Genetics . 2005 Jan 16; {Epub ahead of print} MLH1 and MSH2 promote the symmetry of double-strand break repair events at the HIS4 hotspot in Saccharomyces cerevisiae; Hoffmann ER et al.; Double-strand breaks (DSBs) initiate meiotic recombination . The DSB repair model predicts that two genetic markers spanning the DSB should both be included in heteroduplex DNA and be detectable as non-Mendelian segregations (NMS) . In experiments testing this, a significant fraction of events do not conform to this prediction, as only one of the markers displays NMS (one-sided events) . Two explanations have been proposed to account for the discrepancies between the predictions and experimental observations . One suggests that two-sided events are the norm but are 'hidden' as heteroduplex repair frequently restores the parental configuration of one of the markers . Another explanation posits that one-sided events reflect events in which heteroduplex is formed predominantly on only one side of the DSB . In the absence of heteroduplex repair, the first model predicts two-sided events would be revealed at the expense of one-sided events, while the second predicts no effect on the distribution of events when heteroduplex repair is lost . We tested these predictions by deleting the DNA mismatch repair genes MSH2 or MLH1 and analyzing the proportion of two-sided events . Unexpectedly, the results do not match the predictions of either model . In both mlh1Delta and msh2Delta, the proportion of two-sided events is significantly decreased relative to wild type . These observations can be explained in one of two ways . Either Msh2p/Mlh1p-independent mispair removal leads to restoration of one of the markers flanking the DSB site or Msh2p/Mlh1p actively promote two-sided events. Genetics . 2005 Jan 16; {Epub ahead of print} Trans events associated with crossovers are revealed in the absence of mismatch repair genes in Saccharomyces cerevisiae; Hoffmann ER et al.; Genetic analysis of recombination in Saccharomyces cerevisiae has revealed structures not predicted by the double-strand break repair model of meiotic recombination . A particular type of recombinant containing "trans" heteroduplex DNA has been observed at two loci . Trans events were originally identified only in tetrads in which the non-Mendelian segregations were not associated with a crossover . Because of this, these events were proposed to have arisen from the unwinding of double Holliday Junctions . Previous studies used palindromes as genetic markers whereas we have used a complementary approach of deleting mismatch repair proteins in order to identify heteroduplex DNA . We found that the markers occurred in trans and were associated with crossovers . In both mlh1Delta and msh2Delta strains, the frequency of trans events associated with a crossover exceeded that predicted from the random association of crossovers with noncrossover trans events . We propose two different models to account for trans events associated with crossovers and discuss the relevance to wild-type DSB repair. Genetics . 2005 Jan 16; {Epub ahead of print} Infrequent co-conversion of markers flanking a meiotic recombination initiation site in Saccharomyces cerevisiae; Jessop L et al.; To study the mechanism of meiotic recombination in Saccharomyces cerevisiae, we examined recombination in an interval where the majority of events are initiated at a single hotspot for DNA double strand breaks (DSB) , with little or no expected contribution by outside initiation events . This interval contained infrequently corrected palindromic markers 300 bp to the left and 600 bp to the right of the DSB hotspot . Conversion of single markers occurred frequently, while conversion of both markers occurred rarely, and many of the tetrads in which both markers converted were the products of multiple events . These data indicate that most meiotic recombination intermediates are asymmetrically positioned around the initiating DSB, with a short (less than 300 bp) tract of heteroduplex DNA (hDNA) to one side, and hDNA on the other side frequently extending 600 bp or more . One consequence of this asymmetry is the preferential concentration of crossovers in the vicinity of the initiating DSB. Genetics . 2005 Jan 16; {Epub ahead of print} A genetic screen for top3 suppressors in Saccharomyces cerevisiae identifies SHU1, SHU2, PSY3, and CSM2 - four genes involved in error-free DNA repair; Shor E et al.; Helicases of the RecQ family and topoisomerase III are evolutionarily conserved proteins important for maintenance of genome stability . In Saccharomyces cerevisiae, loss of the TOP3 gene, encoding topoisomerase III, results in a phenotype of slow growth, DNA damage sensitivity, meiotic defects, and hyper-recombination . The sole RecQ helicase in budding yeast, Sgs1, interacts with Top3 both physically and genetically, and the two proteins are thought to act in concert in vivo . Much recent genetic and biochemical evidence points to the role of RecQ helicases and topoisomerase III in regulating homologous recombination (HR) during DNA replication . Previously, we found that mutations in HR genes partially suppress top3 slow growth . Here, we describe the analysis of four additional mutational suppressors of top3 defects: shu1, shu2, psy3, and csm2 . These genes belong to one epistasis group and their protein products interact with each other, strongly suggesting that they function as a complex in vivo . Their mutant phenotype indicates that they are important for error-free repair of spontaneous and induced DNA lesions, protecting the genome from mutation . These mutants exhibit an epistatic relationship with rad52 and show altered dynamics of Rad52-YFP foci, suggesting a role for these proteins in recombinational repair. Genetics . 2005 Jan 16; {Epub ahead of print} The Heme Activator Protein Hap1 Represses Transcription by a Heme-Independent Mechanism in Saccharomyces Cerevisiae; Hon T et al.; The yeast heme activator protein Hap1 binds to DNA and activates transcription of genes encoding functions required for respiration and for controlling oxidative damage, in response to heme . Hap1 contains a DNA-binding domain with a C6 zinc cluster motif and a coiled-coil dimerization element, typical of the members of the yeast Gal4 family, and an acidic activation domain . The regulation of Hap1 transcription-activating activity is controlled by two classes of Hap1 elements, repression modules (RPM1-3) and heme-responsive motifs (HRM1-7) . Previous indirect evidence indicates that Hap1 may repress transcription directly . Here we show, by promoter analysis, by chromatin immunoprecipitation, and by electrophoretic mobility shift assay, that Hap1 binds directly to DNA and represses transcription of its own gene by at least 20-fold . We found that Hap1 repression of the HAP1 gene occurs independently of heme concentrations . While DNA binding is required for transcriptional repression by Hap1, deletion of Hap1 activation domain and heme-regulatory elements has varying effects on repression . Further, we found that repression by Hap1 requires the function of Hsp70 (Ssa), but not Hsp90 . These results show that Hap1 binds to its own promoter and represses transcription in a heme-independent but Hsp70-dependent manner. Spectrochim Acta A Mol Biomol Spectrosc, 2005 Feb, 61(4), 761 - 5 Spectroscopic characterization of Au(3+) biosorption by waste biomass of Saccharomyces cerevisiae; Lin Z et al.; Some spectroscopic characteristics of Au(3+) biosorption by waste biomass of Saccharomyces cerevisiae have been reported in this paper . The effect of temperature on the correlation parameters of chemical kinetics and thermodynamics of the binding reaction was investigated by using AAS . XRD diffraction pattern of gold-loaded biomass revealed that the Au(3+) bound on the cell wall of the biomass had been reduced into gold particle . FTIR spectrophotometry on blank and gold-loaded biomass demonstrated that active groups such as the hydroxyl group of saccharides, and the carboxylate anion of amino-acid residues, from the peptidoglycan layer on the cell wall seem to be the sites for the Au(3+) binding, and the free aldehyde group of the hemiacetalic hydroxyl group from reducing sugars, i.e . the hydrolysates of the polysaccharides on the peptidoglycan layer, serving as the electron donor, in situ reduced the Au(3+) to Au(0) . XPS and IR characterizations of the interaction between glucose and Au(3+) further supported that the reduction of Au(3+) to Au(0) can directly occur at the aldehyde group of the reducing sugars. Cell Mol Biol Lett, 2004, 9(4A), 755 - 63 The aminoesters as inhibitors of plasma membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae; Oblak E et al.; A set of oxalates of alpha-dimethylamino fatty acids n-alkyl esters (MEM-ns and n-MEM-8s) and n-dodecyl-N,N-dimethylalaninate (DMAL-12s) were synthesized . Their activities on the growth, transport, and ATPases from the yeast Saccharomyces cerevisiae were compared . The compounds differ in the number of carbon atoms in their aliphatic chain and in the position of that chain in their molecular structure . The tested aminoesters with twelve carbon atoms (MEM-10s and DMAL-12s) appeared to have the highest level of activity . These drugs inhibited plasma membrane H+-ATPase, but no inhibition of mitochondrial ATPase was observed . Under nitrogen-derepressed conditions, the aminoesters inhibited amino acid uptake by yeast cells. J Biol Chem . 2005 Jan 11; {Epub ahead of print} Identification of a novel class of target genes and a novel type of binding sequence of heat shock transcription factor in Saccharomyces cerevisiae; Yamamoto A et al.; In response to hyperthermia, heat shock transcription factor (HSF) activates transcription of a set of genes encoding heat shock proteins (HSPs) . The promoter regions of HSP genes contain the HSF-binding sequence called the heat shock element (HSE), which consists of contiguous inverted repeats of the sequence 5-nGAAn-3 (where n is any nucleotide) . We have constructed an hsf1 mutant of Saccharomyces cerevisiae and analyzed genome-wide changes in heat shock response in the mutant cells . The results have revealed that Hsf1 is necessary for heat-induced transcription of not only HSP, but also genes encoding proteins involved in diverse cellular processes such as protein degradation, detoxification, energy generation, carbohydrate metabolism, and maintenance of cell wall integrity . Approximately half of the Hsf1-regulated genes lacked the typical HSE in their promoter regions . Instead, several of these genes have a novel Hsf1-binding sequence, which contains three direct repeats of nTTCn (or nGAAn) interrupted by 5-bp . The number and spacing of the repeating units are critical determinants for heat-induced transcription, as well as for recognition by Hsf1 . In the yeast genome, the presence of the sequence is enriched in Hsf1-regulated genes, suggesting that it is generally used as an HSE in the Hsf1 regulon. Yeast . 2005 Jan 11;22(2):111-127 {Epub ahead of print} Influence of low glycolytic activities in gcr1 and gcr2 mutants on the expression of other metabolic pathway genes in Saccharomyces cerevisiae; Sasaki H et al.; A complex of the transcription factors Gcr1p and Gcr2p coordinately regulates the expression of glycolytic genes in Saccharomyces cerevisiae . To understand the effects of gcr mutations on other metabolic pathways, genome-wide gene expression profiles in gcr1 and gcr2 mutants were examined . The biggest effects of gcr1 and gcr2 mutations were observed on the glycolytic genes and the expressions of most of the glycolytic genes were substantially decreased compared to those in the wild-type strain in both glucose and glycerol + lactate growth conditions . On the other hand, the expressions of genes encoding the TCA cycle and respiration were increased in gcr mutants when the cells were grown in glucose . RT-PCR analyses revealed that the expression of SIP4 and HAP5, which are known to affect the expression of some of the gluconeogenic, TCA cycle and respiratory genes, were also increased under this condition . The growth of gcr mutants on glucose was impaired by adding respiration inhibitor antimycin A, whereas the growth of the wild-type strain was not . The conversion of glucose to biomass was higher and, to the contrary, ethanol yield was lower in the gcr2 mutant compared to those in the wild-type strain . These results suggest the possibility that the gcr mutants, in which glycolytic activities are low, changed their metabolic patterns under glucose growth condition to enhance the expression of TCA cycle and respiratory genes to produce more energy . Copyright (c) 2005 John Wiley & Sons, Ltd. Yeast . 