a correlation with the cytosolic GTP/GDP ratio . In addition 'in vitro' mixed-nucleotide exchange experiments done on purified Ras2 protein demonstrated that the GTP and GDP concentrations influence the extent of Ras2-GTP loading giving further support to their possible regulatory role.

Biochem J, 2001 May 15, 356(Pt 1), 207 - 15
Self-association and precursor protein binding of Saccharomyces cerevisiae Tom40p, the core component of the protein translocation channel of the mitochondrial outer membrane; Gordon DM et al.; The precursor protein translocase of the mitochondrial outer membrane (Tom) is a multi-subunit complex containing receptors and a general import channel, of which the core component is Tom40p . Nuclear-encoded mitochondrial precursor proteins are first recognized by surface receptors and then pass through the import channel . The Tom complex has been purified; however, the protein-protein interactions that drive its assembly and maintain its stability have been difficult to study . Here we show that Saccharomyces cerevisiae Tom40p expressed in bacteria and purified to homogeneity associates efficiently with itself . The self-association is very strong and can withstand up to 4 M urea or 1 M salt . The tight self-association does not require the N-terminal segment of Tom40p . Furthermore, purified Tom40p preferentially recognizes the targeting sequence of mitochondrial precursor proteins . Although the binding of the targeting sequence to Tom40p is inhibited by urea concentrations in excess of 1 M, it is moderately resistant to 1 M salt . Simultaneous self-assembly and precursor protein binding suggest that Tom40p contains at least two different domains mediating these processes . The experimental approach described here should be useful for analysing protein-protein interactions involving individual or groups of components of the mitochondrial import machinery.

Genetics, 2001 May, 158(1), 167 - 76
Protosilencers in Saccharomyces cerevisiae subtelomeric regions; Lebrun E et al.; Saccharomyces cerevisiae subtelomeric repeats contain silencing elements such as the core X sequence, which is present at all chromosome ends . When transplaced at HML, core X can enhance the action of a distant silencer without acting as a silencer on its own, thus fulfilling the functional definition of a protosilencer . Here we show that an ACS motif and an Abf1p-binding site participate in the silencing capacity of core X and that their effects are additive . In addition, in a variety of settings, core X was found to bring about substantial gene repression only when a low level of silencing was already detectable in its absence . Adjoining an X-STAR sequence, which naturally abuts core X in subtelomeric regions, did not improve the silencing capacity of core X . We propose that protosilencers play a major role in a variety of silencing phenomena, as is the case for core X, which acts as a silencing relay, prolonging silencing propagation away from telomeres.

Genetics, 2001 May, 158(1), 145 - 54
The Saccharomyces cerevisiae suppressor of choline sensitivity (SCS2) gene is a multicopy Suppressor of mec1 telomeric silencing defects; Craven RJ et al.; Mec1p is a cell cycle checkpoint protein related to the ATM protein kinase family . Certain mec1 mutations or overexpression of Mec1p lead to shortened telomeres and loss of telomeric silencing . We conducted a multicopy suppressor screen for genes that suppress the loss of silencing in strains overexpressing Mec1p . We identified SCS2 (suppressor of choline sensitivity), a gene previously isolated as a suppressor of defects in inositol synthesis . Deletion of SCS2 resulted in decreased telomeric silencing, and the scs2 mutation increased the rate of cellular senescence observed for mec1-21 tel1 double mutant cells . Genetic analysis revealed that Scs2p probably acts through a different telomeric silencing pathway from that affected by Mec1p.

Genetics, 2001 May, 158(1), 109 - 22
Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1; Rattray AJ et al.; Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ) . We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair . Here we describe a genetic screen for mutants that affect the fidelity of DSB repair . The substrate consists of inverted repeats of the trp1 and CAN1 genes . Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants . Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants . In wild-type cells the recombinational repair process is efficient and fairly accurate . Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations . We isolated several mutant strains with altered fidelity of recombination . Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants . The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1 . Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ . Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain . Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.

Genetics, 2001 May, 158(1), 95 - 107
Saccharomyces cerevisiae SMT4 encodes an evolutionarily conserved protease with a role in chromosome condensation regulation; Strunnikov AV et al.; In a search for regulatory genes affecting the targeting of the condensin complex to chromatin in Saccharomyces cerevisiae, we identified a member of the adenovirus protease family, SMT4 . SMT4 overexpression suppresses the temperature-sensitive conditional lethal phenotype of smc2-6, but not smc2-8 or smc4-1 . A disruption allele of SMT4 has a prominent chromosome phenotype: impaired targeting of Smc4p-GFP to rDNA chromatin . Site-specific mutagenesis of the predicted protease active site cysteine and histidine residues of Smt4p abolishes the SMT4 function in vivo . The previously uncharacterized SIZ1 (SAP and Miz) gene, which encodes a protein containing a predicted DNA-binding SAP module and a Miz finger, is identified as a bypass suppressor of the growth defect associated with the SMT4 disruption . The SIZ1 gene disruption is synthetically lethal with the SIZ2 deletion . We propose that SMT4, SIZ1, and SIZ2 are involved in a novel pathway of chromosome maintenance.

Biosci Biotechnol Biochem, 2001 Mar, 65(3), 728 - 31
MUP1, high affinity methionine permease, is involved in cysteine uptake by Saccharomyces cerevisiae; Kosugi A et al.; Using a mutant defective in cysteine uptake, which is resistant to a toxic analog of cysteine, allylglycine, we searched for a gene that complements the defect in cysteine uptake in a yeast genomic library and found a DNA fragment causing the recovery of cysteine uptake and sensitivity to allylglycine . The gene in the fragment was identical to MUP1, the high affinity methionine permease gene . We conclude that Mup1 is a major permease in cysteine uptake.

Cell Biochem Biophys, 2000, 32 Spring, 1 - 8
In silicio search for genes encoding peroxisomal proteins in Saccharomyces cerevisiae; Kal AJ et al.; The biogenesis of peroxisomes involves the synthesis of new proteins that after, completion of translation, are targeted to the organelle by virtue of peroxisomal targeting signals (PTS) . Two types of PTSs have been well characterized for import of matrix proteins (PTS1 and PTS2) . Induction of the genes encoding these matrix proteins takes place in oleate-containing medium and is mediated via an oleate response element (ORE) present in the region preceding these genes . The authors have searched the yeast genome for OREs preceding open reading frames (ORFs), and for ORFs that contain either a PTS1 or PTS2 . Of the ORFs containing an ORE, as well as either a PTS1 or a PTS2, many were known to encode bona fide peroxisomal matrix proteins . In addition, candidate genes were identified as encoding putative new peroxisomal proteins . For one case, subcellular location studies validated the in silicio prediction . This gene encodes a new peroxisomal thioesterase.

J Cell Sci, 2001 May, 114(Pt 10), 1935 - 47
End13p/Vps4p is required for efficient transport from early to late endosomes in Saccharomyces cerevisiae; Zahn R et al.; end13-1 was isolated in a screen for endocytosis mutants and has been shown to have a post-internalisation defect in endocytic transport as well as a defect in vacuolar protein sorting (Vps(-) phenotype), leading to secretion of newly synthesised vacuolar proteins . Here we demonstrate that END13 is identical to VPS4, encoding an AAA (ATPase associated with a variety of cellular activities)-family ATPase . We also report that the end13-1 mutation is a serine 335 to phenylalanine substitution in the AAA-ATPase domain of End13p/Vps4p . It has been reported that mutant cells lacking End13p/Vps4p (end13(vps4)Delta) accumulate endocytosed marker dyes, plasma membrane receptors and newly synthesised vacuolar hydrolase precursors in an endosomal compartment adjacent to the vacuole (prevacuolar compartment, or PVC) . We find, however, that the end13 mutants have defects in transport of endocytosed fluorescent dyes, plasma membrane receptors and ligands from small peripherally located early endosomes to larger late endosomes, which are often located adjacent to the vacuole . Our results indicate that End13p/Vps4p may play an important role in multiple steps of membrane traffic through the endocytic pathway.

Yeast, 2001 May, 18(7), 663 - 70
Alterations of the glucose metabolism in a triose phosphate isomerase-negative Saccharomyces cerevisiae mutant; Compagno C et al.; The absence of triose phosphate isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996) . Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures . The Deltatpi1 null mutant was unable to grow on glucose as the sole carbon source . The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose . The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Deltatpi1 mutant could be a cause of the impaired growth on glucose . The amount of glycerol production in the Deltatpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis . Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Deltatpi1 mutant may occur in relation to the fraction of ethanol present in the media .

J Biochem (Tokyo), 2001 May, 129(5), 827 - 33
Characterization of a Saccharomyces cerevisiae gene that encodes a mitochondrial phosphate transporter-like protein; Takabatake R et al.; The mitochondrial phosphate transporter of Saccharomyces cerevisiae, encoded by MIR1 (YJR077C) gene, shows divergence among the transporters in various eukaryotes . We have characterized another gene, YER053C, that appeared to encode an orthologous mitochondrial phosphate transporter of yeast . The predicted amino acid sequence of the YER053C protein is much more similar to that of mitochondrial phosphate transporters of other species than that of MIR1 . RNA gel blot analysis indicated that, like the MIR1 promoter, the YER053C promoter is functional and that its activity varies according to aeration . An MIR1 gene null mutant did not grow on glycerol medium, whereas a YER053C null mutant grew well on the medium, suggesting that the YER053C gene is not essential for the mitochondrial function . YER053C also did not support the growth of the MIR1 null mutant on glycerol . The MIR1 and YER053C proteins were expressed in Escherichia coli and then reconstituted into liposomes . Unlike the proteoliposomes of MIR1, those of YER053C did not exhibit significant phosphate transport activity . Unexpectedly, it was shown that YER053C is localized in vacuoles, not mitochondria, by immunological electron microscopy . These results suggest that, during evolution, yeast lost the function and/or mitochondrial targeting of YER053C and then recruited an atypical MIR1 as the only transporter.

Biochemistry, 2001 Feb 27, 40(8), 2599 - 605
Dynamics of histone acetylation in Saccharomyces cerevisiae; Waterborg JH; Rates of turnover for the posttranslational acetylation of core histones were measured in logarithmically growing yeast cells by radioactive acetate labeling to near steady-state conditions . On average, acetylation half-lives were approximately 15 min for histone H4, 10 min for histone H3, 4 min for histone H2B, and 5 min for histone H2A . These rates were much faster than the several hours that have previously been reported for the rate of general histone acetylation and deacetylation in yeast . The current estimates are in line with changes in histone acetylation detected directly at specific chromatin locations and the speed of changes in gene expression that can be observed . These results emphasize that histone acetylation within chromatin is subject to constant flux . Detailed analysis revealed that the turnover rates for acetylation of histone H3 are the same from mono- through penta-acetylated forms . A large fraction of acetylated histone H3, including possibly all tetra- and penta-acetylated forms, appears subject to acetylation turnover . In contrast, the rate of acetylation turnover for mono- and di-acetylated forms of histones H4 and H2B, and the fraction subject to acetylation turnover, was lower than for multi-acetylated forms of these histones . This difference may reflect the difference in location of these histones within the nucleosome, a difference in the spectrum of histone-specific acetylating and deacetylating enzymes, and a difference in the role of acetylation in different histones.

Biochem Biophys Res Commun, 2001 May 4, 283(2), 531 - 5
Multiple mechanisms regulate expression of low temperature responsive (LOT) genes in Saccharomyces cerevisiae; Zhang L et al.; Using cDNA subtraction screening, we identified five Saccharomyces cerevisiae genes whose expressions is up-regulated when culture temperature was down-shifted from 30 to 10 degrees C . Among these LOT (low temperature-responsive) genes, three (LOT1, LOT2, and LOT3) were identical to FBA1, RPL2B, and NOP1, encoding a fructose biphosphate aldolase, a ribosomal protein L2B, and a nucleolar protein for rRNA processing, respectively . No functions were assigned for LOT5 and LOT6, which are identical to YKL183w and YLR011w, respectively . Northern hybridization analysis revealed that these genes are not uniformly regulated in response to the change of growth temperature . In addition, all the LOT genes, except for LOT1/FBA1, were induced by a low concentration of cycloheximide . The data indicate that multiple mechanisms, including translational functionality may be involved in the regulation of LOT gene expression in yeast.

Biochem Biophys Res Commun, 2001 May 4, 283(2), 526 - 30
Susceptibility of individual cells of Saccharomyces cerevisiae to the killer toxin K1; Bartunek M et al.; The susceptibility of sensitive yeast to killer toxins is known to depend on various factors, such as the selected killer toxin, the exposed yeast strain, its growth phase and the state of culture under given experimental conditions . The aim of this paper was to find whether individual cells from one culture are equally susceptible to the impact of the killer toxin . For this purpose the rhodamine B assay in a modified form was used . In order to observe the fate of individual cell the method of fluorescence video microscopy with a digital picture analysis was applied . Four selected groups of specific cells (with no, small, medium, and large bud, respectively) were investigated . Different sensitivity of Saccharomyces cerevisiae cells to the killer toxin K1 was observed in these cell groups . The most susceptible appeared to be the cells which were in S-phase (cells with the small buds); the least susceptible were the M-phase cells with large buds . The enhanced susceptibility in S-phase results probably from coincidence in higher porosity of the cell wall, accumulation of surface receptors, and enlarged growth activity at the surface cell structures.

IUBMB Life, 2000 Dec, 50(6), 371 - 7
Molecular analysis of the Saccharomyces cerevisiae YHR076w gene; Ramos CW et al.; Our results demonstrate that open reading frame (ORF) YHR076w on chromosome VIII of Saccharomyces cerevisiae that was previously described as a hypothetical gene is expressed . This ORF is transcribed as an mRNA of approximately 1,100 nucleotides . A 41.2-kDa polypeptide and three others predicted to be modified forms of Yhr076wp are detected by Western blot with a Yhr076wp-specific antibody . Promoter activity assays indicate that YHR076w transcription is regulated by carbon source and primarily by ethanol . Consistent with this observation, we have identified two potential ADR1 regulatory elements in the YHR076w upstream DNA region . Potential YAP1 and HSP elements are also identified in this region, suggesting other forms of regulation . YHR076w knockout strains do not exhibit any measurable growth or morphological phenotype under any conditions tested . However, increased YHR076w gene dosage confers a growth advantage to both wild-type and YHR076w knockout strains on 2% ethanol or 2% galactose medium in a low O2 growth environment . The fluorescence emitted by a Yhr076wp protein fusion to A . aquorin GFP colocalizes with the mitochondria in vivo.

J Bacteriol, 2001 May, 183(10), 3083 - 8
Glucose-induced monoubiquitination of the Saccharomyces cerevisiae galactose transporter is sufficient to signal its internalization; Horak J et al.; In Saccharomyces cerevisiae, the addition of glucose to cells growing on galactose induces internalization of the galactose transporter Gal2p and its subsequent proteolysis in the vacuole . Here we report that the essential step in Gal2p down-regulation is its ubiquitination through the Ubc1p-Ubc4p-Ubc5p triad of ubiquitin-conjugating enzymes and Npi1/Rsp5p ubiquitin-protein ligase . Moreover, Gal2p appears to be stabilized in mutant cells defective in the ubiquitin-hydrolase Npi2p/Doa4p, and the mutant phenotype can be reversed by overexpression of ubiquitin . An analysis of the fate of Gal2p in cells overexpressing wild-type ubiquitin as well as its variants incompetent to form polyubiquitin chains showed that monoubiquitination of Gal2p is sufficient to signal internalization of the protein into the endocytic pathway.

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 207 - 14
The pleitropic drug ABC transporters from Saccharomyces cerevisiae; Rogers B et al.; The Saccharomyces cerevisiae genome contains 16 genes encoding full-size ABC transporters . Each comprises two nucleotide binding folds (NBF) alternating with transmembrane domains (TM) . We have studied in detail three plasma membrane multidrug exporters: Pdr5p (TC3.A.1.205.1) and Snq2p (TC3.A.1.205.2) which share NBF-TM-NBF-TM topology as well as Yor1p (TC3.A.1.208.3) which exhibits the reciprocal TM-NBF-TM-NBF topology . The substrate specificity of Pdr5p, Snq2p and Yor1p are largely, but not totally, overlapping as shown by screening the growth inhibition by 349 toxic compounds of combinatorial deletants of these three ABC genes . Multiple deletion of 7 ABC genes (YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11 and PDR15) and of two transcription activation factors (PDR1 and PDR3) renders the cell from 2 to 200 times more sensitive to numerous toxic coumpounds including antifungals used in agriculture or medicine . The use of the pdr1-3 activating mutation and when necessary of the PDR5 promoter in appropriate multideleted hosts allow high levels of expression of Pdr5p, Snq2p or Yor1 p . These overexpressed proteins exhibit ATPase activity in vitro and confer considerable multiple drug resistance in vivo . The latter property can be used for screening specific inhibitors of fungal and other ABC transporters.

Microbiology, 2001 May, 147(Pt 5), 1149 - 59
Evaluation of in vivo activation of protein kinase A under non-dissociable conditions through the overexpression of wild-type and mutant regulatory subunits in Saccharomyces cerevisiae; Portela P et al.; BCY1-encoded protein kinase A (PKA) wild-type and mutant regulatory (R) subunits from Saccharomyces cerevisiae were inducibly overexpressed in their corresponding background strains containing the same mutation in the bcy1 gene . The aim of this approach was to shift the catalytic activity of PKA within the cell to the undissociated holoenzyme form(s) in order to evaluate whether the wild-type or the mutant forms of the holoenzyme could display catalytic activity . Two mutants of R subunits were used: bcy1-16, with a complete deletion of cAMP-binding domain B; and bcy1-14, with a small deletion in the carboxy terminus of cAMP-binding domain A . Their overexpression caused an increase in the level of R subunits in the range 40-90-fold, as detected by cAMP-binding activity, Coomasie-stained SDS-PAGE and Western blot analysis . The change in PKA activity attained by overexpression of R was assessed in three ways: (i) through the analysis of PKA-dependent phenotypes, and (ii, iii) by measurement of PKA activity -/+ cAMP using the specific substrate kemptide in crude extracts (ii) and permeabilized cells (iii) . Upon overexpression of the R subunits, PKA-dependent phenotypes were less severe when compared with their own background . However, a gradient in the degree of severity of phenotypes bcy1-14>bcy1-16> wild-type was observed in the background strains and was maintained in the strains overexpressing the R subunits . cAMP levels measured in background and in R-overexpressing strains showed an increase of around two orders accompanying the overexpression of the R subunits . Three main conclusions could be drawn from the PKA activity measurements -/+ cAMP in crude extracts: (i) catalytic activity was not increased in compensation for the increase in R subunits in any of the three cases (wild-type, bcy1-16 or bcy1-14 overexpression); (ii) PKA activity assayed in the absence of cAMP was lower in the case of extracts from strains overexpressing wild-type or bcy1-16 R subunits when compared with the corresponding extracts without overexpression; and (iii) in these two cases, the great excess of R subunits in the crude extracts displayed additional inhibitory capacity towards exogenously added catalytic (C) subunits . To provide an estimate of the in vivo activation of PKA, permeabilized cells from control strains and strains transformed with either wild-type, bcy1-16 or bcy1-14 R subunits were used to measure PKA activity in the presence of variable concentrations of cAMP . There were two main observations from the results: (i) the activity of PKA detected in the absence of exogenous cAMP was decreased in the strains overexpressing the R subunits when compared to their corresponding backgrounds, and (ii) the sensitivity to activation by cAMP was decreased or almost nil . The biochemical and genetic results obtained are consistent with the hypothesis that within the cell it is possible to have catalytically active, cAMP-bound, undissociated PKA holoenzyme.

FEMS Microbiol Lett, 2001 Apr 13, 197(2), 179 - 86
FTIR spectroscopic analysis of Saccharomyces cerevisiae cell walls: study of an anomalous strain exhibiting a pink-colored cell phenotype; Galichet A et al.; A new strain, exhibiting an intriguing pink-colored cell phenotype, was obtained after an encoding alpha-glucosidase gene from an archaebacteria Thermococcus hydrothermalis was cloned by functional complementation of a mal11 Saccharomyces cerevisiae mutant TCY70 . The possible implications of the alpha-glucosidase on the cell wall were evaluated by infrared spectroscopy and data indicate a 30% decrease in mannoproteins and an increase in beta-glucans . The loss of mannoproteins was confirmed by experiments on cells deprived of peptidomannans . Modifications in the major components of the cell wall did not jeopardize cell viability . Such rapid optical spectroscopic method can be used to screen a wide range of yeast mutants.

J Biol Chem, 2001 Jul 6, 276(27), 24588 - 93 Epub 2001 Apr 17.
Specific binding of single-stranded telomeric DNA by Cdc13p of Saccharomyces cerevisiae; Lin YC et al.; Cdc13p is a single strand telomere-binding protein of Saccharomyces cerevisiae; its telomere-binding region is within amino acids 451-693, Cdc13(451-693)p . In this study, we used purified Cdc13p and Cdc13(451-693)p to characterize their telomere binding activity . We found that the binding specificity of single-stranded TG(1-3) DNA by these two proteins is similar . However, the affinity of Cdc13(451-693)p to DNA was slightly lower than that of Cdc13p . The binding of telomeric DNA by these two proteins was disrupted at NaCl concentrations higher than 0.3 m, indicating that electrostatic interaction contributed significantly to the binding process . Because both proteins bound to strand TG(1-3) DNA positioned at the 3' end, the 5' end, or in the middle of the oligonucleotide substrates, our results indicated that the location of TG(1-3) in single-stranded DNA does not appear to be important for Cdc13p binding . Moreover, using DNase I footprint analysis, the structure of the telomeric DNA complexes of Cdc13p and Cdc13(451-693)p was analyzed . The DNase I footprints of these two proteins to three different telomeric DNA substrates were virtually identical, indicating that the telomere contact region of Cdc13p is within Cdc13(451-693)p . Together, the binding properties of Cdc13p and its binding domain support the theory that the specific binding of Cdc13p to telomeres is an important feature of telomeres that regulate telomerase access and/or differentiate natural telomeres from broken ends.

J Biol Chem, 2001 Jun 29, 276(26), 24068 - 74 Epub 2001 Apr 16.
Pho23 is associated with the Rpd3 histone deacetylase and is required for its normal function in regulation of gene expression and silencing in Saccharomyces cerevisiae; Loewith R et al.; The Rpd3 histone deacetylase (HDAC) functions in a large complex containing many proteins including Sin3 and Sap30 . Previous evidence indicates that the pho23, rpd3, sin3, and sap30 mutants exhibit similar defects in PHO5 regulation . We report that pho23 mutants like rpd3, sin3, and sap30 are hypersensitive to cycloheximide and heat shock and exhibit enhanced silencing of rDNA, telomeric, and HMR loci, suggesting that these genes are functionally related . Based on these observations, we explored whether Pho23 is a component of the Rpd3 HDAC complex . Our results demonstrate that Myc-Pho23 co-immunoprecipitates with HA-Rpd3 and HA-Sap30 . Furthermore, similar levels of HDAC activity were detected in immunoprecipitates of HA-Pho23, HA-Rpd3, or HA-Sap30 . In contrast, HDAC activity was not detected in immunoprecipitates of HA-Pho23 or HA-Sap30 from strains lacking Rpd3, suggesting that Rpd3 is the HDAC associated with these proteins . However, HDAC activity was detected in immunoprecipitates of HA-Sap30 or HA-Rpd3 from cells lacking Pho23, although levels were significantly lower than those detected in wild-type cells, indicating that Rpd3 activity is compromised in the absence of Pho23 . Together, our genetic and biochemical studies provide strong evidence that Pho23 is a component of the Rpd3 HDAC complex, and is required for the normal function of this complex.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 425 - 34
Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase; Gonzalez E et al.; Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae . The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH) . The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions . The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223 . BDH is inhibited competitively by 4-methylpyrazole with a K(i) of 34 microM . Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism . However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin . This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.

Antimicrob Agents Chemother, 2001 May, 45(5), 1589 - 90
Effect of potassium on Saccharomyces cerevisiae resistance to fluconazole; Stella CA et al.; Susceptibility of strain S288c of Saccharomyces cerevisiae to fluconazole was assayed in the presence and absence of KCl . Addition of 150 mM KCl renders the strain more sensitive to the antifungal agent . The effect is caused by the K(+) ion rather than the anion or the osmolarity of the medium . The increase in sensitivity does not modify the values of intracellular and extracellular pH established in the presence of KCl.

J Pept Res, 2001 Mar, 57(3), 188 - 92
Monomeric destetrapeptide human insulin from a precursor expressed in Saccharomyces cerevisiae; Cui DF et al.; Destetrapeptide insulin (DTI, human insulin with B27-30 removed) was obtained from a monomeric precursor (MIP) expressed in Saccharomyces cerevisiae through tryptic transpeptidation in the presence of synthetic tetrapeptide Gly-Phe-Phe-Tyr . The in vivo biological activity of DTI, determined by mouse convulsion assay, is 22 IU/mg . Its binding activity with insulin receptor on human placental membrane is 80% and its in vitro biological activity, determined by free fat cell assay, is 77% . Compared with native insulin, DTI molecules do not associate in solution but exist in the monomeric form, thus leading to its rapid utilization in vivo.

Eur J Biochem, 2001 Apr, 268(8), 2464 - 79
Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids; Maaheimo H et al.; Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional (13)C labeling of proteinogenic amino acids . This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1-metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine . The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways . In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e . phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive . In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only . The present study shows that fractional (13)C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.

EMBO J, 2001 Apr 17, 20(8), 2097 - 107
Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae; Kamimura Y et al.; Cdc45, which binds to the minichromosomal maintenance (Mcm) proteins, has a pivotal role in the initiation and elongation steps of chromosomal DNA replication in eukaryotes . Here we show that throughout the cell cycle in Saccharomyces cerevisiae, Cdc45 forms a complex with a novel factor, Sld3 . Consistently, Sld3 and Cdc45 associate simultaneously with replication origins in the chromatin immunoprecipitation assay: both proteins associate with early-firing origins in G(1) phase and with late-firing origins in late S phase . Moreover, the origin associations of Sld3 and Cdc45 are mutually dependent . The temperature-sensitive sld3 mutation confers a defect in DNA replication at the restrictive temperature and reduces an interaction not only between Sld3 and Cdc45, but also between Cdc45 and Mcm2 . These results suggest that the Sld3-Cdc45 complex associates with replication origins through Mcm proteins . At the restrictive temperature in sld3-5 cells, replication factor A, a single-strand DNA binding protein, does not associate with origins . Therefore, the origin association of Sld3-Cdc45 complex is prerequisite for origin unwinding in the initiation of DNA replication.

Mol Biol Cell, 2001 Apr, 12(4), 1147 - 60
Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae; Gaigg B et al.; Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:10(5) . A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Delta strain . Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis . In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50-70% . The reduced incorporation of {(3)H}myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport . The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids . Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures . The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

J Bacteriol, 2001 May, 183(9), 2881 - 7
Reciprocal regulation of anaerobic and aerobic cell wall mannoprotein gene expression in Saccharomyces cerevisiae; Abramova N et al.; The DAN/TIR genes encode nine cell wall mannoproteins in Saccharomyces cerevisiae which are expressed during anaerobiosis (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3, TIR4, and TIP1) . Most are expressed within an hour of an anaerobic shift, but DAN2 and DAN3 are expressed after about 3 h . At the same time, CWP1 and CWP2, the genes encoding the major mannoproteins, are down-regulated, suggesting that there is a programmed remodeling of the cell wall in which Cwp1 and Cwp2 are replaced by nine anaerobic counterparts . TIP1, TIR1, TIR2, and TIR4 are also induced during cold shock . Correspondingly, CWP1 is down-regulated during cold shock . As reported elsewhere, Mox4 is a heme-inhibited activator, and Mot3 is a heme-induced repressor of the DAN/TIR genes (but not of TIP1) . We show that CWP2 (but not CWP1) is controlled by the same factors, but in reverse fashion-primarily by Mot3 (which can function as either an activator or repressor) but also by Mox4, accounting for the reciprocal regulation of the two groups of genes . Disruptions of TIR1, TIR3, or TIR4 prevent anaerobic growth, indicating that each protein is essential for anaerobic adaptation . The Dan/Tir and Cwp proteins are homologous, with the greatest similarities shown within three subgroups: the Dan proteins, the Tip and Tir proteins, and, more distantly, the Cwp proteins . The clustering of homology corresponds to differences in expression: the Tip and Tir proteins are expressed during hypoxia and cold shock, the Dan proteins are more stringently repressed by oxygen and insensitive to cold shock, and the Cwp proteins are oppositely regulated by oxygen and temperature.

J Bacteriol, 2001 May, 183(9), 2874 - 80
Interaction of alpha-agglutinin and a-agglutinin, Saccharomyces cerevisiae sexual cell adhesion molecules; Zhao H et al.; alpha-Agglutinin and a-agglutinin are complementary cell adhesion glycoproteins active during mating in the yeast Saccharomyces cerevisiae . They bind with high affinity and high specificity: cells of opposite mating types are irreversibly bound by a few pairs of agglutinins . Equilibrium and surface plasmon resonance kinetic analyses showed that the purified binding region of alpha-agglutinin interacted similarly with purified a-agglutinin and with a-agglutinin expressed on cell surfaces . At 20 degrees C, the K(D) for the interaction was 2 x 10(-9) to 5 x 10(-9) M . This high affinity was a result of a very low dissociation rate ( approximately 2.6 x 10(-4) s(-1)) coupled with a low association rate (= 5 x 10(4) M(-1) s(-1)) . Circular-dichroism spectroscopy showed that binding of the proteins was accompanied by measurable changes in secondary structure . Furthermore, when binding was assessed at 10 degrees C, the association kinetics were sigmoidal, with a very low initial rate . An induced-fit model of binding with substantial apposition of hydrophobic surfaces on the two ligands can explain the observed affinity, kinetics, and specificity and the conformational effects of the binding reaction.

Genetics, 2001 Apr, 157(4), 1569 - 79
Cis-elements governing trinucleotide repeat instability in Saccharomyces cerevisiae; Rolfsmeier ML et al.; Trinucleotide repeat (TNR) instability in humans is governed by unique cis-elements . One element is a threshold, or minimal repeat length, conferring frequent mutations . Since thresholds have not been directly demonstrated in model systems, their molecular nature remains uncertain . Another element is sequence specificity . Unstable TNR sequences are almost always CNG, whose hairpin-forming ability is thought to promote instability by inhibiting DNA repair . To understand these cis-elements further, TNR expansions and contractions were monitored by yeast genetic assays . A threshold of approximately 15--17 repeats was observed for CTG expansions and contractions, indicating that thresholds function in organisms besides humans . Mutants lacking the flap endonuclease Rad27p showed little change in the expansion threshold, suggesting that this element is not altered by the presence or absence of flap processing . CNG or GNC sequences yielded frequent mutations, whereas A-T rich sequences were substantially more stable . This sequence analysis further supports a hairpin-mediated mechanism of TNR instability . Expansions and contractions occurred at comparable rates for CTG tract lengths between 15 and 25 repeats, indicating that expansions can comprise a significant fraction of mutations in yeast . These results indicate that several unique cis-elements of human TNR instability are functional in yeast.

Genetics, 2001 Apr, 157(4), 1531 - 42
The short life span of Saccharomyces cerevisiae sgs1 and srs2 mutants is a composite of normal aging processes and mitotic arrest due to defective recombination; McVey M et al.; Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability . Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging . Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background . In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants . Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena . A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells . This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint . Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles . Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest . This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination . Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.

Genetics, 2001 Apr, 157(4), 1481 - 91
Efficient incorporation of large (>2 kb) heterologies into heteroduplex DNA: Pms1/Msh2-dependent and -independent large loop mismatch repair in Saccharomyces cerevisiae; Clikeman JA et al.; DNA double-strand break (DSB) repair in yeast is effected primarily by gene conversion . Conversion can conceivably result from gap repair or from mismatch repair of heteroduplex DNA (hDNA) in recombination intermediates . Mismatch repair is normally very efficient, but unrepaired mismatches segregate in the next cell division, producing sectored colonies . Conversion of small heterologies (single-base differences or insertions <15 bp) in meiosis and mitosis involves mismatch repair of hDNA . The repair of larger loop mismatches in plasmid substrates or arising by replication slippage is inefficient and/or independent of Pms1p/Msh2p-dependent mismatch repair . However, large insertions convert readily (without sectoring) during meiotic recombination, raising the question of whether large insertions convert by repair of large loop mismatches or by gap repair . We show that insertions of 2.2 and 2.6 kbp convert efficiently during DSB-induced mitotic recombination, primarily by Msh2p- and Pms1p-dependent repair of large loop mismatches . These results support models in which Rad51p readily incorporates large heterologies into hDNA . We also show that large heterologies convert more frequently than small heterologies located the same distance from an initiating DSB and propose that this reflects Msh2-independent large loop-specific mismatch repair biased toward loop loss.

