S. cerevisiae

Nucleic Acids Res, 1998 Jun 15, 26(12), 2865 - 72
Transcription of INO2 and INO4 is regulated by the state of protein N-myristoylation in Saccharomyces cerevisiae; Cok SJ et al.; Inositol regulates transcription of Saccharomyces cerevisiae genes required for de novo synthesis of acylCoAs and phospholipids . Removal of inositol results in transcriptional activation by heterodimeric complexes of two bHLH proteins, Ino2p and Ino4p . In the presence of inositol, transcription is repressed by Opi1p . MyristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme whose activity is influenced by cellular myristoylCoA pool size and availability . nmt451Dp contains a Gly451-->Asp substitution that produces temperature-dependent reductions in affinity for myristoylCoA and associated reductions in acylation of cellular N-myristoylproteins . The conditional lethality produced by nmt1-451D is rescued at temperatures up to 33 degreesC by withdrawal of inositol . We tested the hypothesis that N-myristoylproteins function to regulate INO2, INO4 and/or OPI1 transcription, thereby affecting the expression of inositol-sensitive genes that influence myristoylCoA metabolism . The effect of nmt1-451D on INO2 , INO4 and OPI1 promoter activities was examined by introducing episomes, containing their 5' non-transcribed domains linked to reporters, into isogenic NMT1 and nmt1-451D cells . The activity of INO2 is significantly higher, INO4 significantly lower and OPI1 unaffected in nmt1-451D cells, both in the presence and absence of inositol . These changes are associated with a net increase in expression of some inositol target genes, including FAS1 . FAS1 encodes one of the subunits of the fatty acid synthase complex that catalyzes de novo acylCoA (including myristoylCoA) biosynthesis . Augmented expression of FAS1 overcomes the kinetic defects in nmt451Dp . FAS1 expression is Ino2p-dependent in NMT1 cells at 24-33 degreesC . In contrast, FAS1 expression becomes Ino2p-independent in nmt1-451D cells at temperatures where efficient acylation of cellular N-myristoylproteins is jeopardized . The ability to maintain expression of FAS1 in nmt1-451Dino2 Delta cells suggests the existence of another transcription factor, or factors, whose expression/activity is inversely related to overall levels of cellular protein N-myristoy-lation . This factor is not functionally identical to Ino2p since other inositol-responsive genes (e.g . CHO1 ) maintain INO2 -dependent expression in nmt1-451D cells.

Curr Opin Genet Dev, 1998 Apr, 8(2), 233 - 9
Mechanisms of silencing in Saccharomyces cerevisiae; Lustig AJ; In the yeast Saccharomyces cerevisiae, heterochromatin-like regions are formed at the silent mating type loci and at telomeres . The past year of investigations has led to a clearer understanding of the nature of nucleation and spreading of heterochromatin, as well as uncovering a fascinating link between silencing, the nucleolus and aging.

Biochemistry, 1998 May 19, 37(20), 7572 - 7
Characterization of the copper chaperone Cox17 of Saccharomyces cerevisiae; Srinivasan C et al.; Assembly of functional cytochrome oxidase in yeast requires Cox17, which has been postulated to deliver copper ions to the mitochondrion for insertion into the enzyme . This role for Cox17 is supported by the observation that it binds copper as a binuclear cuprous-thiolate cluster . X-ray absorption spectroscopy, together with UV-visible absorption and emission spectroscopy, indicates the presence of bound cuprous ions, trigonally coordinated by thiolate ligands . Analysis of the EXAFS shows three Cu-S bonds at 2.26 A, plus a short Cu-Cu distance of 2.7 A, indicating a binuclear cluster in Cox17 . The cuprous-thiolate cluster in Cox17 is substantially more labile than structurally related clusters in metallothioneins.

Biochemistry, 1998 May 19, 37(20), 7299 - 312
Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II . 2 . Kinetic mechanism for the sequential hydrolysis of two ATP; Harkins TT et al.; In the preceding paper, we showed that DNA topoisomerase II from Saccharomyces cerevisiae binds two ATP and rapidly hydrolyzes at least one of them before encountering a slow step in the reaction mechanism . These data are potentially consistent with two different types of reaction pathways: (1) sequential ATP hydrolysis or (2) simultaneous hydrolysis of both ATP . Here, we present results that are consistent only with topoisomerase II hydrolyzing its two bound ATP sequentially . Additionally, these results indicate that the products of the first hydrolysis are released from the enzyme before the second ATP is hydrolyzed . Release of products from both the first and second hydrolyses contributes to the rate-determining process . The proposed mechanism for ATP hydrolysis by topoisomerase II is complex, having nine rate constants . To calculate values for each of these rate constants, a technique of kinetic parameter estimation was developed . This technique involved using singular perturbation theory in order to estimate rate constants, and consequently identify kinetic steps following the rate-determining step.

Biochemistry, 1998 May 19, 37(20), 7292 - 8
Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II . 1 . A DNA-dependent burst in ATP hydrolysis; Harkins TT et al.; When bound to DNA, topoisomerase II from Saccharomyces cerevisiae exhibits burst kinetics with respect to ATP hydrolysis . Pre-steady-state analysis shows that the enzyme binds and hydrolyzes two ATP per reaction cycle . Our data indicate that at least one of the two ATP is rapidly hydrolyzed prior to the rate-determining step in the reaction mechanism . When DNA is not bound to topoisomerase II, the rate-determining step shifts to become either ATP binding or hydrolysis . Two possible mechanisms are proposed that agree with our observations.

Biochemistry, 1998 May 19, 37(20), 7268 - 76
Transfer RNA-pseudouridine synthetase Pus1 of Saccharomyces cerevisiae contains one atom of zinc essential for its native conformation and tRNA recognition; Arluison V et al.; RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs . In this paper we demonstrate that the purified recombinant Pus1, expressed in Escherichia coli, contains one atom of zinc per 63-kDa monomer, as determined by atomic absorption spectroscopy . This zinc ion could not be removed by treatment with EDTA or urea . However, a zinc-depleted enzyme was obtained after prolonged dialysis against the specific chelating agent 1,10-phenanthroline . Removal of the zinc ion resulted in inactivation of the enzyme with concomitant loss of its ability to bind tRNA . Dialysis of the zinc-depleted inactive enzyme against buffer containing zinc ions led to recovery of up to 25% of bound zinc in parallel with 25% of its initial activity . Removal of the tightly bound zinc atom resulted in a conformational change of the protein, as determined by analytical ultracentrifugation, with minor changes in the internal structure of the protein, as evidenced by circular dichroism and infrared and fluorescence spectroscopy . Our results are consistent with a structural role for the zinc in the tRNA-pseudouridine synthetase Pus1; zinc ion could maintain the association between domains structurally organized around the coordinated metal ion . Zinc chelation was never demonstrated for any of the tRNA-pseudouridine synthetases characterized so far.