2005 Jan 11;22(2):129-139 {Epub ahead of print} Involvement of actin and polarisome in morphological change during spore germination of Saccharomyces cerevisiae; Kono K et al.; We studied the morphological changes of Saccharomyces cerevisiae ascospores during germination . Initiation of germination is followed by polarization of actin patches, maintaining their localization to the site of cell surface growth . Loss of polarisome components, Spa2p, Pea2p, Bud6p or Bni1p, results in depolarization of actin patches . Green fluorescent protein-fused polarisome components exhibit the polarized localization, implying that polarisome is involved in the polarized outgrowth during germination . At the late stage of germination, we found that actin patches temporally depolarize before bud emergence . The observation that loss of Cla4p extends the polarized growth period suggests that Cla4p is involved in the actin-depolization step . Actin polarization in the initial stage is accelerated by overexpression of Ras2p, whereas hyperpolarization is continuously observed by overexpression of Rho1p . Thus, yeast spore germination is a morphological event that is regulated by a number of factors implicated in mitotic bud morphogenesis . Copyright (c) 2005 John Wiley & Sons, Ltd. Eukaryot Cell, 2005 Jan, 4(1), 209 - 20 Separation of the Saccharomyces cerevisiae Paf1 Complex from RNA Polymerase II Results in Changes in Its Subnuclear Localization; Porter SE et al.; The yeast Paf1 complex (Paf1C), composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, associates with RNA polymerase II (Pol II) at promoters and in the actively transcribed portions of mRNA genes . Loss of Paf1 results in severe phenotypes and significantly reduced levels of the other Paf1C components . In contrast, loss of Rtf1 causes relatively subtle phenotypic changes and no reduction in the other Paf1C factors but disrupts the association of these factors with Pol II and chromatin . To elucidate the fate of the Paf1C when dissociated from Pol II, we examined the localization of the Paf1C components in paf1 and rtf1 mutant yeast strains . We found that although the Paf1C factors remain nuclear in paf1 and rtf1 strains, loss of Paf1 or Rtf1 results in a change in the subnuclear distribution of the remaining factors . In wild-type cells, Paf1C components are present in the nucleoplasm but not the nucleolus . In contrast, in both paf1 and rtf1 strains, the remaining factors are found in the nucleolus as well as the nucleoplasm . Loss of Paf1 affects nucleolar function; we observed that expression of MAK21 and RRP12, important for rRNA processing, is reduced concomitant with an increase in rRNA precursors in a paf1 strain . However, these changes are not the result of relocalization of the Paf1C because loss of Rtf1 does not cause similar changes in rRNA processing . Instead, we speculate that the change in localization may reflect a link between the Paf1C and newly synthesized mRNAs as they exit the nucleus. Eukaryot Cell, 2005 Jan, 4(1), 134 - 46 Novel Interaction between Apc5p and Rsp5p in an Intracellular Signaling Pathway in Saccharomyces cerevisiae; Arnason TG et al.; The ubiquitin-targeting pathway is evolutionarily conserved and critical for many cellular functions . Recently, we discovered a role for two ubiquitin-protein ligases (E3s), Rsp5p and the Apc5p subunit of the anaphase-promoting complex (APC), in mitotic chromatin assembly in Saccharomyces cerevisiae . In the present study, we investigated whether Rsp5p and Apc5p interact in an intracellular pathway regulating chromatin remodeling . Our genetic studies strongly suggest that Rsp5p and Apc5p do interact and that Rsp5p acts upstream of Apc5p . Since E3 enzymes typically require the action of a ubiquitin-conjugating enzyme (E2), we screened E2 mutants for chromatin assembly defects, which resulted in the identification of Cdc34p and Ubc7p . Cdc34p is the E2 component of the SCF (Skp1p/Cdc53p/F-box protein) . Therefore, we analyzed additional SCF mutants for chromatin assembly defects . Defective chromatin assembly extracts generated from strains harboring a mutation in the Cdc53p SCF subunit or a nondegradable SCF target, Sic1(Deltaphos), confirmed that the SCF was involved in mitotic chromatin assembly . Furthermore, we demonstrated that Ubc7p physically and genetically interacts with Rsp5p, suggesting that Ubc7p acts as an E2 for Rsp5p . However, rsp5(CA) and Deltaubc7 mutations had opposite genetic effects on apc5(CA) and cdc34-2 phenotypes . Therefore, the antagonistic interplay between Deltaubc7 and rsp5(CA), with respect to cdc34-2 and apc5(CA), indicates that the outcome of Rsp5p's interaction with Cdc34p and Apc5p may depend on the E2 interacting with Rsp5p. Eukaryot Cell, 2005 Jan, 4(1), 82 - 9 Broad Sensitivity of Saccharomyces cerevisiae Lacking Ribosome-Associated Chaperone Ssb or Zuo1 to Cations, Including Aminoglycosides; Kim SY et al.; The Hsp70 Ssb and J protein Zuo1 of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, proposed to be involved in the folding of newly synthesized polypeptide chains . Cells lacking Ssb and/or Zuo1 have been reported to be hypersensitive to cationic aminoglycoside protein synthesis inhibitors that affect translational fidelity and to NaCl . Since we found that Deltassb1 Deltassb2 (Deltassb1,2), Deltazuo1, and wild-type cells have very similar levels of translational misreading in the absence of aminoglycosides, we asked whether the sensitivities to aminoglycosides and NaCl represent a general increase in sensitivity to cations . We found that Deltassb1,2 and Deltazuo1 cells are hypersensitive to a wide range of cations . This broad sensitivity is similar to that of cells having lowered activity of major plasma membrane transporters, such as the major K(+) transporters Trk1 and Trk2 or their regulators Hal4 and Hal5 . Like Deltahal4,5 cells, Deltassb1,2 and Deltazuo1 cells have increased intracellular levels of Na(+) and Li(+) upon challenge with higher-than-normal levels of these cations, due to an increased rate of influx . In the presence of aminoglycosides, Deltassb1,2, Deltazuo1, and Deltahal 4,5 cells have similarly increased levels of translational misreading . We conclude that, in vivo, the major cause of the aminoglycoside sensitivity of cells lacking ribosome-associated molecular chaperones is a general increase in cation influx, perhaps due to altered maturation of membrane proteins. Eukaryot Cell, 2005 Jan, 4(1), 36 - 45 Molecular Analysis Reveals Localization of Saccharomyces cerevisiae Protein Kinase C to Sites of Polarized Growth and Pkc1p Targeting to the Nucleus and Mitotic Spindle; Denis V et al.; The catalytic activity and intracellular localization of protein kinase C (PKC) are both highly regulated in vivo . This family of kinases contains conserved regulatory motifs, i.e., the C1, C2, and HR1 domains, which target PKC isoforms to specific subcellular compartments and restrict their activity spatially . Saccharomyces cerevisiae contains a single PKC isozyme, Pkc1p, which contains all of the regulatory motifs found in mammalian PKCs . Pkc1p localizes to sites of polarized growth, consistent with its main function in maintaining cell integrity . We dissected the molecular basis of Pkc1p localization by expressing each of its domains individually and in combinations as green fluorescent protein fusions . We find that the Rho1p-binding domains, HR1 and C1, are responsible for targeting Pkc1p to the bud tip and cell periphery, respectively . We demonstrate that Pkc1p activity is required for its normal localization to the bud neck, which also depends on the integrity of the septin ring . In addition, we show for the first time that yeast protein kinase C can accumulate in the nucleus, and we identify a nuclear exit signal as well as nuclear localization signals within the Pkc1p sequence . Thus, we propose that Pkc1p shuttles in and out of the nucleus and consequently has access to nuclear substrates . Surprisingly, we find that deletion of the HR1 domain results in Pkc1p localization to the mitotic spindle and that the C2 domain is responsible for this targeting . This novel nuclear and spindle localization of Pkc1p may provide a molecular explanation for previous observations that suggest a role for Pkc1p in regulating microtubule function. Eukaryot Cell, 2005 Jan, 4(1), 17 - 29 Cyclophilin A Is Localized to the Nucleus and Controls Meiosis in Saccharomyces cerevisiae; Arevalo-Rodriguez M et al.; Cyclophilin A is conserved from yeast to humans and mediates the ability of cyclosporine to perturb signal transduction cascades via inhibition of calcineurin . Cyclophilin A also catalyzes cis-trans peptidyl-prolyl isomerization during protein folding or conformational changes; however, cyclophilin A is not essential in yeast or human cells, and the true biological functions of this highly conserved enzyme have remained enigmatic . In Saccharomyces cerevisiae, cyclophilin A becomes essential in cells compromised for the nuclear prolyl-isomerase Ess1, and cyclophilin A physically interacts with two nuclear histone deacetylase complexes, Sin3-Rpd3 and Set3C, which both control meiosis . Here we show that cyclophilin A is localized to the nucleus in yeast cells and governs the meiotic gene program to promote efficient sporulation . The prolyl-isomerase activity of cyclophilin A is required for this meiotic function . We document that cyclophilin A physically associates with the Set3C histone deacetylase and analyze in detail the structure of this protein-protein complex . Genetic studies support a model in which cyclophilin A controls meiosis via Set3C and an additional target . Our findings reveal a novel nuclear role for cyclophilin A in governing the transcriptional program required for the vegetative to meiotic developmental switch in budding yeast. Biochem J . 2005 Jan 10; {Epub ahead of print} Cellular factors required for protection from hyperoxia toxicity in Saccharomyces cerevisiae; Outten CE et al.; Prolonged exposure to hyperoxia represents a serious danger to cells, yet little is known about the specific cellular factors that affect hyperoxia stress . By screening the yeast deletion library, we have identified genes that protect against high oxygen damage . Out of ~4800 mutants, 84 were identified as hyperoxia-sensitive, representing genes with diverse cellular functions including transcription and translation, vacuole function, NADPH production, and superoxide detoxification . Superoxide plays a significant role since the majority of hyperoxia-sensitive mutants displayed cross-sensitivity to superoxide-generating agents, and mutants with compromised superoxide dismutase (SOD) activity were particularly vulnerable to hyperoxia . By comparison, factors known to guard against hydrogen peroxide toxicity were poorly represented amongst hyperoxia-sensitive mutants . Although many cellular components are potential targets, our studies indicate that mitochondrial glutathione is particularly vulnerable to hyperoxia damage . During hyperoxia stress, mitochondrial glutathione is more susceptible to oxidation than cytosolic glutathione . Furthermore, two factors that help maintain mitochondrial GSH in the reduced form, namely the NADH kinase Pos5p and the mitochondrial glutathione reductase (Glr1p), are critical for hyperoxia resistance, whereas their cytosolic counterparts are not . Our findings are consistent with a model in which hyperoxia toxicity is manifested by superoxide-related damage and changes in the mitochondrial redox state. Biochemistry, 2005 Jan 18, 44(2), 511 - 517 Structure and Function of the GTP Binding Protein Gtr1 and Its Role in Phosphate Transport in Saccharomyces cerevisiae; Lagerstedt JO et al.; The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions . The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function . This idea was based on a displayed deletion phenotype of Deltagtr1 similar to the Deltapho84 phenotype . As of yet, the mode of interaction has not been described . The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84-green fluorescent protein or Pho84-myc chimeras . The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions . EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1-185) harbors the nucleotide responsive elements . In contrast, the spectra obtained for the C-terminal part (residues 186-310) displayed no evidence of conformational changes upon GTP addition. J Biotechnol, 2005 Feb 9, 115(3), 271 - 8 Ascorbate abolishes auxotrophy caused by the lack of superoxide dismutase in Saccharomyces cerevisiae Yeast can be a biosensor for antioxidants; Zyracka E et al.; Yeast (Saccharomyces cerevisiae) mutants lacking cytoplasmic superoxide dismutase (CuZnSOD) show Lys and Met auxotrophy under aerobic conditions . This metabolic defect can be ameliorated by exogenous ascorbate as well as other antioxidants (glutathione, cysteine and N-acetylcysteine) . Restoration of growth of CuZnSOD(-) yeast mutants on media devoid of Met and/or Lys may therefore be a simple and useful means to detect and quantify antioxidants . The protective effect of antioxidants is oxygen-dependent: the lower the oxygen content of the atmosphere, the lower antioxidant concentrations are required to restore prototrophy . Therefore, the sensitivity of the test can be augmented by growing the yeast under lowered partial oxygen pressure . While 6mM, 10mM and 30mM ascorbate was necessary to restore the growth in the absence of Met, in the absence of Lys, and in the absence of Lys and Met, respectively, under 21% oxygen, 3mM and 6mM ascorbate was sufficient for growth restoration in the absence of Lys and in the absence of Lys and Met, respectively, under 3% oxygen . The protective effects of cysteine and N-acetylcysteine peaked at 0.5mM and 6mM, respectively, disappearing at higher concentrations of these compounds, pointing to the detection of not only protective but also toxic cellular effects of the compounds studied by the test proposed. J Biol Chem . 