Genetics, 2001 Apr, 157(4), 1469 - 80
Isolation and characterization of WHI3, a size-control gene of Saccharomyces cerevisiae; Nash RS et al.; WHI3 is a gene affecting size control and cell cycle in the yeast Saccharomyces cerevisiae . The whi3 mutant has small cells, while extra doses of WHI3 produce large cells, and a large excess of WHI3 produces a lethal arrest in G1 phase . WHI3 seems to be a dose-dependent inhibitor of Start . Whi3 and its partially redundant homolog Whi4 have an RNA-binding domain, and mutagenesis experiments indicate that this RNA-binding domain is essential for Whi3 function . CLN3-1 whi3 cells are extremely small, nearly sterile, and largely nonresponsive to mating factor . Fertility is restored by deletion of CLN2, suggesting that whi3 cells may have abnormally high levels of CLN2 function.

Genetics, 2001 Apr, 157(4), 1451 - 67
Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae; van Nues RW et al.; Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes . Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins . DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures . Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome . We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p . The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p . Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.

Genetics, 2001 Apr, 157(4), 1437 - 50
A novel functional domain of Cdc15 kinase is required for its interaction with Tem1 GTPase in Saccharomyces cerevisiae; Asakawa K et al.; Exit from mitosis requires the inactivation of cyclin-dependent kinase (CDK) activity . In the budding yeast Saccharomyces cerevisiae, a number of gene products have been identified as components of the signal transduction network regulating inactivation of CDK (called the MEN, for the mitotic exit network) . Cdc15, one of such components of the MEN, is an essential protein kinase . By the two-hybrid screening, we identified Cdc15 as a binding protein of Tem1 GTPase, another essential regulator of the MEN . Coprecipitation experiments revealed that Tem1 binds to Cdc15 in vivo . By deletion analysis, we found that the Tem1-binding domain resides near the conserved kinase domain of Cdc15 . The cdc15-LF mutation, which was introduced into the Tem1-binding domain, reduced the interaction with Cdc15 and Tem1 and caused temperature-sensitive growth.The kinase activity of Cdc15 was not so much affected by the cdc15-LF mutation . However, Cdc15-LF failed to localize to the SPB at the restrictive temperature . Our data show that the interaction with Tem1 is important for the function of Cdc15 and that Cdc15 and Tem1 function in a complex to direct the exit from mitosis.

Mol Cell Biol, 2001 May, 21(9), 3144 - 58
Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion; Hanna JS et al.; CTF4 and CTF18 are required for high-fidelity chromosome segregation . Both exhibit genetic and physical ties to replication fork constituents . We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint . The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion . We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p . However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p . Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion . Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion . The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 65 - 71
The carboxy-terminal tail of the Ste2 receptor is involved in activation of the G protein in the Saccharomyces cerevisiae alpha-pheromone response pathway; Duran-Avelar MJ et al.; The Ste2 gene encodes the yeast alpha-pheromone receptor that belongs to the superfamily of seven-transmembrane G protein-coupled receptors . Binding of pheromone induces activation of the heterotrimeric G protein triggering growth arrest in G1 phase and induction of genes required for mating . By random PCR-mediated mutagenesis we isolated mutant 8L4, which presents a substitution of an asparagine residue by serine at position 388 of the alpha-factor receptor . The 8L4 mutant strain shows phenotypic defects such as: reduction in growth arrest after pheromone treatment, diminished activation of the Fus1 gene, and impaired mating competence . The asparagine residue lies in the second half of the intracellular protruding C-terminal tail of the receptor, and its replacement by serine affects interaction with both the G(alpha) and Gbeta subunits . Since expression of the receptor as well as its kinetic parameters, i.e., ligand affinity and receptor number, are unaffected in the mutant strain, we propose that association of the C-terminal tail of the receptor with G(alpha) and Gbeta subunits is required for proper activation of the heterotrimeric G protein . Besides its described role in downregulation and in formation of preactivation complex, the results here shown indicate that the C-terminal tail of the receptor plays an active role in transmitting the stimulus of mating pheromone to the heterotrimeric G protein.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 41 - 5
Anaerobiosis induces complex changes in sterol esterification pattern in the yeast Saccharomyces cerevisiae; Valachovic M et al.; Yeast Saccharomyces cerevisiae is auxotrophic for ergosterol in the absence of oxygen . We showed that complex changes in esterification of exogenously supplied sterols were also induced by anaerobiosis . Utilization of oleic acid for sterol esterification was significantly impaired in anaerobic cells . Furthermore, anaerobic cells fed different sterols exhibited striking variation in esterification efficiency (high levels of sterol esters for cholesterol and sitosterol, low levels for ergosterol, lanosterol or stigmasterol) . Relative activities of two yeast acylCoA:sterol acyltransferases (Are1p and Are2p) changed in response to anaerobiosis: while Are2p was dominant under aerobic conditions, Are1p provided the major activity in the absence of oxygen . Our results indicate that sterol esters may fulfil different roles in aerobic and anaerobic cells.

J Biol Chem, 2001 May 18, 276(20), 16936 - 43 Epub 2001 Feb 26.
The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase; Oyedotun KS et al.; The Saccharomyces cerevisiae succinate dehydrogenase (SDH) of the mitochondrial electron transport chain oxidizes succinate and reduces ubiquinone . Using a random mutagenesis approach, we identified functionally important amino acid residues in one of the anchor subunits, Sdh4p . We analyzed three point mutations (F69V, S71A, and H99L) and one nonsense mutation (Y89OCH) that truncates the Sdh4p subunit at the third predicted transmembrane segment . The F69V and the S71A mutations result in greatly impaired respiratory growth in vivo and quinone reductase activities in vitro, with negligible effects on enzyme stability . In contrast, the Y89OCH and the H99L mutations elicit large structural perturbations that impair assembly as evidenced by reduced covalent FAD levels, membrane-associated succinate-phenazine methosulfate reductase activities, and thermal stability . We propose that the Phe-69 and the Ser-71 residues are involved in the formation of a quinone-binding site, whereas the His-99 residue is at the interface of the peripheral and the membrane domains . In addition, the properties of the Y89OCH mutation are consistent with the interpretation that the third transmembrane segment is not involved in catalysis but rather plays an important structural role . The mutant enzymes are differentially sensitive to a quinone analog inhibitor, providing further evidence for a two-quinone binding model in the yeast SDH.

J Biol Chem, 2001 May 11, 276(19), 16265 - 70 Epub 2001 Feb 05.
Helical stalk segments S4 and S5 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae are optimized to impact catalytic site environment; Soteropoulos P et al.; The stalk segments of P-type ion-translocating enzymes are presumed to play important roles in energy coupling . In this work, stalk segments S4 and S5 of the yeast H(+)-ATPase were examined for helical character, optimal length, and segment orientation by a combination of proline substitution, insertion/deletion mutagenesis, and second-site suppressor analyses . The substitution of various residues for helix-disrupting proline in both S4 (L353P,L353G; A354P; and G371P) and S5 (D676P and I684P) resulted in highly defective or inactive enzymes supporting the importance of helical character and/or the maintenance of essential interactions . The contiguous helical nature of transmembrane segment M5 and stalk element S5 was explored and found to be favorable, although not essential . The deletion or addition of one or more amino acids at positions Ala(354) in S4 and Asp(676) in S5, which were intended to either rotate helical faces or extend/reduce the length of helical segments, resulted in enzyme destabilization that abolished most enzyme assembly . Second-site suppressor mutations were obtained to primary site mutations G371A (S4) and D676G (S5) and were analyzed with a molecular structure model of the H(+)-ATPase . Primary site mutations were predicted to alter the site of phosphorylation either directly or indirectly . The suppressor mutations either directly changed packing around the primary site or altered the environment of the site of phosphorylation . Overall, these data support the view that stalk segments S4 and S5 of the H(+)-ATPase are helical elements that are optimized for length and interactions with other stalk elements and can influence the phosphorylation domain.

J Biol Chem, 2001 May 4, 276(18), 15073 - 81 Epub 2001 Jan 30.
Uncoupling of 3'-phosphatase and 5'-kinase functions in budding yeast . Characterization of Saccharomyces cerevisiae DNA 3'-phosphatase (TPP1); Vance JR et al.; Polynucleotide kinase is a bifunctional enzyme containing both DNA 3'-phosphatase and 5'-kinase activities seemingly suited to the coupled repair of single-strand nicks in which the phosphate has remained with the 3'-base . We show that the yeast Saccharomyces cerevisiae is able to repair transformed dephosphorylated linear plasmids by non-homologous end joining with considerable efficiency independently of the end-processing polymerase Pol4p . Homology searches and biochemical assays did not reveal a 5'-kinase that would account for this repair, however . Instead, open reading frame YMR156C (here named TPP1) is shown to encode only a polynucleotide kinase-type 3'-phosphatase . Tpp1p bears extensive similarity to the ancient L-2-halo-acid dehalogenase and DDDD phosphohydrolase superfamilies, but is specific for double-stranded DNA . It is present at high levels in cell extracts in a functional form and so does not represent a pseudogene . Moreover, the phosphatase-only nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabidopsis thaliana K15M2.3 . Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans . Repair of transformed dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more generally, likely proceeds by a cycle of base removal and resynthesis.

J Biol Chem, 2001 Apr 6, 276(14), 10897 - 905 Epub 2001 Jan 16.
Discrete targeting signals direct Pmp47 to oleate-induced peroxisomes in Saccharomyces cerevisiae; Wang X et al.; Pmp47 is a peroxisomal membrane protein consisting of six transmembrane domains (TMDs) . We previously showed that the second matrix loop containing a basic cluster of amino acids is important for peroxisomal targeting, and similar basic targeting motifs have been found in other peroxisomal membrane proteins . However, this basic cluster by itself targets to peroxisomes very poorly . We have developed a sensitive quantitative localization assay based on the targeting of Pmp47-GFP fusion proteins to identify the important elements of the basic cluster and to search for other targeting information on Pmp47 . Our data suggest that side-chain structure and position as well as charge are important for targeting by the basic cluster . Analysis of other regions of Pmp47 indicates that all TMDs except TMD2 can be eliminated or substituted without significant loss of targeting . TMD2 plus an adjacent cytoplasmic-oriented sequence is crucial for targeting . Cytoplasmic-oriented sequences from two other peroxisomal membrane proteins, ScPex15p and ScPmp22, could partially substitute for the analogous sequence in Pmp47 . Targeting with high fidelity to oleate-induced peroxisomes required the following elements: the cytoplasmic-oriented sequence and TMD2, a short matrix loop containing a basic cluster, and a membrane-anchoring TMD.

J Biol Chem, 2001 May 11, 276(19), 15768 - 75 Epub 2001 Feb 05.
Delineation of functional regions within the subunits of the Saccharomyces cerevisiae cell adhesion molecule a-agglutinin; Shen ZM et al.; a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein expressed on the surface of cells of a mating type and consists of an anchorage subunit Aga1p and a receptor binding subunit Aga2p . Cell wall attachment of Aga2p is mediated through two disulfide bonds to Aga1p (Cappellaro, C., Baldermann, C., Rachel, R., and Tanner, W . (1994) EMBO J . 13, 4737-4744) . We report here that purified Aga2p was unstable and had low molar specific activity relative to its receptor alpha-agglutinin . Aga2p co-expressed with a 149-residue fragment of Aga1p formed a disulfide-linked complex with specific activity 43-fold higher than Aga2p expressed alone . Circular dichroism of the complex revealed a mixed alpha/beta structure, whereas Aga2p alone had no periodic secondary structure . A 30-residue Cys-rich Aga1p fragment was partially active in stabilization of Aga2p activity . Mutation of either or both Aga2p cysteine residues eliminated stabilization of Aga2p . Thus the roles of Aga1p include both cell wall anchorage and cysteine-dependent conformational restriction of the binding subunit Aga2p . Mutagenesis of AGA2 identified only C-terminal residues of Aga2p as being essential for binding activity . Aga2p residues 45-72 are similar to sequences in soybean Nod genes, and include residues implicated in interactions with both Aga1p (including Cys(68)) and alpha-agglutinin.

J Biol Chem, 2001 May 11, 276(19), 16328 - 34 Epub 2001 Feb 09.
The crystal structures of Apo and complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of an amino-sugar N-acetyltransferase; Peneff C et al.; The yeast enzymes involved in UDP-GlcNAc biosynthesis are potential targets for antifungal agents . GNA1, a novel member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, participates in UDP-GlcNAc biosynthesis by catalyzing the formation of GlcNAc6P from AcCoA and GlcN6P . We have solved three crystal structures corresponding to the apo Saccharomyces cerevisiae GNA1, the GNA1-AcCoA, and the GNA1-CoA-GlcNAc6P complexes and have refined them to 2.4, 1.3, and 1.8 A resolution, respectively . These structures not only reveal a stable, beta-intertwined, dimeric assembly with the GlcNAc6P binding site located at the dimer interface but also shed light on the catalytic machinery of GNA1 at an atomic level . Hence, they broaden our understanding of structural features required for GNAT activity, provide structural details for related aminoglycoside N-acetyltransferases, and highlight the adaptability of the GNAT superfamily members to acquire various specificities.

J Biol Chem, 2001 Apr 27, 276(17), 14374 - 84 Epub 2001 Jan 23.
Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes . Implications for the conservation of oxygen sensing in eukaryotes; Vasconcelles MJ et al.; An organism's ability to respond to changes in oxygen tension depends in large part on alterations in gene expression . The oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood . To further define these processes, we have studied the Delta9 fatty acid desaturase gene OLE1 in Saccharomyces cerevisiae . We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals . OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline . A 142-base pair (bp) region 3' to the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs . Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia and cobalt . Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation . A 20-base pair oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed upstream of a heterologous promoter in a lacZ reporter construct . Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar to OLE1 . A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators . Hence, the identification of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in eukaryotes.

J Biol Chem, 2001 May 11, 276(19), 15905 - 12 Epub 2001 Feb 22.
Scp160p, an RNA-binding, polysome-associated protein, localizes to the endoplasmic reticulum of Saccharomyces cerevisiae in a microtubule-dependent manner; Frey S et al.; Scp160p is an RNA-binding protein containing 14 tandemly repeated heterogenous nuclear ribonucleoprotein K-homology domains, which are implicated in RNA binding . Scp160p interacts with free and membrane-bound polysomes that are dependent upon the presence of mRNA . Despite its presence on cytosolic polysomes, Scp160p is predominantly localized to the endoplasmic reticulum (ER) . Accumulation of Scp160p-ribosome complexes at the ER requires the function of microtubules but is independent of the actin cytoskeleton . We propose that the multi-K-homology-domain protein Scp160p functions as an RNA binding platform, interacting with polysomes that are transported to the ER.

J Biol Chem, 2001 May 11, 276(19), 15996 - 6007 Epub 2001 Feb 14.
Transcript expression in Saccharomyces cerevisiae at high salinity; Yale J et al.; Transcript expression of Saccharomyces cerevisiae at high salinity was determined by microarray analysis of 6144 open reading frames (ORFs) . From cells grown in 1 m NaCl for 10, 30, and 90 min, changes in transcript abundance >2-fold were classified . Salinity-induced ORFs increased over time: 107 (10 min), 243 (30 min), and 354 (90 min) . Up-regulated, functionally unknown ORFs increased from 17 to 149 over this period . Expression patterns were similar early, with 67% of up-regulated transcripts after 10 min identical to those at 30 min . The expression profile after 90 min revealed different up-regulated transcripts (identities of 13% and 22%, respectively) . Nucleotide and amino acid metabolism exemplified the earliest responses to salinity, followed by ORFs related to intracellular transport, protein synthesis, and destination . Transcripts related to energy production were up-regulated throughout the time course with respiration-associated transcripts strongly induced at 30 min . Highly expressed at 90 min were known salinity stress-induced genes, detoxification-related responses, transporters of the major facilitator superfamily, metabolism of energy reserves, nitrogen and sulfur compounds, and lipid, fatty acid/isoprenoid biosynthesis . We chose severe stress conditions to monitor responses in essential biochemical mechanisms . In the mutant, Deltagpd1/gpd2, lacking glycerol biosynthesis, the stress response was magnified with a partially different set of up-regulated ORFs.

J Biol Chem, 2001 May 18, 276(20), 16731 - 8 Epub 2001 Jan 24.
Nickel resistance and chromatin condensation in Saccharomyces cerevisiae expressing a maize high mobility group I/Y protein; Forzani C et al.; Expression of a maize cDNA encoding a high mobility group (HMG) I/Y protein enables growth of transformed yeast on a medium containing toxic nickel concentrations . No difference in the nickel content was measured between yeast cells expressing either the empty vector or the ZmHMG I/Y2 cDNA . The ZmHMG I/Y2 protein contains four AT hook motifs known to be involved in binding to the minor groove of AT-rich DNA regions . HMG I/Y proteins may act as architectural elements modifying chromatin structure . Indeed, a ZmHMG I/Y2-green fluorescent protein fusion protein was observed in yeast nuclei . Nickel toxicity has been suggested to occur through an epigenetic mechanism related to chromatin condensation and DNA methylation, leading to the silencing of neighboring genes . Therefore, the ZmHMG I/Y2 protein could prevent nickel toxicity by interfering with chromatin structure . Yeast cell growth in the presence of nickel and yeast cells expressing the ZmHMG I/Y2 cDNA increased telomeric URA3 gene silencing . Furthermore, ZmHMG I/Y2 restored a wild-type level of nickel sensitivity to the yeast (Delta)rpd3 mutant . Therefore, nickel resistance of yeast cells expressing the ZmHMG I/Y2 cDNA is likely achieved by chromatin structure modification, restricting nickel accessibility to DNA.

Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 3946 - 51
A large-scale overexpression screen in Saccharomyces cerevisiae identifies previously uncharacterized cell cycle genes; Stevenson LF et al.; We have undertaken an extensive screen to identify Saccharomyces cerevisiae genes whose products are involved in cell cycle progression . We report the identification of 113 genes, including 19 hypothetical ORFs, which confer arrest or delay in specific compartments of the cell cycle when overexpressed . The collection of genes identified by this screen overlaps with those identified in loss-of-function cdc screens but also includes genes whose products have not previously been implicated in cell cycle control . Through analysis of strains lacking these hypothetical ORFs, we have identified a variety of new CDC and checkpoint genes.

Folia Microbiol (Praha), 2000, 45(3), 251 - 4
Protein overexport in a Saccharomyces cerevisiae mutant is not due to facilitated release of cell-surface proteins; Alexieva KI et al.; Saccharomyces cerevisiae strain MW11 is a temperature-sensitive mutant which exports twenty times more proteins at 37 degrees C than parental or wild-type strains do . To understand the mechanism underlying the protein overexport in the mutant the possibility of an altered cell-wall structure leading to facilitated release of cell-surface proteins was studied . Data on calcofluor white and zymolyase sensitivities, resistance to killer 1 toxin and determination of exported acid phosphatase and invertase did not provide evidence for alterations in the cell-wall structure that could explain the protein overexport phenotype . The results were obtained in experiments when transcription of mutated gene was discontinued which permits the full expression of the protein overexport phenotype.

Electrophoresis, 2000 Nov, 21(17), 3717 - 23
Differences between predicted and observed sequences in Saccharomyces cerevisiae; Santucci A et al.; We recently studied the protein composition of a Saccharomyces cerevisiae wine yeast strain (K310) of enological interest . About 2,500 spots of 8-250 kDa observed molecular mass were resolved by two-dimensional gel electrophoresis . Experimental molecular masses and isoelectric points were calculated for most of them . Twenty-seven proteins were subjected to Edman microsequencing . N-terminal sequences of 12/27 proteins were determined, whereas internal sequences of 6/27 proteins were obtained following in situ proteolysis . Comparison between the experimental data and those reported in the SWISS-PROT database revealed some differences between genotypic and phenotypic sequences . These are indicative of the changes a protein can undergo with respect to the primary structure coded by the genomic DNA . Our results highlight the need to complement genomic analysis with detailed proteomics in order to refine the vast amount of information provided by DNA sequencing and to find an exact correlation between genome and proteome.

J Cell Biol, 2001 Feb 19, 152(4), 851 - 6
An essential role for the substrate-binding region of Hsp40s in Saccharomyces cerevisiae; Johnson JL et al.; In addition to regulating the ATPase cycle of Hsp70, a second critical role of Hsp40s has been proposed based on in vitro studies: binding to denatured protein substrates, followed by their presentation to Hsp70 for folding . However, the biological importance of this model is challenged by the fact that deletion of the substrate-binding domain of either of the two major Hsp40s of the yeast cytosol, Ydj1 and Sis1, leads to no severe defects, as long as regions necessary for Hsp70 interaction are retained . As an in vivo test of this model, requirements for viability were examined in a strain having deletions of both Hsp40 genes . Despite limited sequence similarity, the substrate-binding domain of either Sis1 or Ydj1 allowed cell growth, indicating they share overlapping essential functions . Furthermore, the substrate-binding domain must function in cis with a functional Hsp70-interacting domain . We conclude that the ability of cytosolic Hsp40s to bind unfolded protein substrates is an essential function in vivo.

Eur J Clin Invest, 2001 Mar, 31(3), 221 - 5
IgG subclasses of anti Saccharomyces cerevisiae antibody in inflammatory bowel disease; Oshitani N et al.; Elevation of serum anti Saccharomyces cerevisiae antibody (ASCA) has been reported in patients with Crohn's disease . We analysed the subclasses of Immunoglobulin (Ig) G reaction in ASCA in sera from patients with inflammatory bowel disease, healthy controls, and patients with intestinal Behcet's disease . Serum samples were obtained from 29 patients with Crohn's disease, 30 patients with ulcerative colitis, 7 patients with intestinal Behcet's disease, and 12 healthy controls . Serum IgG subclasses IgG1, IgG2, IgG3, and IgG4 of ASCA were analysed using ELISA . IgG4 ASCA was significantly increased in patients with inflammatory bowel disease . In patients with intestinal Behcet's disease, IgG1, IgG3, and IgG4 ASCA were increased . Differential responses, in terms of subclasses in ASCA, were found in patients with inflammatory bowel disease and patients with intestinal Behcet's disease, which may represent different pathophysiologies of these intestinal inflammatory diseases.

J Biol Chem, 2001 Jun 8, 276(23), 20491 - 8 Epub 2001 Mar 22.
Vacuolar localization of oligomeric alpha-mannosidase requires the cytoplasm to vacuole targeting and autophagy pathway components in Saccharomyces cerevisiae; Hutchins MU et al.; One challenge facing eukaryotic cells is the post-translational import of proteins into organelles . This problem is exacerbated when the proteins assemble into large complexes . Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae . The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process . Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway . Many components of the Cvt pathway are also used in the degradative autophagy pathway . alpha-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established . We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways . We have found that Ams1 forms an oligomer in the cytoplasm . The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process . These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.

Biochem Biophys Res Commun, 2001 Mar 23, 282(1), 79 - 83
The Zrc1 is involved in zinc transport system between vacuole and cytosol in Saccharomyces cerevisiae; Miyabe S et al.; The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously we found that the expression of ZRC1 was induced when the intracellular zinc level was decreased . Zrc1 has six putative transmembrane domains and we determined that a Zrc1-GFP fusion protein was localized to the vacuolar membrane . The steady state level of intracellular zinc in a zrc1Delta mutant cultured in the zinc-abundant medium was lower than that in wild type . No distinct difference was observed in the basal activity of glyoxalase I, which is a cytosolic enzyme requiring zinc for catalytic function and is used here as a marker for cytosolic zinc-availability, between wild type and zrc1Delta mutant, although the activity was decreased much greater extent in the zrc1Delta mutant if the cells were exposed to the metal-limited medium . Similarly, the basal expression level of ZRC1-lacZ reporter gene in zrc1Delta mutant was the same as that in wild type; however, the fold of induction of ZRC1-lacZ expression in zrc1Delta mutant under the zinc-limited conditions was higher than that in the wild type . Based on these results, we present a tentative model for the function of Zrc1 as a mechanism to maintain the zinc homeostasis in yeast .

Exp Cell Res, 2001 Apr 1, 264(2), 372 - 87
The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations; Van Hoof C et al.; In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2 . In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains . While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions . These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background . Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation . These results are consistent with an in vivo role of Ypa as a regulator of PP2A . However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest . Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle . These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A .

Mol Microbiol, 2001 Mar, 39(6), 1523 - 32
Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene- and stress type-dependent manner; Amoros M et al.; Saccharomyces cerevisiae possesses several transcription factors involved in the transcriptional activation of stress-induced genes . Among them, the heat shock factor (Hsf1p) and the zinc finger proteins of the general stress response (Msn2p and Msn4p) have been shown to play a major role in stress protection . Some heat shock protein (HSP) genes contain both heat shock elements (HSEs) and stress response elements (STREs), suggesting the involvement of both transcription factors in their regulation . Analysis of the stress-induced expression of two of these genes, HSP26 and HSP104, reveals that the contribution of Hsf1p and Msn2/4p is different depending on the gene and the stress condition.

Mol Cell Biol, 2001 Apr, 21(7), 2359 - 72
Mitochondrial translation of Saccharomyces cerevisiae COX2 mRNA is controlled by the nucleotide sequence specifying the pre-Cox2p leader peptide; Bonnefoy N et al.; The mitochondrial gene encoding yeast cytochrome oxidase subunit II (Cox2p) specifies a precursor protein with a 15-amino-acid leader peptide . Deletion of the entire leader peptide coding region is known to block Cox2p accumulation posttranscriptionally . Here, we examined in vivo the role of the pre-Cox2p leader peptide and the mRNA sequence that encodes it in the expression of a mitochondrial reporter gene, ARG8m, fused to the 91st codon of COX2 . We found within the coding sequence antagonistic elements that control translation: the positive element includes sequences in the first 14 codons specifying the leader peptide, while the negative element appears to be within codons 15 to 91 . Partial deletions, point mutations, and local frameshifts within the leader peptide coding region were placed in both the cox2::ARG8m reporter and in COX2 itself . Surprisingly, the mRNA sequence of the first six codons specifying the leader peptide plays an important role in positively controlling translation, while the amino acid sequence of the leader peptide itself is relatively unconstrained . Two mutations that partially block translation can be suppressed by nearby sequence substitutions that weaken a predicted stem structure and by overproduction of either the COX2 mRNA-specific translational activator Pet111p or the large-subunit mitochondrial ribosomal protein MrpL36p . We propose that regulatory elements embedded in the translated COX2 mRNA sequence could play a role, together with trans-acting factors, in coupling regulated synthesis of nascent pre-Cox2p to its insertion in the mitochondrial inner membrane.

Biophys J, 2001 Apr, 80(4), 1957 - 66
Solution structure of the SL1 RNA of the M1 double-stranded RNA virus of Saccharomyces cerevisiae; Yoo JS et al.; The 20-nucleotide SL1 VBS RNA, 5'-GGAGACGC{GAUUC}GCGCUCC (bulged A underlined and loop bases in brackets), plays a crucial role in viral particle binding to the plus strand and packaging of the RNA . Its structure was determined by NMR spectroscopy . Structure calculations gave a precisely defined structure, with an average pairwise root mean square deviation (RMSD) of 1.28 A for the entire molecule, 0.57 A for the loop region (C8-G14), and 0.46 A for the bulge region (G4-G7, C15-C17) . Base stacking continues for three nucleotides on the 5' side of the loop . The final structure contains a single hydrogen bond involving the guanine imino proton and the carbonyl O(2) of the cytosine between the nucleotides on the 5' and 3' ends of the loop, although they do not form a Watson-Crick base pair . All three pyrimidine bases in the loop point toward the major groove, which implies that Cap-Pol protein may recognize the major groove of the SL1 loop region . The bulged A5 residue is stacked in the stem, but nuclear Overhauser enhancements (NOEs) suggest that A5 spends part of the time in the bulged-out conformation . The rigid conformation of the upper stem and loop regions may allow the SL1 VBS RNA to interact with Cap-Pol protein without drastically changing its own conformation.

DNA Res, 2001 Feb 28, 8(1), 23 - 31
A genome-wide analysis of transcriptional effect of Gal11 in Saccharomyces cerevisiae: an application of "mini-array hybridization technique"; Fukasawa T et al.; The Gal11 protein is a subunit of the Mediator complex . Biochemical as well as genetic studies have strongly suggested that Gal11 is a positive global regulator of transcription . Some reports argue that Gal11 is a negative regulator, however . Here we have adopted the "Mini-array membrane hybridization" to analyze the effect of Gal11 in a genome-wide fashion . This technique has been demonstrated to be reliable to identify genes whose expression is controlled by a specific set of genetic and/or physiological signals . Our experiments indicate that this technique is applicable to profile the gene expression in yeast grown in rich medium . Thus mRNAs of 40% of significantly expressed genes are reduced more than two fold in gal11null yeast, in which only 3% of mRNAs are increased more than two fold . These results strongly suggest that Gal11 functions globally as a positive regulator in vivo.

Electrophoresis, 2001 Feb, 22(3), 566 - 75
Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry; Larsen MR et al.; Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis . The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis . In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated . A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2 . The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1) . Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.

EMBO Rep, 2000 Sep, 1(3), 232 - 8
DNA motif associated with meiotic double-strand break regions in Saccharomyces cerevisiae; Blumental-Perry A et al.; Meiotic recombination in yeast is initiated by DNA double-strand breaks (DSBs) that occur at preferred sites, distributed along the chromosomes . These DSB sites undergo changes in chromatin structure early in meiosis, but their common features at the level of DNA sequence have not been defined until now . Alignment of 1 kb sequences flanking six well-mapped DSBs has allowed us to define a flexible sequence motif, the CoHR profile, which predicts the great majority of meiotic DSB locations . The 50 bp profile contains a poly(A) tract in its centre and may have several gaps of unrelated sequences over a total length of up to 250 bp . The major exceptions to the correlation between CoHRs and preferred DSB sites are at telomeric regions, where DSBs do not occur . The CoHR sequence may provide the basis for understanding meiosis-induced chromatin changes that enable DSBs to occur at defined chromosomal sites.

Yeast, 2001 Mar 30, 18(5), 473 - 80
Disruption and basic phenotypic analysis of six novel genes from the right arm of chromosome XII of Saccharomyces cerevisiae; Watson MD; Six open reading frames (ORFs) of unknown function from the right arm of Saccharomyces cerevisiae chromosome XII were deleted in two genetic backgrounds by disruption cassettes with regions of short flanking homology . This work was carried out within the framework of the EUROFAN consortium . The SFH disruption cassettes, obtained by PCR, were made by amplification of the kanMX marker module with oligonucleotides containing approximately 40 bp of homology to either the promoter or translation terminator regions of the relevant ORF . Transformants resistant to geneticin (G418) were selected . The SFH disruption cassettes were cloned into a bacterial vector . Each cognate gene was also cloned into a yeast centromeric plasmid . Sporulation and tetrad analysis of the disrupted heterozygous strains revealed that ORF YLR153c (now known as ACS2) is essential . Basic phenotypic analysis was performed on haploid deletants of both mating types of the five non-essential ORFs, YLR082c (now known as SRL2), YLR149c, YLR151c, YLR152c and YLR154c . Plate growth tests on different media at 15 degrees C, 30 degrees C and 37 degrees C did not reveal any significant differences between parental and mutant cells . Mating and sporulation efficiencies were not affected in any of the viable disruptants as compared to wild-type cells .