Yeast, 1998 Apr 30, 14(6), 587 - 91
A 9359 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII includes the FOL2 and YTA7 genes and three unknown open reading frames; Agostoni Carbone ML et al.; In the framework of the EU programme for systematic sequencing of the Saccharomyces cervisiae genome we determined the sequence of a 9359 bp fragment of the right arm of chromosome VII . Five open reading frames (ORFs) of at least 300 nucleotides were found in this region . YGR267c encodes a protein with significant similarity to the enzyme GTP-cyclohydrolase I, that controls the first step in the biosynthetic pathway leading to various pterins and shows a high degree of sequence conservation from bacteria to mammals . We have recently demonstrated (Nardese et al., 1996) that YGR267c corresponds to the FOL2 gene, previously localized in the same chromosomal region by genetic mapping . The protein deduced from YGR270w belongs to the superfamily of putative ATPases associated with diverse cellular activities . It corresponds to the YTA7 gene, a member of a set of yeast genes coding for putative ATPases with high similarity to constituents of the 26S protease . The three ORFs YGR266w, YGR268c and YGR269w encode putative products of unknown function, with neither significant similarity to proteins in databases nor recognizable domains . YGR268c and YGR269w are partially overlapping ORFs: YGR268c seems to correspond to a real gene . whereas YGR269w is probably a fortuitous ORF.

Yeast, 1998 Apr 30, 14(6), 551 - 64
A family of laboratory strains of Saccharomyces cerevisiae carry rearrangements involving chromosomes I and III; Casaregola S et al.; In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295 . Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp . A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain . The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome . Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement . We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series.

Mol Gen Genet, 1998 Apr, 257(6), 662 - 71
Switch-domain mutations in the Saccharomyces cerevisiae G protein alpha-subunit Gpa1p identify a receptor subtype-biased mating defect; DeSimone SM et al.; The response to pheromone in Saccharomyces cerevisiae involves a heterotrimeric G protein composed of Gpa1p (alpha subunit), Ste4p (beta) and Ste18p (gamma) . The switch II region of G alpha subunits is involved in several protein-protein interactions and an intrinsic GTPase activity . To investigate the role of this region of Gpa1p, we have analyzed the effect of switch II mutations . The Q323 analog in G alpha subunits and Ras is implicated in GTP hydrolysis . Mutation of the Q323 residue of Gpa1p resulted in constitutive activation of the pheromone response pathway and eliminated the ability to interact with Ste4p, consistent with a defect in GTPase activity . Mutation of residue A59 of Ras and the analogous G alphas residue has had quite different effects . The analogous Gpa1p G321T mutation resulted in phenotypes consistent with a less severe GTPase defect, but also led to an unexpected mating phenotype: mating was decreased in both mating types, but the defect was 1000-fold more severe in alpha cells than in a cells . In addition the G321T mutation resulted in an unusual pheromone response phenotype . We discuss the possibility that these phenotypes may reflect a differential role for the switch II region in activation by the a- and alpha-factor receptors.

Mol Gen Genet, 1998 Apr, 257(6), 624 - 34
Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p; Oki M et al.; A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR . A total of 25 ts gsp1 strains was obtained . Each of these mutants showed between one and seven different amino acid alterations . In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating . All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export . Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export . Following release from alpha-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37 degrees C, the nonpermissive temperature . While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import . Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation.

Mol Gen Genet, 1998 Apr, 257(6), 614 - 23
Mms4, a putative transcriptional (co)activator, protects Saccharomyces cerevisiae cells from endogenous and environmental DNA damage; Xiao W et al.; mms4-1 is one of several Saccharomyces cerevisiae mutants that exhibit an increased sensitivity to methyl methanesulfonate (MMS), but not to UV or X-rays . We have isolated the MMS4 gene by functional complementation of the MMS-sensitive phenotype in the mms4-1 strain . The MMS4 gene encodes a 691-amino acid, 78.7-kDa protein . The deduced Mms4 protein does not show significant homology to any of the known proteins in the database . However, several putative functional domains suggest that it may be a nuclear protein capable of interacting with other proteins . Examination of the mms4delta mutant phenotype indicates that the mutation not only sensitizes DNA to methylating and ethylating agents, but also to other DNA damage that blocks DNA replication . However, the mms4delta mutant appears to be more sensitive to chronic treatment than to acute treatment by DNA-damaging agents . Furthermore, the spontaneous mutation rate increases significantly in the mms4delta mutant . Mms4 alone, when fused to a Gal4 DNA-binding domain, is able to activate P(GAL1)-lacZ and P(GAL1)-HIS3 reporter genes in a two-hybrid system; the Mms4 transactivation domain maps to the highly acidic N-terminal region . These results collectively suggest that Mms4 may function as a transcriptional (co)activator and play an important role in DNA repair and/or synthesis.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6245 - 50
Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae; Ludin K et al.; The Snf1 protein kinase family has been conserved in eukaryotes . In the yeast Saccharomyces cerevisiae, Snf1 is essential for transcription of glucose-repressed genes in response to glucose starvation . The direct interaction between Snf1 and its activating subunit, Snf4, within the kinase complex is regulated by the glucose signal . Glucose inhibition of the Snf1-Snf4 interaction depends on protein phosphatase 1 and its targeting subunit, Reg1 . Here we show that Reg1 interacts with the Snf1 catalytic domain in the two-hybrid system . This interaction increases in response to glucose limitation and requires the conserved threonine in the activation loop of the kinase, a putative phosphorylation site . The inhibitory effect of Reg1 appears to require the Snf1 regulatory domain because a reg1Delta mutation no longer relieves glucose repression of transcription when Snf1 function is provided by the isolated catalytic domain . Finally, we show that abolishing the Snf1 catalytic activity by mutation of the ATP-binding site causes elevated, constitutive interaction with Reg1, indicating that Snf1 negatively regulates its own interaction with Reg1 . We propose a model in which protein phosphatase 1, targeted by Reg1, facilitates the conformational change of the kinase complex from its active state to the autoinhibited state.

J Biol Chem, 1998 Apr 24, 273(17), 10567 - 77
Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein; Chong S et al.; Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds . In this study, we investigated the roles of the last N-extein residue (-1 residue) and the intein penultimate residue in modulating splicing reactions . Most of the 20 amino acid substitutions at the -1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation . A subset of -1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues . Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction . Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step . Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents . Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected.

J Biol Chem, 1998 May 22, 273(21), 12696 - 702
Monitoring the Gcn4 protein-mediated response in the yeast Saccharomyces cerevisiae; Albrecht G et al.; In Saccharomyces cerevisiae the GCN4 gene encodes the transcriptional activator of the "general control" system of amino acid bioynthesis, a network of at least 12 different biosynthetic pathways . We characterized the consequences of the general control response upon the signal "amino acid starvation" induced by the histidine analogue 3-aminotriazole with respect to Gcn4p levels in more detail . Therefore, we established test systems to monitor the time course of different parameters, including GCN4 mRNA, Gcn4 protein, Gcn4p DNA binding activity, as well as Gcn4p transactivation ability . We observed a biphasic response of Gcn4p activity in the cell . At first, translation of GCN4 mRNA is induced within 20 min after switch to starvation conditions . However, an additional increase in GCN4 transcript steady state level was observed, leading to an additional second phase of GCN4 expression after 3-4 h of starvation . The DNA binding activity of Gcn4p, as well as the ability to activate transcription of target genes, correlate with the amount of Gcn4 protein in the cell, suggesting that under the tested conditions there is no additional regulation of DNA binding or transactivation ability of Gcn4p, respectively.