2005 Jan 6; {Epub ahead of print} Characterization and purification of saccharomyces cerevisiae RNase MRP reveals a new unique protein component; Salinas K et al.; In the yeast, Saccharomyces cerevisiae, RNase MRP is an essential endoribonuclease that consists of one RNA component, and at least nine protein components . Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P . To fully characterize the RNase MRP complex we purified it to apparent homogeneity in a highly active state using tandem affinity purification . In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP . Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA . A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants while no defect in tRNA processing was observed . Homologs of the Ylr145w protein were found in most yeasts, fungi and Arabidopsis . Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P . We recommend that this gene be designated RMP1, for RNase MRP Protein 1. J Biol Chem . 2005 Jan 6; {Epub ahead of print} Pkc1 and the upstream elements of the cell integrity pathway in saccharomyces cerevisiae , Rom2 and Mtl1, are required for cellular responses to oxidative stress; Vilella F et al.; In this study we analyse the participation of the PKC1-MAPK cell integrity pathway in cellular responses to oxidative stress in Saccharomyces cerevisiae . Evidence is presented demonstrating that only Pkc1 and the upstream elements of the cell integrity pathway are essential for cell survival upon treatment with two oxidising agents, diamide and hydrogen peroxide . Mtl1 is characterised for the first time as a cell-wall sensor of oxidative stress . We also show that the actin cytoskeleton is a cellular target for oxidative stress . Both diamide and hydrogen peroxide provoke a marked depolarisation of the actin cytoskeleton, being Mtl1, Rom2 and Pkc1 functions all required to restaure the correct actin organisation . Diamide induces the formation of disulphide bonds in newly secreted cell-wall proteins . This mainly provokes structural changes in the cell out-layer which activate the PKC1-MAPK pathway and hence the protein kinase Slt2 . Our results led us to the conclusion that Pkc1 activity is required to overcome the effects of oxidative stress by: (i) enhancing the machinery required to repair the altered cell wall and (ii) restoring actin cytoskeleton polarity by promoting actin cable formation. J Biol Chem . 2005 Jan 4; {Epub ahead of print} Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3' to 5' DNA helicase; Prakash R et al.; The MPH1 (Mutator Phenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of elevated spontaneous mutation rates of haploid cells deleted for this gene . Further studies showed that MPH1 functions to channel DNA lesions into an error-free DNA repair pathway . The Mph1 protein contains the seven conserved motifs of the SF2 family of nucleic acid unwinding enzymes . Genetic analyses have found epistasis of the mph1 deletion with mutations in the RAD52 gene group that mediates homologous recombination and DNA repair by homologous recombination . To begin dissecting the biochemical functions of the MPH1-encoded product, we have expressed it in yeast cells and purified it to near homogeneity . We show that Mph1 has a robust ATPase function that requires ssDNA for activation . Consistent with its homology to members of the SF2 helicase family, we find a DNA helicase activity in Mph1 . We present data to demonstrate that the Mph1 DNA helicase activity is fueled by ATP hydrolysis and has a 3' to 5' polarity with respect to the DNA strand on which this protein translocates . The DNA helicase activity of Mph1 is enhanced by the heterotrimeric ssDNA binding protein RPA . These results thus establish Mph1 as an ATP-dependent DNA helicase, and the availability of purified Mph1 should facilitate efforts at deciphering the role of this protein in homologous recombination and mutation avoidance. Microbiology, 2005 Jan, 151(Pt 1), 91 - 8 Disruption of MRG19 results in altered nitrogen metabolic status and defective pseudohyphal development in Saccharomyces cerevisiae; Das M et al.; It was previously shown that MRG19 downregulates carbon metabolism in Saccharomyces cerevisiae upon glucose exhaustion, and that the gene is glucose repressed . Here, it is shown that glucose repression of MRG19 is overcome upon nitrogen withdrawal, suggesting that MRG19 is a regulator of carbon and nitrogen metabolism . beta-Galactosidase activity fostered by the promoter of GDH1/3, which encode anabolic enzymes of nitrogen metabolism, was altered in an MRG19 disruptant . As compared to the wild-type strain, the MRG19 disruptant showed a decrease in the ratio of 2-oxoglutarate to glutamate under nitrogen-limited conditions . MRG19 disruptants showed reduced pseudohyphal formation and enhanced sporulation, a phenomenon that occurs under conditions of both nitrogen and carbon withdrawal . These studies revealed that MRG19 regulates carbon and nitrogen metabolism, as well as morphogenetic changes, suggesting that MRG19 is a component of the link between the metabolic status of the cell and the corresponding developmental pathway. J Biol Chem . 2005 Jan 4; {Epub ahead of print} YEH2/YLR020c encodes a novel steryl ester hydrolase of the yeast saccharomyces cerevisiae; Muellner H et al.; Previous work from our laboratory (Zinser et al., J . Bacteriol . 175; 1993; 2853-2858) demonstrated steryl ester hydrolase activity in the plasma membrane of the yeast Saccharomyces cerevisiae . Here, we show that the gene product of YEH2/YLR020c, which is homologous to several known mammalian steryl ester hydrolases, is the enzyme catalyzing this reaction . Deletion of yeast YEH2 led to complete loss of plasma membrane steryl ester hydrolase activity whereas overexpression of the gene resulted in a significant elevation of the activity . Finally, purification of enzymatically active Yeh2p close to homogeneity unambiguously identified this protein as a steryl ester hydrolase and thus as the first enzyme of this kind characterized in Saccharomyces cerevisiae . In addition to evidence obtained in vitro experiments in vivo contributed to the characterization of this novel enzyme . Sterol analysis of yeh2 unveiled a slightly elevated level of zymosterol suggesting that the esterified form of this sterol precursor is a preferred substrate of Yeh2p . However, in strains bearing hybrid proteins with strongly enhanced Hys1p activity decreased levels of all steryl esters were observed . Thus, it appears that Yeh2p activity is not restricted to distinct steryl esters but rather has broad substrate specificity . The fact that in a yeh2 deletion strain bulk steryl ester mobilization occurred at a similar rate as in wild type suggested that Yeh2p is not the only steryl ester hydrolase but that other enzymes with overlapping function exist in the yeast. J Biol Chem . 2005 Jan 4; {Epub ahead of print} The saccharomyces cerevisiae peroxisomal import receptor Pex5p is monoubiquitinated in wild type cells; Kragt A et al.; Pex5p is a mobile receptor for peroxisomal targeting signal type-I (PTS1)-containing proteins that is cycling between the cytoplasm and the peroxisome . Here we show that Pex5p is a stable protein, which is monoubiquitinated in wild type cells . By making u se of mutants defective in vacuolar or proteasomal degradation we demonstrate that monoubiquitinated Pex5p is not a breakdown intermediate of either system . Monoubiquitinated Pex5p is localized to peroxisomes and ubiquitination requires the presence of fu nctional docking- and RING-finger complexes, which suggests that it is a late event in peroxisomal matrix protein import . In pex1, pex4, pex6, pex15, and pex22 mutants, all of which are blocked in the terminal steps of peroxisomal matrix protein import, p olyubiquitinated forms of Pex5p accumulate, ubiquitination being dependent on the ubiquitin-conjugating enzyme Ubc4p . However, Ubc4p is not required for Pex5p ubiquitination in wild type cells, and cells lacking Ubc4p are not affected in peroxisome biogen esis . These results indicate that Pex5p monoubiquitination in wild type cells serves to regulate rather than to degrade Pex5p, which is supported by the observed stability of Pex5p . We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, while Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import, may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway. Appl Microbiol Biotechnol . 2005 Jan 4; {Epub ahead of print} Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium; Gorgens JF et al.; Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated . The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions . The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production . Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate . The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism. Biochem Biophys Res Commun, 2005 Feb 4, 327(1), 311 - 9 The high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses; Park JI et al.; The Ras-cyclic AMP (cAMP) pathway is a major determinant of intrinsic stress resistance of the yeast Saccharomyces cerevisiae . Here, we isolated IRA2, encoding the Ras GTPase activator, as a global stress response gene . Subsequently, we studied the other negative regulators on the separate branch of the Ras-cAMP pathway, the low- or high-affinity cAMP phosphodiesterase encoded by PDE1 or PDE2, respectively . Deletion of PDE2, similar to ira2 deletion, rendered cells sensitive to freeze-thawing, peroxides, paraquat, cycloheximide, heavy metals, NaCl, heat, or cold shock . However, deletion of PDE1 did not affect stress tolerance, although it exacerbated stress sensitivity caused by the pde2 deletion, indicating that PDE1 can partly compensate for PDE2 . Deletion of IRA2 uniquely led to high sensitivity to cumene hydroperoxide, suggesting that IRA2 may have a distinct role for the response to this stress . Stress sensitivity of yeast cells in general correlated with the basal level of cAMP . Interestingly, yeast cells lacking PDE2 maintained higher cAMP levels in stationary phase than exponential growth phase, suggesting that Pde2p is the major regulator of cAMP levels in stationary phase . Depletion of Ras activity could not effectively suppress stress sensitivity caused by lack of cAMP phosphodiesterases although it could suppress stress sensitivity caused by lack of IRA2, indicating that cAMP accumulation in stationary phase can be mediated by other signaling proteins in addition to Ras . Our study shows that control of cAMP basal levels is important for determining intrinsic stress tolerance of yeast, and that the cAMP level during stationary phase is a result of a dynamic balance between its rates of synthesis and degradation. Biochemistry, 2005 Jan 11, 44(1), 184 - 192 Cysteine Labeling Studies Detect Conformational Changes in Region 106-132 of the Mitochondrial ADP/ATP Carrier of Saccharomyces cerevisiae; Kihira Y et al.; To know the structural and functional features of the cytosolic-facing first loop (LC1) including its surrounding region of the mitochondrial ADP/ATP carrier (AAC), we prepared 27 mutants, in which each amino acid residue between residues 106 and 132 of the yeast type 2 AAC (yAAC2) was replaced by a cysteine residue . For mutant preparation, we used a Cys-less AAC mutant, in which all four intrinsic cysteine residues were substituted with alanine residues, as a template {Hatanaka, T., Kihira, Y., Shinohara, Y., Majima, E., and Terada, H . (2001) Biochem . Biophys . Res . Commun . 