Yeast, 2001 Mar 30, 18(5), 445 - 54
Use of Saccharomyces cerevisiae in the identification of novel transcription factor DNA binding specificities; Henry KW 2nd et al.; Members of the steroid/hormone nuclear receptor superfamily regulate target gene transcription via recognition and association with specific cis-acting sequences of DNA, called hormone response elements (HREs) . The identification of novel HREs is fundamental to understanding the physiological function of nuclear receptor-mediated signalling pathways . A number of these receptors are transcriptionally active, or can be induced to an active state, when expressed in the yeast strain Saccharomyces cerevisiae . This aspect of nuclear receptor activity was used to screen random rat genomic DNA fragments for their ability to function as a HRE for the farnesoid X-activated receptor (FXR) . An isolated genomic fragment mediated FXR transcriptional activation without the co-expression of the retinoid-X receptor (RXR), a receptor previously thought to be an obligate heterodimer partner for FXR function . This genomic sequence of DNA contained a pair of highly conserved HRE half-sites arranged in an everted orientation and separated by 3 bp (ER3) . Furthermore, it was located 240 bp from a highly conserved TATA box motif . A minimal ER3 sequence of DNA was further demonstrated to function as a FXR HRE and was bound in vitro by FXR-expressing yeast extracts . Using RT-PCR, an expressed mRNA fragment was identified within an 8 kb region downstream of the putative TATA box motif . This sequence of DNA was observed to bear homology to a cDNA found in mouse blastocyst . These findings define a novel FXR DNA binding specificity but, more importantly, these data suggest that this strategy might be universally applied to any transcription system that can be reconstituted in yeast .

Mol Biochem Parasitol, 2001 Mar, 113(1), 139 - 50
Mutagenesis of dihydrofolate reductase from Plasmodium falciparum: analysis in Saccharomyces cerevisiae of triple mutant alleles resistant to pyrimethamine or WR99210; Ferlan JT et al.; Inhibitors of dihydrofolate reductase (DHFR) have been a mainstay of chemotherapy of falciparum malaria for >50 years . Unfortunately, point mutations in DHFR are the major cause of resistance to drugs of this class and mutations have rapidly diminished the clinical effectiveness of these drugs . We designed a simple yeast-based system to produce and analyze point mutations in the Plasmodium falciparum DHFR domain of the DHFR-thymidylate synthase gene that confers resistance to pyrimethamine (PM), the major antifolate currently used in malaria treatment, or to WR99210, an experimental antifolate . We used PCR mutagenesis, screened >1000 DHFR alleles that encoded functional enzymes and studied approximately 100 that were more resistant than a naturally occurring resistant allele (N51I and S108N) . The IC(50) values for both drugs were determined for a subset of 44 alleles that carried only a single new mutation . Mutations that increased resistance to PM 10-100 fold (to >10(-4) M) were identified in three regions of the DHFR domain - around amino acids 50, 188 and 213 . In contrast, mutations that caused WR-resistance were far less common and only conferred approximately 10-fold resistance (to approximately 10(-7) M) . Even more interesting, only the mutations at 188 increased resistance to WR and mutations in the 213 and other regions either had no effect or actually increased sensitivity to WR . This collateral hypersensitivity raises the possibility that opposing selection for resistance/sensitivity to PM and WR might be used to slow selection of populations of P . falciparum resistant to antifolate treatment.

J Biol Chem, 2001 Mar 23, 276(12), 8635 - 8 Epub 2001 Jan 31.
Mnl1p, an alpha -mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic reticulum-associated degradation of glycoproteins; Nakatsukasa K et al.; The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway . Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window . Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure . Here we report a new alpha-mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER . Mnl1p is unlikely to exhibit alpha1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha1,2-mannosidase . However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha-mating pheromone . Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.

Infect Immun, 2001 Apr, 69(4), 2037 - 44
Interactions of surfactant proteins A and D with Saccharomyces cerevisiae and Aspergillus fumigatus; Allen MJ et al.; Surfactant proteins A (SP-A) and D (SP-D) are members of the collectin family of calcium-dependent lectins and are important pulmonary host defense molecules . Human SP-A and SP-D and rat SP-D bind to Aspergillus fumigatus conidia, but the ligand remains unidentified . To identify a fungal ligand for SP-A and/or SP-D, we examined the interactions of the proteins with Saccharomyces cerevisiae . SP-D but not SP-A bound yeast cells, and EDTA inhibited the binding . SP-D also aggregated yeast cells and isolated yeast cell walls . Treating yeast cells to remove cell wall mannoprotein did not reduce SP-D binding, and SP-D failed to aggregate chitin . However, SP-D aggregated yeast glucan before and after treatment with a beta(1-->3)-glucanase, suggesting a specific interaction between the collectin and beta(1-->6)-glucan . In support of this idea, SP-D-induced yeast aggregation was strongly inhibited by pustulan {a beta(1-->6)-linked glucose homopolymer} but was not inhibited by laminarin {a beta(1-->3)-linked glucose homopolymer} . Additionally, pustulan but not laminarin strongly inhibited SP-D binding to A . fumigatus . The pustulan concentration for 50% inhibition of SP-D binding to A . fumigatus is 1.0 +/- 0.3 microM glucose equivalents . Finally, SP-D showed reduced binding to the beta(1-->6)-glucan-deficient kre6 yeast mutant . Taken together, these observations demonstrate that beta(1-->6)-glucan is an important fungal ligand for SP-D and that glycosidic bond patterns alone can determine if an extended carbohydrate polymer is recognized by SP-D.

Nahrung, 2001 Feb, 45(1), 59 - 61
Investigation of kinetics of zinc biosorption by Saccharomyces cerevisiae cells; Dodic SN et al.; The highest amount of zinc (approximately 90%) is bound after 3 h of contact at low initial (total) concentrations of zinc in yeast broth, 2.0-16.0 g/l at 10-30 degrees C . The equilibrium between bound and free zinc ions is established after 6 h of contact time, independently on total zinc concentration in yeast milk . No bigger changes of content of zinc bound to yeast cells was determined at 10 degrees C and 30 degrees C . 40% of bound zinc in the equilibrium state is bound during the first 15 min of contact of zinc ions and yeast cells at all initial (total) zinc concentrations in yeast milk both at 10 degrees C and 30 degrees C . The KEKAM (Kolmogorov-Erofeev-Kozeeva-Avrami-Mampel) equation can be used for the description of kinetics of zinc biosorption by yeast cells, for the ranges of zinc concentration of 2.0-16.0 g/l at 30 degrees C (mean correlation coefficient 0.96) and 10.0-16.0 g/l at 10 degrees C (mean correlation coefficient 0.95).

Mol Microbiol, 2001 Mar, 39(5), 1395 - 405
Expression of the INO2 regulatory gene of Saccharomyces cerevisiae is controlled by positive and negative promoter elements and an upstream open reading frame; Eiznhamer DA et al.; The INO2 gene encodes a transcriptional activator of the phospholipid biosynthetic genes of Saccharomyces cerevisiae . Complete derepression of phospholipid biosynthetic gene expression in response to inositol/choline deprivation requires both INO2 and INO4 . Ino2p dimerizes with Ino4p to bind the upstream activating sequence (UAS)INO element found in the promoters of the target genes . We have demonstrated previously that transcription from the INO2 promoter is autoregulated 12-fold in a manner identical to that of the target genes . Here, we show that this regulation occurs at the levels of transcription and translation . Transcription accounts for fourfold regulation, whereas translation accounts for an additional threefold regulation . Regulation of transcription requires a UAS(INO) element . Additional promoter elements include an upstream essential sequence (UES) located upstream of the UAS(INO) element and a negative regulatory element in the vicinity of the UAS(INO) element . Regulation of translation is dependent on an upstream open reading frame (uORF) in the INO2 leader . These data support the model that regulatory gene promoters may display unusual organizations and may be subject to multiple levels of regulation . We have shown previously that the UME6 gene positively regulates INO2 expression . Here, we limit the UME6-responsive region of the INO2 promoter to nucleotides -217 to -56.

Mol Microbiol, 2001 Mar, 39(5), 1166 - 73
Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis; Laun P et al.; Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells . Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span . The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells . Taken together, these results pointed to a role for oxygen in the yeast ageing process . Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria . A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e . TUNEL (TdT-mediated dUTP nick end labelling) and annexin V staining . Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro-apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild-type yeast mother cells.

Mol Microbiol, 2001 Feb, 39(4), 1022 - 35
Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/Cdc28 activity in Saccharomyces cerevisiae; Belli G et al.; Moderate hyperosmotic stress on Saccharomyces cerevisiae cells produces a temporary delay at the G1 stage of the cell cycle . This is accompanied by transitory downregulation of CLN1, CLN2 and CLB5 transcript levels, although not of CLN3, which codes for the most upstream activator of the G1/S transition . Osmotic shock to cells synchronized in early G1, when Cln3 is the only cyclin present, causes a delay in cell cycle resumption . This points to Cln3 as being a key cell cycle target for osmotic stress . We have observed that osmotic shock causes downregulation of the kinase activity of Cln3-Cdc28 complexes . This is concomitant with a temporary accumulation of Cln3 protein as a result of increased stability . The effects of the osmotic stress in G1 are not suppressed in CLN3-1 cells with increased kinase activity, as the Cln3-Cdc28 activity in this mutant is still affected by the shock . Although Hog1 is not required for the observed cell cycle arrest in hyperosmotic conditions, it is necessary to resume the cell cycle at KCl concentrations higher than 0.4 M.

FEBS Lett, 2001 Mar 9, 492(1-2), 123 - 6
Decreased antioxidant defense during replicative aging of the yeast Saccharomyces cerevisiae studied using the 'baby machine' method; Grzelak A et al.; Replicatively senescent cells of Saccharomyces cerevisiae were obtained using the 'baby machine' method by immobilizing cells on CovaLink NH(2) plates and allowing them to divide while exchanging medium and removing daughter cells . Centrifugation in a Percoll density gradient was employed for further purification of replicatively old yeast cells . Comparison of senescent cells showing more than 20 bud scars with cells from early stationary culture demonstrated a significant reduction of total and reduced glutathione and decrease of superoxide dismutase activity during replicative aging of yeast cells.

J Bacteriol, 2001 Apr, 183(7), 2372 - 5
Conservative duplication of spindle poles during meiosis in Saccharomyces cerevisiae; Wesp A et al.; During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation . Mpc70p is a component of the meiotic plaque and is thus essential for spore formation . We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters . In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters . Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.

Cell, 2001 Feb 9, 104(3), 397 - 408
Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae; Myung K et al.; Cancer cells show increased genome rearrangements, although it is unclear what defects cause these rearrangements . Mutations in Saccharomyces cerevisiae RFC5, DPB11, MEC1, DDC2 MEC3, RAD53, CHK1, PDS1, and DUN1 increased the rate of genome rearrangements up to 200-fold whereas mutations in RAD9, RAD17, RAD24, BUB3, and MAD3 had little effect . The rearrangements were primarily deletion of a portion of a chromosome arm along with TEL1-dependent addition of a new telomere . tel1 mutations increased the proportion of translocations observed, and in some cases showed synergistic interactions when combined with mutations that increased the genome rearrangement rate . These data suggest that one role of S phase checkpoint functions in normal cells is to suppress spontaneous genome rearrangements resulting from DNA replication errors.

Cell, 2001 Feb 9, 104(3), 377 - 86
The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae; Tucker M et al.; The major pathways of mRNA turnover in eukaryotes initiate with shortening of the poly(A) tail . We demonstrate by several criteria that CCR4 and CAF1 encode critical components of the major cytoplasmic deadenylase in yeast . First, both Ccr4p and Caf1p are required for normal mRNA deadenylation in vivo . Second, both proteins localize to the cytoplasm . Third, purification of Caf1p copurifies with a Ccr4p-dependent poly(A)-specific exonuclease activity . We also provide evidence that the Pan2p/Pan3p nuclease complex encodes the predominant alternative deadenylase . These results, and previous work on Pan2p/Pan3p, define the mRNA deadenylases in yeast . The strong conservation of Ccr4p, Caf1p, Pan2p, and Pan3p indicates that they will function as deadenylases in other eukaryotes . Interestingly, because Ccr4p and Caf1p interact with transcription factors, these results suggest an unexpected link between mRNA synthesis and turnover.

Nucleic Acids Res, 2001 Mar 15, 29(6), 1381 - 8
Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants; Singh KK et al.; The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA . We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria . We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type . Using green fluorescent protein (GFP) we show that a GFP-Ogg1 fusion protein is transported to mitochondria . However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP-Ogg1 fusion protein into the mitochondria . This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal . We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage . Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 {encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)} leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type . Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins . Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

Mol Cell Biol, 2001 Mar, 21(5), 1453 - 62
The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis; Basu U et al.; Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit . In Saccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene . To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter . Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis . Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits . Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA . The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells . Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.

Genetics, 2001 Mar, 157(3), 1179 - 89
The Saccharomyces cerevisiae MUM2 gene interacts with the DNA replication machinery and is required for meiotic levels of double strand breaks; Davis L et al.; The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation . Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase alpha-primase suggest that MUM2 influences the function of the DNA replication machinery . Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea . This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant . Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA . Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated . Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type . Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination.

Genetics, 2001 Mar, 157(3), 1169 - 77
Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae; Abramova NE et al.; The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth . Expression of these genes is induced during anaerobiosis and in some cases during cold shock . We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression . The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen) . The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion . MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system . Mox4/Upc2 is required for expression of all the DAN/TIR genes . It appears to act through a consensus sequence termed the AR1 site, as does Mox2 . The noninducible mox4Delta allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion . Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression . MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4 . Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1 . Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1 . In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.

Genetics, 2001 Mar, 157(3), 1107 - 16
Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact with TOM1 mutants defective in ribosome synthesis; Tabb AL et al.; The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA . We show here that the RPS0 genes interact genetically with TOM1 . TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures . Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants . Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B . Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits . Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.

Protein Expr Purif, 2001 Mar, 21(2), 333 - 42
Purification of eukaryotic MutL homologs from Saccharomyces cerevisiae using self-affinity technology; Hall MC et al.; Self-cleaving affinity technology is an effective tool for rapid purification of native sequence recombinant proteins overproduced in Escherichia coli . In this report, we describe the adaptation of this technology to purify DNA mismatch repair proteins overproduced in the eukaryote Saccharomyces cerevisiae . Mlh1 and Pms1 are homologs of the E . coli MutL protein that participate in a variety of DNA transactions in cells, including correction of DNA replication errors, recombination, excision repair, and checkpoint control . Difficulties in preparing substantial quantities of highly purified MutL homologs have impeded descriptions of their biophysical and biochemical properties and mechanisms of action . To overcome this limitation, here we use self-cleaving affinity technology to purify to apparent homogeneity the yeast Mlh1--Pms1 heterodimer and the individual yeast and human Mlh1 subunit . The availability of these proteins should accelerate an understanding of their multiple functions in mismatch repair and other DNA transactions . The general approach is a valid alternative for simple, rapid purification of recombinant proteins in yeast when expression in bacteria is unsuitable .

J Chromatogr B Biomed Sci Appl, 2001 Feb 25, 751(2), 341 - 8
Interaction between phosphofructokinase and aldolase from Saccharomyces cerevisiae studied by aqueous two-phase partitioning; Matic S et al.; Phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) have been highly purified from Saccharomyces cerevisiae by improved protocols . Partitioning of the enzymes in aqueous polymer two-phase systems was used to detect complex formation . The partition of each enzyme was found to be affected by the presence of the other enzyme . AMP affected the partition of the individual enzymes as well as the mixture of the two . The activities of the respective enzymes were stimulated in the putative complex in an AMP-dependent manner . Two strictly conserved residues belonging to an acidic surface loop of class II aldolases, are a potential site for electrostatic interaction with the positively charged regions close to the active site in phosphofructokinase.

FEBS Lett, 2001 Jan 26, 489(1), 92 - 6
Mitochondrial Hsp78, a member of the Clp/Hsp100 family in Saccharomyces cerevisiae, cooperates with Hsp70 in protein refolding; Krzewska J et al.; The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress . To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro . Hsp78 alone was not able to mediate reactivation of firefly luciferase . Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p . Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system . However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins . These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat-inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.

FEBS Lett, 2001 Jan 26, 489(1), 81 - 6
Evidence for the involvement of the GTS1 gene product in the regulation of biological rhythms in the continuous culture of the yeast Saccharomyces cerevisiae; Wang J et al.; In the yeast Saccharomyces cerevisiae, ultradian oscillations of energy metabolism have been observed in continuous cultures . Here, we found that the level of the GTS1 gene product oscillated in concert with the ultradian rhythm of energy metabolism . When GTS1 was inactivated by gene disruption, the metabolic oscillation was affected severely, mostly disappearing within a day, in the absence of synchronized stress-response oscillations throughout the continuous culture . The disappearance of biological rhythms in the GTS1-deleted mutant was substantially rescued by transformation with chimera plasmids carrying GTS1 under the control of GTS1's own promoter . On the other hand, this disappearance was not rescued by constitutive expression of GTS1 under the control of the triose phosphate isomerase promoter.

Appl Environ Microbiol, 2001 Mar, 67(3), 1280 - 3
Enhanced iron uptake of Saccharomyces cerevisiae by heterologous expression of a tadpole ferritin gene; Shin YM et al.; We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin . A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity . We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter . We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron . The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate . The cell-associated concentration of iron was 500 microg per gram (dry cell weight) of the recombinant yeast but was 210 microg per gram (dry cell weight) in the wild type . These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.

Yeast, 2001 Mar 15, 18(4), 325 - 34
Functional Analysis of six novel ORFs on the left arm of Chromosome XII of Saccharomyces cerevisiae reveals three of them responding to S-starvation; Zhang N et al.; Six novel Open Reading Frames (ORFs) located on the left arm of the chromosome XII (YLL061w, YLL060c, YLL059c, YLL058w, YLL057c and YLL056c) have been analysed using either short-flanking homology (SFH) or long-flanking homology (LFH) gene replacement . Sporulation and tetrad analysis showed none of these ORFs to be essential for vegetative growth . The standard EUROFAN growth tests failed to reveal any obvious phenotypes resulting from deletion of each of the ORFs . Bioinformatic analysis revealed that YLL061w is probably an amino acid permease for S-methylmethionine and that YLL060c encodes a glutathione transferase which is involved in cellular detoxification, while YLL058w may play a role in sulphur-containing amino-acid metabolism, YLL057c in sulphonate catabolism and YLL056c in stress response . The transcription of three ORFs (YLL061w, YLL057c and YLL056c) has been shown to increase more than 10-fold under sulphate starvation . Replacement cassettes, comprising the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7 . All the cognate clones, were generated using direct PCR products amplified from genomic DNA or using gap-repair . All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt) .

Yeast, 2001 Mar 15, 18(4), 291 - 300
Replication of minichromosomes in Saccharomyces cerevisiae is sensitive to histone gene copy number and strain ploidy; Holmes SG et al.; We have characterized a defect in the mitotic transmission of plasmid minichromosomes in yeast strains deleted for the more highly expressed pair of histone H3 and H4 genes . Several observations indicate that an impairment in DNA replication contributes to the decrease in minichromosome stability . First, the maintenance of ARS plasmids that lack centromeres was also defective . Second, the addition of multiple ARS elements suppressed the defect in plasmid maintenance . Third, a synergistic increase in plasmid loss rate was seen when a plasmid containing an inefficient mutated ARS was tested in a strain deleted for histone genes, implying an interaction between ARS activity and the histone gene deletion . These results support the existence of a histone-dependent step in the initiation of DNA replication . We find that the stability of native chromosomes is not affected in strains deleted for histone genes . We propose that reduced histone H3 and H4 protein decreases the efficiency of initiation at ARS elements on plasmids and chromosomes, but that the presence of multiple origins on chromosomes compensates for the reduced efficiency . We find that decreased minichromosome stability is suppressed by increases in strain ploidy . The greater stability due to ploidy increases is not due to a relative increase in the expression of histone genes . We discuss models for the effect of strain ploidy on minichromosome maintenance .

Mol Gen Genet, 2001 Jan, 264(5), 702 - 8
Involvement of SGS1 in DNA damage-induced heteroallelic recombination that requires RAD52 in Saccharomyces cerevisiae; Onoda F et al.; The SGS1 gene of Saccharomyces cerevisiae is homologous to the genes that are mutated in Bloom's syndrome and Werner's syndrome in humans . Disruption of SGS1 results in high sensitivity to methyl methanesulfonate (MMS), poor sporulation, and a hyper-recombination phenotype including recombination between heteroalleles . In this study, we found that SGS1 forms part of the RAD52 epistasis group when cells are exposed to MMS . Exposure to DNA-damaging agents causes a striking, Rad52-dependent, increase in heteroallelic recombination in wild-type cells, but not in sgs1 disruptants . However, in the absence of DNA damage, the frequency of heteroallelic recombination in sgs1 disruptants was several-fold higher than in wild-type cells, as described previously . These results imply a function for Sgs1: it acts to suppress spontaneous heteroallelic recombination, and to promote DNA damage-induced heteroallelic recombination.

Mol Gen Genet, 2001 Jan, 264(5), 613 - 22
Transcriptional regulation of the Saccharomyces cerevisiae amino acid permease gene BAP2; Nielsen PS et al.; Uptake of branched-chain amino acids by Saccharomyces cerevisiae from media containing a preferred nitrogen source is mediated by the permeases encoded by BAP2, BAP3, and VAP1/TAT1 . The transcriptional activity of the BAP2 promoter is affected by a number of genes, including SSY1, which encodes an amino acid permease homologue that is necessary for transcription of BAP2 . Other genes that control BAP2 encode known (Leu3p, Tup1p) and putative (Stp1p, Stp2p) transcription factors . We present evidence that the zinc-finger proteins Stp1p and Stp2p bind directly to the BAP2 promoter . Binding of Stplp to the BAP2 promoter in vivo and in vitro indicates that the STP gene family indeed encodes transcription factors . The presence of a Leu3p binding site in the BAP2 promoter is required for full promoter activity on synthetic complete medium . The capacity of Leu3p to activate BAP2 transcription correlates with conditions that affect the level of alpha-isopropyl malate . The effect of a tup1 deletion on BAP2 transcription depends on SSY1 . In an ssy1 strain, the phenotype of tup1 conforms to the well-established role of Tup1p as part of a repressor complex, but in the SSY1 strain deletion of TUP1 causes a decrease in transcription, indicating that Tup1p may also have an activating role at the BAP2 promoter . Our results thus suggest a complex interplay between several transcription factors in the expression of BAP2.

Antonie Van Leeuwenhoek, 2000 Aug, 78(2), 117 - 22
Electron microscopy of the K2 killer effect of Saccharomyces cerevisiae T206 on a mesophilic wine yeast; Vadasz AS et al.; A mesophilic wine yeast, Saccharomyces cerevisiae CSIR Y217 K-R- was subjected to the K2 killer effect of Saccharomyces cerevisiae T206 K+R+ in a liquid grape medium . The lethal effect of the K2 mycoviral toxin was confirmed by methylene blue staining . Scanning electron microscopy of cells from challenge experiments revealed rippled cell surfaces, accompanied by cracks and pores, while those unaffected by the toxin, as in the control experiments, showed a smooth surface . Transmission electron microscopy revealed that the toxin damaged the cell wall structure and perturbed cytoplasmic membranes to a limited extent.

Ann N Y Acad Sci, 2000, 922, 65 - 75
Mechanisms of DNA topoisomerase I-induced cell killing in the yeast Saccharomyces cerevisiae; Fiorani P et al.; DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a mechanism of transient DNA strand cleavage characterized by the formation of a phosphotyrosyl bond between the DNA end and active site tyrosine . Camptothecin reversibly stabilizes the covalent enzyme-DNA intermediate by inhibiting DNA religation . During S-phase, collisions with advancing replication forks convert these complexes into potentially lethal lesions . To define the DNA damage induced by alterations in Top1p catalysis and the cellular processes that mediate the repair of such lesions, the yeast Saccharomyces cerevisiae was used . Substitution of conserved residues N-terminal to the active site tyrosine (Tyr-727) produced alterations in the camptothecin sensitivity or catalytic cycle of DNA Top1 . For example, substituting Ala for Thr-722 in Top1T722A increased the stability of the covalent enzyme DNA intermediate . As with camptothecin, Top1T722A-induced cytotoxicity was ascribed to a reduction in DNA religation . By contrast, enhanced covalent complex formation by Top1N726H resulted from a relative increase in the rate of DNA cleavage . Conditional yeast mutants were also selected that exhibit temperature-sensitive growth only in the presence of the self-poisoning Top1T722A enzyme . Subsequent analyses of these tah mutants identified 9 genes whose function suppresses the cytotoxic action of camptothecin and Top1T722A . These include genes encoding essential DNA replication proteins (CDC45 and DPB11) and proteins involved in SUMO- or ubiquitination (UBC9 and DOA4).

Curr Genet, 2000 Dec, 38(5), 256 - 63
Genetic analysis of the in vivo role of DNA polymerases in Saccharomyces cerevisiae; Cooley M et al.; In Saccharomyces cerevisiae, the suggested functions of DNA polymerases (DNApol) have been based primarily on the characterization of the wild-type and mutant enzymes via in vitro studies . Here we describe a novel replication system to decipher the role of different DNA polymerases in in vivo DNA replication . Using this system, {alpha-32P}dNTP is allowed to cross the membrane of permeabilized cells; then the nature of the radiolabeled products of DNA synthesis is analyzed by gel electrophoresis and densitometry . Results of such analyses show that these replication intermediates are synthesized in the range 50-1,300 bp, which are then rapidly elongated and then ligated into longer DNA chains, and that the in vivo synthesis of yeast DNA fragments is dependent essentially on DNApolalpha and DNApoldelta, but not necessarily on DNApolepsilon . Results presented here support the views that DNApolepsilon is dispensable for yeast DNA replication or that DNA polalpha and DNApoldelta are epistatic to DNApolepsilon in yeast.

Genetika, 2000 Dec, 36(12), 1629 - 33
{Repair of dual-stranded DNA in Saccharomyces cerevisiae cells: homolog-dependent ligation and role of the RAD55 gene}; Glazunov AV et al.; In our previous works, a mutation in the RAD57 gene was shown to induce the plasmid DNA double-strand gap (DSG) repair via a special recombinational repair mechanism: homolog-dependent ligation responsible for reuniting disrupted plasmid ends without reconstructing the sequence lost because of the DSG . In this work, the role of the RAD55 gene in the plasmid DNA DSG repair was studied . A cold-sensitive rad55-3 mutation markedly decreased the precision of plasmid DNA DSG repair under conditions of restrictive temperature (23 degrees C): only 5-7% of plasmids can repair DSG, whereas under permissive conditions (36 degrees C), DSGs were repaired in approximately 50% of the cells . In the cold-sensitive mutation rad57-1, the proportion of plasmids in which DSGs were repaired was nearly the same under both permissive and restrictive conditions (5-10%) . The results indicate that a disturbance in the function of the RAD55 gene, as in the RAD57 gene, leads to a drastic increase in the contribution of homolog-dependent ligation to the repair of double-strand DNA breaks.

Genetika, 2000 Dec, 36(12), 1622 - 8
{Effect of mutations in PHO85 and PHO4 genes on utilization of proline in Saccharomyces cerevisiae yeasts}; Popova IuG et al.; Yeast Saccharomyces cerevisiae can utilize proline as a single nitrogen source . We have found that the strains with mutations in gene PH085, a structural gene encoding cyclin-dependent phosphoprotein kinase, cannot grow on the proline-containing media . The ability to utilize proline is restored in strains of the pho85pho4 genotype . We suggest that phosphoprotein kinase Pho85p is involved in either phosphorylation of a highly specific proline permease, Put4p, or in signaling proline concentration . The Pho4p protein that activates transcription of the PH05 gene, a structural gene of acid phosphatase, seems to participate in the negative regulation of the PUT1 and PUT2 genes encoding enzymes of proline catabolism, proline oxidase and delta-pyrroline-5-carboxylate dehydrogenase . Thus, regulation of phosphorus and nitrogen metabolism have common elements.

J Bacteriol, 2001 Feb, 183(3), 830 - 4
Identification of a UPC2 homolog in Saccharomyces cerevisiae and its involvement in aerobic sterol uptake; Shianna KV et al.; Saccharomyces cerevisiae normally will not take up sterols from the environment under aerobic conditions . A specific mutant, upc2-1, of the predicted transcriptional activator UPC2 (YDR213w) has been recognized as a strain that allows a high level of aerobic sterol uptake . Another predicted transcriptional activator, the YLR228c gene product, is highly homologous to Upc2p . In fact, at the carboxy terminus 130 of the last 139 amino acids are similar between the two proteins . Since these proteins are very similar, the effect of mutations in the YLR228c open reading frame (ORF) was compared with like alterations in UPC2 . First, the YLR228c ORF was insertionally inactivated and crossed with various UPC2 constructs . Deletion of YLR228c and UPC2 in combination resulted in nonviability, suggesting that the two proteins have some essential overlapping function . The upc2-1 point mutation responsible for aerobic sterol uptake was duplicated in the homologous carboxy region of the YLR228c ORF using site-directed mutagenesis . This mutation on a high-copy vector resulted in an increase in sterol uptake compared to an isogenic wild-type strain . The combination of both point mutations resulted in the greatest level of aerobic sterol uptake . When the YLR228c point mutation was expressed from a low-copy vector there was little if any effect on sterol uptake . Gas chromatographic analysis of the nonsaponifiable fractions of the various strains showed that the major sterol for all YLR228c and UPC2 combinations was ergosterol, the consensus yeast sterol.

J Bacteriol, 2001 Feb, 183(3), 821 - 9
Hyperactive glycogen synthase mutants of Saccharomyces cerevisiae suppress the glc7-1 protein phosphatase mutant; Anderson C et al.; A yeast glc7-1 mutant expressing a variant of protein phosphatase type 1 fails to accumulate glycogen . This defect is associated with hyperphosphorylated and inactive glycogen synthase, consistent with Glc7p acting directly to dephosphorylate and activate glycogen synthase . To characterize the glycogen synthesis defect of this mutant in more detail, we isolated 26 pseudorevertants of the glc7-1 mutant . All pseudoreversion events were due to missense mutations in GSY2, the gene encoding the major isoform of glycogen synthase . A majority of the mutations responsible for the suppression were in the 3' end of the gene, corresponding to the phosphorylated COOH terminus of Gsy2p . Phosphorylation of the mutant proteins was reduced, suggesting that they are poor substrates for glycogen synthase kinases . Suppressor mutations outside this domain did not decrease the phosphorylation of the resulting proteins, indicating that these proteins are immune to the regulatory effects of phosphorylation . Since no growth defect has been observed for strains with altered glycogen levels, the relative levels of fitness of GSY2 mutants that fail to accumulate glycogen and that hyperaccumulate glycogen were assayed by cocultivation experiments . A wild-type strain outcompeted both hypo- and hyperaccumulating strains, suggesting that glycogen levels contribute substantially to the fitness of yeast.

Plant J, 2001 Feb, 25(3), 271 - 80
Resistance to the plant PR-5 protein osmotin in the model fungus Saccharomyces cerevisiae is mediated by the regulatory effects of SSD1 on cell wall composition; Ibeas JI et al.; The capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi . Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant . Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR-5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene . Expression of the SSD1-v allele afforded resistance to the antifungal protein . Conversely, yeast strains carrying the SSD1-d allele or a null ssd1Delta mutation displayed high sensitivity to osmotin . The SSD1-v protein mediates osmotin resistance in a cell wall-dependent manner . Deletion of SSD1-v or SSD1-d impeded sorting of the PIR proteins (osmotin-resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post-transcriptional regulation of gene expression . The sensitivity of ssd1Delta cells to osmotin was only partially suppressed by over-accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall-mediated resistance . Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell-wall morphology and lower levels of alkali-insoluble cell-wall glucans . Therefore SSD1 is an important regulator of fungal cell-wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR-5 proteins.

Yeast, 2001 Feb, 18(3), 251 - 60
Construction of phosphatidylethanolamine-less strain of Saccharomyces cerevisiae . Effect on amino acid transport; Robl I et al.; A triple yeast mutant was constructed which lacks BST1, the gene for sphingosine lyase, besides the phosphatidylserine decarboxylases PSD1 and PSD2 . In this yeast mutant, which can only be grown in the presence of exogenous ethanolamine, phosphatidylethanolamine can be depleted to very low levels . Under those conditions, respiration as well as glucose and 3-O-methylglucose uptake proceed unaffected . Plasma membrane ATPase is as active in these cells as that of control cells grown in the presence of ethanolamine . Drastically decreased, however, are H+/amino acid symporters . The activities of arginine (Can1p), proline (Put4p) and general amino acid permease (Gap1p) are decreased more than 20-fold . Amino acid transport in yeast is dependent on coupling to the proton motive force . It can be envisaged that phosphatidylethanolamine might play a role in this process or in the early steps of the secretion pathway common for all amino acid permeases or, eventually, it could affect the transport proteins directly at the plasma membrane Transformation of the triple mutant with a CEN plasmid harbouring BST1 wild-type gene totally reversed its phenotype to that observed in the double mutant.