Curr Genet, 1998 Apr, 33(4), 239 - 47
The Tup1-Ssn6 general repressor is involved in repression of IME1 encoding a transcriptional activator of meiosis in Saccharomyces cerevisiae; Mizuno T et al.; Ime1 plays a pivotal role in the initiation of meiosis in a/alpha diploid cells of Saccharomyces cerevisiae . In the absence of glucose and nitrogen, IME1 expression is greater in a/alpha cells than in either a or alpha cells and therefore only a/alpha, but not a/a or alpha/alpha, cells are committed to sporulation . It is known that IME1 expression is positively regulated by Mck1, Rim1, Ime4 and the Swi-Snf complex but other factors may also be involved . In addition, Rme1 is assumed to repress IME1 expression . To provide more details of the repression of expression of IME1, we have isolated mutants in which the IME1p-PHO5 fusion gene integrated at the ura3 locus is expressed in alpha cells under nutritionally rich conditions . We found that mutations occurred in TUP1, SSN6, SIN4 and RGR1, among which TUP1 and SSN6 were identified for the first time as negative regulators of IME1 expression . Deletion of the Rme1-binding site from the IME1 promoter did not result in activation of the expression of IME1 under nutritionally rich conditions, suggesting that Rme1 does not function as a DNA-binding protein with the Tup1-Ssn6 repression complex . We also demonstrated that the 294-bp fragment from nucleotide position -914 to -621 and the 301-bp fragment from nucleotide position -1215 to -915 of the IME1 promoter region contain elements acting as URS and UAS in TUP1+ and tup1 mutant cells, respectively . These findings indicate that IME1 is negatively regulated by the Tup1-Ssn6 repressor complex through two distinct upstream regions in conjunction with unidentified DNA-binding proteins.

Glycobiology, 1998 May, 8(5), 455 - 62
The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation; Burda P et al.; The biosynthesis of the lipid-linked oligosaccharide substrate for N-linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum . Based on the synthetic growth defect in combination with a reduced oligosaccharyltransferase activity (wbp1), we have identified alg10 mutant strains which accumulate lipid-linked Glc2Man9GlcNAc2 . We cloned the corresponding wild-type gene and show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide . Hypoglycosylation of secreted proteins in alg10 deletion strains demonstrates that the terminal alpha-1,2-linked glucose residue is a key element in substrate recognition by the oligosaccharyltransferase . This ensures that primarily completely assembled oligosaccharide is transferred to protein.

Biochim Biophys Acta, 1998 Apr 10, 1380(2), 232 - 8
Adenosine 5'-tetraphosphate phosphohydrolase activity is an inherent property of soluble exopolyphosphatase from yeast Saccharomyces cerevisiae; Guranowski A et al.; Homogeneous soluble exopolyphosphatase (EC 3.6.1.11) from yeast Saccharomyces cerevisiae, (scPPX1) behaves as an adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14) . The hydrolysis of adenosine 5'-tetraphosphate (p4A) to ATP and orthophosphate absolutely depends on one of the following cations: Co2+>Mn2+>Mg2+>Ni2+ . Optimum pH is around 4.75 and the Km for p4A estimated at that pH in 50 mM sodium acetate and at 5 mM CoCl2 is 80+/-10 microM . Adenosine 5'-pentaphosphate (p5A) is degraded under these conditions 18-fold more slowly than p4A . Assuming that the mass of scPPX1 is 45 kDa, the calculated kcat values for p4A and for p5A are 723 and 40 s-1, respectively . Two other nucleoside 5'-tetraphosphates (p4N), guanosine tetraphosphate (p4G) and inosine tetraphosphate (p4I), were hydrolyzed to Pi and either GTP or ITP, respectively, at the same rate as that observed for the hydrolysis of p4A . Ammonium molybdate, sodium o-vanadate and zinc chloride inhibit the hydrolysis of p4A (I50 values are 0.08, 0.3 and 0.4 mM, respectively) . This newly recognized 'acidic' adenosine tetraphosphatase activity from yeast is compared with two 'pH 8' adenosine tetraphosphatases described earlier in rabbit and yellow lupin .

Biochemistry, 1998 Apr 28, 37(17), 6033 - 40
Saccharomyces cerevisiae possesses two functional homologues of Escherichia coli endonuclease III; You HJ et al.; We previously identified two distinct genes of Saccharomyces cerevisiae redoxyendonuclease (SCR1 and SCR2) which possess a high degree of sequence similarity to Escherichia coli endonuclease III {Augeri, L., Lee, Y . M., Barton, A . B., and Doetsch, P . W . (1997) Biochemistry 36, 721-729} . The proteins encoded by SCR1 and SCR2 were overexpressed in E . coli and purified to apparent homogeneity . Both proteins recognized and cleaved DNA substrates containing dihydrouracil, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (FaPy-7-MeGua), and abasic sites but not DNA substrates containing uracil or 8-oxoguanine . Purified Scr2, but not Scr1, possesses spectral properties which indicate the presence of an iron-sulfur center . Kinetic parameters for Scr1 and Scr2 were determined by using an oligonucleotide containing a single dihydrouracil . Analysis of the deduced amino acid sequences of Scr1 and Scr2 suggests that Scr2 bears an iron-sulfur motif, while Scr1 does not have this motif . However, Scr1 has a long, positively charged N-terminus that could be a mitochondrial transit sequence . Targeted gene disruption of SCR1 and SCR2 produced a double mutant that had no detectable enzymatic activity against the dihydrouracil-containing substrate . Northern blot analysis showed that SCR1 was induced by menadione, but SCR2 was not . These results indicate that although Scr1 and Scr2 are both functional homologues of E . coli endonuclease III, they differ from each other with respect to their amino acid sequences and inducibility by DNA damaging agents, suggesting that their precise biological roles may be different.

Biochim Biophys Acta, 1998 May 8, 1380(3), 320 - 8
Proteolytic processing of a secreted glycoprotein and O-glycosylation of mannoproteins are affected in the N-glycosylation mutant Saccharomyces cerevisiae ldb1; Manas P et al.; In a previous work {P.I . Manas, I . Olivero, M . Avalos, L.M . Hernandez, Glycobiology, 7 (1997) 487-497}, we described the isolation and characterization of the Saccharomyces cerevisiae ldb1 mutant which is affected in several steps of the N-glycosylation of mannoproteins probably due to a malfunction of the Golgi apparatus . Here, we found that two further functions assigned to the Golgi cisternae are also affected in the mutant: proteolytic processing of a secreted protein and O-glycosylation . We found that around 70% of the exoglucanase activity that is secreted into the culture medium by ldb1 bears an extra tetrapeptide in its NH2-terminus due to incomplete proteolytic processing . The O-linked oligosaccharides from ldb1 mnn1 were indistinguishable from those synthesized by the parental strain mnn1 . However, when the O-oligosaccharides from the wild type and ldb1 were compared, we found a significant decrease in the tetrasaccharide in the latter, as well as a concomitant increase in the disaccharide, suggesting a defect in the Kre2p/Mnt1p involved in the transfer of the third mannose of these residues .