286, 936-942} . From the labeling intensities of the membrane-impermeable SH-reagent eosin-5-maleimide (EMA), sequence Lys(108)-Phe(127) was suggested to constitute the LC1 . The N-terminal half of this region (Lys(108)-Phe(115)) was suggested to change its location from the cytosol to a region close to the membrane on conversion from the c-state to the m-state in association with disruption or unwinding of its alpha-helical structure, whereas the C-terminal half region (Gly(116)-Phe(127)) was considered to extrude essentially into the cytosol, while keeping its alpha-helical structure . Hence, the conformation of m-state LC1 is greatly different from that of c-state LC1 . Possibly the LC1 changes its location between the membranous region and the cytosol during ADP/ATP transport . Lys(108) in the LC1 of the yAAC2 was found to be associated with binding of the transport substrates, and its -NH(3)(+) moiety, to be of importance for the transport function . On the basis of these results, possible roles of the conformational changes of the LC1 in the transport activity are discussed. Biochim Biophys Acta, 2005 Jan 11, 1681(2-3), 166 - 74 Epub 2004 Dec 15. Translational control by internal ribosome entry site in Saccharomyces cerevisiae; Seino A et al.; To confirm the active involvement of the internal ribosome entry site (IRES)-dependent translation in living eukaryotes, Saccharomyces cerevisiae and HAP4 IRES were used for in vitro and in vivo experiments . Since HAP4 protein might be required for activating mRNAs transcription in yeast cells when they are released from catabolite repression, the translational efficiency of HAP4 mRNA is presumed to increase under such a condition . The in vitro experiment showed clearly that the translational mechanism was shifted from the cap-dependent to the completely IRES-dependent translation when the yeast cells were derepressed from catabolite repression . From in vivo experiment, it was confirmed that the IRES-dependent translational efficiency was in a low level at the beginning of the stationary growth phase, and was enhanced at the glucose-exhausted phase . These results indicate that yeast cells on the catabolite derepressed condition could get a large amount of HAP4 protein for the completely IRES-dependent translation, while the IRES-dependent translational efficiency is increased . It has been proven that IRES functions directing the initiation of translation in living yeast cells. Proc Natl Acad Sci U S A, 2005 Jan 11, 102(2), 402 - 6 Epub 2004 Dec 29. Advances in measuring lifespan in the yeast Saccharomyces cerevisiae; Minois N et al.; Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Saccharomyces cerevisiae . Unfortunately, yeast researchers use a definition of longevity not applied to other species . We propose here a method that makes it possible to estimate for yeast the same measures of longevity calculated for other species . We also show that the conventional method (equating longevity with the number of offspring) is only an approximate measure of true chronological lifespan . Our method will allow results for yeast to be compared more correctly with those for other species. Proteins . 2004 Dec 28; {Epub ahead of print} Crystal structure of an alpha/beta serine hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 A resolution; Arndt JW et al.; No abstract. J Biol Chem . 2004 Dec 28; {Epub ahead of print} Toward biochemical understanding of a transcriptionally silenced chromosomal domain in Saccharomyces cerevisiae; Fox CA et al.; Silencing of the HMR domain in Saccharomyces cerevisiae requires the action of a DNA element called a silencer that binds sequence-specific DNA binding proteins to form a silencer-protein complex (SPC) . The SPC recruits specialized chromatin proteins, the Sir proteins that deacetylate and bind nucleosomes within HMR . Sir-dependent chromatin is absolutely necessary to repress transcription of a gene within HMR and causes repression at a step after recruitment of the RNA Pol II holoenzyme . Specific mechanisms that include specialized nucleosomes help confine Sir-dependent chromatin to HMR. BMC Genet . 2004 Dec 23;5(1):34 {Epub ahead of print} The exceptionally high rate of spontaneous mutations in the polymerase delta proofreading exonuclease-deficient Saccharomyces cerevisiae strain starved for adenine; Achilli A et al.; BACKGROUND: Mutagenesis induced in the yeast Saccharomyces cerevisiae by starvation for nutrilites is a well-documented phenomenon of an unknown mechanism . We have previously shown that the polymerase delta proofreading activity controls spontaneous mutagenesis in cells starved for histidine . To obtain further information, we compared the effect of adenine starvation on mutagenesis in wild-type cells and, in cells lacking the proofreading activity of polymerase delta (phenotype Exo-, mutation pol3-01) . RESULTS: Ade+ revertants accumulated at a very high rate on adenine-free plates so that their frequency on day 16 after plating was 1.5 x 10-4 for wild-type and 1.0 x 10-2 for the Exo- strain . In the Exo- strain, all revertants arising under adenine starvation are suppressors of the original mutation, most possessed additional nutritional requirements, and 50% of them were temperature sensitive . CONCLUSIONS: Adenine starvation is highly mutagenic in yeast . The deficiency in the polymerase delta proofreading activity in strains with the pol3-01 mutation leads to a further 66-fold increase of the rate of mutations . Our data suggest that adenine starvation induces genome-wide hyper-mutagenesis in the Exo- strain. Curr Genet . 2004 Dec 21; {Epub ahead of print} Grr1p is required for transcriptional induction of amino acid permease genes and proper transcriptional regulation of genes in carbon metabolism of Saccharomyces cerevisiae; Eckert-Boulet N et al.; The F-box protein Grr1p is involved in cell cycle regulation, glucose repression and transcriptional induction of the amino acid permease (AAP) gene AGP1 . We investigated the role of Grr1p in amino acid-mediated induction of AAP genes by performing batch cultivations with a wild-type strain and a grr1Delta strain and adding citrulline in the exponential phase . Whole-genome transcription analyses were performed on samples from each cultivation, both immediately before and 30 min after citrulline addition . Transcriptional induction of the AAP genes AGP1, BAP2, BAP3, DIP5, GNP1 and TAT1 is fully dependent on Grr1p . Comparison of the grr1Delta strain with the reference strain in the absence of citrulline revealed that GRR1 disruption leads to increased transcription of numerous genes . These encode enzymes in the tricarboxylic acid cycle, the pentose-phosphate pathway and both glucose and starch metabolism . Promoter analysis showed that many of the genes with increased transcription display Mig1p- and/or Msn2p/Msn4p-binding sites . Increased expression of glucose-repressed genes in the grr1Delta strain may be explained by the reduced expression of the hexose transporter genes HXT1, HXT2, HXT3 and HXT4 and a subsequent lowering of the glucose uptake; and the effect of GRR1 deletion on general carbon metabolism may therefore be indirect . Finally, none of the genes known to be primarily involved in cell cycle regulation displayed different expression levels in the grr1Delta cells as compared with the reference strain, suggesting that the role of Grr1p in cell cycle regulation does not include any transcriptional component. Genetics, 2004 Dec, 168(4), 1855 - 65 Examination of the Roles of Sgs1 and Srs2 Helicases in the Enforcement of Recombination Fidelity in Saccharomyces cerevisiae; Spell RM et al.; Mutation in SGS1, which encodes the yeast homolog of the human Bloom helicase, or in mismatch repair (MMR) genes confers defects in the suppression of mitotic recombination between similar but nonidentical (homeologous) sequences . Mutational analysis of SGS1 suggests that the helicase activity is required for the suppression of both homologous and homeologous recombination and that the C-terminal 200 amino acids may be required specifically for the suppression of homeologous recombination . To clarify the mechanism by which the Sgs1 helicase enforces the fidelity of recombination, we examined the phenotypes associated with SGS1 deletion in MMR-defective and recombination-defective backgrounds . Deletion of SGS1 caused no additional loss of recombination fidelity above that associated with MMR defects, indicating that the suppression of homeologous recombination by Sgs1 may be dependent on MMR . However, the phenotype of the sgs1 rad51 mutant suggests a MMR-independent role of Sgs1 in the suppression of RAD51-independent recombination . While homologous recombination levels increase in sgs1Delta and in srs2Delta strains, the suppression of homeologous recombination was not relaxed in the srs2 mutant . Thus, although both Sgs1 and Srs2 limit the overall level of mitotic recombination, there are distinct differences in the roles of these helicases with respect to enforcement of recombination fidelity. Genetics, 2004 Dec, 168(4), 1817 - 25 Spontaneous Mutations in Diploid Saccharomyces cerevisiae: More Beneficial Than Expected; Joseph SB et al.; We performed a 1012-generation mutation-accumulation (MA) experiment in the yeast, Saccharomyces cerevisiae . The MA lines exhibited a significant reduction in mean fitness and a significant increase in variance in fitness . We found that 5.75% of the fitness-altering mutations accumulated were beneficial . This finding contradicts the widely held belief that nearly all fitness-altering mutations are deleterious . The mutation rate was estimated as 6.3 x 10(-5) mutations per haploid genome per generation and the average heterozygous fitness effect of a mutation as 0.061 . These estimates are compatible with previous estimates in yeast. Genetics, 2004 Dec, 168(4), 1805 - 16 Competing Crossover Pathways Act During Meiosis in Saccharomyces cerevisiae; Argueso JL et al.; In Saccharomyces cerevisiae the MSH4-MSH5, MLH1-MLH3, and MUS81-MMS4 complexes act to promote crossing over during meiosis . MSH4-MSH5, but not MUS81-MMS4, promotes crossovers that display interference . A role for MLH1-MLH3 in crossover control is less clear partly because mlh1Delta mutants retain crossover interference yet display a decrease in crossing over that is only slightly less severe than that seen in msh4Delta and msh5Delta mutants . We analyzed the effects of msh5Delta, mlh1Delta, and mms4Delta single, double, and triple mutants on meiotic crossing over at four consecutive genetic intervals on chromosome XV using newly developed computer software . mlh1Delta mms4Delta double mutants displayed the largest decrease in crossing over (13- to 15-fold) of all mutant combinations, yet these strains displayed relatively high spore viability (42%) . In contrast, msh5Delta mms4Delta and msh5Delta mms4Delta mlh1Delta mutants displayed smaller decreases in crossing over (4- to 6-fold); however, spore viability (18-19%) was lower in these strains than in mlh1Delta mms4Delta strains . These data suggest that meiotic crossing over can occur in yeast through three distinct crossover pathways . In one pathway, MUS81-MMS4 promotes interference-independent crossing over; in a second pathway, both MSH4-MSH5 and MLH1-MLH3 promote interference-dependent crossovers . A third pathway, which appears to be repressed by MSH4-MSH5, yields deleterious crossovers. J Biol Chem . 