Yeast, 2001 Feb, 18(3), 195 - 205
The cloning and characterization of the CDC50 gene family in Saccharomyces cerevisiae; Radji M et al.; We have cloned a gene that complements the cold-sensitive growth of cdc50-1 mutant strain of Saccharomyces cerevisiae at 14 degrees C . The CDC50 gene was found to be identical to YCR094w on chromosome III and contains 1173 nucleotides encoding 391 amino acids . We found a missense mutation at the first initiation codon of cdc50-1 . The disruption of the CDC50 gene revealed that it is not essential for growth, but the disruptant caused the same cold-sensitive phenotype as cdc50-1, suggesting that the cdc50-1 is a null mutation resulted from the mutation in the first codon . The cdc50-1 mutant arrests at START in G1 phase at the non-permissive temperature . The CDC50 gene product has strong structural similarity to two other proteins in Saccharomyces cerevisiae encoded by YNR048w and YNL323w . The over-expression of either YNR048w or YNL323w suppressed the cdc50-1 mutant and the double disruption of either CDC50 and YNR048w or CDC50 and YNL323w resulted in a severe slow-growth phenotype . We conclude that these three genes constitute a family with redundant function . We also found that the CDC39 gene was a multicopy suppressor of cdc50-1 mutation, suggesting that the CDC50 family is involved in regulation of transcription via CDC39.

Eur J Biochem, 2001 Feb, 268(4), 914 - 24
A novel sequence element is involved in the transcriptional regulation of expression of the ERG1 (squalene epoxidase) gene in Saccharomyces cerevisiae; Leber R et al.; Squalene epoxidase is an essential enzyme in the ergosterol-biosynthesis pathway . It catalyzes the epoxidation of squalene to 2,3-oxidosqualene and is the specific target of the antifungal drug terbinafine . Treatment of yeast cells with this inhibitor leads to squalene accumulation and sterol depletion . As ergosterol fulfils several essential functions, each requiring optimal sterol concentrations, synthesis of sterols in yeast must be tightly regulated . This study focuses on the sterol-mediated regulation of expression of the ERG1 gene, which codes for squalene epoxidase in Saccharomyces cerevisiae . Inhibition of ergosterol biosynthesis with terbinafine increases the expression of ERG1 in a concentration-dependent manner to a maximum of sevenfold . Inhibition of later steps in the ergosterol-biosynthetic pathway by ketoconazole, an inhibitor of the lanosterol-14alpha-demethylase, and U18666A, an inhibitor of the squalene-2,3-epoxide-lanosterol cyclase, also induce expression of ERG1, suggesting that ERG1 expression is positively regulated by diminished intracellular ergosterol levels . The regulatory effect of sterols is manifested at the level of transcription . Deletion analysis of the ERG1 promoter identified a novel regulatory DNA sequence element . Two 6-bp direct repeats, separated by 4 bp, AGCTCGGCCGAGCTCG, are unique to the ERG1 promoter . A DNA fragment containing this region confers ergosterol-regulated expression on an otherwise unregulated CYC1 promoter construction . One copy of the 6-bp element, AGCTCG, is sufficient to confer regulation, albeit less effectively than when both elements are present, whereas the removal of both elements from the ERG1 promoter leads to the loss of sterol-dependent ERG1 regulation.

FEMS Microbiol Lett, 2001 Feb 20, 195(2), 245 - 51
Relation between growth dynamics and diffusional limitations in Saccharomyces cerevisiae cells growing as entrapped in an insolubilised gelatin gel; de Alteriis E et al.; Flow-cytometric analysis was employed to investigate growth dynamics of a yeast cell population immobilised in an insolubilised gelatin gel by means of the quantitative determination of the average protein content per cell . This analysis was carried out on both the immobilised cell population considered as a whole and the subpopulations colonising the gelatin matrix at different depths . The results show that growth of the gelatin-immobilised yeast population was affected by the existence of a gradient of nutrient concentrations through the matrix and are in agreement with the unsteady-state diffusion model employed for the description of glucose transfer in the gel.

EMBO J, 2001 Feb 15, 20(4), 905 - 13
The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase; Johnson FB et al.; Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN . Precisely how loss of WRN leads to the phenotypes of WS is unknown . Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase . Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase . Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten . In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors . Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1531 - 6
Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells; Zhou W et al.; In higher eukaryotes, translation of some mRNAs occurs by internal initiation . It is not known, however, whether this mechanism is used to initiate the translation of any yeast mRNAs . In this report, we identify naturally occurring nucleotide sequences that function as internal ribosome entry sites (IRESes) within the 5' leader sequences of Saccharomyces cerevisiae YAP1 and p150 mRNAs . When tested in the 5' untranslated regions of monocistronic reporter genes, both leader sequences enhanced translation efficiency in vegetatively growing yeast cells . Moreover, when tested in the intercistronic region of dicistronic mRNAs, both sequences were shown to contain IRESes that functioned in living cells . The activity of the p150 leader was much greater than that of the YAP1 leader . The second cistron was not expressed in control dicistronic constructs that lacked these sequences or contained the 5' leader sequence of the CLN3 mRNA in the intercistronic region . Further analyses of the p150 IRES revealed that it contained several nonoverlapping segments that were able independently to mediate internal initiation . These results suggested a modular composition for the p150 IRES that resembled the composition of IRESes contained within some cellular mRNAs of higher eukaryotes . Both YAP1 and p150 leaders contain several complementary sequence matches to yeast 18S rRNA . The findings are discussed in terms of our understanding of internal initiation in higher eukaryotes.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1483 - 8
Jac1, a mitochondrial J-type chaperone, is involved in the biogenesis of Fe/S clusters in Saccharomyces cerevisiae; Voisine C et al.; A minor Hsp70 chaperone of the mitochondrial matrix of Saccharomyces cerevisiae, Ssq1, is involved in the formation or repair of Fe/S clusters and/or mitochondrial iron metabolism . Here, we report evidence that Jac1, a J-type chaperone of the mitochondrial matrix, is the partner of Ssq1 in this process . Reduced activity of Jac1 results in a decrease in activity of Fe/S containing mitochondrial proteins and an accumulation of iron in mitochondria . Fe/S enzyme activities remain low in both jac1 and ssq1 mutant mitochondria even if normal mitochondrial iron levels are maintained . Therefore, the low activities observed are not solely due to oxidative damage caused by excess iron . Rather, these molecular chaperones likely play a direct role in the normal assembly process of Fe/S clusters.

J Cell Sci, 2001 Feb, 114(Pt 3), 589 - 97
The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants; Hood JK et al.; The cyclin-dependent kinase Cdc28p associates with the cyclin Clb2p to induce mitosis in the yeast Saccharomyces cerevisiae . Several cell cycle regulatory proteins have been shown to require specific nuclear transport events to exert their regulatory functions . Therefore, we investigated the subcellular localization of wild-type Clb2p and several mutant versions of the protein using green fluorescent protein (GFP) fusion constructs . Wild-type Clb2p is primarily nuclear at all points of the cell . A point mutation in a potential leucine-rich nuclear export signal (NES) enhances the nuclear localization of the protein, and delta-yrb2 cells exhibit an apparent Clb2p nuclear export defect . Clb2p contains a bipartite nuclear localization signal (NLS), and its nuclear localization requires the alpha and beta importins (Srp1p and Kap95p), as well as the yeast Ran GTPase and its regulators . Deletion of the Clb2p NLS causes increased cytoplasmic localization of the protein, as well as accumulation at the bud neck . These data indicate that Clb2p exists in multiple places in the yeast cell, possibly allowing Cdc28p to locally phosphorylate substrates at distinct subcellular sites.

Biochem Soc Trans, 2000 Dec, 28(6), 700 - 2
An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae; Sandager L et al.; The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT) . Genes encoding DAGAT have been identified in both plant and animal tissues . These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT) . So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome . Both these genes have been suggested to be involved in the synthesis of sterol esters . We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.

Biochemistry, 2001 Jan 30, 40(4), 1102 - 8
Probing the binding domain of the Saccharomyces cerevisiae alpha-mating factor receptor with rluorescent ligands; Ding FX et al.; Three analogues of the alpha-mating factor pheromone of Saccharomyces cerevisiae containing the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group were synthesized that had high binding affinity to the receptor and retained biological activity . The fluorescence emission maximum of the NBD group in {K7(NBD),Nle(12)}-alpha-factor was blue shifted by 35 nm compared to buffer when the pheromone bound to its receptor . Fluorescence quenching experiments revealed that the NBD group in {K7(NBD),Nle(12)}-alpha-factor bound to the receptor was shielded from collision with iodide anion when in aqueous buffer . In contrast, the emission maximum of NBD in {K7(ahNBD),Nle(12)}-alpha-factor or {Orn7(NBD),Nle(12)}-alpha-factor was not significantly shifted and iodide anion efficiently quenched the fluorescence of these derivatives when they were bound to receptor . The fluorescence investigation suggests that when the alpha-factor is bound to its receptor, K7 resides in an environment that has both hydrophobic and hydrophilic groups within a few angstroms of each other.

Biochemistry, 2001 Jan 30, 40(4), 1083 - 90
Regulation of phosphotransferase activity of hexokinase 2 from Saccharomyces cerevisiae by modification at serine-14; Golbik R et al.; Isoenzyme 2 of hexokinase functions in sugar sensing and glucose repression in Saccharomyces cerevisiae . The degree of in vivo phosphorylation of hexokinase 2 at serine-14 is inversely related to the extracellular glucose concentration {Vojtek, A . B., and Fraenkel, D . G . (1990) Eur . J . Biochem . 190, 371-375}; however, a physiological role of the modification causing the dissociation of the dimeric enzyme in vitro {as effected by a serine-glutamate exchange at position 14; Behlke et al . (1998) Biochemistry 37, 11989-11995} is unclear . This paper describes a comparative stopped-flow kinetic and sedimentation equilibrium analysis performed with native unphosphorylated hexokinase 2 and a permanently pseudophosphorylated glutamate-14 mutant enzyme to determine the functional consequences of phosphorylation-induced enzyme dissociation . The use of a dye-linked hexokinase assay monitoring proton generation allowed the investigation of the kinetics of glucose phosphorylation over a wide range of enzyme concentrations . The kinetic data indicated that monomeric hexokinase represents the high-affinity form of isoenzyme 2 for both glycolytic substrates . Inhibition of glucose phosphorylation by ATP {Moreno et al . (1986) Eur . J . Biochem . 161, 565-569} was only observed at a low enzyme concentration, whereas no inhibition was detected at the high concentration of hexokinase 2 presumed to occur in the cell . Pseudophosphorylation by glutamate substitution for serine-14 increased substrate affinity at high enzyme concentration and stimulated the autophosphorylation of isoenzyme 2 . The possible role of hexokinase 2 in vivo phosphorylation at serine-14 in glucose signaling is discussed.

Yeast, 2001 Jan 30, 18(2), 125 - 35
Ultradian metabolic oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide, a population synchronizer, is produced by sulphite reductase; Sohn H et al.; We have reported that the consecutive cyclic production of H(2)S resulted in population synchrony of ultradian metabolic oscillation (Sohn et al., 2000) . In order to understand the origin of H(2)S and its nature of periodic production, changes of sulphur compounds concentration and responsible enzymes were investigated . The concentrations of extracellular sulphate, intracellular glutathione and cysteine oscillated during metabolic oscillation but only the oscillation of sulphate concentration was out of phase with H(2)S production . The sulphate concentration in culture directly affected the amplitude and the period of metabolic oscillation: (a) the period of metabolic oscillation shortened from 50 min to 30 min when sulphate concentration in the medium was reduced from 46 mM to 2.5 mM; (b) the metabolic oscillation disappeared under sulphate-depletion conditions and arose again by the addition of sulphate . Pulse injection of sulphite (10 microM) perturbed metabolic oscillation with a burst production of H(2)S, while thiosulphate (up to 500 microM) was without apparent effect . Furthermore, addition of S-adenosyl methionine (100 microM) or azoxybacilin (3 mg/kg) decreased H(2)S production with perturbation of metabolic oscillation . The results presented here suggest that H(2)S, a population synchronizer, is produced by sulphite reductase in the sulphate assimilation pathway, and dynamic regulation of sulphate uptake plays an important role in ultradian metabolic oscillation .

Mol Microbiol, 2001 Feb, 39(3), 595 - 605
Role of thioredoxin reductase in the Yap1p-dependent response to oxidative stress in Saccharomyces cerevisiae; Carmel-Harel O et al.; The Saccharomyces cerevisiae Yap1p transcription factor is required for the H2O2-dependent activation of many antioxidant genes including the TRX2 gene encoding thioredoxin 2 . To identify factors that regulate Yap1p activity, we carried out a genetic screen for mutants that show elevated expression of a TRX2-HIS3 fusion in the absence of H2O2 . Two independent mutants isolated in this screen carried mutations in the TRR1 gene encoding thioredoxin reductase . Northern blot and whole-genome expression analysis revealed that the basal expression of most Yap1p targets and many other H2O2-inducible genes is elevated in Deltatrr1 mutants in the absence of external stress . In Deltatrr1 mutants treated with H2O2, the Yap1p targets, as well as genes comprising a general environmental stress response and genes encoding protein-folding chaperones, are hyperinduced . However, despite the elevated expression of genes encoding antioxidant enzymes, Deltatrr1 mutants are extremely sensitive to H2O2 . The results suggest that cells lacking thioredoxin reductase have diminished capacity to detoxify oxidants and/or to repair oxidative stress-induced damage and that the thioredoxin system is involved in the redox regulation of Yap1p transcriptional activity.

Lett Appl Microbiol, 2001 Jan, 32(1), 42 - 6
Effect of hydrostatic pressure on the morphology and ultrastructure of wild-type and trehalose synthase mutant cells of Saccharomyces cerevisiae; Fernandes PM et al.; AIMS: Saccharomyces cerevisiae was used for studying the physiological effects of hydrostatic pressure . METHODS AND RESULTS: The effects of hydrostatic pressure on the ultrastructure of wild-type and trehalose-6-phosphate synthase (tps1) mutant cells were investigated by transmission electron microscopy . Pressure induced several morphological changes in wild-type and tps1 cells, the latter showing greater structural alterations . When the cells were submitted to a preheat treatment they both acquired resistance to the pressure treatment . CONCLUSION: As the tps1 mutant was 1000-fold more barosensitive than its parental strain, it showed greater structural alterations compared with the wild-type . Microscopic images of the yeast cells suggested that hydrostatic pressure induced changes in the cytoskeleton and therefore, on the cell wall and in the dynamics of the organelles . SIGNIFICANCE AND IMPACT OF THE STUDY: This work presents the effects of hydrostatic pressure on the morphology of yeast cells and confirms the importance of several different factors in the protection of cells against stress.

J Appl Microbiol, 2001 Feb, 90(2), 216 - 22
Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition; Kapat A et al.; AIMS: To obtain an optimal combination of agitation speed and aeration rate for maximization of specific glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between kLa vis-a-vis oxygen transfer condition and specific glucose oxidase production . METHODS AND RESULTS: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD) . The dissolved oxygen concentration and the volumetric oxygen transfer coefficient (kLa) were determined at corresponding combinations of impeller speed and aeration rate . The maximal specific extracellular glucose oxidase production (3.17 U mg-1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6.83 mg l-1 at the impeller speed of 420 rev min-1 and at the rate of aeration of 0.25 vvm . It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between kLa and specific GOD production could not be established . CONCLUSION: At the agitation speed of 420 rev min-1 and at 0.25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme . SIGNIFICANCE AND IMPACT OF THE STUDY: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature . Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production . It also suggests that since there is no correlation between kLa and specific production of GOD, kLa should not be used as one of the scale-up parameters.

Genes Cells, 2000 Dec, 5(12), 975 - 89
Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae; Kawasaki Y et al.; BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae . Previous work showed that Mcm10p interacts with the Mcm2-7 protein complex that may be functioning as the replication-licensing factor . In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks . RESULTS: We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures . Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases epsilon and delta . Mcm10p is an abundant protein (approximately 4 x 10(4) copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle . When it is visualized by the chromosome-spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC . CONCLUSIONS: These results suggest that Mcm10p, like the Mcm2-7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication.

Eur J Biochem, 2001 Feb, 268(3), 761 - 7
Major tyrosine identity determinants in Methanococcus jannaschii and Saccharomyces cerevisiae tRNA(Tyr) are conserved but expressed differently; Fechter P et al.; Using in vitro tRNA transcripts and minihelices it was shown that the tyrosine identity for tRNA charging by tyrosyl-tRNA synthetase (TyrRS) from the archaeon Methanococcus jannaschii is determined by six nucleotides: the discriminator base A73 and the first base-pair C1-G72 in the acceptor stem together with the anticodon triplet . The anticodon residues however, participate only weakly in identity determination, especially residues 35 and 36 . The completeness of the aforementioned identity set was verified by its tranfer into several tRNAs which then become as efficiently tyrosylatable as the wild-type transcript from M . jannaschii . Temperature dependence experiments on both the structure and the tyrosylation properties of M . jannaschii and yeast tRNA(Tyr) transcripts show that the archaeal transcript has greater structural stability and enhanced aminoacylation behaviour than the yeast transcript . Tyrosine identity in M . jannaschii is compared to that in yeast, and the conservation of the major determinant in both organisms, namely the C1-G72 pair, gives additional support to the existence of a functional connection between archaeal and eukaryotic aminoacylation systems.

Eur J Biochem, 2001 Jan, 268(2), 334 - 43
Existence of a tightly regulated water channel in Saccharomyces cerevisiae; Meyrial V et al.; The Saccharomyces cerevisiae strain Sigma1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p . Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte . However, no function could be attributed to Aqy2-1p in this system . Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast . Aqy1-1p was never detected whatever the growth phase and culture conditions tested . In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose . Aqy2-1p expression was repressed by hyper-osmotic culture conditions . Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane . In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis . Our results show that the expression of aquaporins is tightly controlled . The physiological relevance of aquaporin-mediated water transport in yeast is discussed.

Mutat Res, 2001 Feb 20, 473(2), 151 - 61
Mutagenic specificity of the base analog 6-N-hydroxylaminopurine in the LYS2 gene of yeast Saccharomyces cerevisiae; Kulikov VV et al.; We used the LYS2 gene mutational system to study mutation specificity of the base analog 6-N-hydroxylaminopurine (HAP) in yeast . We characterized phenotypes of mutations using codon-specific nonsense suppressors and the test employing inactivation of the release factor Sup35 due to overexpression and formation of prion-like derivative {PSI} . We have shown that HAP induces predominantly nonsense mutations . While the tests using codon-specific nonsense-suppressors allowed to identify only about 50% of nonsense-mutations, all the nonsense-mutations were identified in the test with defective Sup35 . We determined and analyzed the spectrum of HAP-induced nucleotide changes in two regions of the gene . HAP induces predominantly GC-->AT transitions in a hotspots of a central position of trinucleotide GGA or AGG . Directionality of these transitions is consistent with the idea that initial dHAPMP incorporation in the leading strand is more genetically dangerous than in lagging DNA strand . We revealed a specific context inhibitory for HAP mutagenesis, a "T" in -1 position to mutation site.

Mutat Res, 2001 Jan 25, 473(1), 101 - 7
Adaptive reversions of a frameshift mutation in arrested Saccharomyces cerevisiae cells by simple deletions in mononucleotide repeats; Heidenreich E et al.; Adaptive mutations are characterised as the outcome of an as yet unknown mechanism, which allows a few individuals of a cell population to overcome a starvation-induced cell cycle arrest and to proliferate . A release from such a non-lethal growth limitation is accomplished by mutations generated without DNA replication . Originally adaptive mutations were described in Escherichia coli, but more recently also in a simple eukaryote, the budding yeast Saccharomyces cerevisiae . We are studying the adaptive reversion of a frameshift allele which occurs when an auxotrophic yeast strain is starved for the amino acid essential for its proliferation . In this communication, we report on the DNA sequences from the locus concerned . Comparison between sequences from revertant clones which arose several days after growth arrest by starvation and those from revertants produced during proliferation shows significantly different mutation spectra: for replication-dependent revertants nucleotide gains and losses in a variety of sequence contexts are reasonably balanced, whereas for the replication-independent, i.e . adaptive, revertants mainly simple deletions in mononucleotide repeats were observed . These mutations resemble those known to originate from DNA polymerase slippage errors which were miscorrected or had escaped correction by the mismatch repair machinery . Our data present strong evidence for differences in the mechanistic origins of adaptive versus DNA replication-dependent mutations in a eukaryote . Most probably, mutations in non-replicating cells contribute to evolution, and if conserved in mammals, to human carcinogenesis.

FEBS Lett, 2001 Feb 2, 489(2-3), 197 - 201
Kcc4 associates with septin proteins of Saccharomyces cerevisiae; Okuzaki D et al.; Kcc4, a kinase of the budding yeast Saccharomyces cerevisiae, is homologous to the bud neck protein kinases Hsl1/Nik1 and Gin4 . We report here that a GFP-Kcc4 fusion protein is localized at the bud neck and that the non-kinase domain is required for this localization . We also demonstrate that Kcc4 associates with septin proteins in vitro and in vivo by two-hybrid analysis, GST pull-down experiments, immunoprecipitation, and analysis of direct association with affinity-purified GST-Kcc4 and MBP-Septin proteins . From the results obtained here, we suggest that Cdc11 is the primary association partner of Kcc4.

Mol Cell, 2000 Dec, 6(6), 1491 - 9
DNA polymerase zeta introduces multiple mutations when bypassing spontaneous DNA damage in Saccharomyces cerevisiae; Harfe BD et al.; Spontaneous DNA damage can be dealt with by multiple repair/bypass pathways that have overlapping specificities . We have used a frameshift reversion assay to examine spontaneous mutations that accumulate in yeast strains defective for the high-fidelity nucleotide excision repair or recombination pathways . In contrast to the simple frameshift mutations that occur in wild-type strains, the reversion events in mutant strains are often complex in nature, with the selected frameshift mutation being accompanied by one or more base substitutions . Genetic analyses demonstrate that the complex events are dependent on the Pol zeta translesion polymerase, thus implicating the DNA damage bypass activity of low-fidelity translesion polymerases in hypermutation phenomena.

Virology, 2001 Feb 15, 280(2), 169 - 75
The human papillomavirus type 16 E5 protein alters vacuolar H(+)-ATPase function and stability in Saccharomyces cerevisiae; Briggs MW et al.; The human papillomavirus 16 (HPV-16) E5 oncoprotein is a small integral membrane protein that binds to the 16-kDa subunit of the vacuolar H(+)-ATPase (v-ATPase) . Conservation within the family of v-ATPases prompted us to look to Saccharomyces cerevisiae as a potential model organism for E5 study . The E5 open reading frame, driven by a galactose-inducible promoter, was integrated into the yeast genome, and the resulting strain demonstrated a nearly complete growth arrest at neutral pH, consistent with defects associated with yeast v-ATPase mutants . Furthermore, this strain demonstrated a severe reduction in pH-dependent and v-ATPase-dependent vacuolar localization of fluorescent markers . Overexpression of the yeast 16-kDa subunit homolog partially suppressed E5-associated growth defects . E5 expression was correlated with a disassociation of the integral (V(o)) and peripheral (V(i)) v-ATPase sub-complexes, as well as a dramatic reduction of the steady-state levels of one mature V(o) subunit and the concomitant accumulation of its major proteolytic fragment, with unchanged levels of two V(i) subunits . Similar analyses of selected E5 mutants in yeast demonstrated a correlation between E5 biology and v-ATPase disruption . Our observations suggest that wild-type HPV-16 E5 acts during the assembly of the v-ATPase to inhibit, either directly or indirectly, V(o) stability and complex formation .

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 216 - 22
Transcriptional activation of FLR1 gene during Saccharomyces cerevisiae adaptation to growth with benomyl: role of Yap1p and Pdr3p; Tenreiro S et al.; The adaptation of Saccharomyces cerevisiae to growth in the presence of the antimitotic fungicide benomyl involves the dramatic activation of FLR1 transcription, taking place during benomyl-induced latency following sudden exposure to the fungicide . FLR1 gene encodes a plasma membrane transporter of the major facilitator superfamily (MFS) conferring resistance to multiple drugs, in particular to benomyl . FLR1 activation is completely abolished in a mutant devoided of YAP1 gene being exerted by Yap1p either directly or via Pdr3p . YAP1 gene was proved to be a determinant of benomyl resistance; the duration of the adaptation period preceding cell division under benomyl stress was longer for the Deltayap1 population, presumably due to the abolishment of FLR1 activation during latency . Although benomyl resistance mediated by Yap1p is reduced in a FLR1 deletion mutant, results also indicate that Yap1p may have other target genes that confer benomyl resistance in yeast .

Nucleic Acids Res, 2001 Feb 1, 29(3), 799 - 808
Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites; Cohen BD et al.; The DAN/TIR mannoprotein genes of Saccharomyces cerevisiae (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3 and TIR4) are expressed in anaerobic cells while the predominant cell wall proteins Cwp1 and Cwp2 are down-regulated . Elements involved in activation and repression of the DAN/TIR genes were defined in this study, using the DAN1 promoter as a model . Nested deletions in a DAN1/lacZ reporter pinpointed regions carrying activation and repression elements . Inspection revealed two consensus sequences subsequently shown to be independent anaerobic response elements (AR1, consensus TCGTTYAG; AR2, consensus AAAAATTGTTGA) . AR1 is found in all of the DAN/TIR promoters; AR2 is found in DAN1, DAN2 and DAN3 . A 120 bp segment carrying two copies of AR1 preferentially activated transcription of lacZ under anaerobic conditions . A fusion of three synthetic copies of AR1 to MEL1 was also expressed anaerobically . Mutations in either AR1 site within the 120 bp segment caused a drastic loss of expression, indicating that both are necessary for activation and implying cooperativity between adjacent transcriptional activation complexes . A single AR2 site carried on a 46 bp fragment from the DAN1 promoter activated lacZ transcription under anaerobic conditions, as did a 26 bp synthetic AR2 fragment fused to MEL1 . Nucleotide substitutions within the AR2 sequence eliminated the activity of the 46 bp segment . Ablation of the AR2 sequences in the full promoter caused a partial reduction of expression . The presence of the ATTGTT core (recognized by HMG proteins) in the AR2 sequence suggests that an HMG protein may activate through AR2 . One region was implicated in aerobic repression of DAN1 . It contains sites for the heme-induced Mot3 and Rox1 repressors.

Biophys J, 2001 Feb, 80(2), 626 - 34
Control of glycolytic dynamics by hexose transport in Saccharomyces cerevisiae; Reijenga KA et al.; It is becoming accepted that steady-state fluxes are not necessarily controlled by single rate-limiting steps . This leaves open the issue whether cellular dynamics are controlled by single pacemaker enzymes, as has often been proposed . This paper shows that yeast sugar transport has substantial but not complete control of the frequency of glycolytic oscillations . Addition of maltose, a competitive inhibitor of glucose transport, reduced both average glucose consumption flux and frequency of glycolytic oscillations . Assuming a single kinetic component and a symmetrical carrier, a frequency control coefficient of between 0.4 and 0.6 and an average-flux control coefficient of between 0.6 and 0.9 were calculated for hexose transport activity . In a second approach, mannose was used as the carbon and free-energy source, and the dependencies on the extracellular mannose concentration of the transport activity, of the frequency of oscillations, and of the average flux were compared . In this case the frequency control coefficient and the average-flux control coefficient of hexose transport activity amounted to 0.7 and 0.9, respectively . From these results, we conclude that 1) transport is highly important for the dynamics of glycolysis, 2) most but not all control resides in glucose transport, and 3) there should at least be one step other than transport with substantial control.

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 1107 - 12 Epub 2001 Jan 16.
Environmental stress and mutational load in diploid strains of the yeast Saccharomyces cerevisiae; Szafraniec K et al.; The negative effect of permanent contamination of populations because of spontaneous mutations does not appear to be very high if judged from the relatively good health of humans or many wild and domesticated species . This is partly explained by the fact that, in diploids, the new mutations are usually located in heterozygous loci and therefore are masked by wild-type alleles . The expression of mutations at the phenotypic level may also strongly depend on environmental factors if, for example, deleterious alleles are more easily compensated under favorable conditions . The present experiment uses diploid strains of yeast in which mutations arise at high rates because a mismatch-repair protein is missing . This mutagenesis resulted in a number of new alleles that were in heterozygous loci . They had no detectable effect on fitness when the environment was benign . A very different outcome was seen when thermal shock was applied, where fitness of the mutation-contaminated clones was lower and more diverse than that of the nonmutagenized clones . This shows that the genetic load conferred by spontaneous mutations can be underestimated or even overlooked in favorable conditions . Therefore, genetic variation can be higher and natural selection more intense when environmental conditions are getting poorer . These conclusions apply, at least, to that component of variation that directly originates from spontaneous mutations (as opposed to the variation resulting from the history of selection).

Proc Natl Acad Sci U S A, 2001 Jan 30, 98(3), 1018 - 23
Characterization of Rny1, the Saccharomyces cerevisiae member of the T2 RNase family of RNases: unexpected functions for ancient enzymes?
MacIntosh GC, Bariola PA, Newbigin E, Green PJ.
The T(2) family of nonspecific endoribonucleases (EC ) is a widespread family of RNases found in every organism examined thus far . Most T(2) enzymes are secretory RNases and therefore are found extracellularly or in compartments of the endomembrane system that would minimize their contact with cellular RNA . Although the biological functions of various T(2) RNases have been postulated on the basis of enzyme location or gene expression patterns, the cellular roles of these enzymes are generally unknown . In the present work, we characterized Rny1, the only T(2) RNase in Saccharomyces cerevisiae . Rny1 was found to be an active, secreted RNase whose gene expression is controlled by heat shock and osmotic stress . Inactivation of RNY1 leads to unusually large cells that are temperature-sensitive for growth . These phenotypes can be complemented not only by RNY1 but also by both structurally related and unrelated secretory RNases . Additionally, the complementation depends on RNase activity . When coupled with a recent report on the effect of specific RNAs on membrane permeability {Khvorova, A., Kwak, Y-G., Tamkun, M., Majerfeld, I . & Yarus, M . (1999) Proc . Natl . Acad . Sci . USA 96, 10649-10654}, our work suggests an unexpected role for Rny1 and possibly other secretory RNases . These enzymes may regulate membrane permeability or stability, a hypothesis that could present an alternative perspective for understanding their functions.

Microbiology, 2001 Feb, 147(Pt 2), 289 - 98
Siderophore uptake and use by the yeast Saccharomyces cerevisiae; Lesuisse E et al.; The non-reductive uptake of several siderophores (ferrioxamine B, ferrichrome, triacetylfusarinine C and ferricrocin) by various strains of Saccharomyces cerevisiae was studied . Several aspects of siderophore transport were examined, including specificity of transport, regulation of transport and intracellular localization of the ferri-siderophores . Ferrioxamine B was taken up preferentially via the products of the SIT1 gene and triacetylfusarinine C by the TAF1 gene product, but the specificity was not absolute . Ferrichrome and ferricrocin uptake was not dependent on a single major facilitator superfamily (MFS) gene product . The apparent specificity of transport was strongly dependent on the genetic background of the cells . Non-reductive uptake of siderophores was induced under more stringent conditions (of iron deprivation) than was the reductive uptake of ferric citrate . Regulation of transport depended on the transcriptional factors Aft1 and Tup1/Ssn6 . Cells disrupted for the TUP1 or SSN6 genes were constitutively derepressed for the uptake of ferrichrome, ferricrocin or ferrioxamine B, but not for the uptake of triacetylfusarinine C . Cells bearing the AFT1(up) mutation accumulated large amounts of ferric siderophores . Intracellular decomplexation of the siderophores occurred when transcription of the AFT1(up) gene was repressed . Ferrioxamine B and ferrichrome seemed to accumulate in an endosomal compartment, as shown by biochemical studies and by confocal microscopy study of cells loaded with a fluorescent derivative of ferrichrome . Endocytosis was, however, not involved in the non-reductive uptake of siderophores.