J Biol Chem, 1998 Apr 17, 273(16), 9912 - 20
Palindrome with spacer of one nucleotide is characteristic of the cis-acting unfolded protein response element in Saccharomyces cerevisiae; Mori K et al.; When unfolded proteins are accumulated in the endoplasmic reticulum (ER), an intracellular signaling pathway termed the unfolded protein response (UPR) is activated to induce transcription of ER-localized molecular chaperones and folding enzymes in the nucleus . In Saccharomyces cerevisiae, at least six lumenal proteins including essential Kar2p and Pdi1p are known to be regulated by the UPR . We and others recently demonstrated that the basic-leucine zipper protein Hac1p/Ern4p functions as a trans-acting factor responsible for the UPR . Hac1p binds directly to the cis-acting unfolded protein response element (UPRE) responsible for Kar2p induction . Moreover, we showed that the KAR2 UPRE contains an E box-like palindrome separated by one nucleotide (CAGCGTG) that is essential for its function . We report here that the promoter regions of each of five target proteins (Kar2p, Pdi1p, Eug1p, Fkb2p, and Lhs1p) contain a single UPRE sequence that is necessary and sufficient for induction and that binds specifically to Hac1p in vitro . All of the five functional UPRE sequences identified contain a palindromic sequence that has, in four cases, a spacer of one C nucleotide . This unique characteristic of UPRE explains why only a specific set of proteins are induced in the UPR to cope with ER stress.

J Biol Chem, 1998 Apr 17, 273(16), 9644 - 50
Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae; Bennett RJ et al.; The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a family of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes . To study the enzymatic characteristics of the protein, a recombinant Sgs1 fragment (amino acids 400-1268 of the 1447-amino acid full-length protein) was overexpressed in yeast and purified to near homogeneity . The purified protein exhibits an ATPase activity in the presence of single- or double-stranded DNA . In the presence of ATP or dATP, unwinding of duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragment was readily observed . Similar to the E . coli RecQ helicase, displacement of the DNA strand occurs in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex . The efficiency of unwinding was found to correlate inversely with the length of the duplex region and was enhanced by the presence of E . coli single-stranded DNA-binding protein . In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single- or double-stranded DNA.

J Biol Chem, 1998 Apr 17, 273(16), 9534 - 8
Functional correlation among Gal11, transcription factor (TF) IIE, and TFIIH in Saccharomyces cerevisiae . Gal11 and TFIIE cooperatively enhance TFIIH-mediated phosphorylation of RNA polymerase II carboxyl-terminal domain sequences; Sakurai H et al.; Saccharomyces cerevisiae Gal11, a component of the holoenzyme of RNA polymerase II, interacts through its functional domains A and B with the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE, respectively . We have recently suggested that Gal11 functions through a common pathway with TFIIE in transcriptional regulation (Sakurai, H., and Fukasawa, T . (1997) J . Biol . Chem . 272, 32663-32669) . Here, we report that the activity of the TFIIH-associated kinase, responsible for phosphorylation of the largest subunit of RNA polymerase II at the carboxyl-terminal domain (CTD), is enhanced cooperatively by Gal11 and TFIIE . The enhancement of CTD phosphorylation was observed in the holoenzyme of RNA polymerase II, but not in its core enzyme . The stimulatory effect was completely abolished in the absence of either domain B of Gal11 or the Tfa1 subunit of TFIIE, suggesting that the domain B-Tfa1 interaction is necessary, if not sufficient, for an extensive phosphorylation of the CTD by TFIIH . Stimulation of basal transcription by Gal11 was coupled with enhancement of TFIIH-catalyzed CTD phosphorylation in a cell-free transcription system, suggesting that Gal11 activates transcription by stimulating the CTD phosphorylation in the cell.

J Biol Chem, 1998 Apr 17, 273(16), 9527 - 33
Flavohemoglobin expression and function in Saccharomyces cerevisiae . No relationship with respiration and complex response to oxidative stress; Buisson N et al.; The yeast Saccharomyces cerevisiae contains a flavohemoglobin, encoded by the gene YHB1, whose function is unclear . Previous reports presented evidence that its maximal expression requires disruption of mitochondrial respiration and that it plays a role in the response to oxidative stress . We have studied the expression of YHB1 in respiratory deficient cells and in cells exposed to various compounds causing oxidative stress . Several different strains and approaches (spectroscopic detection of the oxygenated form of Yhb1p, beta-galactosidase activity of a YHB1-lacZ fusion, and Northern blot analysis) were used to demonstrate that YHB1 expression and Yhb1p production are not increased by respiration deficiency . YHB1 expression was unchanged in cells challenged with antimycin A or menadione, while it decreased in cells exposed to H2O2, diamide, dithiothreitol, and Cu2+ . Transcription of YHB1 is not under the control of the transcriptional factor Yap1p . These results do not support a participation of YHB1 in the genetic response to oxidative stress . We also analyzed the growth phenotypes associated with altered Yhb1p production using YHB1-deleted strains and strains that greatly overproduced Yhb1p . Yhb1p appears to protect cells against the damage caused by Cu2+ and dithiothreitol, while sensitizing them to H2O2 . Yhb1p overproduction in a glucose-6-phosphate dehydrogenase-deficient mutant decreased its growth rate . These data indicate that there is a complex relationship(s) between Yhb1p function(s) and cell defense reactions against various stresses.

J Biol Chem, 1998 Apr 17, 273(16), 9472 - 9
Amino acid residues that define both the isoprenoid and CAAX preferences of the Saccharomyces cerevisiae protein farnesyltransferase . Creating the perfect farnesyltransferase; Caplin BE et al.; Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C = cysteine, A = aliphatic residue, X = cysteine, serine, methionine, alanine) and to a lesser degree CAAL . Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine- and leucine-ending substrates both in vitro and in vivo . Substrate overlap of FTase and GGTase I has not been determined to be biologically significant . In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis . Three of the mutations altered the substrate preferences of the wild type enzyme significantly . The ram1p-74D FTase farnesylated only Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three substrates as well or better than wild type . The ram1p-206DDLF FTase farnesylated Ras-CII(S,M,L) at wild type levels but could no longer geranylgeranylate the Ras-CII(M,L) substrates . The ram1p-351FSKN FTase farnesylated Ras-CIIS and Ras-CIIM but not Ras-CIIL . The ram1p-351FSKN FTase was not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving this mutant the attributes of the dogmatic FTase that only farnesylates non-leucine-ending CAAX substrates and does not geranylgeranylate any substrate . These results suggest that the isoprenoid and protein substrate specificities of FTase are interrelated . The availability of a mutant FTase that lacked substrate overlap with the protein GGTase-I made possible an analysis of the role of substrate overlap in normal cellular processes of yeast, such as mating and growth at elevated temperatures . Our findings suggest that neither farnesylation of leucine-ending CAAX substrates nor geranylgeranylation by the FTase is necessary for these cellular processes.

Curr Genet, 1998 Mar, 33(3), 171 - 7
Dip5p mediates high-affinity and high-capacity transport of L-glutamate and L-aspartate in Saccharomyces cerevisiae; Regenberg B et al.; Genes encoding homologues of known amino-acid permeases were deleted in a strain also deficient in the general amino-acid permease . The uptake capacity of the mutants was investigated for several L-alpha-amino acids . Deletion of a gene denoted DIP5 results in the loss of L-aspartate and L-glutamate uptake . The dip5 mutation caused a several hundred-fold reduction of uptake of the two amino acids, both in cells grown on proline as a nitrogen source and in cells grown on ammonium . DIP5-dependent uptake of L-aspartate and L-glutamate was somewhat lower in ammonium-grown cells than in proline-grown cells . Transcriptional regulation is at least partially responsible for this difference, as shown by assaying the DIP5 promoter fused to lacZ . This suggests that the promoter is subject to nitrogen catabolite repression . Transport of a few other amino acids was moderately affected by dip5 but was not competed by L-aspartate in the DIP5 parental strain; transport of these amino acids is therefore unlikely to be mediated by Dip5p . Our results suggest that DIP5 encodes an amino-acid permease with a high transport capacity and a high affinity for L-glutamate and L-aspartate, with a Kt of about 50 microM for both.