2004 Dec 15; {Epub ahead of print} Functional analysis of Rpn6p, a lid component of the 26S proteasome, using temperature-sensitive rpn6 mutants of the yeast Saccharomyces cerevisiae; Isono E et al.; Rpn6p is a component of the lid of the 26S proteasome . We isolated and analyzed two temperature-sensitive rpn6 mutants in the yeast, Saccharomyces cerevisiae . Both mutants showed defects in protein degradation in vivo . However, the affinity-purified 26S proteasome of the rpn6 mutants grown at the permissive temperature degraded poly-ubiquitinated Sic1p efficiently, even at a higher temperature . Interestingly, their enzyme activity was even higher at a higher temperature, indicating that once made, mutant proteasomes are stable and have little defect in the proteolytic function . These results suggest that the deficiency in protein degradation observed in vivo be rather due to a defect in the assembly of a holoenzyme at the restrictive temperature . Indeed, both rpn6 mutants grown at the restrictive temperature were defective in assembling the 26S proteasome . A striking feature of the rpn6 mutants at the restrictive temperature was that there appeared a protein complex, composed of only four out of the nine lid components, Rpn5p, Rpn8p, Rpn9p and Rpn11p . Altogether, we conclude that Rpn6p is essential for the integrity/assembly of the lid, in the sense that it is necessary for the incorporation of Rpn3p, Rpn7p, Rpn12p and Sem1p (Rpn15p) into the lid, thereby playing an essential role in the proper function of the 26S proteasome. Protein Sci, 2005 Jan, 14(1), 209 - 15 Crystal structure of the YML079w protein from Saccharomyces cerevisiae reveals a new sequence family of the jelly-roll fold; Zhou CZ et al.; We determined the three-dimensional crystal structure of the protein YML079wp, encoded by a hypothetical open reading frame from Saccharomyces cerevisiae to a resolution of 1.75 A . The protein has no close homologs and its molecular and cellular functions are unknown . The structure of the protein is a jelly-roll fold consisting of ten beta-strands organized in two parallel packed beta-sheets . The protein has strong structural resemblance to the plant storage and ligand binding proteins (canavalin, glycinin, auxin binding protein) but also to some plant and bacterial enzymes (epimerase, germin) . The protein forms homodimers in the crystal, confirming measurements of its molecular mass in solution . Two monomers have their beta-sheet packed together to form the dimer . The presence of a hydrophobic ligand in a well conserved pocket inside the barrel and local sequence similarity with bacterial epimerases may suggest a biochemical function for this protein. Proc Natl Acad Sci U S A, 2004 Dec 28, 101(52), 18006 - 11 Epub 2004 Dec 17. Global network analysis of phenotypic effects: protein networks and toxicity modulation in Saccharomyces cerevisiae; Said MR et al.; Using genome-wide information to understand holistically how cells function is a major challenge of the postgenomic era . Recent efforts to understand molecular pathway operation from a global perspective have lacked experimental data on phenotypic context, so insights concerning biologically relevant network characteristics of key genes or proteins have remained largely speculative . Here, we present a global network investigation of the genotype/phenotype data set we developed for the recovery of the yeast Saccharomyces cerevisiae from exposure to DNA-damaging agents, enabling explicit study of how protein-protein interaction network characteristics may be associated with phenotypic functional effects . We show that toxicity-modulating proteins have similar topological properties as essential proteins, suggesting that cells initiate highly coordinated responses to damage similar to those needed for vital cellular functions . We also identify toxicologically important protein complexes, pathways, and modules . These results have potential implications for understanding toxicity-modulating processes relevant to a number of human diseases, including cancer and aging. Eur J Biochem, 2004 Dec, 271(23-24), 4855 - 64 Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae; Elbing K et al.; The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression . Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms . The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates . Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing . The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation . Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway . Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes . Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates . Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates . This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal . At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes . Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses. Nucleic Acids Res, 2004 Dec 15, 32(22), 6605 - 16 Print 2004. Genome-wide prediction of stop codon readthrough during translation in the yeast Saccharomyces cerevisiae; Williams I et al.; In-frame stop codons normally signal termination during mRNA translation, but they can be read as 'sense' (readthrough) depending on their context, comprising the 6 nt preceding and following the stop codon . To identify novel contexts directing readthrough, under-represented 5' and 3' stop codon contexts from Saccharomyces cerevisiae were identified by genome-wide survey in silico . In contrast with the nucleotide bias 3' of the stop codon, codon bias in the two codon positions 5' of the termination codon showed no correlation with known effects on stop codon readthrough . However, individually, poor 5' and 3' context elements were equally as effective in promoting stop codon readthrough in vivo, readthrough which in both cases responded identically to changes in release factor concentration . A novel method analysing specific nucleotide combinations in the 3' context region revealed positions +1,2,3,5 and +1,2,3,6 after the stop codon were most predictive of termination efficiency . Downstream of yeast open reading frames (ORFs), further in-frame stop codons were significantly over-represented at the +1, +2 and +3 codon positions after the ORF, acting to limit readthrough . Thus selection against stop codon readthrough is a dominant force acting on 3', but not on 5', nucleotides, with detectable selection on nucleotides as far downstream as +6 nucleotides . The approaches described can be employed to define potential readthrough contexts for any genome. Mol Cell Biol, 2005 Jan, 25(1), 461 - 71 The multiple biological roles of the 3'-->5' exonuclease of Saccharomyces cerevisiae DNA polymerase delta require switching between the polymerase and exonuclease domains; Jin YH et al.; Until recently, the only biological function attributed to the 3'-->5' exonuclease activity of DNA polymerases was proofreading of replication errors . Based on genetic and biochemical analysis of the 3'-->5' exonuclease of yeast DNA polymerase delta (Pol delta) we have discerned additional biological roles for this exonuclease in Okazaki fragment maturation and mismatch repair . We asked whether Pol delta exonuclease performs all these biological functions in association with the replicative complex or as an exonuclease separate from the replicating holoenzyme . We have identified yeast Pol delta mutants at Leu523 that are defective in processive DNA synthesis when the rate of misincorporation is high because of a deoxynucleoside triphosphate (dNTP) imbalance . Yet the mutants retain robust 3'-->5' exonuclease activity . Based on biochemical studies, the mutant enzymes appear to be impaired in switching of the nascent 3' end between the polymerase and the exonuclease sites, resulting in severely impaired biological functions . Mutation rates and spectra and synergistic interactions of the pol3-L523X mutations with msh2, exo1, and rad27/fen1 defects were indistinguishable from those observed with previously studied exonuclease-defective mutants of the Pol delta . We conclude that the three biological functions of the 3'-->5' exonuclease addressed in this study are performed intramolecularly within the replicating holoenzyme. Mol Cell Biol, 2005 Jan, 25(1), 114 - 23 The Saccharomyces cerevisiae Srb8-Srb11 complex functions with the SAGA complex during Gal4-activated transcription; Larschan E et al.; The Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase) complex functions as a coactivator during Gal4-activated transcription . A functional interaction between the SAGA component Spt3 and TATA-binding protein (TBP) is important for TBP binding at Gal4-activated promoters . To better understand the role of SAGA and other factors in Gal4-activated transcription, we selected for suppressors that bypass the requirement for SAGA . We obtained eight complementation groups and identified the genes corresponding to three of the groups as NHP10, HDA1, and SRB9 . In contrast to the srb9 suppressor mutation that we identified, an srb9Delta mutation causes a strong defect in Gal4-activated transcription . Our studies have focused on this requirement for Srb9 . Srb9 is part of the Srb8-Srb11 complex, associated with the Mediator coactivator . Srb8-Srb11 contains the Srb10 kinase, whose activity is important for GAL1 transcription . Our data suggest that Srb8-Srb11, including Srb10 kinase activity, is directly involved in Gal4 activation . By chromatin immunoprecipitation studies, Srb9 is present at the GAL1 promoter upon induction and facilitates the recruitment or stable association of TBP . Furthermore, the association of Srb9 with the GAL1 upstream activation sequence requires SAGA and specifically Spt3 . Finally, Srb9 association also requires TBP . These results suggest that Srb8-Srb11 associates with the GAL1 promoter subsequent to SAGA binding, and that the binding of TBP and Srb8-Srb11 is interdependent. FEMS Microbiol Lett, 2004 Dec 15, 241(2), 221 - 8 Glucose repression of PRX1 expression is mediated by Tor1p and Ras2p through inhibition of Msn2/4p in Saccharomyces cerevisiae; Monteiro G et al.; Expression of mitochondrial thioredoxin peroxidase (Prx1p) from Saccharomyces cerevisiae is subjected to complex transcriptional regulation and is responsive to the levels of several compounds such as glucose and peroxides . We have previously shown that glucose represses the expression of mitochondrial thioredoxin peroxidase gene (PRX1) in a process mediated by cAMP/protein kinase A (PKA) and Msn2/4p . Here, we show by northern blot and reporter gene (beta-galactosidase) assays that deletion of genes encoding Tor1p and Ras2p resulted in increased PRX1 expression, indicating that these proteins are also mediators of the glucose repression effect . We also identified the position of the stress transcription responsive element (STRE) in the PRX1 promoter, which is recognized by Msn2p and Msn4p activators . Mutation of AGGGG sequence at position -116 to -112 caused a high drop in PRX1 expression under respiratory conditions and in strains containing deletions of TOR1 or RAS2, confirming the finding that this sequence is a STRE. FEMS Microbiol Lett, 2004 Dec 15, 241(2), 193 - 9 The Saccharomyces cerevisiae gene ECM11 is a positive effector of meiosis; Zavec AB et al.; Ecm11 is classified as a protein involved in yeast cell wall biogenesis and organization, but in this paper, we provide evidence that it is involved in meiosis as well . Mutants with deleted ECM11 exhibit complex defects in meiosis: replication, recombination and chromosome segregation are affected . The ecm11Delta diploid strains sporulate more slowly and less efficiently than parental strains with wild type copies of ECM11 . Fluorescence activated cell sorter scans of DNA content during sporulation showed that meiotic DNA synthesis is initiated at the same time in parental and ecm11Delta strains, but is less efficient in the knockout strain . By recombination tests, we demonstrated that ECM11 is required for crossing-over, but not for gene conversion . In the absence of ECM11 gene product, viability of spores is reduced to 50% and predominantly two viable spores per tetrad are formed . Our results suggest that ECM11 is required in early stages of meiosis where its function is related to DNA replication and crossing-over. Acta Clin Belg, 2004 Jul-Aug, 59(4), 220 - 2 Saccharomyces cerevisiae fungemia in a head and neck cancer patient: a case report and review of the literature; Henry S et al.; We report the case of a 65-year old male who developed Saccharomyces cerevisiae fungemia after completing a course of concomitant chemotherapy and radiation therapy for head and neck carcinoma . He had grade IV oral mucositis, and received Saccharomyces boulardii (Perenterol) orally as treatment for aseptic diarrhoea just before the onset of fungemia . We discuss the epidemiology and pathology of Saccharomyces cerevisiae in the cancer patient population. J Virol, 2005 Jan, 79(1), 495 - 502 Nodamura virus RNA replication in Saccharomyces cerevisiae: heterologous gene expression allows replication-dependent colony formation; Price BD et al.; Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae . The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor . A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication . Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells . FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters . Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals . Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae . Induced NoV RNA1 replication was robust . Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells . Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids . Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 10(4)-fold lower than in the analogous FHV system . Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA . In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies. Eukaryot Cell, 2004 Dec, 3(6), 1627 - 38 Gid8p (Dcr1p) and Dcr2p function in a common pathway to promote START completion in Saccharomyces cerevisiae; Pathak R et al.; How cells determine when to initiate DNA replication is poorly understood . Here we report that in Saccharomyces cerevisiae overexpression of the dosage-dependent cell cycle regulator genes DCR2 (YLR361C) and GID8 (DCR1/YMR135C) accelerates initiation of DNA replication . Cells lacking both GID8 and DCR2 delay initiation of DNA replication . Genetic analysis suggests that Gid8p functions upstream of Dcr2p to promote cell cycle progression . DCR2 is predicted to encode a gene product with phosphoesterase activity . Consistent with these predictions, a DCR2 allele carrying a His338 point mutation, which in known protein phosphatases prevents catalysis but allows substrate binding, antagonized the function of the wild-type DCR2 allele . Finally, we report genetic interactions involving GID8, DCR2, and CLN3 (which encodes a G(1) cyclin) or SWI4 (which encodes a transcription factor of the G(1)/S transcription program) . Our findings identify two gene products with a probable regulatory role in the timing of initiation of cell division. Eukaryot Cell, 2004 Dec, 3(6), 1544 - 56 Role for the Ran binding protein, Mog1p, in Saccharomyces cerevisiae SLN1-SKN7 signal transduction; Lu JM et al.; Yeast Sln1p is an osmotic stress sensor with histidine kinase activity . Modulation of Sln1 kinase activity in response to changes in the osmotic environment regulates the activity of the osmotic response mitogen-activated protein kinase pathway and the activity of the Skn7p transcription factor, both important for adaptation to changing osmotic stress conditions . Many aspects of Sln1 function, such as how kinase activity is regulated to allow a rapid response to the continually changing osmotic environment, are not understood . To gain insight into Sln1p function, we conducted a two-hybrid screen to identify interactors . Mog1p, a protein that interacts with the yeast Ran1 homolog, Gsp1p, was identified in this screen . The interaction with Mog1p was characterized in vitro, and its importance was assessed in vivo . mog1 mutants exhibit defects in SLN1-SKN7 signal transduction and mislocalization of the Skn7p transcription factor . The requirement for Mog1p in normal localization of Skn7p to the nucleus does not fully account for the mog1-related defects in SLN1-SKN7 signal transduction, raising the possibility that Mog1p may play a role in Skn7 binding and activation of osmotic response genes. Eukaryot Cell, 2004 Dec, 3(6), 1504 - 12 Deletion of the three distal S1 motifs of Saccharomyces cerevisiae Rrp5p abolishes pre-rRNA processing at site A(2) without reducing the production of functional 40S subunits; Vos HR et al.; Yeast Rrp5p, one of the few trans-acting proteins required for the biogenesis of both ribosomal subunits, has a remarkable two-domain structure . Its C-terminal region consists of seven tetratricopeptide motifs, several of which are crucial for cleavages at sites A(0) to A(2) and thus for the formation of 18S rRNA . The N-terminal region, on the other hand, contains 12 S1 RNA-binding motifs, most of which are required for processing at site A(3) and thus for the production of the short form of 5.8S rRNA . Yeast cells expressing a mutant Rrp5p protein that lacks S1 motifs 10 to 12 (mutant rrp5Delta6) have a normal growth rate and wild-type steady-state levels of the mature rRNA species, suggesting that these motifs are irrelevant for ribosome biogenesis . Here we show that, nevertheless, in the rrp5Delta6 mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A(2) . Instead, the 32S precursor is processed directly at site A(3), producing exclusively 21S rather than 20S pre-rRNA . This is the first evidence that the 21S precursor, which was observed previously only in cells showing a substantial growth defect or as a minor species in addition to the normal 20S precursor, is an efficient substrate for 18S rRNA synthesis . Maturation of the 21S precursor occurs via the same endonucleolytic cleavage at site D as that used for 20S pre-rRNA maturation . The resulting D-A(3) fragment, however, is degraded by both 5'-->3' and 3'-->5' exonuclease digestions, the latter involving the exosome, in contrast to the exclusively 5'-->3' exonucleolytic digestion of the D-A(2) fragment . We also show that rrp5Delta6 cells are hypersensitive to both hygromycin B and cycloheximide, suggesting that, despite their wild-type growth rate, their preribosomes or ribosomes may be structurally abnormal. Eukaryot Cell, 2004 Dec, 3(6), 1492 - 503 Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretion of amino acids; Velasco I et al.; Excretion of amino acids by yeast cells was reported long ago but has not been characterized in molecular terms . It is typically favored by overproduction of the amino acid and/or impairment of its uptake . Here we describe the construction of a yeast strain excreting threonine and homoserine . Using this excretor strain, we then applied a reverse-genetics approach and found that the transporter encoded by the YNL065w/AQR1 gene, a protein thought to mediate H(+) antiport, is involved in homoserine and threonine excretion . Furthermore, overexpression of AQR1 led to increased excretion of several amino acids (alanine, aspartate, and glutamate) known to be relatively abundant in the cytosol . Transcription of the AQR1 gene is induced severalfold by a number of amino acids and appears to be under the negative control of Gcn4 . An Aqr1-green fluorescent protein fusion protein is located in multiple internal membrane structures and appears to cycle continuously between these compartments and the plasma membrane . The Aqr1 sequence is significantly similar to the vesicular amine transporters of secretory vesicles of neuronal cells . We propose that Aqr1 catalyzes transport of excess amino acids into vesicles, which then release them in the extracellular space by exocytosis. Eukaryot Cell, 2004 Dec, 3(6), 1464 - 75 Morphogenetic pathway of spore wall assembly in Saccharomyces cerevisiae; Coluccio A et al.; The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation . A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor . The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays . Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly . The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins . The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure. Eukaryot Cell, 2004 Dec, 3(6), 1381 - 90 Anaerobicity prepares Saccharomyces cerevisiae cells for faster adaptation to osmotic shock; Krantz M et al.; Yeast cells adapt to hyperosmotic shock by accumulating glycerol and altering expression of hundreds of genes . This transcriptional response of Saccharomyces cerevisiae to osmotic shock encompasses genes whose products are implicated in protection from oxidative damage . We addressed the question of whether osmotic shock caused oxidative stress . Osmotic shock did not result in the generation of detectable levels of reactive oxygen species (ROS) . To preclude any generation of ROS, osmotic shock treatments were performed in anaerobic cultures . Global gene expression response profiles were compared by employing a novel two-dimensional cluster analysis . The transcriptional profiles following osmotic shock under anaerobic and aerobic conditions were qualitatively very similar . In particular, it appeared that expression of the oxidative stress genes was stimulated upon osmotic shock even if there was no apparent need for their function . Interestingly, cells adapted to osmotic shock much more rapidly under anaerobiosis, and the signaling as well as the transcriptional response was clearly attenuated under these conditions . This more rapid adaptation is due to an enhanced glycerol production capacity in anaerobic cells, which is caused by the need for glycerol production in redox balancing . Artificially enhanced glycerol production led to an attenuated response even under aerobic conditions . These observations demonstrate the crucial role of glycerol accumulation and turgor recovery in determining the period of osmotic shock-induced signaling and the profile of cellular adaptation to osmotic shock. J Biol Chem . 2004 Dec 7; {Epub ahead of print} Modulation of DNA synthesis in saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex; Mitkova AV et al.; We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system . Anti-sera specific against the four-subunit pol a, the catalytic subunit of pol d, and the Mcm467 complex each inhibited DNA synthesis . However, DNA synthesis in this system appeared to be independent of pole . Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, a and d, as well as the Mcm467 DNA helicase . This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I . In addition, we also report here a novel finding that purified recombinant six-subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1. Nucleic Acids Res, 2004, 32(21), 6414 - 24 Print 2004. Global protein function annotation through mining genome-scale data in yeast Saccharomyces cerevisiae; Chen Y et al.; As we are moving into the post genome-sequencing era, various high-throughput experimental techniques have been developed to characterize biological systems on the genomic scale . Discovering new biological knowledge from the high-throughput biological data is a major challenge to bioinformatics today . To address this challenge, we developed a Bayesian statistical method together with Boltzmann machine and simulated annealing for protein functional annotation in the yeast Saccharomyces cerevisiae through integrating various high-throughput biological data, including yeast two-hybrid data, protein complexes and microarray gene expression profiles . In our approach, we quantified the relationship between functional similarity and high-throughput data, and coded the relationship into 'functional linkage graph', where each node represents one protein and the weight of each edge is characterized by the Bayesian probability of function similarity between two proteins . We also integrated the evolution information and protein subcellular localization information into the prediction . Based on our method, 1802 out of 2280 unannotated proteins in yeast were assigned functions systematically. J Cell Sci, 2005 Jan 1, 118(Pt 1), 65 - 77 Epub 2004 Dec 07. Ice2p is important for the distribution and structure of the cortical ER network in Saccharomyces cerevisiae; de Martin PE et al.; In Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found along the cell periphery (cortical ER) and nucleus (perinuclear ER) . In this study, we characterize a novel ER protein called Ice2p that localizes to the cortical and perinuclear ER . Ice2p is predicted to be a type-III transmembrane protein . Cells carrying a genomic disruption of ICE2 display defects in the distribution of cortical ER in mother and daughter cells . Furthermore, fluorescence imaging of ice2Delta cells reveals an abnormal cortical ER tubular network morphology in both the mother cell and the developing bud . Subcellular fractionation analysis using sucrose gradients corroborate the data from the fluorescence studies . Our findings indicate that Ice2p plays a role in forming and/or maintaining the cortical ER network in budding yeast. Proc Natl Acad Sci U S A, 2004 Dec 14, 101(50), 17422 - 7 Epub 2004 Dec 06. The Saccharomyces cerevisiae aquaporin Aqy1 is involved in sporulation; Sidoux-Walter F et al.; Aquaporins mediate rapid selective water transport across biological membranes . Elucidation of their precise physiological roles promises important insight into cellular and organismal osmoregulation . The genome of the yeast Saccharomyces cerevisiae encodes two similar but differentially regulated aquaporins . Here, we show that expression of AQY1 is stimulated during sporulation and that the Aqy1 protein is detectable exclusively in spore membranes . When spores are rapidly frozen, those that lack Aqy1 survive better, providing for a functional test of active spore water channels . Under ambient conditions, lack of Aqy1 reduces spore fitness . Because this reduction is independent from germination conditions, Aqy1 may be important during spore formation rather than subsequent maintenance or germination . Indeed, it seems that Aqy1 is degraded after spores have been formed and during germination . Taken together, Aqy1 is developmentally controlled and may play a role in spore maturation, probably by allowing water outflow . Taken together, we demonstrate a functional role of an aquaporin in gametogenesis, as well as in the formation of