J Clin Microbiol, 2001 Feb, 39(2), 551 - 9
Microsatellite typing as a new tool for identification of Saccharomyces cerevisiae strains; Hennequin C et al.; Since Saccharomyces cerevisiae appears to be an emerging pathogen, there is a need for a valuable molecular marker able to distinguish among strains . In this work, we investigated the potential value of microsatellite length polymorphism with a panel of 91 isolates, including 41 clinical isolates, 14 laboratory strains, and 28 strains with industrial relevance . Testing seven polymorphic regions (five trinucleotide repeats and two dinucleotide repeats) in a subgroup of 58 unrelated strains identified a total of 69 alleles (6 to 13 per locus) giving 52 different patterns with a discriminatory power of 99.03% . We found a cluster of clinical isolates sharing their genotype with a bakery strain, suggesting a digestive colonization following ingestion of this strain with diet . With the exception of this cluster of isolates and isolates collected from the same patient or from patients treated with Saccharomyces boulardii, all clinical isolates gave different and unique patterns . The genotypes are stable, and the method is reproducible . The possibility to make the method portable is of great interest for further studies using this technique . This work shows the possibility to readily identify S . boulardii (a strain increasingly isolated from invasive infections) using a unique and specific microsatellite allele.

J Cell Biol, 2001 Feb 5, 152(3), 519 - 30
Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae; Kihara A et al.; Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY) . Although Vps30p is known to interact with Apg14p, its precise role remains unclear . We found that two proteins copurify with Vps30p . They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase . Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p . These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es) . Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes . Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting . The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es) . We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.

J Cell Biol, 2001 Feb 5, 152(3), 451 - 69
A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis; Song S et al.; Coordination of mitotic exit with timely initiation of cytokinesis is critical to ensure completion of mitotic events before cell division . The Saccharomyces cerevisiae polo kinase Cdc5 functions in a pathway leading to the degradation of mitotic cyclin Clb2, thereby permitting mitotic exit . Here we provide evidence that Cdc5 also plays a role in regulating cytokinesis and that an intact polo-box, a conserved motif in the noncatalytic COOH-terminal domain of Cdc5, is required for this event . Depletion of Cdc5 function leads to an arrest in cytokinesis . Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN), but not the corresponding polo-box mutant, resulted in connected cells . These cells shared cytoplasms with incomplete septa, and possessed aberrant septin ring structures . Provision of additional copies of endogenous CDC5 remedied this phenotype, suggesting a dominant-negative inhibition of cytokinesis . The polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways . Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box-dependent cytokinetic pathway.

EMBO J, 2001 Feb 1, 20(3), 520 - 31
RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae; Donze D et al.; The chromosomes of eukaryotes are organized into structurally and functionally discrete domains . Several DNA elements have been identified that act to separate these chromatin domains . We report a detailed characterization of one of these elements, identifying it as a unique tRNA gene possessing the ability to block the spread of silent chromatin in Saccharomyces cerevisiae efficiently . Transcriptional potential of the tRNA gene is critical for barrier activity, as mutations in the tRNA promoter elements, or in extragenic loci that inhibit RNA polymerase III complex assembly, reduce barrier activity . Also, we have reconstituted the Drosophila gypsy element as a heterochromatin barrier in yeast, and have identified other yeast sequences, including the CHA1 upstream activating sequence, that function as barrier elements . Extragenic mutations in the acetyltransferase genes SAS2 and GCN5 also reduce tRNA barrier activity, and tethering of a GAL4/SAS2 fusion creates a robust barrier . We propose that silencing mediated by the Sir proteins competes with barrier element-associated chromatin remodeling activity.

EMBO J, 2001 Feb 1, 20(3), 316 - 29
Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation; Ullrich TC et al.; ATP sulfurylases (ATPSs) are ubiquitous enzymes that catalyse the primary step of intracellular sulfate activation: the reaction of inorganic sulfate with ATP to form adenosine-5'-phosphosulfate (APS) and pyrophosphate (PPi) . With the crystal structure of ATPS from the yeast Saccharomyces cerevisiae, we have solved the first structure of a member of the ATP sulfurylase family . We have analysed the crystal structure of the native enzyme at 1.95 Angstroms resolution using multiple isomorphous replacement (MIR) and, subsequently, the ternary enzyme product complex with APS and PPi bound to the active site . The enzyme consists of six identical subunits arranged in two stacked rings in a D:3 symmetric assembly . Nucleotide binding causes significant conformational changes, which lead to a rigid body structural displacement of domains III and IV of the ATPS monomer . Despite having similar folds and active site design, examination of the active site of ATPS and comparison with known structures of related nucleotidylyl transferases reveal a novel ATP binding mode that is peculiar to ATP sulfurylases.

Genetics, 2001 Feb, 157(2), 567 - 77
Tof1p regulates DNA damage responses during S phase in Saccharomyces cerevisiae; Foss EJ; A tof1 mutant was recovered in a screen aimed at identifying genes involved specifically in the S phase branch of the MEC1-dependent DNA damage response pathway . The screen was based on the observation that mutants missing this branch are particularly dependent on the cell cycle-wide branch and, therefore, on RAD9, for surviving DNA damage . tof1 and rad9 conferred synergistic sensitivity to MMS, UV, and HU, and the double mutant was incapable of slowing S phase in response to MMS, inducing RNR3 transcription in response to UV, and phosphorylating Rad53p in response to HU . TOF1's contribution to DNA damage response appeared to be restricted to S phase, since TOF1 did not contribute to UV-induced transcription during G1 or to the cdc13-1-induced block to anaphase in G2/M . I suggest a model in which Tof1p functions to link Mec1p with Rad53p.

Genetics, 2001 Feb, 157(2), 557 - 65
Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae; Klein HL; The SRS2 gene of Saccharomyces cerevisiae encodes a DNA helicase that is active in the postreplication repair pathway and homologous recombination . srs2 mutations are lethal in a rad54Delta background and cause poor growth or lethality in rdh54Delta, rad50Delta, mre11Delta, xrs2Delta, rad27Delta, sgs1Delta, and top3Delta backgrounds . Some of these genotypes are known to be defective in double-strand break repair . Many of these lethalities or poor growth can be suppressed by mutations in other genes in the DSB repair pathway, namely rad51, rad52, rad55, and rad57, suggesting that inhibition of recombination at a prior step prevents formation of a lethal intermediate . Lethality of the srs2Delta rad54Delta and srs2Delta rdh54Delta double mutants can also be rescued by mutations in the DNA damage checkpoint functions RAD9, RAD17, RAD24, and MEC3, indicating that the srs2 rad54 and srs2 rdh54 mutant combinations lead to an intermediate that is sensed by these checkpoint functions . When the checkpoints are intact the cells never reverse from the arrest, but loss of the checkpoints releases the arrest . However, cells do not achieve wild-type growth rates, suggesting that unrepaired damage is still present and may lead to chromosome loss.

Genetics, 2001 Feb, 157(2), 545 - 56
Coupling of Saccharomyces cerevisiae early meiotic gene expression to DNA replication depends upon RPD3 and SIN3; Lamb TM et al.; It has been established that meiotic recombination and chromosome segregation are inhibited when meiotic DNA replication is blocked . Here we demonstrate that early meiotic gene (EMG) expression is also inhibited by a block in replication . Since early meiotic genes are required to promote meiotic recombination and DNA division, the low expression of these genes may contribute to the block in meiotic progression . We have identified three Hur- (HU reduced recombination) mutants that fail to couple meiotic recombination and gene expression with replication . One of these mutations is in RPD3, a gene required to maintain meiotic gene repression in mitotic cells . Complete deletions of RPD3 and the repression adapter SIN3 permitted recombination and early meiotic gene expression when replication was inhibited with hydroxyurea (HU) . Biochemical analysis showed that the Rpd3p-Sin3p-Ume6p repression complex does exist in meiotic cells . These observations suggest that repression of early meiotic genes by SIN3 and RPD3 is critical for the normal response to inhibited replication . A second response to inhibited replication has also been discovered . HU-inhibited replication reduced the accumulation of phospho-Ume6p in meiotic cells . Phosphorylation of Ume6p normally promotes interaction with the meiotic activator Ime1p, thereby activating EMG expression . Thus, inhibited replication may also reduce the Ume6p-dependent activation of EMGs . Taken together, our data suggest that both active repression and reduced activation combine to inhibit EMG expression when replication is inhibited.

J Biol Chem, 2000 Apr 14, 275(15), 11174 - 80
Elongin from Saccharomyces cerevisiae; Koth CM et al.; Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C . Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome . Neither yeast elongin A nor C sequence homologues was required for cell viability . The two gene products could be purified from yeast as a complex . A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps . The complex did not stimulate transcription elongation by yeast RNA polymerase II . Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C . The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy . These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.

J Biol Chem, 2000 Apr 14, 275(15), 10937 - 42
An investigation of the metabolism of isoleucine to active Amyl alcohol in Saccharomyces cerevisiae; Dickinson JR et al.; The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of (13)C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants . From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible . All involved the initial decarboxylation of isoleucine to alpha-keto-beta-methylvalerate . The first, via branched chain alpha-ketoacid dehydrogenase to alpha-methylbutyryl-CoA, was eliminated because abolition of branched-chain alpha-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol . However, the lpd1 mutant still produced large amounts of alpha-methylbutyrate which initially seemed contradictory because it had been assumed that alpha-methylbutyrate was derived from alpha-methylbutyryl-CoA via acyl-CoA hydrolase . Subsequently it was observed that alpha-methylbutyrate arises from the non-enzymic oxidation of alpha-methylbutyraldehyde (the immediate decarboxylation product of alpha-keto-beta-methylvalerate) . Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize alpha-keto-beta-methylvalerate . Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol . This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each alpha-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways . In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of alpha-ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate alpha-ketoisovalerate.

Mol Cell Biol, 2001 Feb, 21(3), 827 - 39
Dynamic localization and function of Bni1p at the sites of directed growth in Saccharomyces cerevisiae; Ozaki-Kuroda K et al.; Formin homology (FH) proteins are implicated in cell polarization and cytokinesis through actin organization . There are two FH proteins in the yeast Saccharomyces cerevisiae, Bni1p and Bnr1p . Bni1p physically interacts with Rho family small G proteins (Rho1p and Cdc42p), actin, two actin-binding proteins (profilin and Bud6p), and a polarity protein (Spa2p) . Here we analyzed the in vivo localization of Bni1p by using a time-lapse imaging system and investigated the regulatory mechanisms of Bni1p localization and function in relation to these interacting proteins . Bni1p fused with green fluorescent protein localized to the sites of cell growth throughout the cell cycle . In a small-budded cell, Bni1p moved along the bud cortex . This dynamic localization of Bni1p coincided with the apparent site of bud growth . A bni1-disrupted cell showed a defect in directed growth to the pre-bud site and to the bud tip (apical growth), causing its abnormally spherical cell shape and thick bud neck . Bni1p localization at the bud tips was absolutely dependent on Cdc42p, largely dependent on Spa2p and actin filaments, and partly dependent on Bud6p, but scarcely dependent on polarized cortical actin patches or Rho1p . These results indicate that Bni1p regulates polarized growth within the bud through its unique and dynamic pattern of localization, dependent on multiple factors, including Cdc42p, Spa2p, Bud6p, and the actin cytoskeleton.

Mol Cell Biol, 2001 Feb, 21(3), 755 - 64
Pie1, a protein interacting with Mec1, controls cell growth and checkpoint responses in Saccharomyces cerevisiae; Wakayama T et al.; In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls . These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins . In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth . We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo . Like MEC1, PIE1 is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Delta mutants . Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Delta cells, as in mec1Delta cells . Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3 . Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of the pie1Delta mutation . Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth . However, Mec1 kinase activity is unaffected by the pie1Delta mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity . Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.

FEMS Microbiol Rev, 2001 Jan, 25(1), 125 - 45
Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae; Francois J et al.; Glycogen and trehalose are the two glucose stores of yeast cells . The large variations in the cell content of these two compounds in response to different environmental changes indicate that their metabolism is controlled by complex regulatory systems . In this review we present information on the regulation of the activity of the enzymes implicated in the pathways of synthesis and degradation of glycogen and trehalose as well as on the transcriptional control of the genes encoding them . cAMP and the protein kinases Snf1 and Pho85 appear as major actors in this regulation . From a metabolic point of view, glucose-6-phosphate seems the major effector in the net synthesis of glycogen and trehalose . We discuss also the implication of the recently elucidated TOR-dependent nutrient signalling pathway in the control of the yeast glucose stores and its integration in growth and cell division . The unexpected roles of glycogen and trehalose found in the control of glycolytic flux, stress responses and energy stores for the budding process, demonstrate that their presence confers survival and reproductive advantages to the cell . The findings discussed provide for the first time a teleonomic value for the presence of two different glucose stores in the yeast cell.

Environ Mol Mutagen, 2000, 36(4), 325 - 34
Mutations in RAD3, MSH2, and RAD52 affect the rate of gene amplification in the yeast Saccharomyces cerevisiae; Peterson C et al.; We report here the use of the ADH4:CUP1 amplification detection system to identify five high amplification rate (HAR) strains of Saccharomyces cerevisiae that display 40- to 600-fold higher amplification rates than those of parental strains . We have identified a mutation in RAD3 DNA repair helicase gene in HAR strain B9-40 that results in a 40-fold increase in amplification rate . RAD3 is the functional homolog of the human XPD gene, suggesting that this model system will provide important candidates for genes that affect gene amplification in human cells . Isolation of the HAR strains has allowed us to test whether RAD52, which is essential for recombinational repair of DNA double-strand breaks, is also essential for amplification . Deletion of RAD52 in HAR strains B3-10 and B11-60 decreases amplification approximately 100-fold . In contrast, deletion of MSH2, which increases recombination between sequences with limited similarity, increases the amplification rate about 10-fold . These results suggest that recombination is an important step in amplification .

FEBS Lett, 2000 Dec 29, 487(2), 307 - 12
Ecm10, a novel hsp70 homolog in the mitochondrial matrix of the yeast Saccharomyces cerevisiae; Baumann F et al.; Members of the heat shock protein 70 (Hsp70) family are found in most of the compartments of eukaryotic cells where they play essential roles in protein metabolism . In yeast mitochondria, two Hsp70 proteins are known: Ssc1 and Ssq1 . We identified Ecm10 as a third Hsp70 protein in the mitochondrial matrix . Ecm10 shares 82% amino acid identity with Ssc1 and 54% with Ssq1 . Overexpression of Ecm10 mitigates protein import defects in ssc1 mutants suggesting that Ecm10 can play a role in protein translocation . Like Ssc1, Ecm10 interacts with the nucleotide exchange factor Mge1 in an ATP-dependent manner . Deletion of ecm10 leads to synthetic growth defects with ssc1 mutations at low temperature . Our data suggest an overlapping function of Ecm10 and Ssc1.

Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 445 - 50 Epub 2001 Jan 09.
Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae; Barilla D et al.; The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors . We show here that the cleavage/polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD . First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD . This interaction is confirmed for Rna15p by yeast two-hybrid analysis . Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins . These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD . Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p . Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript.

Yi Chuan Xue Bao, 2000, 27(4), 369 - 76
{Yeast (Saccharomyces cerevisiae) secretory expression vector maintained stably in Pro3+ transformants in rich medium}; Xie HY et al.; A yeast (Saccharomyces cerevisiae) secretory expression vector containing PRO3 gene was constructed (pCBy310) . beta HCG(Human choriogonadotropin beta subunit)-cDNA was inserted into pCBy310 to form a recombinant plasmid pCBy314 . Since yeast proline auxotroph will not survive in rich medium (YPD), YPD could be used as a selection pressure, and pCBy314 could be maintained mitotically stable in transformants of yeast Pro3- auxotroph (MB299-7A) in rich medium . At an improved, yet not optimized cultural condition, the expression of beta HCG in culture medium was 650 micrograms/L . Our results showed not only that YPD could be used as a selection medium, but also that yeast grew better in YPD so as to increase the gene expression product, and that the component of YPD was simple and cheap . Our data suggested that PRO genes might be used widely in constructing vectors to clone and express foreign genes in yeast so that rich medium can be used as a selection pressure for direct selection.

Redox Rep, 2000, 5(5), 277 - 85
Adaptive response of the yeast Saccharomyces cerevisiae to reactive oxygen species: defences, damage and death; Moradas-Ferreira P et al.; The yeast Saccharomyces cerevisiae has been extensively utilised to address the mechanisms underlying the oxidative stress response . The antioxidant defences can be induced either by respiratory growth or in the presence of pro-oxidants . The cell response involves the transcriptional control of genes by protein regulators that have been recently identified and post-translational activation of pre-existing defences . The current state of the art regarding the induction of antioxidant defences during respiratory growth and by exposure to hydrogen peroxide is reviewed.

RNA, 2000 Dec, 6(12), 1762 - 72
The mRNA export in Caenorhabditis elegans is mediated by Ce-NXF-1, an ortholog of human TAP/NXF and Saccharomyces cerevisiae Mex67p; Tan W et al.; Human TAP and Saccharomyces cerevisiae Mex67p belong to a family of proteins that mediate mRNA export . Computer searches identified previously two Caenorhabditis elegans genes, C15H11.3 and C115H11.6, that encode putative homologs of hTAP and Mex67p (Segref et al., EMBO J, 1997, 16:3256-3271) . Using RNA interference experiments in C . elegans, we found that functional knockout of C15H11.3 resulted in nuclear accumulation of poly(A)-containing RNAs and was lethal for both embryos and adult nematodes . No embryonic or progeny abnormality was observed in functional knockout of C15H11.6 . Taken together, these data established that the C15H11.3 gene product is an ortholog of hTAP and Mex67p; thus, it was named Ce-NXF-1 . Ce-NXF-1 binds RNA directly and is a nucleocytoplasmic shuttle protein accumulating in the nucleoplasm and at the nuclear rim . The rim association is mediated via unique signals present in the C-terminal portion of all TAP/NXF and Mex67p proteins . This region was shown to interact with the FG-repeat domains of nucleoporins Nup98, Nup153, and Nup214, indicating that the rim association occurs through components of the nuclear pore complex . In summary, Ce-NXF-1 belongs together with hTAP and Mex67p to a family of proteins that participate in mRNA export and can provide a direct molecular link between mRNAs and components of the nuclear pore complex . Therefore, despite differences in mRNA metabolism between these species, they utilize a conserved mRNA transport mechanism.

RNA, 2000 Dec, 6(12), 1698 - 703
The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'-->3' exonucleases; Geerlings TH et al.; The final stage in the formation of the two large subunit rRNA species in Saccharomyces cerevisiae is the removal of internal transcribed spacer 2 (ITS2) from the 27SB precursors . This removal is initiated by endonucleolytic cleavage approximately midway in ITS2 . The resulting 7S pre-rRNA, which is easily detectable, is then converted into 5.8S rRNA by the concerted action of a number of 3'-->5' exonucleases, many of which are part of the exosome . So far the complementary precursor to 25S rRNA resulting from the initial cleavage in ITS2 has not been detected and the manner of its conversion into the mature species is unknown . Using various yeast strains that carry different combinations of wild-type and mutant alleles of the major 5'-->3' exonucleases Rat1p and Xrn1p, we now demonstrate the existence of a short-lived 25.5S pre-rRNA whose 5' end is located closely downstream of the previously mapped 3' end of 7S pre-rRNA . The 25.5S pre-rRNA is converted into mature 25S rRNA by rapid exonucleolytic trimming, predominantly carried out by Rat1p . In the absence of Rat1p, however, the removal of the ITS2 sequences from 25.5S pre-rRNA can also be performed by Xrn1p, albeit somewhat less efficiently.

Anal Biochem, 2001 Jan 1, 288(1), 44 - 51
Quantification of plasma membrane ergosterol of Saccharomyces cerevisiae by direct-injection atmospheric pressure chemical ionization/tandem mass spectrometry; Toh TH et al.; A method for the quantification of ergosterol by atmospheric pressure chemical ionization (APcI) mass spectrometry with direct injection is described . Ergosterol and squalene were ionizable with methanol as the carrier solvent . Using positive-mode tandem mass spectrometry (MS/MS), ergosterol could be identified unambiguously without interference from structurally related compounds such as lanosterol, cholesterol, and squalene . Molecular ions of ergosterol, lanosterol, and cholesterol were detected as the {M + H - H(2)O}(+) ion species, while squalene appeared as the {M + H}(+) ion species . Upon fragmentation of the three sterols and squalene, the product ion at m/z 69 was present as one of the major fragments in all four compounds . This product ion was used for the quantification of ergosterol in multiple-reaction-monitoring acquisition mode . The relationship between signal intensity and ergosterol concentration was linear over the concentration range of 0.15 to 5 microg/ml, or 7 . 56-252 pmol ergosterol per 20 microl injection . The plasma membrane ergosterol of the yeast Saccharomyces cerevisiae could be quantified reproducibly without the need for prior separation from other lipids or derivatization . Six repeated injections of ergosterol standards at concentrations of 0.95 and 4.25 microg/ml gave standard deviations of 0.031 and 0.084, respectively, and coefficients of variation of 3.33 and 1.98%, respectively . The coefficient of variation for the four independently extracted membrane ergosterol samples was 11.18% . The presence of other lipids in a crude lipid extract did not interfere with the ergosterol determination . Direct injection APcI with multiple reaction monitoring is aconvenient and sensitive method for ergosterol quantification requiring no prior fractionation .

Nature, 2000 Dec 21-28, 408(6815), 1001 - 4
A role for Saccharomyces cerevisiae histone H2A in DNA repair; Downs JA et al.; Histone proteins associate with and compact eukaryotic nuclear DNA to form chromatin . The basic unit of chromatin is the nucleosome, which is made up of 146 base pairs of DNA wrapped around two of each of four core histones, H2A, H2B, H3 and H4 . Chromatin structure and its regulation are important in transcription and DNA replication . We therefore thought that DNA-damage signalling and repair components might also modulate chromatin structure . Here we have characterized a conserved motif in the carboxy terminus of the core histone H2A from Saccharomyces cerevisiae that contains a consensus phosphorylation site for phosphatidylinositol-3-OH kinase related kinases (PIKKs) . This motif is important for survival in the presence of agents that generate DNA double-strand breaks, and the phosphorylation of this motif in response to DNA damage is dependent on the PIKK family member Mec1 . The motif is not necessary for Mec1-dependent cell-cycle or transcriptional responses to DNA damage, but is required for efficient DNA double-strand break repair by non-homologous end joining . In addition, the motif has a role in determining higher order chromatin structure . Thus, phosphorylation of a core histone in response to DNA damage may cause an alteration of chromatin structure that facilitates DNA repair.

Genetics, 2001 Jan, 157(1), 103 - 18
Requirement for three novel protein complexes in the absence of the Sgs1 DNA helicase in Saccharomyces cerevisiae; Mullen JR et al.; The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability . To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed . We obtained mutations in six complementation groups that we refer to as SLX genes . Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species . None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase . Analysis of the null mutants identified a pair of genes in each of three phenotypic classes . Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1 . Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex . Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology . The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts . The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts . We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

Genetics, 2001 Jan, 157(1), 91 - 101
WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae; Gajewska B et al.; Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions . To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains . The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees . Thus, individual WW domains are not essential . Each rsp5-w mutation caused temperature-sensitive growth . Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion . Thus, the WW3 domain is sufficient for Rsp5p essential functions . To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined . The WW3 domain is important for both processes . WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis . In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis . Thus, various WW domains play different roles in the endocytosis of these two substrates . Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle . Altering WW domains does not change the location of Rsp5p.

Genetics, 2001 Jan, 157(1), 53 - 61
Estimates of the rate and distribution of fitness effects of spontaneous mutation in Saccharomyces cerevisiae; Zeyl C et al.; The per-genome, per-generation rate of spontaneous mutation affecting fitness (U) and the mean fitness cost per mutation (s) are important parameters in evolutionary genetics, but have been estimated for few species . We estimated U and sh (the heterozygous effect of mutations) for two diploid yeast strains differing only in the DNA mismatch-repair deficiency used to elevate the mutation rate in one (mutator) strain . Mutations were allowed to accumulate in 50 replicate lines of each strain, during 36 transfers of randomly chosen single colonies (approximately 600 generations) . Among wild-type lines, fitnesses were bimodal, with one mode showing no change in mean fitness . The other mode showed a mean 29.6% fitness decline and the petite phenotype, usually caused by partial deletion of the mitochondrial genome . Excluding petites, maximum-likelihood estimates adjusted for the effect of selection were U = 9.5 x 10(-5) and sh = 0.217 for the wild type . Among the mutator lines, the best fit was obtained with 0.005 < or = U < or = 0.94 and 0.049 > or = sh > or = 0.0003 . Like other recently tested model organisms, wild-type yeast have low mutation rates, with high mean fitness costs per mutation . Inactivation of mismatch repair increases the frequency of slightly deleterious mutations by approximately two orders of magnitude.

Genetics, 2001 Jan, 157(1), 39 - 51
Genetic evidence for a morphogenetic function of the Saccharomyces cerevisiae Pho85 cyclin-dependent kinase; Lenburg ME et al.; The Saccharomyces cerevisiae PHO85 gene encodes a nonessential cyclin-dependent kinase that associates with 10 cyclin subunits . To survey the functions provided by Pho85, we identified mutants that require PHO85 for viability . We identified mutations that define seven Pho Eighty-Five Requiring or Efr loci, six of which are previously identified genes-BEM2 (YER155C), SPT7 (YBR081C), GCR1 (YPL075W), SRB5 (YGR104C), HFI1 (YPL254W), and BCK1 (YJL095W)-with one novel gene (YMR212C) . We found that mutations in the EFR genes involved in morphogenesis are specifically inviable when the Pho85-associated G1 cyclins encoded by PCL1 and PCL2 are absent . pcl1 Delta bem2, pcl1 Delta pcl2 Delta cla4 Delta, and pcl1 Delta pcl2 Delta cdc42-1 strains are inviable . pcl1 Delta pcl2 Delta mpk1 Delta, pcl1 Delta pcl2 Delta bck1, and pcl1 Delta pcl2 Delta cln1 Delta cln2 Delta strains are also inviable, but are rescued by osmotic stabilization with 1 m sorbitol . We propose that the G1 cyclins encoded by PCL1 and PCL2 positively regulate CDC42 or another morphogenesis promoting function.

Genetics, 2001 Jan, 157(1), 27 - 37
Two related proteins, Edc1p and Edc2p, stimulate mRNA decapping in Saccharomyces cerevisiae; Dunckley T et al.; The major mRNA decay pathway in Saccharomyces cerevisiae occurs through deadenylation, decapping, and 5' to 3' degradation of the mRNA . Decapping is a critical control point in this decay pathway . Two proteins, Dcp1p and Dcp2p, are required for mRNA decapping in vivo and for the production of active decapping enzyme . To understand the relationship between Dcp1p and Dcp2p, a combination of both genetic and biochemical approaches were used . First, we demonstrated that when Dcp1p is biochemically separated from Dcp2p, Dcp1p was active for decapping . This observation confirmed that Dcp1p is the decapping enzyme and indicated that Dcp2p functions to allow the production of active Dcp1p . We also identified two related proteins that stimulate decapping, Edc1p and Edc2p (Enhancer of mRNA DeCapping) . Overexpression of the EDC1 and EDC2 genes suppressed conditional alleles of dcp1 and dcp2, respectively . Moreover, when mRNA decapping was compromised, deletion of the EDC1 and/or EDC2 genes caused significant mRNA decay defects . The Edc1p also co-immunoprecipitated with Dcp1p and Dcp2p . These results indicated that Edc1p and Edc2p interact with the decapping proteins and function to enhance the decapping rate.

Genetics, 2001 Jan, 157(1), 17 - 26
The rye mutants identify a role for Ssn/Srb proteins of the RNA polymerase II holoenzyme during stationary phase entry in Saccharomyces cerevisiae; Chang YW et al.; Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation . We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase . These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression . In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway . In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth . The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation . These data indicated that the RYE genes might encode important regulators of yeast cell growth . Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme . Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

Nat Genet, 2001 Jan, 27(1), 113 - 6
SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination; Myung K et al.; The Escherichia coli gene recQ was identified as a RecF recombination pathway gene . The gene SGS1, encoding the only RecQ-like DNA helicase in Saccharomyces cerevisiae, was identified by mutations that suppress the top3 slow-growth phenotype . Relatively little is known about the function of Sgs1p because single mutations in SGS1 do not generally cause strong phenotypes . Mutations in genes encoding RecQ-like DNA helicases such as the Bloom and Werner syndrome genes, BLM and WRN, have been suggested to cause increased genome instability . But the exact DNA metabolic defect that might underlie such genome instability has remained unclear . To better understand the cellular role of the RecQ-like DNA helicases, sgs1 mutations were analyzed for their effect on genome rearrangements . Mutations in SGS1 increased the rate of accumulating gross chromosomal rearrangements (GCRs), including translocations and deletions containing extended regions of imperfect homology at their breakpoints . sgs1 mutations also increased the rate of recombination between DNA sequences that had 91% sequence homology . Epistasis analysis showed that Sgs1p is redundant with DNA mismatch repair (MMR) for suppressing GCRs and for suppressing recombination between divergent DNA sequences . This suggests that defects in the suppression of rearrangements involving divergent, repeated sequences may underlie the genome instability seen in BLM and WRN patients and in cancer cases associated with defects in these genes.

Mol Microbiol, 2001 Jan, 39(2), 469 - 79
Low external pH induces HOG1-dependent changes in the organization of the Saccharomyces cerevisiae cell wall; Kapteyn JC et al.; Low environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to beta1,3-glucanase . At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins, namely an alkali-labile linkage to beta1,3-glucan . This novel type of modification for Cwp1p did not require the presence of a GPI-derived structure connecting the protein with beta1,6-glucan . In addition, we found high levels of Cwp1p, which was double-anchored through both the novel alkali-sensitive bond to beta1,3-glucan and the alkali-resistant GPI-derived linkage to beta1,6-glucan . Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the beta1,3-glucan framework by an alkali-sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5 . Consequently, the alkali-sensitive type of linkage of cell wall proteins to beta1,3-glucan was induced by low pH . The low pH-induced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p-mediated MAP kinase pathway . Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high-osmolarity glycerol (HOG) pathway-dependent genes, including four cell wall-related genes, namely CWP1, HOR7, SPI1 and YGP1.

Mol Cell Biol, 2001 Jan, 21(2), 488 - 500
The phosphotyrosyl phosphatase activator, Ncs1p (Rrd1p), functions with Cla4p to regulate the G(2)/M transition in Saccharomyces cerevisiae; Mitchell DA et al.; The Saccharomyces cerevisiae p21-activated kinases, Ste20p and Cla4p, have individual functions but appear to share an essential function(s) as well because a strain lacking both kinases is inviable . To learn more about the shared function, we sought new mutations that were lethal in the absence of CLA4 . This approach led to the identification of at least 10 complementation groups designated NCS (need CLA4 to survive) . As for ste20 cla4-75 mutants, most ncs cla4-75 double mutants were defective for septin localization during budding . One group, NCS1/RRD1 (YIL153w), did not confer this defect, however, and we investigated its function further . ncs1Delta cla4Delta cells arrested with elongated buds and short mitotic spindles . The morphological defects and lethality were suppressed by mutations that abrogate the cell cycle morphogenetic checkpoint, CDC28Y19F or swe1Delta . The connection to the cell cycle may be direct, as we detected a Cla4p-Cdc28p complex . NCS1 encodes a protein with significant similarity to a mammalian phosphotyrosyl phosphatase activator (PTPA) regulatory subunit for type 2A protein phosphatases (PP2As) . Genetic and biochemical evidence suggested that the phosphatase Sit4p is a target for Ncs1p . First, CLA4 and SIT4 were synthetically lethal . Second, Ncs1p and its yeast paralog, Noh1p (Rrd2p), bound to the catalytic domain of Sit4p in vitro, and Ncs1p could be immunoprecipitated with Sit4p but not with another PP2A (Pph21p) from yeast cell extracts . Strains lacking both NCS1 and NOH1 were inviable and arrested as unbudded cells, implying that PTPA function is required for proper G(1) progression.

J Bacteriol, 2001 Jan, 183(2), 795 - 9
Characterization of the promoter of PRS1 in Saccharomyces cerevisiae identifies three regions potentially involved in control of expression; Hernando Y et al.; The transcription initiation site of the Saccharomyces cerevisiae PRS1 gene was mapped at -179 bp . Measurement of beta-galactosidase activity of the successively deleted PRS1 promoter linked to lacZ and integrated at the ura3 locus defined three DNA regions involved in the control of PRS1 expression . Gel shift analysis confirmed the data.