Curr Genet, 1998 Feb, 33(2), 110 - 6
Ts mutations in mitochondrial tRNA genes: characterization and effects of two point mutations in the mitochondrial gene for tRNAphe in Saccharomyces cerevisiae; Francisci S et al.; Two new mitochondrial mutations conferring heat sensitivity on glycerol medium to the cells that carry them and affecting mitochondrial protein synthesis were investigated . Both map in the mitochondrial tRNAphe gene and have C-to-U transitions, one at position 2 (ts22b16) and the other at 62 (ts1345) . The latter mutation clearly affects the 3' end-maturation of tRNAphe, while the former presents normal patterns of both tRNA processing and amino-acylation . The defective phenotype resulting from the ts22b16 mutation can be corrected by over-expressing either the mitochondrial elongation factor EF-Tu or the mutated form of the tRNA . These results suggest that this mutation's primary effect might involve modified interactions during the ternary complex formation.

Curr Genet, 1998 Feb, 33(2), 100 - 9
RPG1: an essential gene of saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase; Kovarik P et al.; In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G1 phase . We characterized a novel gene, RPG1, which is also involved in this phase . RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein . Elutriated or alpha-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature . The cells remained unbudded and neither grew nor duplicated DNA . rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G1 cells after a shift to a non-permissive temperature . Similarly, the asynchronous rpg1-1 cells accumulated in G1 at the non-permissive temperature, but mother and daughter cells did not separate . A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum . Our data show that Rpg1p is required for passage through the G1 phase and may be involved in growth control . Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3.

Biochem J, 1998 Mar 15, 330 ( Pt 3), 1333 - 40
Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway; Copley KS et al.; A system is described for the heterologous expression of peptides in Saccharomyces cerevisiae . A synthetic gene encoding a precursor of the 41 amino acid Manduca sexta diuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide . A precursor of a mutant peptide of Mas-DH, Mas-DH{K22Q} was also expressed . The peptides were purified, then treated with peptidylglycine alpha-amidating enzyme to generate the alpha-amidated, mature, form of Mas-DH or Mas-DH{K22Q}, which were biologically active . Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain . In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors . Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases . This protease-deficient strain may prove of general utility for expression of peptides . Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites . Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys downward arrow-Glu-Arg site . Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys . This paper is the first report on the in vivo activity and specificity of Yap3 and Mkc7 expressed at physiological levels.

Mol Biol Cell, 1998 May, 9(5), 1221 - 33
Rho1p-Bni1p-Spa2p interactions: implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae; Fujiwara T et al.; Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein . Rho1p is localized at the growth sites and required for bud formation . We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein . Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p . This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity . The C-terminal 254 amino acid region of Spa2p, Spa2p(1213-1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826-987) . Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p . Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells . However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm . A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip . These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

Mol Biol Cell, 1998 May, 9(5), 1081 - 91
The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species; Luikenhuis S et al.; Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases . Two genes, designated GRX1 and GRX2, which share 40-52% identity and 61-76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae . Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using beta-hydroxyethylene disulfide as a substrate . Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo . Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2 . In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide . Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains . The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth . Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins.

Mol Biol Cell, 1998 May, 9(5), 977 - 91
Saccharomyces cerevisiae cells with defective spindle pole body outer plaques accomplish nuclear migration via half-bridge-organized microtubules; Brachat A et al.; Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB) . Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%) . Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules . Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Delta1 cells that lack a detectable outer plaque . These microtubules are attached to the SPB half- bridge throughout the cell cycle . This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque . Although CNM67 is not strictly required for mitosis, it is essential for sporulation . Time-lapse microscopy of cnm67Delta1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation . This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

Mol Microbiol, 1998 Apr, 28(1), 69 - 79
A role for the Pcl9-Pho85 cyclin-cdk complex at the M/G1 boundary in Saccharomyces cerevisiae; Tennyson CN et al.; PHO85 is a cyclin-dependent kinase (CDK) with roles in phosphate and glycogen metabolism and cell cycle progression . As a CDK, Pho85 is activated by association with Pho85 cyclins (Pcls), of which 10 are known . PCL1, PCL2 and PCL9 are the only members of the Pho85 cyclin family that are expressed in a cell cycle-regulated pattern . We found that PCL9 is expressed in late M/early G1 phase of the cell cycle and is activated by the transcription factor, Swi5 . This pattern of regulation is different from PCL1 and PCL2, which are expressed later in G1 phase and are regulated primarily by the transcription factor SBF . Co-immunoprecipitation experiments using in vitro translated proteins showed that Pcl9 and Pho85 form a complex . Furthermore, immunoprecipitated Pcl9 complexes from yeast lysates were capable of phosphorylating the exogenous substrate Pho4 . The Pcl9-associated kinase activity was dependent on PHO85, showing that Pcl9 and Pho85 form a functionally active kinase complex in vivo . Deletion of PCL9 in diploid cells caused random, rather than bipolar, budding in 18% of cells . In contrast, deletion of PCL2, the closest relative of PCL9, had no effect on the budding pattern . Deleting more members of the PCL1,2 subfamily (which includes PCL9) increased the percentage of random budding in the cell population . When all members of the PCL1,2 subfamily were deleted, 73% of cells budded randomly, a value similar to that obtained when the CDK partner PHO85 was deleted . Our results show that PCL9 and PHO85 form a functional kinase complex and suggest a role for Pho85 CDKs at the M/G1 boundary.

Oncogene, 1998 Apr 16, 16(15), 2011 - 6
Interaction of Rho1p target Bni1p with F-actin-binding elongation factor 1alpha: implication in Rho1p-regulated reorganization of the actin cytoskeleton in Saccharomyces cerevisiae; Umikawa M et al.; The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae . We have shown that Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein . A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified to be Tef1p/Tef2p, translation elongation factor 1alpha (EF1alpha) . EF1alpha is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)-binding and -bundling activities . EF1alpha bound to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, of which function is not known . The binding of Bni1p to EF1alpha inhibited its F-actin-binding and -bundling activities . The BNI1 gene deleted in the EF1alpha-binding region did not suppress the bni1 bnr1 mutation in which the actin organization was impaired . These results suggest that the Rho1p-Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1alpha and profilin.

Genetika, 1998 Feb, 34(2), 190 - 7
{New phenotypic manifestation of the ad2 mutation in Saccharomyces cerevisiae yeast--the inability to grow on a synthetic medium with glycerol and hypoxanthine}; Zekhnov AM et al.; The ADE2 gene of Saccharomyces cerevisiae yeast encodes aminoimidazole ribonucleotide-carboxylase (AIR-carboxylase), an enzyme catalyzing the sixth stage of purine nucleotide biosynthesis . Strains bearing the ade2 mutation are able to grow on a glucose-containing synthetic medium with the addition of adenine or hypoxanthine, which under the action of the cellular phosphoribosyltransferases are converted into adenosine monophosphate and inosine monophosphate, respectively . Our studies showed that ade2 mutants were unable to grow on a synthetic medium with glycerol and hypoxanthine . This newly described feature is not constitutively manifested, because some strains can contain suppressor mutations which restore the ability to grow on a synthetic medium with glycerol and hypoxanthine . The ade4, ade5, ade8, ade6, and ade7 mutations were found to suppress the phenotypic manifestation of the ade2 mutations via inactivation of enzymes catalyzing the first, second, third, fourth and fifth stages of purine biosynthesis, while the ade1 mutation, which inactivates enzyme of the seventh stage, lacks suppressive activity . Strains with single adenine mutations, ade4, ade5, ade8, ade6, ade7, or ade1 grow on glycerol- and hypoxanthine-containing media . Our data suggest that the new property of the ade2 mutations could be associated with the accumulation of the AIR-carbole-ribonucleotide . A mutation resulting in the requirement for serine on the medium with glycerol, but not glucose, is described.