durable structures such as spores, a role that may have wider biological and medical implications. J Mol Biol, 2005 Jan 21, 345(3), 513 - 20 ATP synthase from Saccharomyces cerevisiae: location of subunit h in the peripheral stalk region; Rubinstein JL et al.; Subunit h is a component of the peripheral stalk region of ATP synthase from Saccharomyces cerevisiae . It is weakly homologous to subunit F6 in the bovine enzyme, and F6 can replace the function of subunit h in a yeast strain from which the gene for subunit h has been deleted . The removal of subunit h (or F6) uncouples ATP synthesis from the proton motive force . A biotinylation signal has been introduced following the C terminus of subunit h . It becomes biotinylated in vivo, and allows avidin to be bound quantitatively to the purified enzyme complex in vitro . By electron microscopy of the ATP synthase-avidin complex in negative stain and by subsequent image analysis, the C terminus of subunit h has been located in a region of the peripheral stalk that is close to the Fo membrane domain of ATP synthase . Models of the peripheral stalk are proposed that are consistent with this location and with reconstitution experiments conducted with isolated peripheral stalk subunits. FEBS Lett, 2004 Dec 3, 578(1-2), 152 - 6 Decreased cellular permeability to H2O2 protects Saccharomyces cerevisiae cells in stationary phase against oxidative stress; Sousa-Lopes A et al.; The higher resistance of stationary-phase Saccharomyces cerevisiae to H2O2 when compared with exponential phase is well characterized, but the molecular mechanisms underlying it remain mostly unknown . By applying the steady-state H2O2-delivery model, we show that (a) cellular permeability to H2O2 is five times lower in stationary--than in exponential phase; (b) cell survival to H2O2 correlates with H2O2 cellular gradients for a variety of cells; and, (c) cells in stationary phase are predicted to be more susceptible to intracellular H2O2 than in exponential phase . In conclusion, limiting H2O2 diffusion into cells is a key protective mechanism against extracellular H2O2. Genetics, 2004 Nov, 168(3), 1205 - 18 The amino terminus of the Saccharomyces cerevisiae DNA helicase Rrm3p modulates protein function altering replication and checkpoint activity; Bessler JB et al.; The Pif1 family of DNA helicases is conserved from yeast to humans . Although the helicase domains of family members are well conserved, the amino termini of these proteins are not . The Saccharomyces cerevisiae genome encodes two Pif1 family members, Rrm3p and Pif1p, that have very different functions . To determine if the amino terminus of Rrm3p contributes to its role in promoting fork progression at >1000 discrete chromosomal sites, we constructed a deletion series that lacked portions of the 249-amino-acid amino terminus . The phenotypes of cells expressing alleles that lacked all or most of the amino terminus were indistinguishable from those of rrm3Delta cells . Rrm3p deletion derivatives that lacked smaller portions of the amino terminus were also defective, but the extent of replication pausing at tRNA genes, telomeres, and ribosomal DNA (rDNA) was not as great as in rrm3Delta cells . Deleting only 62 amino acids from the middle of the amino terminus affected only rDNA replication, suggesting that the amino terminus can confer locus-specific effects . Cells expressing a fusion protein consisting of the Rrm3p amino terminus and the Pif1p helicase domain displayed defects similar to rrm3Delta cells . These data demonstrate that the amino terminus of Rrm3p is essential for Rrm3p function . However, the helicase domain of Rrm3p also contributes to its functional specificity. Genetics, 2004 Nov, 168(3), 1159 - 76 Host factors that affect Ty3 retrotransposition in Saccharomyces cerevisiae; Aye M et al.; The retrovirus-like element Ty3 of Saccharomyces cerevisiae integrates at the transcription initiation region of RNA polymerase III . To identify host genes that affect transposition, a collection of insertion mutants was screened using a genetic assay in which insertion of Ty3 activates expression of a tRNA suppressor . Fifty-three loci were identified in this screen . Corresponding knockout mutants were tested for the ability to mobilize a galactose-inducible Ty3, marked with the HIS3 gene . Of 42 mutants tested, 22 had phenotypes similar to those displayed in the original assay . The proteins encoded by the defective genes are involved in chromatin dynamics, transcription, RNA processing, protein modification, cell cycle regulation, nuclear import, and unknown functions . These mutants were induced for Ty3 expression and assayed for Gag3p protein, integrase, cDNA, and Ty3 integration upstream of chromosomal tDNA(Val(AAC)) genes . Most mutants displayed differences from the wild type in one or more intermediates, although these were typically not as severe as the genetic defect . Because a relatively large number of genes affecting retrotransposition can be identified in yeast and because the majority of these genes have mammalian homologs, this approach provides an avenue for the identification of potential antiviral targets. Nucleic Acids Res, 2004, 32(21), 6268 - 75 Print 2004. DNA polymerase delta, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae; Corrette-Bennett SE et al.; Small looped mispairs are corrected by DNA mismatch repair (MMR) . In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells . Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components . This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR . Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase delta efficiently inhibited LLR in extracts by blocking repair just prior to gap filling . Gap filling was inhibited regardless of whether the loop was retained or removed . These experiments suggest polymerase delta is uniquely required in yeast extracts for LLR-associated synthesis . Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e . LLR was inhibited just prior to gap filling for both loop removal and loop retention . Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR . These biochemical experiments support the idea that yeast polymerase delta, RFC and PCNA are required for large loop DNA repair synthesis. Mol Cell Biol, 2004 Dec, 24(24), 10857 - 67 Counting of Rif1p and Rif2p on Saccharomyces cerevisiae telomeres regulates telomere length; Levy DL et al.; Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex . Rap1p in Saccharomyces cerevisiae binds the telomeric TG(1-3) repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p . We investigated how these three proteins negatively regulate telomere length . We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p . Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins . Thus, Rap1p counting is in fact Rif protein counting . In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation . We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control . We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length. J Biol Chem . 2004 Nov 30; {Epub ahead of print} A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae; Shimazu M et al.; Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport of the basic amino acids histidine, lysine and arginine into vacuoles . ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a VBA1-deleted strain, whereas uptake of tyrosine or calcium was little affected . This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane . A defect in the uptake of histidine, lysine or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3) . These three VBA genes are closely related phylogenetically, and constitute a new family of basic amino acid transporters in the yeast vacuole. Annu Rev Genet, 2004, 38, 725 - 48 Principles of MAP kinase signaling specificity in Saccharomyces cerevisiae; Schwartz MA et al.; Cells respond to a plethora of signals using a limited set of intracellular signal transduction components . Surprisingly, pathways that transduce distinct signals can share protein components, yet avoid erroneous cross-talk . A highly tractable model system in which to study this paradox is the yeast Saccharomyces cerevisiae, which harbors three mitogen-activated protein kinase (MAPK) signal transduction cascades that share multiple signaling components . In this review we first describe potential mechanisms by which specificity could be achieved by signaling pathways that share components . Second, we summarize key features and components of the yeast MAPK pathways that control the mating pheromone response, filamentous growth, and the response to high osmolarity . Finally, we review biochemical analyses in yeast of mutations that cause cross-talk between these three MAPK pathways and their implications for the mechanistic bases for signaling specificity . Although much remains to be learned, current data indicate that scaffolding and cross pathway inhibition play key roles in the maintenance of fidelity. Yeast, 2005 Jan 15, 22(1), 1 - 12 New modules for the repeated internal and N-terminal epitope tagging of genes in Saccharomyces cerevisiae; Gauss R et al.; Epitope tagging is a powerful method for the rapid analysis of protein function . In Saccharomyces cerevisiae epitope tags are introduced easily into chromosomal loci by homologous recombination using a simple PCR-based strategy . Although quite a number of tools exist for C-terminal tagging as well as N-terminal tagging of proteins expressed by heterologous promoters, there are only very limited possibilities to tag proteins at the N-terminus and retain the endogenous expression level . Furthermore, no PCR-templates for internal tagging have been reported . Here we describe new modules that are suitable for both the repeated N-terminal and internal tagging of proteins, leaving their endogenous promoters intact . The tags include 6xHA, 9xMyc, yEGFP, TEV-GST-6xHIS, ProtA, TEV-ProtA and TEV-ProtA-7xHIS in conjunction with different heterologous selection markers . Copyright (c) 2004 John Wiley & Sons, Ltd. Yeast, 2004 Dec, 21(16), 1359 - 63 GUD1 (YDL238c) encodes Saccharomyces cerevisiae guanine deaminase, an enzyme expressed during post-diauxic growth; Saint-Marc C et al.; Purine salvage is a complex pathway allowing a correct balance between adenylic and guanylic derivatives . In this paper, we show that GUD1 (YDL238c) encodes guanine deaminase, a catabolic enzyme producing xanthine and ammonia from guanine . Importantly, Gud1p activity was higher during post-diauxic growth, suggesting that a decrease of the guanylic nucleotide pool could be required when cells shift from proliferation to quiescence. Yeast, 2004 Dec, 21(16), 1365 - 74 YAP4 gene expression is induced in response to several forms of stress in Saccharomyces cerevisiae; Nevitt T et al.; Exposure of Saccharomyces cerevisiae to several environmental insults, including conditions of oxidative, heavy metal, metalloid and heat stress, induces the expression of the YAP4 gene, previously shown to play a role in the response to hyperosmotic stress . Expression analyses in several mutant strains under pro-oxidant conditions have determined that YAP4 is regulated by the transactivators Yap1p and Msn2p . Mutation of either the Yap1p-response element (YRE), located at - 517 bp from the ATG, or the most proximal stress response element (STRE) at -430 bp, is shown to strongly compromise YAP4 gene expression under these conditions . Furthermore, these two mutations in combination lead to a severe depletion of detectable mRNA levels, indicating interplay between the transcription factors Yap1p and Msn2p in the regulation of YAP4 transcription . Transcriptional activation of this gene reflects a concomitant increase in Yap4p protein levels that appear phosphorylated upon stress and negatively regulated by protein kinase A . Yap4p amino acid residues Ser89, Ser196 and Thr241 are shown to be required for protein phosphorylation and/or protein stability. Biosci Biotechnol Biochem, 2004 Nov, 68(11), 2306 - 12 Purification and characterization of alpha-keto amide reductase from Saccharomyces cerevisiae; Ishihara K et al.; An NADPH-dependent alpha-keto amide reductase was purified from Saccharomyces cerevisiae . The molecular mass of the native enzyme was estimated to be 33 and 36 kDa by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively . The purified enzyme showed a reducing activity not only for aromatic alpha-keto amides but also for aliphatic and aromatic alpha-keto esters . The internal sequence of the enzyme was identical with that of a hypothetical protein (ORF YDL 124w) coded by yeast chromosome IV. J Biol Chem . 