J Bacteriol, 2001 Jan, 183(2), 745 - 51
O2R, a novel regulatory element mediating Rox1p-independent O(2) and unsaturated fatty acid repression of OLE1 in Saccharomyces cerevisiae; Nakagawa Y et al.; Fatty acid desaturation catalyzed by fatty acid desaturases requires molecular oxygen (O(2)) . Saccharomyces cerevisiae cells derepress expression of OLE1 encoding Delta9 fatty acid desaturase under hypoxic conditions to allow more-efficient use of limited O(2) . It has been proposed that aerobic conditions lead to repression of OLE1 by well-established O(2)-responsive repressor Rox1p, since putative binding sequences for Rox1p are present in the promoter of OLE1 . However, we revealed in this study that disruption of ROX1 unexpectedly did not affect the O(2) repression of OLE1, indicating that a Rox1p-independent novel mechanism operates for this repression . We identified by promoter deletion analysis the 50-bp O(2)-regulated (O2R) element in the OLE1 promoter approximately 360 bp upstream of the start codon . Site-directed mutagenesis of the O2R element showed that the putative binding motif (5'-GATAA-3') for the GATA family of transcriptional factors is important for O(2) repression . Anaerobic derepression of OLE1 transcription was repressed by unsaturated fatty acids (UFAs), and interestingly the O2R element was responsible for this UFA repression despite not being included within the fatty acid-regulated (FAR) element previously reported . The fact that such a short 50-bp O2R element responds to both O(2) and UFA signals implies that O(2) and UFA signals merge in the ultimate step of the pathways . We discuss the differential roles of FAR and O2R elements in the transcriptional regulation of OLE1.

J Biol Chem, 2001 Mar 30, 276(13), 10218 - 23 Epub 2000 Dec 18.
The role of the FRE family of plasma membrane reductases in the uptake of siderophore-iron in Saccharomyces cerevisiae; Yun CW et al.; Saccharomyces cerevisiae takes up siderophore-bound iron through two distinct systems, one that requires siderophore transporters of the ARN family and one that requires the high affinity ferrous iron transporter on the plasma membrane . Uptake through the plasma membrane ferrous iron transporter requires that the iron first must dissociate from the siderophore and undergo reduction to the ferrous form . FRE1 and FRE2 encode cell surface metalloreductases that are required for reduction and uptake of free ferric iron . The yeast genome contains five additional FRE1 and FRE2 homologues, four of which are regulated by iron and the major iron-dependent transcription factor, Aft1p, but whose function remains unknown . Fre3p was required for the reduction and uptake of ferrioxamine B-iron and for growth on ferrioxamine B, ferrichrome, triacetylfusarinine C, and rhodotorulic acid in the absence of Fre1p and Fre2p . By indirect immunofluorescence, Fre3p was expressed on the plasma membrane in a pattern similar to that of Fet3p, a component of the high affinity ferrous transporter . Enterobactin, a catecholate siderophore, was not a substrate for Fre3p, and reductive uptake required either Fre1p or Fre2p . Fre4p could facilitate utilization of rhodotorulic acid-iron when the siderophore was present in higher concentrations . We propose that Fre3p and Fre4p are siderophore-iron reductases and that the apparent redundancy of the FRE genes confers the capacity to utilize iron from a variety of siderophore sources.

Curr Genet, 2000 Nov, 38(4), 178 - 87
POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase delta, defines a link between DNA replication and the mutagenic bypass repair pathway; Huang ME et al.; Pol32 is a subunit of Saccharomyces cerevisiae DNA polymerase delta required in DNA replication and repair . To gain insight into the function of Pol32 and to determine in which repair pathway POL32 may be involved, we extended the analysis of the pol32delta mutant with respect to UV and methylation sensitivity, UV-induced mutagenesis; and we performed an epistasis analysis of UV sensitivity by combining the pol32delta with mutations in several genes for postreplication repair (RA D6 group), nucleotide excision repair (RAD3 group) and recombinational repair (RAD52 group) . These studies showed that pol32delta is deficient in UV-induced mutagenesis and place POL32 in the error-prone RAD6/ REV3 pathway . We also found that the increase in the CAN1 spontaneous forward mutation of different rad mutators relies entirely or partially on a functional POL32 gene . Moreover, in a two-hybrid screen, we observed that Pol32 interacts with Srs2, a DNA helicase required for DNA replication and mutagenesis . Simultaneous deletion of POL32 and SRS2 dramatically decreases cellular viability at 15 degrees C and greatly increases cellular sensitivity to hydroxyurea at the permissive temperature . Based on these findings, we propose that POL32 defines a link between the DNA polymerase and helicase activities, and plays a role in the mutagenic bypass repair pathway.

Biochemistry, 2000 Dec 26, 39(51), 15807 - 16
Transient-state kinetic analysis of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase reveals that a step after chemical transformation is rate limiting; Farazi TA et al.; MyristoylCoA:protein N-myristoyltransferase is a member of the superfamily of GCN5-related N-acetyltransferases and catalyzes the covalent attachment of myristate to the N-terminal Gly residue of proteins with diverse functions . Saccharomyces cerevisiae Nmt1p is a monomeric protein with an ordered bi-bi reaction mechanism: myristoylCoA is bound prior to peptide substrate; after catalysis, CoA is released followed by myristoylpeptide . Analysis of the X-ray structure of Nmt1p with bound substrate analogues indicates that the active site contains an oxyanion hole and a catalytic base and that catalysis proceeds through the nucleophilic addition-elimination mechanism . To determine the rate-limiting step in the enzyme reaction, pre-steady-state kinetic analyses were performed using a new, sensitive nonradioactive assay that detects CoA . Multiple turnover quenched flow studies disclosed that a step after the chemical transformation limits the overall rate of the reaction . Multiple and single turnover analyses revealed that the rate for the chemical transformation step is 13.8+/-0.6 s(-1) while the slower steady-state phase is 0.10+/-0.01 s(-1) . Stopped flow kinetic studies of substrate acquisition indicated that binding of myristoylCoA to the apo-enzyme occurs through at least a two-step process, with a fast phase rate of 3.2 x 10(8) M(-1) s(-1) and a slow phase rate of 23+/-2 s(-1) (defined at 5 degrees C) . Binding of an octapeptide substrate, representing the N-terminal sequence of a known yeast N-myristoylprotein (Cnb1p), to a binary complex composed of Nmt1p and a nonhydrolyzable myristoylCoA analogue (S-(2-oxo)pentadecylCoA) has a second-order rate constant of 2.1+/-0.3 x 10(6) M(-1) s(-1) and a dissociation rate of 26+/-15 s(-1) (defined at 10 degrees C) . These results are interpreted in light of the X-ray structures of this enzyme.

Curr Opin Microbiol, 2000 Dec, 3(6), 567 - 72
Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae; Pan X et al.; In response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous pseudohyphal growth . At least two signaling pathways regulate filamentation . One involves components of the MAP kinase cascade that also regulates mating of haploid cells . The second involves a nutrient-sensing G-protein-coupled receptor that signals via an unusual G(alpha) protein, cAMP and protein kinase A . Recent studies reveal crosstalk between these pathways during pseudohyphal growth . Related MAP kinase and cAMP pathways regulate filamentation and virulence of human and plant fungal pathogens, and represent novel targets for antifungal drug design.

Trends Microbiol, 2000 Nov, 8(11), 508 - 13
Mitochondrial DNA inheritance in Saccharomyces cerevisiae; Berger KH et al.; Respiratory metabolism depends on mitochondrial DNA, yet the mechanisms that ensure the inheritance of the mitochondrial genome are largely obscure . Recent studies with Saccharomyces cerevisiae suggest that distinct factors mediate the active segregation of mitochondrial DNA during mitotic growth . The identification of the proteins required for the maintenance of the mitochondrial genome provides clues to the mechanisms of, and molecular machinery involved in, mitochondrial DNA inheritance.

Biochim Biophys Acta, 2000 Dec 11, 1499(1-2), 63 - 73
Oligomerization properties of the acidic ribosomal P-proteins from Saccharomyces cerevisiae: effect of P1A protein phosphorylation on the formation of the P1A-P2B hetero-complex; Tchorzewski M et al.; Acidic ribosomal P-proteins form, in all eukaryotic cells, a lateral protuberance, the so-called 'stalk', which is directly involved in translational activity of the ribosomes . In Saccharomyces cerevisiae cells, there are four distinct P-proteins: P1A, P1B, P2A and P2B . In spite of the high level of their structural homology, they are not completely equivalent and may perform different functions . As yet, the protein-protein interactions between yeast P-proteins have not been fully defined . In this paper, the interplay between yeast P-proteins has been investigated by means of a two-hybrid system, chemical cross-linking and gel filtration . The data presented herein show that all P-proteins are able to form homo-oligomeric complexes . By analyzing hetero-interactions, we were able to detect strong interactions between P1A and P2B proteins . Additionally, the pair of P1B and P2A proteins is also able to form a hetero-complex, though at a very low efficiency . All P-proteins are phosphorylated by numerous protein kinases . Using the multifunctional protein kinase CK II, we have shown that incorporation of phosphate into P1A protein can exert its effect on the hetero-oligomerization process, namely by preventing the formation of the hetero-oligomer P1A-P/P2B . These findings are the first to show differences in the oligomerization behavior of the yeast P-proteins; moreover, they emphasize a significant impact of the phosphorylation on the formations of P-protein complex.

Enzyme Microb Technol, 2001 Jan 2, 28(1), 89 - 99
Two-stage cultivation of recombinant Saccharomyces cerevisiae to enhance plasmid stability under non-selective conditions: experimental study and modeling; Gupta JC et al.; A leucine auxotroph strain of Saccharomyces cerevisiae was used to study plasmid stability and expression using a recombinant plasmid, which contained a foreign gene for firefly luciferase (luc) . This recombinant yeast was tested in a series of continuous cultures in semi-defined media with varying concentrations of yeast extract in order to study its effect on stability . While the biomass concentration and luciferase activity increased with increasing concentrations of yeast extract, the plasmid stability declined . An analysis of the growth rates showed that the recombinants enjoyed a growth rate advantage over the plasmid-free cells at critically low yeast extract concentrations, possibly due to leucine starvation in the media . A two-stage cultivation strategy was designed in order to create a yeast extract limited environment so that plasmid-free cells could not grow and overtake the recombinant cells . The cells were cultivated in selective media in the first stage, and then transferred continuously to the second stage where the media was enriched by feeding yeast extract . The feed rate was kept low in order to ensure yeast extract and hence leucine starvation, thereby selecting against the plasmid-free cells . This strategy resulted in a stable existence of recombinant cells, which stabilized around 60% at steady state during the tested period of cultivation . The complex nitrogen feed helped in increasing the cell density and volumetric activity by approximately 9 and 18-fold respectively with respect to that achieved in minimal medium . The experimental data was used to formulate a mathematical model to predict cell growth and plasmid stability in two-stage cultivation, which correctly explained the experimental data.

Curr Microbiol, 2001 Jan, 42(1), 65 - 9
Cgr1p, a novel nucleolar protein encoded by Saccharomyces cerevisiae orf YGL0292w; Sun J et al.; Saccharomyces cerevisiae open reading frame (ORF) YGL029w (CGR1) encodes a small hydrophilic protein of unknown function . To investigate the role of this gene, we have determined the intracellular localization of the encoded product and examined the effects of Cgr1p depletion on cell growth . Tagging Cgr1p with the green fluorescent protein (GFP) or the myc epitope showed focal accumulation of the fusion protein in the yeast nucleolus, and this localization overlapped with the distribution of the nucleolar protein Nop1p . Cells depleted of CGR1 mRNA were growth impaired and hypersensitive to the translational inhibitor paromomycin, and this phenotype was complemented by episomal expression of the CGR1-GFP fusion gene . These results identify Cgr1p as a novel component of the yeast nucleolus and suggest a potential role in ribosome biogenesis.

Yeast, 2000 Dec, 16(16), 1469 - 81
Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae; Tenreiro S et al.; In this work, we report results on the functional analysis of Saccharomyces cerevisiae ORF YGR224w, predicted to code for an integral membrane protein, with 14 potential transmembrane segments, belonging to the major facilitator superfamily (MFS) of transporters which are required for multiple-drug resistance (MDR) . This MFS-MDR homologue is required for yeast adaptation to high stress imposed by low-chain organic acids, in particular by acetic acid, and for resistance to azoles, especially to ketoconazole and fluconazole; the encoding gene was thus named the AZR1 gene . These conclusions were based on the higher susceptibility to these compounds of an azr1Delta deletion mutant strain compared with the wild-type and on the increased resistance of both azr1Delta and wild-type strains upon increased expression of the AZR1 gene from a centromeric plasmid clone . AZR1 gene expression reduces the duration of acetic acid-induced latency, although the growth kinetics of adapted cells under acetic acid stress is apparently independent of AZR1 expression level . Fluorescence microscopy observation of the distribution of the Azr1-GFP fusion protein in yeast living cells indicated that Azr1 is a plasma membrane protein . Studies carried out to gain some understanding of how this plasma membrane putative transporter facilitates yeast adaptation to acetic acid did not implicate Azr1p in the alteration of acetic acid accumulation into the cell through the active efflux of acetate .

Yeast, 2000 Dec, 16(16), 1457 - 68
Functional analysis of eight open reading frames on chromosomes XII and XIV of Saccharomyces cerevisiae; Ahmed Khan S et al.; Deletion, together with basic functional and bioinformatic analyses has been carried out on eight novel ORFs discovered during the sequencing of the Saccharomyces cerevisiae genome . Six ORFs (YLL049w, YLL051c, YLL052c, YLL053c, YLL054c and YLL055w) located on the left arm, and one (YLR130c) on the right arm, of chromosome XII, and an eighth ORF (YNL331c) on the left arm of the chromosome XIV, have been investigated . ORFs were deleted by the SFH-PCR gene-replacement strategy . Basic functional analysis revealed no obvious phenotype for any of the eight ORFs . Bioinformatic analysis, however, revealed possible functions for seven of the ORFs on the basis of the amino acid sequence similarity of their predicted protein products to those of proteins with known functions . ORF YLL051c (FRE6) shows similarity to iron transport proteins, such as ferric reductase . YLL052c and YLL053c appear to be aquaporins . The product of YLL054c (Yll054p) is highly similar to the oleate-specific transcriptional activator protein (Pip2p), which is involved in the peroxisomal induction pathway (pip) . ORF YLL055w is similar to Dal5p, allantoate permease, and may play role in allantoin transport . YLR130c (ZRT2) is a low-affinity zinc transporter protein . YNL331c is also named AAD14, which is induced by chemicals that induce oxidative stress by depleting the cell of glutathione .

Mol Cell Biol, 2001 Jan, 21(1), 235 - 48
Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae; Mosch HU et al.; In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth . Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth . In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression . In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth . Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions . Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.

Mol Cell Biol, 2001 Jan, 21(1), 189 - 95
Cross talk between tRNA and rRNA synthesis in Saccharomyces cerevisiae; Briand JF et al.; Temperature-sensitive RNA polymerase III (rpc160-112 and rpc160-270) mutants were analyzed for the synthesis of tRNAs and rRNAs in vivo, using a double-isotopic-labeling technique in which cells are pulse-labeled with {(33)P}orthophosphate and coextracted with {(3)H}uracil-labeled wild-type cells . Individual RNA species were monitored by Northern blot hybridization or amplified by reverse transcription . These mutants impaired the synthesis of RNA polymerase III transcripts with little or no influence on mRNA synthesis but also largely turned off the formation of the 25S, 18S, and 5.8S mature rRNA species derived from the common 35S transcript produced by RNA polymerase I . In the rpc160-270 mutant, this parallel inhibition of tRNA and rRNA synthesis also occurred at the permissive temperature (25 degrees C) and correlated with an accumulation of 20S pre-rRNA . In the rpc160-112 mutant, inhibition of rRNA synthesis and the accumulation of 20S pre-rRNA were found only at 37 degrees C . The steady-state rRNA/tRNA ratio of these mutants reflected their tRNA and rRNA synthesis pattern: the rpc160-112 mutant had the threefold shortage in tRNA expected from its preferential defect in tRNA synthesis at 25 degrees C, whereas rpc160-270 cells completely adjusted their rRNA/tRNA ratio down to a wild-type level, consistent with the tight coupling of tRNA and rRNA synthesis in vivo . Finally, an RNA polymerase I (rpa190-2) mutant grown at the permissive temperature had an enhanced level of pre-tRNA, suggesting the existence of a physiological coupling between rRNA synthesis and pre-tRNA processing.

Mol Cell Biol, 2001 Jan, 21(1), 175 - 84
Screening for modulators of spermine tolerance identifies Sky1, the SR protein kinase of Saccharomyces cerevisiae, as a regulator of polyamine transport and ion homeostasis; Erez O et al.; Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown . Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants of Saccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport . Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine . Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine . SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine . In addition to spermine tolerance, sky1Delta cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock . The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport . We demonstrate that these two kinases act in two different signaling pathways . While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity of PPZ1 but not of ENA1.

Mol Cell Biol, 2001 Jan, 21(1), 136 - 47
Identification of DNA cis elements essential for expansion of ribosomal DNA repeats in Saccharomyces cerevisiae; Kobayashi T et al.; Saccharomyces cerevisiae carries approximately 150 ribosomal DNA (rDNA) copies in tandem repeats . Each repeat consists of the 35S rRNA gene, the NTS1 spacer, the 5S rRNA gene, and the NTS2 spacer . The FOB1 gene was previously shown to be required for replication fork block (RFB) activity at the RFB site in NTS1, for recombination hot spot (HOT1) activity, and for rDNA repeat expansion and contraction . We have constructed a strain in which the majority of rDNA repeats are deleted, leaving two copies of rDNA covering the 5S-NTS2-35S region and a single intact NTS1, and whose growth is supported by a helper plasmid carrying, in addition to the 5S rRNA gene, the 35S rRNA coding region fused to the GAL7 promoter . This strain carries a fob1 mutation, and an extensive expansion of chromosomal rDNA repeats was demonstrated by introducing the missing FOB1 gene by transformation . Mutational analysis using this system showed that not only the RFB site but also the adjacent approximately 400-bp region in NTS1 (together called the EXP region) are required for the FOB1-dependent repeat expansion . This approximately 400-bp DNA element is not required for the RFB activity or the HOT1 activity and therefore defines a function unique to rDNA repeat expansion (and presumably contraction) separate from HOT1 and RFB activities.

Mol Cell Biol, 2001 Jan, 21(1), 109 - 25
Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae; Kohlwein SD et al.; The TSC13/YDL015c gene was identified in a screen for suppressors of the calcium sensitivity of csg2Delta mutants that are defective in sphingolipid synthesis . The fatty acid moiety of sphingolipids in Saccharomyces cerevisiae is a very long chain fatty acid (VLCFA) that is synthesized by a microsomal enzyme system that lengthens the palmitate produced by cytosolic fatty acid synthase by two carbon units in each cycle of elongation . The TSC13 gene encodes a protein required for elongation, possibly the enoyl reductase that catalyzes the last step in each cycle of elongation . The tsc13 mutant accumulates high levels of long-chain bases as well as ceramides that harbor fatty acids with chain lengths shorter than 26 carbons . These phenotypes are exacerbated by the deletion of either the ELO2 or ELO3 gene, both of which have previously been shown to be required for VLCFA synthesis . Compromising the synthesis of malonyl coenzyme A (malonyl-CoA) by inactivating acetyl-CoA carboxylase in a tsc13 mutant is lethal, further supporting a role of Tsc13p in VLCFA synthesis . Tsc13p coimmunoprecipitates with Elo2p and Elo3p, suggesting that the elongating proteins are organized in a complex . Tsc13p localizes to the endoplasmic reticulum and is highly enriched in a novel structure marking nuclear-vacuolar junctions.

J Org Chem, 2000 Dec 15, 65(25), 8552 - 63
Synthesis and biological evaluation of the geometric farnesylated analogues of the a-factor mating peptide of Saccharomyces cerevisiae; Xie H et al.; The a-factor of Saccharomyces cerevisiae is a dodecapeptide pheromone (YIIKGVFWDPAC(Farnesyl)-OCH(3), 1), in which post-translational modification with a farnesyl isoprenoid and carboxymethyl group is required for full biological activity . This peptide has been used as a model system to explore the biological function of the farnesylcysteine moiety, which is found on and required for the biological activity of many key mammalian proteins . The objective of this particular study was the determination of the biological effect of double bond isomerization of the natural E, E-farnesyl moiety on the biological activity of the a-factor . A unified, stereoselective synthetic route to the three geometric isomers of E,E-farnesol (12, 13, and 14) has been developed . The key feature of this synthesis is the ability to control the stereochemistry of triflation of the beta-ketoester 22 to give either 23 or 25 . The three farnesol isomers were converted to the corresponding isomeric a-factors (9, 10 and 11) via a modified version of a previously utilized synthetic route . Biological evaluation of these peptides indicates that, surprisingly, all three possess nearly equivalent activity to the natural a-factor bearing the E,E-farnesyl moiety.

Biochemistry, 2000 Dec 19, 39(50), 15462 - 74
Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor; Xie H et al.; The Ste2p receptor for alpha-factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of receptors . In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor . The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers . Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers . All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures . In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers . Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species . The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form beta-structures . These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function . The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.

Biochem Biophys Res Commun, 2000 Dec 9, 279(1), 17 - 22
delta(6)-desaturase of Mucor rouxii with high similarity to plant delta(6)-desaturase and its heterologous expression in Saccharomyces cerevisiae; Laoteng K et al.; Gamma-linolenic acid (GLA, gamma-C18:3) is an essential fatty acid that plays a vital role in biological structures and cellular functions . Based on available sequence information and using polymerase chain reaction (PCR) technique, we cloned from the fungus Mucor rouxii the entire coding sequence of a delta(6)-desaturase enzyme, which is responsible for the transformation of linoleic acid into GLA . The deduced amino acid sequence of M . rouxii gene showed the highest homology with the plant delta(6)-desaturase . It comprises the characteristics of membrane-bound desaturases, including histidine-rich motifs and hydrophobic regions . A cytochrome b(5)-like domain was observed at the N-terminus . In addition to three conserved histidine-rich motifs, we found an additional histidine-rich motif, HKHHSH, downstream of the cytochrome b(5)-like domain, which is not present in previously cloned delta(6)-desaturase genes . Heterologous expression of the M . rouxii cDNA in Saccharomyces cerevisiae resulted in the synthesis and accumulation of GLA .

Can J Microbiol, 2000 Nov, 46(11), 1058 - 65
Use of lycorine and DAPI staining in Saccharomyces cerevisiae to differentiate between rho0 and rho- cells in a cce1/delta cce1 nuclear background; Massardo DR et al.; In the yeast Saccharomyces cerevisiae, mutants are viable with large deletions (rho-), or even complete loss of the mitochondrial genome (rho0) . One class of rho- mutants, which is called hypersuppressive, is characterised by a high transmission of the mutated mitochondrial genome to the diploid progeny when mated to a wild-type (rho+) haploid . The nuclear gene CCE1 encodes a cruciform cutting endonuclease, which is located in the mitochondrion and is responsible for the highly biased transmission of the hypersuppressive rho- genome . CCE1 is a Holliday junction specific endonuclease that resolves recombination intermediates in mitochondrial DNA . The cleavage activity shows a strong preference for cutting after a 5'-CT dinucleotide . In the absence of the CCE1 gene product, the mitochondrial genomes remain interconnected and have difficulty segregating to the daughter cells . As a consequence, there is an increase in the fraction of daughter cells that are rho0 . In this paper we demonstrate the usefulness of lycorine, together with staining by 4',6-diamidino-2-phenylindole (DAPI), to assay for the mitotic stability of a variety of mitochondrial genomes . We have found that rho+ and rho- strains that contain CT sequences produce a large fraction of rho0 progeny in the absence of CCE1 activity . Only those rho- mitochondrial genomes lacking the CT recognition sequence are unaffected by the cce1 allele.

J Biol Chem, 2001 Mar 2, 276(9), 6392 - 7 Epub 2000 Dec 05.
Pet111p, an inner membrane-bound translational activator that limits expression of the Saccharomyces cerevisiae mitochondrial gene COX2; Green-Willms NS et al.; The protein specified by the Saccharomyces cerevisiae nuclear gene PET111 specifically activates translation of the mitochondrially coded mRNA for cytochrome c oxidase subunit II (Cox2p) . We found Pet111p specifically in mitochondria of both wild-type cells and cells expressing a chromosomal gene for a functional epitope-tagged form of Pet111p . Pet111p was associated with mitochondrial membranes and was highly resistant to extraction with alkaline carbonate . Pet111p was protected from proteolytic digestion by the mitochondrial inner membrane . Thus, it is exposed only on the matrix side, where it could participate directly in organellar translation and localize Cox2p synthesis by virtue of its functional interaction with the COX2 mRNA 5'-untranslated leader . We also found that Pet111p is present at levels limiting the synthesis of Cox2p by examining the effect of altered PET111 gene dosage in the nucleus on expression of a reporter gene, cox2::ARG8(m), that was inserted into mitochondrial DNA . The level of the reporter protein, Arg8p, was one-half that of wild type in a diploid strain heterozygous for a pet111 deletion mutation, whereas it was increased 2.8-fold in a strain bearing extra copies of PET111 on a high-copy plasmid . Thus, Pet111p could play dual roles in both membrane localization and regulation of Cox2p synthesis within mitochondria.

RNA, 2000 Nov, 6(11), 1565 - 72
Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae; Russell CS et al.; Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty) . Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing . In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition . Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes . We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

RNA, 2000 Nov, 6(11), 1498 - 508
Bypassing the rRNA processing endonucleolytic cleavage at site A2 in Saccharomyces cerevisiae; Torchet C et al.; Rrp5p is the only ribosomal RNA processing trans-acting factor that is required for the synthesis of both 18S and 5.8S rRNAs in Saccharomyces cerevisiae . Mutational analyses have characterized modified forms of Rrp5p that either affect formation of 18S rRNA by inhibiting cleavage at sites A0/A1/A2, or synthesis of 5.8S rRNA by inhibiting cleavage at site A3 . Here, we examine the rRNA maturation process associated with a RRP5 bipartite allele that codes for two noncontiguous parts of the protein . This slow-growing bipartite mutant has a unique rRNA-processing phenotype that proceeds without endonucleolytic cleavage at site A2 . In wild-type cells, the A2 cleavage takes place on the 32S pre-rRNA and is responsible for the formation of 20S and 27SA2 species, the precursors of mature 18S and 5.8S/25S rRNAs, respectively . In the bipartite strain, such precursors were not detectable as judged by Northern analysis or in vivo labeling . They were replaced by the aberrant 21S species and the bypassing 27SA3 precursor, both descended from direct cleavage of 32S pre-rRNA at site A3, which provides an alternative rRNA maturation pathway in this strain . The 21S pre-rRNA is the sole detectable and most likely available precursor of 18S rRNA in this particular strain, indicating that 18S rRNA can be directly produced from 21S . Furthermore, 21S species were found associated with 43S preribosomal particles as similarly observed for the 20S pre-rRNA in the wild-type cells.

Bioseparation, 2000, 9(3), 167 - 72
Production and purification of recombinant human alpha 2C2 adrenergic receptor using Saccharomyces cerevisiae; Kapat A et al.; The objective is to generate milligram quantities of recombinant human alpha 2C2 adrenergic receptor for X-ray crystallographic studies . It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay . The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles . The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.

Biochem J, 2000 Dec 15, 352 Pt 3, 893 - 8
A carbon-source-responsive element is required for regulation of the hypoxic ADP/ATP carrier (AAC3) isoform in Saccharomyces cerevisiae; Sokolikova B et al.; The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three genes that are differentially expressed under different physiological conditions . We investigated the transcriptional control of AAC3, an oxygen-repressed isoform . By deletion analysis, DNA electrophoretic mobility-shift assays, DNase I footprinting and site-directed mutagenesis, we have identified a promoter region (upstream repressing sequence 1, URS(1)) involved in a carbon-source-dependent repression of AAC3 . It is different from the previously characterized oxygen-dependent ROX1 (regulation by oxygen 1) repressor-binding region (URS(2)) . The complex character of URS(1) includes the presence of two different cis-acting sequences: (i) a RAP1 (repressor activator protein 1)-binding site that is capable of binding the RAP1 protein in vitro and (ii) two putative ethanol-repression sequences, the modification of which derepresses the AAC3 gene . These findings demonstrate that the hypoxic AAC3 gene is regulated by two upstream repressor sites; one controlled by oxygen and haem, the other by the carbon source . Both sites function to completely switch off the expression of the AAC3 isoform when ATP is made by oxidative phosphorylation, and they modulate AAC3 expression when import of glycolytic ATP into mitochondria is required.

Mol Biol Cell, 2000 Dec, 11(12), 4309 - 21
New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis; Ogawa N et al.; The PHO regulatory pathway is involved in the acquisition of phosphate (P(i)) in the yeast Saccharomyces cerevisiae . When extracellular P(i) concentrations are low, several genes are transcriptionally induced by this pathway, which includes the Pho4 transcriptional activator, the Pho80-Pho85 cyclin-CDK pair, and the Pho81 CDK inhibitor . In an attempt to identify all the components regulated by this system, a whole-genome DNA microarray analysis was employed, and 22 PHO-regulated genes were identified . The promoter regions of 21 of these genes contained at least one copy of a sequence that matched the Pho4 recognition site . Eight of these genes, PHM1-PHM8, had no previously defined function in phosphate metabolism . The amino acid sequences of PHM1 (YFL004w), PHM2 (YPL019c), PHM3 (YJL012c), and PHM4 (YER072w) are 32-56% identical . The phm3 and phm4 single mutants and the phm1 phm2 double mutant were each severely deficient in accumulation of inorganic polyphosphate (polyP) and P(i) . The phenotype of the phm5 mutant suggests that PHM5 (YDR452w) is essential for normal catabolism of polyP in the yeast vacuole . Taken together, the results reveal important new features of a genetic system that plays a critical role in P(i) acquisition and polyP metabolism in yeast.

Mol Biol Cell, 2000 Dec, 11(12), 4277 - 94
The sodium/proton exchanger Nhx1p is required for endosomal protein trafficking in the yeast Saccharomyces cerevisiae; Bowers K et al.; We show that the vacuolar protein sorting gene VPS44 is identical to NHX1, a gene that encodes a sodium/proton exchanger . The Saccharomyces cerevisiae protein Nhx1p shows high homology to mammalian sodium/proton exchangers of the NHE family . Nhx1p is thought to transport sodium ions into the prevacuole compartment in exchange for protons . Pulse-chase experiments show that approximately 35% of the newly synthesized soluble vacuolar protein carboxypeptidase Y is missorted in nhx1 delta cells, and is secreted from the cell . nhx1 delta cells accumulate late Golgi, prevacuole, and lysosome markers in an aberrant structure next to the vacuole, and late Golgi proteins are proteolytically cleaved more rapidly than in wild-type cells . Our results show that efficient transport out of the prevacuolar compartment requires Nhx1p, and that nhx1 delta cells exhibit phenotypes characteristic of the "class E" group of vps mutants . In addition, we show that Nhx1p is required for protein trafficking even in the absence of the vacuolar ATPase . Our analysis of Nhx1p provides the first evidence that a sodium/proton exchange protein is important for correct protein sorting, and that intraorganellar ion balance may be important for endosomal function in yeast.

Genetics, 2000 Dec, 156(4), 1531 - 48
Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells; Hiraoka M et al.; To obtain a broad perspective of the events leading to spontaneous loss of heterozygosity (LOH), we have characterized the genetic alterations that functionally inactivated the URA3 marker hemizygously or heterozygously situated either on chromosome III or chromosome V in diploid Saccharomyces cerevisiae cells . Analysis of chromosome structure in a large number of LOH clones by pulsed-field gel electrophoresis and PCR showed that chromosome loss, allelic recombination, and chromosome aberration were the major classes of genetic alterations leading to LOH . The frequencies of chromosome loss and chromosome aberration were significantly affected when the marker was located in different chromosomes, suggesting that chromosome-specific elements may affect the processes that led to these alterations . Aberrant-sized chromosomes were detected readily in approximately 8% of LOH events when the URA3 marker was placed in chromosome III . Molecular mechanisms underlying the chromosome aberrations were further investigated by studying the fate of two other genetic markers on chromosome III . Chromosome aberration caused by intrachromosomal rearrangements was predominantly due to a deletion between the MAT and HMR loci that occurred at a frequency of 3.1 x 10(-6) . Another type of chromosome aberration, which occurred at a frequency slightly higher than that of the intrachromosomal deletion, appeared to be caused by interchromosomal rearrangement, including unequal crossing over between homologous chromatids and translocation with another chromosome.