Arch Biochem Biophys, 1998 Apr 15, 352(2), 175 - 81
One of the fumarate reductase isoenzymes from Saccharomyces cerevisiae is encoded by the OSM1 gene; Muratsubaki H et al.; Soluble fumarate reductase from yeast irreversibly catalyzes the reduction of fumarate to succinate and has noncovalently bound flavin adenine dinucleotide . In yeast, there are two isoenzymes of fumarate reductase, which can be distinguished on the basis of their absorption or nonabsorption to DE-52 columns . Previously, we have purified FRDS1 and isolated its gene (FRDS) from Saccharomyces cerevisiae . In the present study, FRDS2 was purified to homogeneity by four chromatography steps . The N-terminal and C-terminal amino acid sequences of FRDS2 were identical to the deduced amino acid sequence of the OSM1 gene (EMBL Database Accession No . L-26347), whose isolation and biochemical properties have not been studied up until now . From these results, we conclude that FRDS2 is encoded by the OSM1 gene . The deduced amino acid sequence of the OSM1 gene revealed that FRDS2 is synthesized as a precursor protein containing a presequence composed of 32 amino acid residues . The mature enzyme consists of a protein of 469 amino acid residues with a molecular weight of 51,370 . The N-terminal extension had the characteristics of a typical signal sequence required for targeting and sorting to a noncytosolic destination . In fact, FRDS2 was found to be located in promitochondria.

Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5584 - 9
Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae; Srinivasa SP et al.; RGS4, a mammalian GTPase activating protein for G protein alpha subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae . To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system . Deletion of the N-terminal 33 aa of RGS4 (Delta1-33) yielded a nonfunctional protein and loss of plasma membrane localization . These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (Delta1-33) . Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling . Fusion of the N-terminal 33 aa of RGS4 to green fluorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain . RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation . Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling . Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.

Cell Stress Chaperones, 1998 Mar, 3(1), 37 - 43
Identification of an integral membrane 80 kDa protein of Saccharomyces cerevisiae induced in response to dehydration; Eleutherio EC et al.; Using SDS-PAGE gels we observed the induced synthesis of a protein with a molecular mass of 80 kDa when cells of strains of Saccharomyces cerevisiae were subjected to dehydration . Physiological analysis showed that this protein is not present during growth on glucose but was found in derepressed cells from stationary phase . Furthermore, its synthesis was induced when cells were grown on medium containing alpha-methyl-glucoside as carbon source . However, the 80 kDa protein was not found in cells of mutants unable to transport trehalose . This protein was localized in the cytoplasmic membrane and showed trehalose-binding activity, determined by its partial purification on a trehalose-Sepharose 6B affinity column . The possible involvement of the 80 kDa protein with the trehalose transport system is discussed.

Biochem Mol Biol Int, 1998 Apr, 44(4), 747 - 59
Menadione toxicity in Saccharomyces cerevisiae cells: activation by conjugation with glutathione; Zadzinski R et al.; Menadione (2-methyl-1,4-naphthoquinone) has been used extensively as an oxidant stressor at the cellular level . However, the mechanism of cytotoxicity of this compound still remains controversial . This study deals with the role of intracellular glutathione in the resistance of the yeast Saccharomyces cerevisiae to menadione . Incubation with 0.5 mM menadione resulted in a decrease of total glutathione concentration in yeast cells, intracellular formation of menadione S-glutathione conjugate and export of the conjugate from cells . GSH-deficient mutants showed lower stimulation of superoxide and hydrogen peroxide production upon exposure to menadione and were more resistant to menadione than wild-type isogenic strains . These results indicate that in yeast cells the formation of S-glutathione conjugate is a major pathway of menadione metabolism and that this reaction leads to redox activation of menadione but permits its removal from the cells.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4264 - 9
The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae; Berset C et al.; Initiation factor eIF4G is an essential protein required for initiation of mRNA translation via the 5' cap-dependent pathway . It interacts with eIF4E (the mRNA 5' cap-binding protein) and serves as an anchor for the assembly of further initiation factors . With treatment of Saccharomyces cerevisiae with rapamycin or with entry of cells into the diauxic phase, eIF4G is rapidly degraded, whereas initiation factors eIF4E and eIF4A remain stable . We propose that nutritional deprivation or interruption of the TOR signal transduction pathway induces eIF4G degradation.

J Biol Chem, 1998 Apr 10, 273(15), 9306 - 11
The Gag domain of the Gag-Pol fusion protein directs incorporation into the L-A double-stranded RNA viral particles in Saccharomyces cerevisiae; Ribas JC et al.; The L-A double-stranded RNA virus of yeast encodes its major coat protein, Gag, and a Gag-Pol fusion protein made by a -1 ribosomal frameshift, a coding strategy used by many retroviruses . We find that cells expressing only Gag from one plasmid and only Gag-Pol (in frame) from a separate plasmid can support the propagation of M1 double-stranded RNA, encoding the killer toxin . We use this system to separately investigate the functions of Gag and the Gag part of Gag-Pol . L-A contains two fusion protein molecules per particle, and although N-terminal acetylation of Gag is essential for viral assembly, it is completely dispensable for function of Gag-Pol . In general, the requirements on Gag for viral assembly and propagation are more stringent than on the Gag part of Gag-Pol . Finally, we directly show that it is Gag that instructs the incorporation of Gag-Pol into the viral particles.

J Biol Chem, 1998 Apr 10, 273(15), 9249 - 54
A homologue of Saccharomyces cerevisiae Dpm1p is not sufficient for synthesis of dolichol-phosphate-mannose in mammalian cells; Tomita S et al.; Dolichol-phosphate-mannose (Dol-P-Man) serves as a donor of mannosyl residues in major eukaryotic glycoconjugates . It donates four mannosyl residues in the N-linked oligosaccharide precursor and all three mannosyl residues in the core of the glycosylphosphatidylinositol anchor . In yeasts it also donates one mannose to the O-linked oligosaccharide . The yeast DPM1 gene encodes a Dol-P-Man synthase that is a transmembrane protein expressed in the endoplasmic reticulum . We cloned human and mouse homologues of DPM1, termed hDPM1 and mDPM1, respectively, both of which encode proteins of 260 amino acids, having 30% amino acid identity with yeast Dpm1 protein but lacking a hydrophobic transmembrane domain, which exists in the yeast synthase . Human and mouse DPM1 cDNA restored Dol-P-Man synthesis in mouse Thy-1-deficient mutant class E cells . Mouse class E mutant cells had an inactivating mutation in the mDPM1 gene, indicating that mDPM1 is the gene for class E mutant . In contrast, hDPM1 and mDPM1 cDNA did not complement another Dol-P-Man synthesis mutant, hamster Lec15 cells, whereas yeast DPM1 restored both mutants . Therefore, in contrast to yeast, mammalian cells require hDPM1/mDPM1 protein and a product of another gene that is defective in Lec15 mutant cells for synthesis of Dol-P-Man.