2004 Nov 22; {Epub ahead of print} Competition of electrons to enter the respiratory chain : A new regulatory mechanism of oxidative metabolism in Saccharomyces cerevisiae; Bunoust O et al.; In the yeast Saccharomyces cerevisiae, the most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are the external NADH dehydrogenases (Nde1p and Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle . In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol-3-phosphate by the cytosolic Gpd1p . Subsequently, glycerol-3-phosphate donates electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p) . At saturating concentrations of NADH, the activation of external NADH dehydrogenases completely inhibits glycerol-3-phosphate oxidation . Studies on the functionally isolated enzymes demonstrated that neither Nde1p nor Nde2p directly inhibit Gut2p . Thus, the inhibition of glycerol-3-phosphate oxidation may be due to competition for entrance of electrons into the respiratory chain . Using single deletion mutants of Nde1p or Nde2p, we have shown that glycerol-3-phosphate oxidation via Gut2p is fully inhibited when NADH is oxidized via Nde1p, whereas only 50% of glycerol-3-phosphate oxidation is inhibited when Nde2p is functioning . By comparing respiratory rates with different respiratory substrates, we show that : (i) electrons from Nde1p are favored over electrons coming from Ndip (internal NADH dehydrogenase); (ii) when electrons are coming from either Nde1p or Nde2p, and succino-dehydrogenase, their use by the respiratory chain is shared up to a comparable extent . This suggests a very specific competition for electron entrance into the respiratory chain, which may be due to supramolecular organization of the respiratory chain . The physiological consequences of such regulation are discussed. FEMS Yeast Res, 2004 Dec, 5(3), 193 - 204 Elucidation of the role of Grr1p in glucose sensing by Saccharomyces cerevisiae through genome-wide transcription analysis; Westergaard SL et al.; The role of Grr1p in glucose sensing in Saccharomyces cerevisiae was elucidated through genome-wide transcription analysis . From triplicate analysis of a strain with deletion of the GRR1-gene from the genome and an isogenic reference strain, 68 genes were identified to have significantly altered expression using a Student's t-test with Bonferroni correction . These 68 genes were widely distributed across different parts of the cellular metabolism and GRR1-deletion is therefore concluded to result in polytrophic effects, indicating multiple roles for Grr1p . Using a less conservative statistical test, namely the SAM test, 232 genes were identified as having significantly altered expression, and also these genes were widely distributed across different parts of the cellular metabolism . Promoter analyses on a genome-wide scale and on the genes with significant changes revealed an over-representation of DNA-binding motifs for the transcriptional regulators Mig1p and Rgt1p in the promoter region of the significantly altered genes, indicating that Grr1p plays an important role in the regulatory pathways that ultimately lead to transcriptional regulation by each of the components Mig1p and Rgt1p. Curr Opin Microbiol, 2004 Dec, 7(6), 647 - 54 Ubiquitin-dependent control of development in Saccharomyces cerevisiae; Laney JD et al.; In response to external environmental stimuli and intrinsic developmental cues, yeast cells reset their gene expression programs and change phenotype . These switches in cellular state require the dismantling of an initial regulatory program, in addition to the induction of different sets of genes to specify the new cell phenotype . Recent experiments examining the role of protein degradation in these transitions have highlighted the importance of inactivating previously utilized regulators and have led to advances in our understanding of how cells change from one phenotypic state to another. Mikrobiol Z, 2004 Sep-Oct, 66(5), 48 - 56 {Protective action of electromagnetic radiation (40.68 MHz) on Saccharomyces cerevisiae UCM Y-517}; Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae; Edward A . Doisy Department of Biochemistry and Molecular Biology, St . Louis University Health Sciences Center, 1402 S . Grand Blvd., St . Louis, Missouri 63104Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged . In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2 . Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro . Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth . Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth . Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function . (c) 2004 Wiley-Liss, Inc. Genetics . 2004 Nov 15; {Epub ahead of print} Protein Kinase A Regulates Constitutive Expression of Small Heat Shock Genes in an Msn2p/4p-Independent and Hsf1p-Dependent Manner in Saccharomyces cerevisiae; Ferguson SB et al.; Hsf1p, the heat shock transcription factor from Saccharomyces cerevisiae, has a low level of constitutive transcriptional activity and is kept in this state through negative regulation . In an effort to understand this negative regulation, we developed a novel genetic selection that detects altered expression from the HSP26 promoter . Using this reporter strain, we identified mutations and dosage compensators in the Ras/cAMP signaling pathway that decrease cAMP levels and increase expression from the HSP26 promoter . In yeast, low cAMP levels reduce the catalytic activity of the cAMP-dependent kinase, PKA . Previous studies had proposed that the stress response transcription factors Msn2p/4p, but not Hsf1p, are repressed by PKA . However, we found that reduction or elimination of PKA activity strongly derepresses transcription of the small heat shock genes HSP26 and HSP12, even in the absence of MSN2/4 . In a strain deleted for MSN2/4 and the PKA catalytic subunits, expression of HSP12 and HSP26 depends on HSF1 expression . Our findings indicate that Hsf1p functions downstream of PKA and suggest that PKA might be involved in negative regulation of Hsf1p activity . These results represent a major change in our understanding of how PKA signaling influences the heat shock response and heat shock protein expression. Genetics . 2004 Nov 15; {Epub ahead of print} Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion {PSI+}; Allen KD et al.; {PSI(+)} is a prion isoform of the yeast release factor Sup35 . In some assays, cytosolic chaperones Ssa1 and Ssb1/2 of Hsp70 family were previously shown to exhibit "pro-{PSI(+)}" and "anti-{PSI+}" effects, respectively . Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of {PSI(+)}, and that pro-{PSI(+)} effects of Ssa1 are shared by all other Ssa proteins . Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on {PSI(+)} . Surprisingly, overproduction of either chaperone increases loss of {PSI(+)} when Sup35 is simultaneously overproduced . Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein . Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins . The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, that serve as substrates for prion conversion . In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity . In contrast, Ssb either stimulates refolding into non-prion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation. J Cell Biol, 2004 Nov 22, 167(4), 599 - 604 Epub 2004 Nov 22. Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae; Nair DM et al.; Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence . We observe that appending GFP to Pex7p's COOH terminus shifts Pex7p's intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae . Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol . The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid . These results support the "extended shuttle" model of peroxisome import receptor function and open the way to future studies of receptor export. J Biol Chem . 2004 Nov 15; {Epub ahead of print} Rom2p, the Rho1 GTP/GDP exchange factor of Saccharomyces cerevisiae, can mediate stress responses via the Ras-cAMP pathway; Park JI et al.; The Ras-cyclic AMP (Ras-cAMP) pathway is connected to other nutrient-regulated signaling pathways, and mediates global stress responses of Saccharomyces cerevisiae . Here, we show that Rom2p, the Rho1 GTP/GDP exchange factor, can mediate stress responses and cell growth via the Ras-cAMP pathways . ROM2 was isolated as a suppresser of heat and NaCl sensitivity caused by lack of the Ras-GTPase activator, Ira2p or cAMP phosphodiesterases . Subsequent analysis of strains with a rom2 deletion showed that Rom2p is essential for resistance to a variety of stresses caused by freeze-thawing, oxidants, cycloheximide, NaCl, or cobalt ions . Stress sensitivity and the growth defect caused by the rom2 deletion could be suppressed by depleting Ras or protein kinase A (PKA) activity, or by overexpressing the high-affinity cAMP phosphodiesterase Pde2p . In addition, overexpression of ROM2 could not rescue cells lacking the regulatory subunit of PKA, indicating that the Ras-adenylate cyclase-PKA cascade is essential for Rom2p-mediated stress responses and cell growth . Deletion of IRA2 exacerbated the freeze-thaw sensitivity and growth defect of the rom2 mutant, indicating that Rom2p signaling may control Ras independently of IRA2 . Increases in cAMP levels were detected in the rom2 deletion mutants, and these were comparable to the effects of an ira2 mutation . The effects of deletion of ROM2 on sensitivity to hydrogen peroxide, paraquat and cobalt ions, but not to caffeine, were reduced when a constitutive allele of RHO1 was introduced on a single copy plasmid . However, the effects of deletion of ROM2 on sensitivity to diamide and NaCl were exacerbated . Taken together, our data indicate that Rom2p can regulate PKA activity by controlling cAMP levels via the Ras-cAMP pathway, and that for those stresses related to oxidative stress, this cross-talk is probably mediated via the Rho1p-activated MAPK pathway. Yeast, 2004 Nov, 21(15), 1241 - 52 Analysis of the meiotic role of the mitochondrial ribosomal proteins Mrps17 and Mrpl37 in Saccharomyces cerevisiae; Hanlon SE et al.; Sporulation in the yeast Saccharomyces cerevisiae is a complex and tightly regulated pathway that involves the induction of a large number of genes . We have identified MRPS17 in a cDNA library enriched for sporulation-specific genes . Homology searches show that the first one-third of Mrps17 has strong sequence similarity to bacterial S17 proteins, suggesting that Mrps17 is a potential mitochondrial ribosomal protein . This is further supported by the fact that mrps17Delta cells are respiratory-deficient and that a Mrps17-GFP fusion localizes to the mitochondria . We have confirmed by Northern blot analysis that both MRPS17 and MRPL37 are strongly induced during the middle stages of sporulation and that this induction is dependent on the presence of a middle sporulation element (MSE) in the promoters of these genes . Interestingly, we found that Mrps17 and Mrpl37, but not other mitochondrial ribosomal proteins, accumulate during the middle stages of sporulation . These results suggest that Mrps17 and Mrpl37 may have additional meiosis-specific roles. Mol Cell Biol, 2004 Dec, 24(23), 10437 - 47 A mammalian ortholog of Saccharomyces cerevisiae Vac14 that associates with and up-regulates PIKfyve phosphoinositide 5-kinase activity; Sbrissa D et al.; Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P(2), produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P . Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity . In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14 . PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated . A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P(2) production . Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions . Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity . Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P(2) conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14 . These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P(2) synthesis and endomembrane homeostasis in mammalian cells. Mol Cell Biol, 2004 Dec, 24(23), 10208 - 22 Diminished S-phase cyclin-dependent kinase function elicits vital Rad53-dependent checkpoint responses in Saccharomyces cerevisiae; Gibson DG et al.; Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes . In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing . Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins . We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function . We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage . Deficient late origin firing in clb5Delta cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased . Moreover, the viability of clb5Delta cells depends on Rad53 . Activation of Rad53 by either Mrc1 or Rad9 contributes to the sur