Genetics, 2000 Dec, 156(4), 1519 - 29
Accumulation of phosphorylated sphingoid long chain bases results in cell growth inhibition in Saccharomyces cerevisiae; Kim S et al.; Sphingolipid metabolites in mammals can function as signaling molecules with cell-specific functions . In Saccharomyces cerevisiae, phosphorylated long chain bases, such as dihydrosphingosine 1-phosphate and phytosphingosine 1-phosphate, have also been implicated in stress responses . To further explore the biological roles of these molecules, we created disruption mutants for LCB4, LCB5, DPL1, YSR2, YSR3, and SUR2 . LCB4 and LCB5 encode kinases that phosphorylate long chain bases . DPL1 and YSR2/YSR3 are involved in degradation of the phosphorylated long chain bases . SUR2 catalyzes conversion of dihydrosphingosine to phytosphingosine . We adapted an HPLC method to measure intracellular concentrations of the phosphorylated long chain bases . Double mutants of dpl1 and ysr2 were inviable, whereas dpl1 ysr2 lcb4 triple mutants were viable . Further, growth inhibition associated with accumulated phosphorylated long chain bases was observed in the triple mutant dpl1 ysr2 lcb4 overexpressing LCB4 or LCB5 . These results indicate that phosphorylated long chain bases can inhibit cell growth . Mutants defective in both YSR2 and SUR2, which accumulated dihydrosphingosine 1-phosphate only, grew poorly . The phenotypes of the ysr2 sur2 mutants were suppressed by overexpression of DPL1 . Our results clearly show that elevated levels of phosphorylated long chain bases have an antiproliferative effect in yeast.

Genetics, 2000 Dec, 156(4), 1503 - 17
Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae; Ben-Yehuda S et al.; The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression . The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle . Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele . Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA . The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing . Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR . This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif . Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p . We discuss the role played by these proteins in splicing and cell cycle progression.

Microbiology, 2000 Dec, 146 Pt 12, 3269 - 78
The importance of the five phosphoribosyl-pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p; Schneiter R et al.; Phosphoribosyl-pyrophosphate synthetase (Prs) catalyses the synthesis of phosphoribosyl pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan . The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5 . Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures . This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p . Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway . Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type . The fact that Deltaprs1 and Deltaprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.

EMBO J, 2000 Dec 1, 19(23), 6489 - 97
The pachytene checkpoint in Saccharomyces cerevisiae requires the Sum1 transcriptional repressor; Lindgren A et al.; Saccharomyces cerevisiae mutants that fail to complete meiotic recombination are blocked by the RAD17/RAD24/MEC1 checkpoint signaling pathway in pachytene when early sporulation genes are expressed . Middle genes are not activated in checkpoint-arrested cells because the Ndt80 transcription factor is inhibited . We find that the pachytene checkpoint requires Sum1, a transcriptional repressor that recognizes a subset of Ndt80-binding sites . Mutants lacking Sum1 or Rad17 partially bypass the block to the nuclear divisions but do not form spores, while mutants lacking both Sum1 and Rad17 completely bypass the block and form morphologically normal spores . The level of Sum1 protein decreases as middle genes are expressed, and this decrease is blocked in checkpoint-arrested cells . These data suggest that Sum1 levels are regulated by the checkpoint and that progression of the meiotic divisions and spore differentiation can be differentially controlled by competition of the Sum1 repressor and Ndt80 activator for occupancy at key middle promoters.

Appl Environ Microbiol, 2000 Dec, 66(12), 5477 - 9
Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells; Vai M et al.; We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells . The hIGF-1 gene was fused to the mating factor alpha prepro- leader sequence under the control of the constitutive ACT1 promoter . We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.

Appl Environ Microbiol, 2000 Dec, 66(12), 5182 - 5
Evidence for contribution of neutral trehalase in barotolerance of Saccharomyces cerevisiae; Iwahashi H et al.; In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance . We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance . To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains . Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain . Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells . Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose . The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction . Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13749 - 54
Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase; Mallory JC et al.; The Saccharomyces cerevisiae proteins Tel1p and Mec1p are involved in telomere length regulation and cellular responses to DNA damage . The closest relative of these proteins is the human Ataxia Telangiectasia Mutated (ATM) protein, a wortmannin-sensitive protein kinase that primarily phosphorylates serines in an SQ motif . We constructed yeast strains containing functional epitope-tagged versions of Tel1p and Mec1p . We showed that immunoprecipitated Tel1p and Mec1p were capable of in vitro phosphorylation of the mammalian protein PHAS-I (Phosphorylated Heat and Acid Stable protein) . These activities are sensitive to wortmannin . Tel1p phosphorylates serine in an SQ motif in PHAS-I . Mutations in the kinase domains of Tel1p and Mec1p result in loss of in vitro kinase activity and the in vivo phenotypes associated with the null tel1 and mec1 mutations.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13585 - 90
Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae; Krappmann S et al.; Control of transcription and enzyme activities are two interwoven regulatory systems essential for the function of a metabolic node . Saccharomyces cerevisiae strains differing in enzyme activities at the chorismate branch point of aromatic amino acid biosynthesis were constructed by recombinant DNA technology . Expression of an allosterically unregulated, constitutively activated chorismate mutase encoded by the ARO7(T226I) (ARO7(c)) allele depleted the chorismate pool . The resulting tryptophan limitation caused growth defects, which could be counteracted only by transcriptional induction of TRP2 encoding the competing enzyme anthranilate synthase . ARO7 expression is not transcriptionally regulated by amino acids . Transcriptional activation of the ARO7(c) allele led to stronger growth retardation upon tryptophan limitation . The same effect was achieved by removing the competing enzyme anthranilate synthase, which is encoded by the TRP2 gene, from the transcriptional control . The allelic situation of ARO7(c) being under general control instead of TRP2 resulted in severe growth defects when cells were starved for tryptophan . In conclusion, the specific regulatory pattern acting on enzymatic activities at the first metabolic node of aromatic amino acid biosynthesis is necessary to maintain proper flux distribution . Therefore, the evolution of the sophisticated allosteric regulation of yeast chorismate mutase requires as prerequisite (i) that the encoding ARO7 gene is not transcriptionally regulated, whereas (ii) the transcription of the competing feedback-regulated anthranilate synthase-encoding gene is controlled by availability of amino acids.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4733 - 41
Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p; Wang MJ et al.; Yeast Saccharomyces cerevisiae Cdc13p is the telomere-binding protein that protects telomeres and regulates telomere length . It is documented that Cdc13p binds specifically to single-stranded TG(1-3) telomeric DNA sequences and interacts with Stn1p . To localize the region for single-stranded TG(1-3) DNA binding, Cdc13p mutants were constructed by deletion mutagenesis and assayed for their binding activity . Based on in vitro electrophoretic mobility shift assay, a 243-amino-acid fragment of Cdc13p (amino acids 451-693) was sufficient to bind single-stranded TG(1-3) with specificity similar to that of the native protein . Consistent with the in vitro observation, in vivo one-hybrid analysis also indicated that this region of Cdc13p was sufficient to localize itself to telomeres . However, the telomere-binding region of Cdc13p (amino acids 451-693) was not capable of complementing the growth defects of cdc13 mutants . Instead, a region comprising the Stn1p-interacting and telomere-binding region of Cdc13p (amino acids 252-924) complemented the growth defects of cdc13 mutants . These results suggest that binding to telomeres by Cdc13p is not sufficient to account for the cell viability, interaction with Stn1p is also required . Taken together, we have defined the telomere-binding domain of Cdc13p and showed that both binding to telomeres and Stn1p by Cdc13p are required to maintain cell growth.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4649 - 56
Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae; Symington LS et al.; A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair . In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap . Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events . In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks . However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases . The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates . The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over . In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.

Support Care Cancer, 2000 Nov, 8(6), 504 - 5
Saccharomyces cerevisiae fungemia in a neutropenic patient treated with Saccharomyces boulardii; Cesaro S et al.; A case of Saccharomyces fungemia in an 8-month-old baby affected by acute myeloid leukemia while receiving intensive chemotherapy is reported . The patient was receiving prophylaxis treatment with Saccharomyces boulardii capsules (Codex) to prevent diarrhea, which is commonly associated with this type of chemotherapy . Fever spiked just the day after ending the chemotherapy course, and a strain of Saccharomyces cerevisiae was isolated from blood culture although the patient was also receiving antifungal prophylaxis with fluconazole . The patient recovered, though still neutropenic, with amphotericin-B and removal of the central venous catheter . The common biochemical characteristics make it difficult to differentiate between the strain of Saccharomyces cerevisiae and that of Saccharomyces boulardii with routine methods . In other cases, authors demonstrated an identity between the two strains with a more detailed analysis . These reports raise concern about the potential side effects of such biotherapeutic agents.

FEMS Microbiol Lett, 2000 Dec 1, 193(1), 143 - 7
Analysis of the morphologic changes of Monascus sp . J101 cells cocultured with Saccharomyces cerevisiae; Suh JH et al.; Changes in cell life cycle and intracellular structure of Monascus sp . J101 by coculture with Saccharomyces cerevisiae were investigated . Cocultured Monascus cells showed accelerated cell growth and reproduction . Production of asexual and sexual spores was used as an efficient method of cell proliferation . Formation of meiotic (sexual) spores was more frequently observed in the cocultured Monascus cells . The interior structure of a cocultured cell was characterized by increased numbers and sizes of vacuoles . The vacuoles probably serve as repositories for pigment storage . Pigments produced by the cocultured Monascus cells were more hydrophobic than pigments produced by control cells with no coculture.

FEMS Microbiol Lett, 2000 Dec 1, 193(1), 105 - 10
The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme; Teste MA et al.; The YPR184w gene encodes a 1536-amino acid protein that is 34-39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme . The N-terminal part of the protein possesses the four conserved sequences of the alpha-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes . Reliable measurement of alpha-1,4-glucanotransferase and alpha-1, 6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w . The alpha-1, 4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl . Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown . In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

Mol Cell Biol, 2000 Dec, 20(24), 9162 - 72
Aberrant double-strand break repair in rad51 mutants of Saccharomyces cerevisiae; Kang LE et al.; A number of studies of Saccharomyces cerevisiae have revealed RAD51-independent recombination events . These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication . Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism . We propose a model for RAD51-independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing . The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required for RAD51-independent events by this model . Double-strand break repair between plasmid-borne inverted repeats was less efficient in rad1 rad51 double mutants than in rad1 and rad51 strains . In addition, repair events were delayed and frequently associated with plasmid loss . Furthermore, the repair products recovered from the rad1 rad51 strain were primarily in the crossover configuration, inconsistent with conservative models for mitotic double-strand break repair.

J Biol Chem, 2001 Jan 19, 276(3), 1701 - 6 Epub 2000 Nov 28.
The human homolog of Saccharomyces cerevisiae Apg7p is a Protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3; Tanida I et al.; Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system . In the yeast, Saccharomyces cerevisiae, an autophagosome is formed in the cytosol . The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole . The autophagic body is subsequently degraded by vacuolar hydrolases . Taking advantage of yeast genetics, apg (autophagy-defective) mutants were isolated that are defective in terms of formation of autophagic bodies under nutrient starvation conditions . One of the APG gene products, Apg12p, is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation, and this conjugation is essential for autophagy . Apg7p is a novel E1 enzyme essential for the Apg12p-conjugation system . In mammalian cells, the human Apg12p homolog (hApg12p) also conjugates with the human Apg5p homolog . In this study, the unique characteristics of hApg7p are shown . A two-hybrid experiment indicated that hApg12p interacts with hApg7p . Site-directed mutagenesis revealed that Cys(572) of hApg7p is an authentic active site cysteine residue essential for the formation of the hApg7p.hApg12p intermediate . Overexpression of hApg7p enhances the formation of the hApg5p.hApg12p conjugate, indicating that hApg7p is an E1-like enzyme essential for the hApg12p conjugation system . Cross-linking experiments and glycerol-gradient centrifugation analysis showed that the mammalian Apg7p homolog forms a homodimer as in yeast Apg7p . Each of three human Apg8p counterparts, i.e . the Golgi-associated ATPase enhancer of 16 kDa, GABA(A) receptor-associated protein, and microtubule-associated protein light chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant hApg7p(C572S) to form a stable intermediate via an ester bond . These results indicate that hApg7p is an authentic protein-activating enzyme for hApg12p and the three Apg8p homologs.

J Virol, 2000 Dec, 74(24), 11724 - 33
DNA-Directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis; Price BD et al.; Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions . To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo . Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells . The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs . The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA . As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3'-terminal RNA1 sequences but not viral protein B2 . Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3 . These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA . Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication . Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.

Mech Ageing Dev, 2000 Dec 1, 120(1-3), 1 - 22
The budding yeast, Saccharomyces cerevisiae, as a model for aging research: a critical review; Gershon H et al.; In this review we discuss the yeast as a paradigm for the study of aging . The budding yeast Saccharomyces cerevisiae, which can proliferate in both haploid and diploid states, has been used extensively in aging research . The budding yeast divides asymmetrically to form a 'mother' cell and a bud . Two major approaches, 'budding life span' and 'stationary phase' have been used to determine 'senescence' and 'life span' in yeast . Discrepancies observed in metabolic behavior and longevity between cells studied by these two systems raise questions of how 'life span' in yeast is defined and measured . Added to this variability in experimental approach and results is the variety of yeast strains with different genetic make up used as 'wild type' and experimental organisms . Another problematic genetic point in the published studies on yeast is the use of both diploid and haploid strains . We discuss the inherent, advantageous attributes that make the yeast an attractive choice for modern biological research as well as certain pitfalls in the choice of this model for the study of aging . The significance of the purported roles of the Sir2 gene, histone deacetylases, gene silencing, rDNA circles and stress genes in determination of yeast 'life span' and aging is evaluated . The relationship between cultivation conditions and longevity are assessed . Discrepancies between the yeast and mammalian systems with regard to aging are pointed out . We discuss unresolved problems concerning the suitability of the budding yeast for the study of basic aging phenomena.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13203 - 8
A computationally directed screen identifying interacting coiled coils from Saccharomyces cerevisiae; Newman JR et al.; Computational methods can frequently identify protein-interaction motifs in otherwise uncharacterized open reading frames . However, the identification of candidate ligands for these motifs (e.g., so that partnering can be determined experimentally in a directed manner) is often beyond the scope of current computational capabilities . One exception is provided by the coiled-coil interaction motif, which consists of two or more alpha helices that wrap around each other: the ligands for coiled-coil sequences are generally other coiled-coil sequences, thereby greatly simplifying the motif/ligand recognition problem . Here, we describe a two-step approach to identifying protein-protein interactions mediated by two-stranded coiled coils that occur in Saccharomyces cerevisiae . Coiled coils from the yeast genome are first predicted computationally, by using the multicoil program, and associations between coiled coils are then determined experimentally by using the yeast two-hybrid assay . We report 213 unique interactions between 162 putative coiled-coil sequences . We evaluate the resulting interactions, focusing on associations identified between components of the spindle pole body (the yeast centrosome).

FEBS Lett, 2000 Nov 17, 485(1), 29 - 34
A novel strategy for constructing N-terminal chromosomal fusions to green fluorescent protein in the yeast Saccharomyces cerevisiae; Prein B et al.; A novel rapid polymerase chain reaction (PCR)-based technique for N-terminal attachment of green fluorescent protein (GFP) to a yeast protein is described . Genomic integration of a PCR-generated loxPkanMX4loxP-yEGFP fusion cassette immediately upstream of the open reading frame (ORF) allows for selection of G418 resistant transformants carrying GFP fused N-terminally to the protein of interest . In a subsequent step, the loxPkanMX4loxP selection marker that is inserted between the tagged ORF and the endogenous promoter is excised upon site-specific recombination between the loxP sites by Cre recombinase, leaving behind in the promoter one loxP site, immediately upstream of the GFP start codon . The essential protein Ydl193wp of unknown function and the oleate-inducible fatty acid activation protein, encoded by FAA2, were N-terminally tagged using the novel technique . Both experiments yielded viable haploid strains with growth phenotypes indistinguishable from the wild type strain . The subcellular localization pattern for the chromosomally expressed GFP-Ydl193wp to the endoplasmic reticulum and lipid particles was identical to the pattern observed for a plasmid-borne GFP construct expressed under control of the MET25(p) promoter, albeit at a lower level and with a more homogeneous distribution among the cell population . GFP-FAA2 was inducible by oleate, as is the wild type gene, demonstrating that specific expression patterns are not grossly affected by the promoter manipulation . In agreement with previous reports, GFP-Faa2p was found to localize to peroxisomes.

J Cell Biol, 2000 Nov 27, 151(5), 973 - 84
Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae; Shimoni Y et al.; Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat . We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae . A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer . We found that cytosol from an lst1-null strain supported the packaging of alpha-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase . Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles . When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p . Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles . Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs . 75-nm diameter) . Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles . These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles . Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles . By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles.

Mol Gen Genet, 2000 Oct, 264(3), 317 - 24
Post-translational regulation of Saccharomyces cerevisiae proteins tagged with the hormone-binding domains of mammalian nuclear receptors; Launhardt H et al.; In the post-genome sequencing era the functional analysis of newly discovered proteins becomes more and more important . In this report we describe a genetic approach to the post-translational regulation of protein function in Saccharomyces cerevisiae by creating conditional lethal mutants . The yeast ORFs YDL139c, YDL147w, ERG3 and ERG11 were tagged with sequences encoding the hormone-binding domains of mammalian steroid receptors by PCR-mediated, targeted integration into the yeast genome . We found that the function of the chimeric proteins is regulated in a hormone-dependent way . This technique provides another important tool for the functional analysis of the yeast proteome.

Curr Biol, 2000 Nov 2, 10(21), 1375 - 8
The spindle checkpoint of Saccharomyces cerevisiae responds to separable microtubule-dependent events; Daum JR et al.; The spindle checkpoint regulates microtubule-based chromosome segregation and helps to maintain genomic stability {1,2} . Mutational inactivation of spindle checkpoint genes has been implicated in the progression of several types of human cancer . Recent evidence from budding yeast suggests that the spindle checkpoint is complex . Order-of-function experiments have defined two separable pathways within the checkpoint . One pathway, defined by MAD2, controls the metaphase-to-anaphase transition and the other, defined by BUB2, controls the exit from mitosis {3-6} . The relationships between the separate branches of the checkpoint, and especially the events that trigger the pathways, have not been defined . We localized a Bub2p-GFP fusion protein to the cytoplasmic side of the spindle pole body and used a kar9 mutant to show that cells with misoriented spindles are arrested in anaphase of mitosis . We used a kar9 bub2 double mutant to show that the arrest is BUB2 dependent . We conclude that the separate pathways of the spindle checkpoint respond to different classes of microtubules . The MAD2 branch of the pathway responds to kinetochore microtubule interactions and the BUB2 branch of the pathway operates within the cytoplasm, responding to spindle misorientation.

Genome, 2000 Oct, 43(5), 881 - 6
Transcript analysis of 203 novel genes from Saccharomyces cerevisiae in hap1 and rox1 mutant backgrounds; Lombardia LJ et al.; Hap1 and Rox1 are transcriptional regulators that bind regulatory sites in the promoters of oxygen-regulated genes in Saccharomyces cerevisiae . Hap1 is a heme-responsive activator of genes induced in aerobic conditions and Rox1 is a repressor of hypoxic genes in aerobic conditions . We have studied transcriptional regulation of a pool of 203 open reading frames (ORFs) from chromosomes IV, VII, and XIV in wild-type, hap1, and rox1 mutant genetic backgrounds in an attempt to extend the family of oxygen and heme regulated genes . Only three ORFs are significantly repressed by Rox1 but they cannot be considered as typical hypoxic genes because they are not overexpressed during hypoxia.

Electrophoresis, 2000 Oct, 21(16), 3396 - 410
A proteomic approach for the study of Saccharomyces cerevisiae cell wall biogenesis; Pardo M et al.; In fungi, cell shape is determined by the presence of a rigid cell wall which separates the cell from the extracellular medium . This highly dynamic structure is essential for the maintenance of cell integrity and is involved in several phenomena such as flocculation, adherence and pathogenicity . The composition of the fungal cell wall is well known, but issues such as the assembly and remodeling of its components remain poorly understood . In an attempt to study the de novo construction of the yeast cell wall, we have undertaken a large-scale proteomic approach to analyze the proteins secreted by regenerating protoplasts . Upon incubation of protoplasts in regenerating conditions, numerous proteins are secreted into the culture medium . These presumably include proteins destined for the cell wall, comprising both structural proteins as well as enzymes involved in cell wall biogenesis . This work reports the establishment of a reference map of proteins secreted by regenerating protoplasts by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and their identification by mass spectrometry . Thirty-two different proteins have been identified, including known cell wall proteins, glycolytic enzymes, heat shock proteins, and proteins involved in several other processes . Using this approach, novel proteins possibly involved in cell wall construction have also been identified . This reference map will allow comparative analyses to be carried out on a selected collection of mutants affected in the cell wall.

J Biol Chem, 2001 Mar 16, 276(11), 8469 - 74 Epub 2000 Nov 14.
A proteome analysis of the cadmium response in Saccharomyces cerevisiae; Vido K et al.; Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood . We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins . A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway . Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment . Several proteins with antioxidant properties were also induced . The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain . Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain . Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal . Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.

J Biol Chem, 2001 Feb 9, 276(6), 3756 - 63 Epub 2000 Nov 14.
Turnover of phosphatidylcholine in Saccharomyces cerevisiae . The role of the CDP-choline pathway; Dowd SR et al.; The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae . In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C . In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature . In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e . CHO2/PEM1 and OPI3/PEM2) . Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift . These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway . Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.

Biol Chem, 2000 Sep-Oct, 381(9-10), 1025 - 9
Sec61p is the main ribosome receptor in the endoplasmic reticulum of Saccharomyces cerevisiae; Prinz A et al.; A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane . Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells . Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex . However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER . We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane . The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.

Mol Cell Biol, 2000 Dec, 20(23), 8916 - 22
{URE3} prion propagation in Saccharomyces cerevisiae: requirement for chaperone Hsp104 and curing by overexpressed chaperone Ydj1p; Moriyama H et al.; The {URE3} nonchromosomal genetic element is an infectious form (prion) of the Ure2 protein, apparently a self-propagating amyloidosis . We find that an insertion mutation or deletion of HSP104 results in inability to propagate the {URE3} prion . Our results indicate that Hsp104 is a common factor in the maintenance of two independent yeast prions . However, overproduction of Hsp104 does not affect the stability of {URE3}, in contrast to what is found for the {PSI(+)} prion, which is known to be cured by either overproduction or deficiency of Hsp104 . Like Hsp104, the Hsp40 class chaperone Ydj1p, with the Hsp70 class Ssa1p, can renature proteins . We find that overproduction of Ydj1p results in a gradual complete loss of {URE3} . The involvement of protein chaperones in the propagation of {URE3} indicates a role for protein conformation in inheritance.

Mol Cell Biol, 2000 Dec, 20(23), 8836 - 44
DNA integration by Ty integrase in yku70 mutant Saccharomyces cerevisiae cells; Kiechle M et al.; In the present work we examined nonhomologous integration of plasmid DNA in a yku70 mutant . Ten of 14 plasmids integrated as composite elements, including Ty sequences probably originating from erroneous strand-switching and/or priming events . Three additional plasmids integrated via Ty integrase without cointegrating Ty sequences, as inferred from 5-bp target site duplication and integration site preferences . Ty integrase-mediated integration of non-Ty DNA has never been observed in wild-type cells, although purified integrase is capable of using non-Ty DNA as a substrate in vitro . Hence our data implicate yKu70 as the cellular function preventing integrase from accepting non-Ty DNA as a substrate.

J Bacteriol, 2000 Dec, 182(23), 6584 - 91
The level of DAL80 expression down-regulates GATA factor-mediated transcription in Saccharomyces cerevisiae; Cunningham TS et al.; Nitrogen-catabolic gene expression in Saccharomyces cerevisiae is regulated by the action of four GATA family transcription factors: Gln3p and Gat1p/Nil1p are transcriptional activators, and Dal80 and Deh1p/Gzf3p are repressors . In addition to the GATA sequences situated upstream of all nitrogen catabolite repression-sensitive genes that encode enzyme and transport proteins, the promoters of the GAT1, DAL80, and DEH1 genes all contain multiple GATA sequences as well . These GATA sequences are the binding sites of the GATA family transcription factors and are hypothesized to mediate their autogenous and cross regulation . Here we show, using DAL80 fused to the carbon-regulated GAL1,10 or copper-regulated CUP1 promoter, that GAT1 expression is inversely regulated by the level of DAL80 expression, i.e., as DAL80 expression increases, GAT1 expression decreases . The amount of DAL80 expression also dictates the level at which DAL3, a gene activated almost exclusively by Gln3p, is transcribed . Gat1p was found to partially substitute for Gln3p in transcription . These data support the contention that regulation of GATA-factor gene expression is tightly and dynamically coupled . Finally, we suggest that the complicated regulatory circuit in which the GATA family transcription factors participate is probably most beneficial as cells make the transition from excess to limited nitrogen availability.

Nucleic Acids Res, 2000 Nov 15, 28(22), 4497 - 505
Conformational changes induced in the Saccharomyces cerevisiae GTPase-associated rRNA by ribosomal stalk components and a translocation inhibitor; Briones C et al.; The yeast ribosomal GTPase associated center is made of parts of the 26S rRNA domains II and VI, and a number of proteins including P0, P1alpha, P1beta, P2alpha, P2beta and L12 . Mapping of the rRNA neighborhood of the proteins was performed by footprinting in ribosomes from yeast strains lacking different GTPase components . The absence of protein P0 dramatically increases the sensitivity of the defective ribosome to degradation hampering the RNA footprinting . In ribosomes lacking the P1/P2 complex, protection of a number of nucleotides is detected around positions 840, 880, 1100, 1220-1280 and 1350 in domain II as well as in several positions in the domain VI alpha-sarcin region . The protection pattern resembles the one reported for the interaction of elongation factors in bacterial systems . The results exclude a direct interaction of these proteins with the rRNA and are compatible with an increase in the ribosome affinity for EF-2 in the absence of the acidic P proteins . Interestingly, a sordarin derivative inhibitor of EF-2 causes an opposite effect, increasing the reactivity in positions protected by the absence of P1/P2 . Similarly, a deficiency in protein L12 exposes nucleotides G1235, G1242, A1262, A1269, A1270 and A1272 to chemical modification, thus situating the protein binding site in the most conserved part of the 26S rRNA, equivalent to the bacterial protein L11 binding site.

Nucleic Acids Res, 2000 Nov 15, 28(22), 4435 - 43
The two Saccharomyces cerevisiae SUA7 (TFIIB) transcripts differ at the 3'-end and respond differently to stress; Hoopes BC et al.; Despite much information as to the structure and function of the general transcription factors, little is known about the regulation of their expression . Transcription of the Saccharomyces cerevisiae SUA7 (TFIIB) gene results in the formation of two discrete transcripts . It was originally reported that the two transcripts were derived from two promoters separated by approximately 80 bp . We have found that the two transcripts are instead derived from a common promoter and differ at the 3'-end by approximately 115 bp . The longer of the two transcripts has an unusually long 3'-untranslated region . We have analyzed the levels of these transcripts under different cell growth conditions and find that the relative amounts of the two transcripts vary . Approximately equal amounts of each transcript are observed during exponential growth, but stresses and growth limiting conditions lead to a decrease in the relative amount of the larger transcript . These results suggest that the expression of the SUA7 gene may be controlled by regulation of 3'-end formation or mRNA stability . One of the general transcription factors, then, may be subject to regulation by a general response of the mRNA processing machinery.

Mol Biol Cell, 2000 Nov, 11(11), 3963 - 76
Saccharomyces cerevisiae PTS1 receptor Pex5p interacts with the SH3 domain of the peroxisomal membrane protein Pex13p in an unconventional, non-PXXP-related manner; Bottger G et al.; A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p . In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain . Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p . Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way . To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3 . Such mutations were all located in a small region in the N-terminal half of Pex5p . One of the altered residues (F208) was part of the sequence W(204)XXQF(208), that is conserved between Pex5 proteins of different species . Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain . The Pex5p mutants could only partially restore PTS1-protein import in pex5Delta cells in vivo . In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3 . In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes . Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function.

Mol Biol Cell, 2000 Nov, 11(11), 3689 - 702
Bud6 directs sequential microtubule interactions with the bud tip and bud neck during spindle morphogenesis in Saccharomyces cerevisiae; Segal M et al.; In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule-cortex interactions throughout spindle assembly . Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality . Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule-neck interactions to spindle orientation inherent to haploids . These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids . Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly . bud6Delta mutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture . Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule-cortex interactions accordingly . These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity.

J Cell Sci, 2000 Dec, 113 Pt 23, 4301 - 11
The novel protein Ccz1p required for vacuolar assembly in Saccharomyces cerevisiae functions in the same transport pathway as Ypt7p; Kucharczyk R et al.; CCZ1 was previously identified by the sensitivity of ccz1(delta) mutants to high concentrations of Caffeine and the divalent ions Ca(2+ )and Zn(2+) . In this paper we show that deletion of CCZ1 leads to aberrant vacuole morphology, similar to the one reported for the family of vacuolar protein sorting (vps) mutants of class B . The ccz1(&Dgr;) cells display severe vacuolar protein sorting defects for both the soluble carboxipeptidase Y and the membrane-bound alkaline phosphatase, which are delivered to the vacuole by distinct routes . Ccz1p is a membranous protein and the vast majority of Ccz1p resides in late endosomes . These results, along with a functional linkage found between the CCZ1 and YPT7 genes, indicate that the site of Ccz1p function is at the last step of fusion of multiple transport intermediates with the vacuole.

Mol Microbiol, 2000 Nov, 38(3), 552 - 64
In vivo N-glycosylation of the mep2 high-affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N-terminus; Marini AM et al.; Saccharomyces cerevisiae possesses three related ammonium transporters, Mep1, Mep2 and Mep3, differing in their kinetic properties and in the level and regulation of their gene expression . The three Mep proteins belong to a family conserved in bacteria, plants and animals, which also includes proteins of the rhesus blood group family . In addition to its role in scavenging extracellular ammonium, the Mep2 protein has been proposed to act as an ammonium sensor, essential to pseudohyphal differentiation in response to ammonium limitation . To pursue the biochemical study of the Mep transporters, we raised polyclonal antibodies against the C-terminal tail of each Mep protein . When electrophoresed on SDS-polyacrylamide gel, the Mep1 and Mep3 proteins migrate as expected from their predicted size, whereas the Mep2 protein migrates as a high-molecular-weight smear . Protein deglycosylation with peptide-N-glycosidase F (PNGase F) indicates that, in contrast to Mep1 and Mep3, Mep2 is an asparagine-linked glycoprotein . Site-directed mutagenesis of the four potential N-glycosylation sites of Mep2 shows that Asn-4 of the protein's N-terminal tail is the only site that binds oligosaccharides . This provides evidence for the extracytosolic location of the Mep2 N-terminus . Consistently, treatment of intact protoplasts with proteinase K leads to specific proteolysis of the N-terminal tail of Mep2 . The protein's C-terminus, on the other hand, is protected against protease degradation under these conditions, but digested after protoplast permeabilization, suggesting a cytoplasmic location for this part of the protein . Mep2 glycosylation is not required for pseudohyphal differentiation in response to ammonium starvation, and its absence causes only a slight reduction in the affinity of the transporter for its substrate.

Genetics, 2000 Nov, 156(3), 963 - 72
A genetic analysis of glucocorticoid receptor signaling . Identification and characterization of ligand-effect modulators in Saccharomyces cerevisiae; Sitcheran R et al.; To find novel components in the glucocorticoid signal transduction pathway, we performed a yeast genetic screen to identify ligand-effect modulators (LEMs), proteins that modulate the cellular response to hormone . We isolated several mutants that conferred increased glucocorticoid receptor (GR) activity in response to dexamethasone and analyzed two of them in detail . These studies identify two genes, LEM3 and LEM4, which correspond to YNL323w and ERG6, respectively . LEM3 is a putative transmembrane protein of unknown function, and ERG6 is a methyltransferase in the ergosterol biosynthetic pathway . Analysis of null mutants indicates that LEM3 and ERG6 act at different steps in the GR signal transduction pathway.