J Biol Chem, 1998 Apr 10, 273(15), 8564 - 71
Hydrogen peroxide causes RAD9-dependent cell cycle arrest in G2 in Saccharomyces cerevisiae whereas menadione causes G1 arrest independent of RAD9 function; Flattery-O'Brien JA et al.; This study shows differences at the level of cell cycle arrest between the response of yeast cells to hydrogen peroxide and superoxide stress . These include both cell cycle phases at which arrest occurs and the involvement of the RAD9 checkpoint gene . Wild-type and rad9 cells were treated with hydrogen peroxide or the superoxide-generating agent menadione . rad9 mutants were up to 100-fold more sensitive to hydrogen peroxide but not affected in their resistance to menadione . Hydrogen peroxide caused G2-phase arrest, whereas menadione-treated cells arrested in G1 . G2 arrest, induced by methyl 2-benzimidazil carbamate, increased cellular resistance to hydrogen peroxide but not to menadione . G1 arrest mediated by alpha-factor caused an increase in survival of wild-type cells treated with menadione but not with hydrogen peroxide . A cdc28 mutant arrested in G1 was significantly more sensitive to hydrogen peroxide than other cdc mutants arrested in later phases, including G2 . rad9 cells have normal stationary phase resistance to hydrogen peroxide, the ability to adapt to it, glutathione content and induction of genes via the stress responsive element . Although rad9-dependent G2 arrest is important, other rad9-dependent factors may be involved in the resistance of cells to hydrogen peroxide since arrest in G2 did not make rad9 cells fully resistant.

J Biol Chem, 1998 Apr 10, 273(15), 8556 - 9
The role of Cdc42 in signal transduction and mating of the budding yeast Saccharomyces cerevisiae; Oehlen LJ et al.; The small G-protein Cdc42 functions in many eukaryotic signal transduction pathways . In the budding yeast Saccharomyces cerevisiae, cells with defective Cdc42 fail to induce mating-specific genes in response to mating factor and to adopt the proper morphology for conjugation . Here we show that the failure of mating factor-induced transcription is largely the indirect result of arrest at a specific cell cycle position and/or the accumulation of high levels of the Cln1/2-Cdc28 kinase, a known repressor of mating factor signal transduction . Cdc42-defective cells with restored transcriptional induction have a partially restored mating ability but are still defective in the morphological response to mating factor . These results show that Cdc42 is not required for transduction of the mating factor signal per se but that it is essential for proper mating factor-induced morphogenesis.

Mol Biol Cell, 1998 Apr, 9(4), 917 - 30
Mitochondrial inheritance is delayed in Saccharomyces cerevisiae cells lacking the serine/threonine phosphatase PTC1; Roeder AD et al.; In wild-type yeast mitochondrial inheritance occurs early in the cell cycle concomitant with bud emergence . Cells lacking the PTC1 gene initially produce buds without a mitochondrial compartment; however, these buds later receive part of the mitochondrial network from the mother cell . Thus, the loss of PTC1 causes a delay, but not a complete block, in mitochondrial transport . PTC1 encodes a serine/threonine phosphatase in the high-osmolarity glycerol response (HOG) pathway . The mitochondrial inheritance delay in the ptc1 mutant is not attributable to changes in intracellular glycerol concentrations or defects in the organization of the actin cytoskeleton . Moreover, epistasis experiments with ptc1delta and mutations in HOG pathway kinases reveal that PTC1 is not acting through the HOG pathway to control the timing of mitochondrial inheritance . Instead, PTC1 may be acting either directly or through a different signaling pathway to affect the mitochondrial transport machinery in the cell . These studies indicate that the timing of mitochondrial transport in wild-type cells is genetically controlled and provide new evidence that mitochondrial inheritance does not depend on a physical link between the mitochondrial network and the incipient bud site.

Gene, 1998 Mar 16, 209(1-2), 247 - 54
Activation of rat androgen receptor by androgenic ligands is unaffected by antiandrogens in Saccharomyces cerevisiae; Rana S et al.; The E . coli lacZ has been utilized as a reporter to evaluate ligand-mediated activation of the rat androgen receptor (AR) in Saccharomyces cerevisiae strain YCR1 . beta-galactosidase activity was androgen-specific and was found to be inducible approximately 260-fold by dihydrotestosterone (DHT), testosterone and R1881 . None of the antiandrogens tested was able to antagonize the DHT-dependent induction of beta-galactosidase activity . In the gel retardation assay, exposure of the receptor to DHT in vitro led to the formation of a protein-DNA complex that was not detected in yeast extracts unexposed to hormone . However, activation of AR by a steroidal (cyproterone acetate) and a non-steroidal antiandrogen (flutamide) either alone or in combination with DHT also results in a similar migration pattern . Additionally, LEM1, the ABC transporter that selectively modulates the biological potency of steroids in yeast, although operative in YCR1, was not responsible for antiandrogen resistance . These results thus indicate the involvement of other non-receptor factor(s) in mediating the effect of antiandrogens in yeast.

RNA, 1998 May, 4(5), 566 - 81
Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly; Daugeron MC et al.; Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism . Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae . The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype . In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes . Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs . Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p . Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus . We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit.

RNA, 1998 May, 4(5), 551 - 65
Conservation of functional domains involved in RNA binding and protein-protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1; Rain JC et al.; The modular structure of splicing factor SF1 is conserved from yeast to man and SF1 acts at early stages of spliceosome assembly in both organisms . The hnRNP K homology (KH) domain of human (h) SF1 is the major determinant for RNA binding and is essential for the activity of hSF1 in spliceosome assembly, supporting the view that binding of SF1 to RNA is essential for its function . Sequences N-terminal to the KH domain mediate the interaction between hSF1 and U2AF65, which binds to the polypyrimidine tract upstream of the 3' splice site . Moreover, yeast (y) SF1 interacts with Mud2p, the presumptive U2AF65 homologue in yeast, and the interaction domain is conserved in ySF1 . The C-terminal degenerate RRMs in U2AF65 and Mud2p mediate the association with hSF1 and ySF1, respectively . Analysis of chimeric constructs of hSF1 and ySF indicates that the KH domain may serve a similar function in both systems, whereas sequences C-terminal to the KH domain are not exchangeable . Thus, these results argue for hSF1 and ySF1, as well as U2AF65 and Mud2p, being functional homologues.

Genetics, 1998 Apr, 148(4), 1763 - 76
Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae; Zelenaya-Troitskaya O et al.; Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization . Here we report new functions for Abf2p in mtDNA transactions . We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses . Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium . The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control . Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium . Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells . The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability . In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein.These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination.

Mol Cell Biochem, 1998 Jan, 178(1-2), 27 - 31
The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases; de Souza Pereira R et al.; This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products . The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program . These artificial substrates have been widely used to produce chiral synthons . Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates . Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have.