Genetics, 2000 Nov, 156(3), 943 - 51
Synthetic interactions of the post-Golgi sec mutations of Saccharomyces cerevisiae; Finger FP et al.; In the budding yeast Saccharomyces cerevisiae, synthetic lethality has been extensively used both to characterize interactions between genes previously identified as likely to be involved in similar processes as well as to uncover new interactions . We have performed a large study of the synthetic lethal interactions of the post-Golgi sec mutations . Included in this study are the interactions of the post-Golgi sec mutations with each other, with mutations affecting earlier stages of the secretory pathway, with selected mutations affecting the actin cytoskeleton, and with selected cell division cycle (cdc) mutations affecting processes thought to be important for or involving secretion, such as polarity establishment and cytokinesis . Synthetic negative interactions of the post-Golgi sec mutations appear (as predicted) to be largely stage specific, although there are some notable exceptions . The significance of these results is discussed in the context of both secretory pathway function and the utility of synthetic lethality studies and their interpretation.

J Cell Biol, 2000 Oct 30, 151(3), 613 - 26
Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae; Hartman T et al.; The PDS5 gene (precocious dissociation of sisters) was identified in a genetic screen designed to identify genes important for chromosome structure . PDS5 is an essential gene and homologues are found from yeast to humans . Pds5p function is important for viability from S phase through mitosis and localizes to chromosomes during this cell cycle window, which encompasses the times when sister chromatid cohesion exists . Pds5p is required to maintain cohesion at centromere proximal and distal sequences . These properties are identical to those of the four cohesion complex members Mcd1p/Scc1p, Smc1p, Smc3p, and Scc3p/Irr1p (Guacci, V., D . Koshland, and A . Strunnikov . 1997 . Cell . 91:47-57; Michaelis, C., R . Ciosk, and K . Nasmyth . 1997 . Cell . 91:35-45; Toth, A., R . Ciosk, F . Uhlmann, M . Galova, A . Schleiffer, and K . Nasmyth . 1999 . Genes Dev . 13:307-319) . Pds5p binds to centromeric and arm sequences bound by Mcd1p . Furthermore, Pds5p localization to chromosomes is dependent on Mcd1p . Thus, Pds5p, like the cohesin complex members, is a component of the molecular glue that mediates sister chromatid cohesion . However, Mcd1p localization to chromosomes is independent of Pds5p, which may reflect differences in their roles in cohesion . Finally, Pds5p is required for condensation as well as cohesion, which confirms the link between these processes revealed through analysis of Mcd1p (Guacci, V., D . Koshland, and A . Strunnikov . 1997 . Cell . 91:47-57) . Therefore, the link between cohesion and condensation is a general property of yeast chromosomes.

Biochem J, 2000 Nov 15, 352 Pt 1, 71 - 8
Role of glutathione in heat-shock-induced cell death of Saccharomyces cerevisiae; Sugiyama K et al.; Previously we reported that expression of GSH1 (gamma-glutamylcysteine synthetase) and GSH2 (glutathione synthetase) of the yeast Saccharomyces cerevisiae was increased by heat-shock stress in a Yap1p-dependent fashion and consequently intracellular glutathione content was increased {Sugiyama, Izawa and Inoue (2000) J . Biol . Chem . 275, 15535-15540} . In the present study, we discuss the physiological role of glutathione in the heat-shock stress response in this yeast . Both gsh1 and gsh2 mutants could acquire thermotolerance by mild heat-shock stress and induction of Hsp104p in both mutants was normal; however, mutant cells died faster by heat shock than their parental wild-type strain . After pretreatment at a sublethal temperature, the number of respiration-deficient mutants increased in a gsh1 mutant strain in the early stages of exposure to a lethal temperature, although this increase was partially suppressed by the addition of glutathione . These results lead us to suspect that an increase of glutathione synthesis during heat-shock stress is to protect mitochondrial DNA from oxidative damage . To investigate the correlation between mitochondrial DNA damage and glutathione, mitochondrial Mn-superoxide dismutase (the SOD2 gene product) was disrupted . As a result, the rate of generation of respiration-deficient mutants of a sod2 delta strain was higher than that of the isogenic wild-type strain and treatment of the sod2 delta mutant with buthionine sulphoximine, an inhibitor of glutathione synthesis, inhibited cell growth . These results suggest that glutathione synthesis is induced by heat shock to protect the mitochondrial DNA from oxidative damage that may lead to cell death.

EMBO J, 2000 Nov 1, 19(21), 5824 - 34
A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae; Chavez S et al.; Transcription-induced recombination has been reported in all organisms from bacteria to mammals . We have shown previously that the yeast genes HPR1 and THO2 may be keys to the understanding of transcription-associated recombination, as they both affect transcription elongation and hyper-recombination in a concerted manner . Using a yeast strain that has the wild-type THO2 gene replaced by one encoding a His(6)-HA-tagged version, we have isolated an oligomeric complex containing four proteins: Tho2, Hpr1, Mft1 and a novel protein that we have named Thp2 . We have reciprocally identified a complex containing Hpr1, Tho2 and Mft1 using anti-Mft1 antibodies in immunoprecipitation experiments . The protein complex is mainly nuclear; therefore, Tho2 and Hpr1 are physically associated . Like hpr1Delta and tho2Delta cells, mft1Delta and thp2Delta cells show mitotic hyper- recombination and impaired transcription elongation, in particular, through the bacterial lacZ sequence . Hyper-recombination conferred by mft1Delta and thp2Delta is only observed in DNA regions under transcription conditions . We propose that this protein complex acts as a functional unit connecting transcription elongation with the incidence of mitotic recombination.

Nat Cell Biol, 2000 Nov, 2(11), 812 - 8
Functional dissection of in vivo interchromosome association in Saccharomyces cerevisiae; Aragon-Alcaide L et al.; Homologue pairing mediates both recombination and segregation of chromosomes at meiosis I . The recognition of nucleic-acid-sequence homology within the somatic nucleus has an impact on DNA repair and epigenetic control of gene expression . Here we investigate interchromosomal interactions using a non-invasive technique that allows tagging and visualization of DNA sequences in vegetative and meiotic live yeast cells . In non-meiotic cells, chromosomes are ordered in the nucleus, but preferential pairing between homologues is not observed . Association of tagged chromosomal domains occurs irrespective of their genomic location, with some preference for similar chromosomal positions . Here we describe a new phenomenon that promotes associations between sequence-identical ectopic tags with a tandem-repeat structure . These associations, termed interchromosome trans-associations, may underlie epigenetic phenomena.

Metab Eng, 2000 Jul, 2(3), 252 - 75
Tendency modeling: a new approach to obtain simplified kinetic models of metabolism applied to Saccharomyces cerevisiae; Visser D et al.; A novel approach to construct kinetic models of metabolic pathways, to be used in metabolic engineering, is presented: the tendency modeling approach . This approach greatly facilitates the construction of these models and can easily be applied to complex metabolic networks . The resulting models contain a minimal number of parameters; identification of their values is straightforward . Use of in vitro obtained information in the identification of the kinetic equations is minimized . The tendency modeling approach has been used to derive a dynamic model of primary metabolism for aerobic growth of Saccharomyces cerevisiae on glucose, in which compartmentation is included . Simulation results obtained with the derived model are satisfying for most of the carbon metabolites that have been measured . Compared to a more detailed model, the simulations of our model are less accurate, but taking into account the much smaller number of kinetic parameters (35 instead of 84), the tendency the modeling approach is considered promising .

J Biol Chem, 2001 Mar 16, 276(11), 8574 - 81 Epub 2000 Oct 30.
Role for de novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae; Jenkins GM et al.; The recent findings of sphingolipids as potential mediators of yeast heat stress responses led us to investigate their possible role in the heat-induced cell cycle arrest and subsequent recovery . The sphingolipid-deficient yeast strain 7R4 was found to lack the cell cycle arrest seen in the isogenic wild type . Furthermore, strain lcb1-100, which harbors a temperature-sensitive serine palmitoyltransferase, lacked increased de novo generated sphingoid bases upon heat stress . Importantly, this strain was found to lack the transient heat-induced G0/G1 arrest . These results indicate a role for sphingolipids and specifically those generated in the de novo pathway in the cell cycle arrest response to heat . To determine the bioactive sphingolipid regulating this response, an analysis of key mutants in the sphingolipid biosynthetic and degradation pathways was performed . Strains deleted in sphingoid base kinases, sphingoid phosphate phosphatase, lyase, or dihydrosphingosine hydroxylase were found to display the cell cycle arrest . Also, the knockout of a fatty acyl elongation enzyme, which severely attenuates ceramide production, displayed the arrest . These experiments suggested that the active species for cell cycle arrest were the sphingoid bases . In further support of these findings, exogenous phytosphingosine (10 microM) was found to induce transient arrest . Stearylamine did not induce an arrest, demonstrating chemical specificity, and L-erythro- was not as potent as D-erythro-dihydrosphingosine showing stereospecificity . To investigate a possible arrest mechanism, we studied the hyperstable Cln3 (Cln3-1) strain LDW6A that has been previously shown to be resistant to heat stress-induced cell cycle arrest . The strain containing Cln3-1 was found to be resistant to cell cycle arrest induced by exogenous phytosphingosine, indicating that Cln3 acts downstream of the sphingoid bases in this response . Interestingly, cell cycle recovery from the transient arrest was found to be dependent upon the sphingoid base kinases (LCB4, LCB5) . Overall, this combination of genetic and pharmacologic results demonstrates a role for de novo sphingoid base biosynthesis by serine palmitoyltransferase in the transient G0/G1 arrest mediated through Cln3 via a novel mechanism.

Appl Environ Microbiol, 2000 Nov, 66(11), 4883 - 9
Screening of genes involved in isooctane tolerance in Saccharomyces cerevisiae by using mRNA differential display; Miura S et al.; A Saccharomyces cerevisiae strain, KK-211, isolated by the long-term bioprocess of stereoselective reduction in isooctane, showed extremely high tolerance to the solvent, which is toxic to yeast cells, but, in comparison with its wild-type parent, DY-1, showed low tolerance to hydrophilic organic solvents, such as dimethyl sulfoxide and ethanol . In order to detect the isooctane tolerance-associated genes, mRNA differential display (DD) was employed using mRNAs isolated from strains DY-1 and KK-211 cultivated without isooctane, and from strain KK-211 cultivated with isooctane . Thirty genes were identified as being differentially expressed in these three types of cells and were classified into three groups according to their expression patterns . These patterns were further confirmed and quantified by Northern blot analysis . On the DD fingerprints, the expression of 14 genes, including MUQ1, PRY2, HAC1, AGT1, GAC1, and ICT1 (YLR099c) was induced, while the expression of the remaining 16 genes, including JEN1, PRY1, PRY3, and KRE1, was decreased, in strain KK-211 cultivated with isooctane . The genes represented by HAC1, PRY1, and ICT1 have been reported to be associated with cell stress, and AGT1 and GAC1 have been reported to be involved in the uptake of trehalose and the production of glycogen, respectively . MUQ1 and KRE1, encoding proteins associated with cell surface maintenance, were also detected . Based on these results, we concluded that alteration of expression levels of multiple genes, not of a single gene, might be the critical determinant for isooctane tolerance in strain KK-211.

Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1937 - 41
Defect in cell wall integrity of the yeast saccharomyces cerevisiae caused by a mutation of the GDP-mannose pyrophosphorylase gene VIG9; Yoda K et al.; The Saccharomyces cerevisiae VIG9 gene encodes GDP-mannose pyrophosphorylase, which synthesizes GDP-mannose from GTP and mannose-1-phosphate . Although the null mutant was lethal, the vig9 mutants so far obtained showed no growth defect but immature protein glycosylation and drug hypersensitivity . During our search for cell-wall mutants, we found a novel temperature-sensitive mutant, JS30, which required an osmotic stabilizer for viability . JS30 excreted cell surface proteins in the medium without any indication of cell lysis . Although conventional genetic analysis using mating was impossible, by detailed characterization of JS30 including an in vitro enzyme assay and nucleotide sequencing, we found the defect of JS30 was due to a mutation in the VIG9 gene . These results indicated a critical role of GDP-mannose in maintenance of cell-wall integrity.

Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1930 - 6
Gene regulation in response to overexpression of cytochrome P450 and proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae; Zimmer T et al.; (CYP52A4) in Saccharomyces cerevisiae . Using the mRNA differential display technique, six genes were found to be up-regulated: ASN2, MDJ1, YLR194c, YNL208w, YER175, and YGL121c . Genes coding for Dur1.2p, Dal2p, and Sps19p were down-regulated . Two strongly induced genes, which were found to accommodate the peroxisome box (YLR194c) and a 10-bp consensus sequence of genes involved in lipid metabolism (YNL208w) in their promoter regions, were further analyzed with respect to the course of induction, the necessity of the P450 membrane anchor for induction, and the effects of gene disruption on P450Cm2 overexpression . We found that both genes are not essential to overproduce P450Cm2, but their induction was dependent on P450Cm2 membrane integration.

Yeast, 2000 Nov, 16(15), 1429 - 35
Disruption and functional analysis of six ORFs on chromosome XII of saccharomyces cerevisiae: YLR124w, YLR125w, YLR126c, YLR127c, YLR128w and YLR129w; Willer M et al.; In the context of the EUROFAN programme, we report the deletion and functional analysis of six open reading frames (ORFs) on the right arm of chromosome XII of Saccharomyces cerevisiae . Using a PCR-based gene replacement strategy, we have systematically deleted individual ORFs and subjected the heterozygous diploids and haploid knockout strains to basic genetic and phenotypic characterization . Two ORFs, YLR127c and YLR129w, are essential for viability, whereas no growth phenotype could be detected following deletion of YLR124w, YLR125w, YLR126c or YLR128w . For each of the individual ORFs, a kanMX4 replacement cassette and the corresponding cognate wild-type gene were cloned into appropriate plasmids .

Yeast, 2000 Nov, 16(15), 1421 - 7
Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae; Westermann B et al.; We describe the construction and characterization of a novel set of plasmids for expression of mitochondria-targeted green fluorescent protein (GFP) in Saccharomyces cerevisiae . The vectors include constructs with strong regulatable and constitutive promoters, four different auxotrophic markers for yeast transformation, and a green (S65T) and a blue-shifted (P4-3) variant of GFP . Mitochondria are brightly fluorescent in living yeast cells grown on different carbon sources and at different temperatures, with virtually no background staining . Specific staining of mitochondria is also shown for a respiratory-deficient mutant with abnormal mitochondrial morphology . The plasmids facilitate convenient analysis of mutants defective in mitochondrial morphology or inheritance and, at the same time, are suitable vectors for easy construction of different kinds of GFP fusion proteins to study various aspects of organelle biogenesis in living yeast cells .

Yeast, 2000 Nov, 16(15), 1365 - 75
Constitutive activation of the Saccharomyces cerevisiae transcriptional regulator Ste12p by mutations at the amino-terminus; Crosby JA et al.; The transcriptional activator Ste12p is required for the expression of genes induced by mating pheromone in the yeast Saccharomyces cerevisiae . We identified mutations in the amino-terminal DNA-binding domain of Ste12p that lead to constitutively high-level transcription of pheromone-induced genes . The behaviour of these mutant proteins is consistent with an enhanced DNA-binding ability . Cells carrying these hyperactive proteins retain their sensitivity to pheromone treatment, and their phenotype is largely dependent on the presence of at least one of the MAP kinases (Fus3p or Kss1p) and the scaffold protein Ste5p . Deletion of either FUS3 or KSS1 leads to a marked increase in Ste12p activity, consistent with a negative regulatory role for Fus3p, similar to that described for Kss1p . The properties of the constitutive mutants support the idea that the pheromone response pathway plays a role in basal as well as pheromone-induced transcription .

Eur J Biochem, 2000 Nov, 267(22), 6571 - 9
Molecular cloning, functional complementation in Saccharomyces cerevisiae and enzymatic properties of phosphatidylinositol synthase from the protozoan parasite Toxoplasma gondii; Seron K et al.; The obligate intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, switches between the rapidly dividing tachyzoite and the slowly replicating bradyzoite in intermediate hosts such as humans and domestic animals . We have recently identified a bradyzoite cDNA encoding a putative phosphatidylinositol (PtdIns) synthase using a subtractive library {Yahiaoui, B., Dzierszinski, F., Bernigaud, A., Slomianny, C., Camus, D., and Tomavo, S . (1999) Mol . Biochem . Parasitol . 99, 223-235} . Here, we report the cloning of another cDNA encoding PtdIns synthase that is exclusively expressed in the tachyzoite stage . The two transcripts are encoded by two different genes, which are stage-specifically regulated . The deduced amino-acid sequence (258 amino acids with a calculated total molecular mass of 27.8 kDa) of the tachyzoite-specific cDNA shares a significant degree of identity (between 26.5 and 30.1%) to the PtdIns synthases from human, rat, Arabidopsis thaliana and yeast . Interestingly, the putative protein encompasses an N-terminal extension that is approximately 40 amino-acids longer than that of PtdIns synthases from other organisms . Functional complementation realized by tetrad analysis of segregants of a Saccharomyces cerevisiae PtdIns synthase-deficient mutant (PIS1/pis1:kanMX4) showed that only the T . gondii putative PtdIns synthase truncated at its N-terminal extension is able to restore the viability of the cells . We demonstrate that this protein expressed in yeast transformants is functionally active in the membrane preparation and requires manganese and magnesium ions for activity . To our knowledge, this is the first report on the molecular cloning and functional analysis of a gene encoding a PtdIns synthase in protozoan parasites.

Acta Biochim Pol, 2000, 47(2), 355 - 64
Effect of stress on the life span of the yeast Saccharomyces cerevisiae; Swiecilo A et al.; A correlation is known to exist in yeast and other organisms between the cellular resistance to stress and the life span . The aim of this study was to examine whether stress treatment does affect the generative life span of yeast cells . Both heat shock (38 degrees C, 30 min) and osmotic stress (0.3 M NaCl, 1 h) applied cyclically were found to increase the mean and maximum life span of Saccharomyces cerevisiae . Both effects were more pronounced in superoxide dismutase-deficient yeast strains (up to 50% prolongation of mean life span and up to 30% prolongation of maximum life span) than in their wild-type counterparts . These data point to the importance of the antioxidant barrier in the stress-induced prolongation of yeast life span.

J Biol Chem, 2001 Jan 19, 276(3), 1850 - 6 Epub 2000 Oct 24.
Alkaline response genes of Saccharomyces cerevisiae and their relationship to the RIM101 pathway; Lamb TM et al.; Environmental pH exerts broad control over growth and differentiation, but the molecular responses to external pH changes are poorly understood . Here we have used open reading frame macroarray hybridization to identify alkaline response genes in Saccharomyces cerevisiae . Northern or lacZ fusion assays confirmed the alkaline induction of two ion pump genes (ENA1 and VMA4), several ion limitation genes (CTR3, FRE1, PHO11/12, and PHO84), a siderophore-iron transporter gene (ARN4/ENB1), two transcription factor genes (NRG2 and TIS11), and two predicted membrane protein genes (YAR068W/YHR214W and YOL154W) . Unlike ENA1 and SHC1, these new alkaline response genes are not induced by high salinity . The known pH-responsive genes in other fungi depend on the conserved PacC/Rim101p transcription factor, but induction of several of these new genes relied upon Rim101p-independent pH signaling mechanisms . Rim101p-dependent genes were also dependent on Rim13p, a protease required for Rim101p processing . The Rim101p-dependent gene VMA4 is required for growth in alkaline conditions, illustrating how Rim101p may control adaptation . Because Rim101p activates ion pump genes, we tested the role of RIM101 in ion homeostasis and found that RIM101 promotes resistance to elevated cation concentrations . Thus, gene expression surveys can reveal new functions for characterized transcription factors in addition to uncovering physiological responses to environmental conditions.

Mol Cell Biol, 2000 Nov, 20(22), 8548 - 59
Saccharomyces cerevisiae cdc42p GTPase is involved in preventing the recurrence of bud emergence during the cell cycle; Richman TJ et al.; The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators and downstream effectors through an approximately 25-amino-acid effector domain . Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into Cdc42p and characterized for their effects on these interactions . Each mutant protein showed differential interactions with a number of downstream effectors and regulators and various levels of functionality . Specifically, Cdc42(D38E)p showed reduced interactions with the Cla4p p21-activated protein kinase and the Bem3p GTPase-activating protein and cdc42(D38E) was the only mutant allele able to complement the Deltacdc42 null mutant . However, the mutant protein was only partially functional, as indicated by a temperature-dependent multibudded phenotype seen in conjunction with defects in both septin ring localization and activation of the Swe1p-dependent morphogenetic checkpoint . Further analysis of this mutant suggested that the multiple buds emerged consecutively with a premature termination of bud enlargement preceding the appearance of the next bud . Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and GFP-Cdc24p all predominantly localized to one bud at a time per multibudded cell . These data suggest that Cdc42(D38E)p triggers a morphogenetic defect post-bud emergence, leading to cessation of bud growth and reorganization of the budding machinery to another random budding site, indicating that Cdc42p is involved in prevention of the initiation of supernumerary buds during the cell cycle.

Mol Cell Biol, 2000 Nov, 20(22), 8343 - 51
Functional interaction between Ssu72 and the Rpb2 subunit of RNA polymerase II in Saccharomyces cerevisiae; Pappas DL Jr et al.; SSU72 is an essential gene encoding a phylogenetically conserved protein of unknown function that interacts with the general transcription factor TFIIB . A recessive ssu72-1 allele was identified as a synthetic enhancer of a TFIIB (sua7-1) defect, resulting in a heat-sensitive (Ts(-)) phenotype and a dramatic downstream shift in transcription start site selection . Here we describe a new allele, ssu72-2, that confers a Ts(-) phenotype in a SUA7 wild-type background . In an effort to further define Ssu72, we isolated suppressors of the ssu72-2 mutation . One suppressor is allelic to RPB2, the gene encoding the second-largest subunit of RNA polymerase II (RNAP II) . Sequence analysis of the rpb2-100 suppressor defined a cysteine replacement of the phylogenetically invariant arginine residue at position 512 (R512C), located within homology block D of Rpb2 . The ssu72-2 and rpb2-100 mutations adversely affected noninduced gene expression, with no apparent effects on activated transcription in vivo . Although isolated as a suppressor of the ssu72-2 Ts(-) defect, rpb2-100 enhanced the transcriptional defects associated with ssu72-2 . The Ssu72 protein interacts directly with purified RNAP II in a coimmunoprecipitation assay, suggesting that the genetic interactions between ssu72-2 and rpb2-100 are a consequence of physical interactions . These results define Ssu72 as a highly conserved factor that physically and functionally interacts with the RNAP II core machinery during transcription initiation.

FEBS Lett, 2000 Oct 20, 483(2-3), 87 - 92
SGD1 encodes an essential nuclear protein of Saccharomyces cerevisiae that affects expression of the GPD1 gene for glycerol 3-phosphate dehydrogenase; Akhtar N et al.; We here report the identification of the previously uncharacterized SGD1 gene, encoding a 102.8-kDa protein containing a leucine zipper region and a bipartite nuclear localization signal . Deletion of SGD1 results in loss of cell viability, while an increased dosage of SGD1 partially suppresses the osmosensitivity of pbs2 delta and hog1 delta mutants that are defective in the osmosignaling high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway . The rescued mutants display a partially re-established transcriptional control of the osmostress-induced expression of GPD1, a target gene of the HOG pathway encoding NAD(+)-dependent glycerol 3-phosphate dehydrogenase, and a partially recovered hyperosmolarity-induced production of glycerol . Consistent with Sgd1p affecting the transcriptional control of GPD1, a functional green fluorescent protein tagged Sgd1p is localized to the cell nucleus.

J Biol Chem, 2001 Jan 19, 276(3), 2023 - 30 Epub 2000 Oct 19.
The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae; Phylip LH et al.; The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure . IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species . Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence . Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme . This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence . Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A . The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A . This results, paradoxically, in potent selective inhibition of the target enzyme.

Chin J Biotechnol, 1999, 15(4), 219 - 24
Expression of rainbow trout growth hormone cDNA in Saccharomyces cerevisiae; Jin M et al.; Rainbow trout growth hormone cDNA was modified by polymerase chain reaction (PCR) . The modified cDNA was subcloned into the E . coli-yeast shuttle vector pMA91 under the yeast PGK promoter, and transformed into Saccharomyces cerevisiae Y33 to construct an expression strain Y33 (pMArGH16) . The recombinant gene could express the growth hormone peptide (about 3% of the total yeast proteins) in Y33 (pMArGH16) . The expression product was used as a supplement to feed Tilapia fingerlings . The result showed that the recombinant fish GH could significantly enhance the growth of Tilapia fingerlings.

J Biotechnol, 2001 Nov 17, 84(1), 87 - 91
Improved resistance to transition metals of a cobalt-substituted alcohol dehydrogenase 1 from Saccharomyces cerevisiae; Cavaletto M et al.; Cobalt-substituted alcohol dehydrogenase 1 was purified from a yeast culture of Saccharomyces cerevisiae . Its reactivity towards different transition metals was tested and compared with the native zinc enzyme . The cobalt enzyme displayed a catalytic efficiency 100-fold higher than that of the zinc enzyme . Copper, nickel and cadmium exerted a mixed-type inhibition, with a scale of inhibition efficiency: Cu(2+)>Ni(2+)>Cd(2+) . In general, a higher resistance of the modified protein to the inhibitory action of transition metals was observed, with two orders of magnitude for copper I(50) . The presence of nickel in the complexes enzyme-coenzyme-inhibitor-substrate resulted in a decrease of the ampholytic nature of the catalytic site . On the contrary, cadmium and copper exerted an enhancement of this parameter . Electrostatic or other types of interactions may be involved in conferring a good resistance in the basic pH range, making cobalt enzyme very suitable for biotechnological processes.

J Biol Chem, 2001 Jan 5, 276(1), 395 - 405
Mutations in the TATA-binding protein, affecting transcriptional activation, show synthetic lethality with the TAF145 gene lacking the TAF N-terminal domain in Saccharomyces cerevisiae; Kobayashi A et al.; The general transcription factor TFIID, which is composed of the TATA box-binding protein (TBP) and a set of TBP-associated factors (TAFs), is crucial for both basal and regulated transcription by RNA polymerase II . The N-terminal small segment of yeast TAF145 (yTAF145) binds to TBP and thereby inhibits TBP function . To understand the physiological role of this inhibitory domain, which is designated as TAND (TAF N-terminal domain), we screened mutations, synthetically lethal with the TAF145 gene lacking TAND (taf145 Delta TAND), in Saccharomyces cerevisiae by exploiting a red/white colony-sectoring assay . Our screen yielded several recessive nsl (Delta TAND synthetic lethal) mutations, two of which, nsl1-1 and nsl1-2, define the same complementation group . The NSL1 gene was found to be identical to the SPT15 gene encoding TBP . Interestingly, both temperature-sensitive nsl1/spt15 alleles, which harbor the single amino acid substitutions, S118L and P65S, respectively, were defective in transcriptional activation in vivo . Several other previously characterized activation-deficient spt15 alleles also displayed synthetic lethal interactions with taf145 Delta TAND, indicating that TAND and TBP carry an overlapping but as yet unidentified function that is specifically required for transcriptional regulation.

Genetika, 2000 Aug, 36(8), 1025 - 32
{RAD29 and RAD31--new genes from Saccharomyces cerevisiae yeasts, participating in control of DNA repair . II . Clarification of possible functions of these genes}; Kozhin SA et al.; Possible functions of previously described genes RAD29 and RAD31 involved in DNA repair were determined by analyzing the interaction between these genes and mutations in the genes of the three basic epistatic groups: RAD3 (nucleotide excision repair), RAD6 (error-prone mutagenic repair system), RAD52 (recombination repair pathway), and also the apn1 mutation that blocks the synthesis of major AP endonuclease (base excision repair) . The results obtained in these studies and the estimation of the capability for excision repair of lesions induced by 8-metoxipsoralen and subsequent exposure to long-wavelength UV light in mutants for these genes led to the assumption that the RAD29 and RAD31 genes are involved in yeast DNA repair control.

Genetika, 2000 Aug, 36(8), 1017 - 24
{Cryptic heterokaryons in strains of Saccharomyces cerevisiae . Conditions for detecting a cryptic nucleus}; Nevzgliadova OV et al.; A phenomenon discovered earlier, cryptic heterokaryosis in Saccharomyces yeast, has been further investigated . A phenotypically silent nucleus in a yeast cell may resume its expression after fusion with another parental cell . The resulting hybrid is capable of sporulation . By the growth of a cytoductant with an expressing nucleus from one parent and a silent nucleus from the other parent on suitable selective media, the silent nucleus can be activated . The presence of deletion or insertion mutations in several genes in YPH strains allows nuclei of the YPH type to be traced not only genetically but also by blotting.

J Biol Chem, 2001 Jan 12, 276(2), 895 - 903
Peroxisomal degradation of trans-unsaturated fatty acids in the yeast Saccharomyces cerevisiae; Gurvitz A et al.; Degradation of trans-unsaturated fatty acids was studied in the yeast Saccharomyces cerevisiae . Propagation of yeast cells on trans-9 elaidic acid medium resulted in transcriptional up-regulation of the SPS19 gene, whose promoter contains an oleate response element . This up-regulation depended on the Pip2p-Oaf1p transcription factor and was accompanied by induction of import-competent peroxisomes . Utilization of trans fatty acids as a single carbon and energy source was evaluated by monitoring the formation of clear zones around cell growth on turbid media containing fatty acids dispersed with Tween 80 . For metabolizing odd-numbered trans double bonds, cells required the beta-oxidation auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase Eci1p . Metabolism of the corresponding even-numbered double bonds proceeded in the absence of Sps19p (2,4-dienoyl-CoA reductase) and Dci1p (Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase) . trans-2,trans-4-Dienoyl-CoAs could enter beta-oxidation directly via Fox2p (2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase) without the involvement of Sps19p, whereas trans-2,cis-4-dienoyl-CoAs could not . This reductase-independent metabolism of trans-2,trans-4-dienoyl-CoAs resembled the situation postulated for mammalian mitochondria in which oleic acid is degraded through a di-isomerase-dependent pathway . In this hypothetical process, trans-2,trans-4-dienoyl-CoA metabolites are generated by Delta(3)-Delta(2)-enoyl-CoA isomerase and Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and are degraded by 2-enoyl-CoA hydratase 1 in the absence of 2,4-dienoyl-CoA reductase . Growth of a yeast fox2sps19Delta mutant in which Fox2p was exchanged with rat peroxisomal multifunctional enzyme type 1 on trans-9,trans-12 linolelaidic acid medium gave credence to this theory . We propose an amendment to the current scheme of the carbon flux through beta-oxidation taking into account the dispensability of beta-oxidation auxiliary enzymes for metabolizing trans double bonds at even-numbered positions.

Hum Mol Genet, 2000 Oct 12, 9(17), 2523 - 30
Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae; Cavadini P et al.; Frataxin is a nuclear-encoded mitochondrial protein widely conserved among eukaryotes . Human frataxin (fxn) is severely reduced in Friedreich ataxia (FRDA), a frequent autosomal recessive neuro- and cardio-degenerative disease . Whereas the function of fxn is unknown, the yeast frataxin homolog (Yfh1p) has been shown to be involved in mitochondrial iron homeostasis and protection from free radical toxicity . Evidence of iron accumulation and oxidative damage in cardiac tissue from FRDA patients suggests that fxn may have a similar function, but whether yeast and human frataxin actually have interchangeable roles in mitochondrial iron homeostasis is unknown . We show that a wild-type FRDA cDNA can complement Yfh1p-deficient yeast (yfh1 delta) by preventing the mitochondrial iron accumulation and oxidative damage associated with loss of Yfh1p . We analyze the functional effects of two FRDA point mutations, G130V and W173G, associated with a mild and a severe clinical presentation, respectively . The G130V mutation affects protein stability and results in low levels of mature (m) fxn, which are nevertheless sufficient to rescue yfh1 delta yeast . The W173G mutation affects protein processing and stability and results in severe m-fxn deficiency . Expression of the FRDA (W173G) cDNA in yfh1 delta yeast leads to increased levels of mitochondrial iron which are not as elevated as in Yfh1p-deficient cells but are above the threshold for oxidative damage of mitochondrial DNA and iron-sulfur centers, causing a typical yfh1 delta phenotype . These results demonstrate that fxn functions like Yfh1p, providing experimental support to the hypothesis that FRDA is a disorder of mitochondrial iron homeostasis.

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