Microbiology, 1998 Apr, 144 ( Pt 4), 1103 - 11
Lack of correlation between trehalose accumulation, cell viability and intracellular acidification as induced by various stresses in Saccharomyces cerevisiae; Alexandre H et al.; A pma1-1 mutant of Saccharomyces cerevisiae with reduced H(+)-ATPase activity and the isogenic wild-type strain accumulated high levels of trehalose in response to a temperature upshift to 40 degrees C and after addition of 10% ethanol, but only modest levels in response to a rapid drop in external pH and after addition of decanoic acid . There was, however, no correlation between the absolute levels of trehalose in the stressed cells and their viability . All these treatments induced a significant decrease in intracellular pH, and surprisingly, this decrease was very similar in both strains, indicating that intracellular acidification could not be the triggering mechanism for trehalose accumulation in response to stress . A careful investigation of metabolic parameters was carried out to explain how trehalose accumulated under the four different stress conditions tested . No single and common mechanism for trehalose accumulation could be put forward and the transcriptional activation of TPS1 was not unequivocally related to trehalose accumulation . Another finding was that a pma1-1 mutant exhibited a two- to threefold greater capacity to accumulate trehalose than the isogenic wild-type . This enhanced disaccharide synthesis could be attributed to a twofold higher trehalose-6-phosphate synthase activity, together with a fourfold higher content of intracellular UDP-Glc . In addition, this mutant showed 1.5-fold higher levels of ATP compared to the wild-type . The various stress treatments studied showed that a drop in intracellular pH does not correlate with trehalose accumulation . It is suggested that plasma membrane alteration could be the physiological trigger inducing trehalose accumulation in yeast.

J Exp Biol, 1998 Jun, 201 ( Pt 11), 1729 - 37
Neither respiration nor cytochrome c oxidase affects mitochondrial morphology in Saccharomyces cerevisiae; Church C et al.; Previous studies have reported that mitochondrial morphology and volume in yeast cells are linked to cellular respiratory capacity . These studies revealed that mitochondrial morphology in glucose-repressed or anaerobically grown cells, which lack or have reduced levels of respiration, is different from that in fully respiring cells . Although both oxygen deprivation and glucose repression decrease the levels of respiratory chain proteins, they decrease the expression of many non-mitochondrial proteins as well, making it difficult to determine whether it is a defect in respiration or something else that effects mitochondrial morphology . To determine whether mitochondrial morphology is dependent on respiration per se, we used a strain with a null mutation in PET100, a nuclear gene that is specifically required for the assembly of cytochrome c oxidase . Although this strain lacks respiration, the mitochondrial morphology and volumes are both comparable to those found in its respiration-proficient parent . These findings indicate that respiration is not involved in the establishment or maintenance of yeast mitochondrial morphology, and that the previously observed effects of oxygen availability and glucose repression on mitochondrial morphology are not exerted through the respiratory chain . By applying the principle of symmorphosis to these findings, we conclude that the shape and size of the mitochondrial reticulum found in respiring yeast cells is maintained for reasons other than respiration.

Biochem J, 1998 May 15, 332 ( Pt 1), 153 - 9
Saccharomyces cerevisiae GPI10, the functional homologue of human PIG-B, is required for glycosylphosphatidylinositol-anchor synthesis; Sutterlin C et al.; An increasing number of plasma membrane proteins have been shown to be attached to the membrane via a glycosylphosphatidylinositol (GPI) moiety . All eukaryotes share a highly conserved GPI-core structure EthN-P-Man3-GlcN-PI, where EthN is ethanolamine . We have identified a protein encoded by the yeast open reading frame YGL142C that shares 33% identity with the human Pig-B protein . Deletion of this essential gene leads to a block in GPI anchor biosynthesis . We therefore named the gene GPI10 . Gpi10p and Pig-B are functional homologues and the lethal deletion of GPI10 can be rescued by expression of the PIG-B cDNA . As found for PIG-B mutant cells, gpi10 deletant cells cannot attach the third mannose in an alpha-1,2 linkage to the GPI core-structure intermediate . Overexpression of GPI10 gives partial resistance to the GPI-synthesis inhibitor YW3548, suggesting that this gene product may affect the target of the inhibitor.

J Basic Microbiol, 1998, 38(1), 27 - 31
Depression by miconazole of heat-induced respiratory-deficiency in Saccharomyces cerevisiae; Madeira-Lopes A et al.; Miconazole, at 0.2 microM, decreased, by two orders of magnitude, the specific mutation rate of Saccharomyces cerevisiae to respiratory-deficient mutants (petites), which had been induced at either 37 degrees C or 39 degrees C . Identical concentrations of ketoconazole did not change the mutation rate . The results fit in the mechanisms of action which have been proposed for imidazole antimycotics, and constitute further support for the hypothesis that the targets of thermal death, in petite-positive associately-profiled yeasts, lie in the mitochondria.

J Bacteriol, 1998 May, 180(9), 2556 - 9
The Saccharomyces cerevisiae YCC5 (YCL025c) gene encodes an amino acid permease, Agp1, which transports asparagine and glutamine; Schreve JL et al.; The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease) . Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression.

J Biol Chem, 1998 Apr 3, 273(14), 8425 - 33
Increased camptothecin toxicity induced in mammalian cells expressing Saccharomyces cerevisiae DNA topoisomerase I; Hann C et al.; The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I . To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells . Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant . Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not . Thus, the enzyme retained catalytic activity while tethered to a support . Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin . This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug . The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells.

RNA, 1998 Feb, 4(2), 167 - 80
Genetic interaction between U6 snRNA and the first intron nucleotide in Saccharomyces cerevisiae; Luukkonen BG et al.; Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites . It is known that 5' splice site selection requires base pairing of U6 snRNA with intron positions 4-6 . However, no factor recognizing the highly conserved 5' splice site GU has yet been identified . We have tested if the known U6 snRNA-pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA . We observe that some combinations of 5' splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step . In addition, the U6-G52U allele can switch between two competing 5' splice sites harboring different nucleotides following the cleavage site . These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5' splice site cleavage . Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation . From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence . Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.

FEMS Microbiol Lett, 1998 Apr 15, 161(2), 359 - 64
Identification of the mgc1 mutation which affects mating-pheromone-induced morphogenesis in the yeast Saccharomyces cerevisiae; Fujimura HA; Cells of the yeast Saccharomyces cerevisiae undergo morphogenesis in response to the mating pheromones a- and alpha-factors . The 'shmoo' morphology involves localized cell surface projection formation and cytoskeleton protein synthesis . This polarization is presumed to be a prelude to mating between mating partners with opposite mating types, a and alpha . To identify genes involved in pheromone-induced morphogenesis, a system in which a gpa1 fus3 double mutant was used as a parent strain was developed, and mutants which showed altered morphogenesis in response to mating pheromone were identified . The mutation was designated mgc1 for morphogenesis control by mating pheromones . The mgc1 mutant arrested cell division in response to alpha-factor and mated with cells of the opposite mating type, but did not form a typical projection in response to pheromones.

FEMS Microbiol Lett, 1998 Apr 15, 161(2), 345 - 9
Transcription of multiple cell wall protein-encoding genes in Saccharomyces cerevisiae is differentially regulated during the cell cycle; Caro LH et al.; The yeast cell wall consists of an internal skeletal layer and an outside protein layer . The synthesis of both beta-1,3-glucan and chitin, which together from the cell wall skeleton, is cell cycle-regulated . We show here that the expression of five cell wall protein-encoding genes (CWP1, CWP2, SED1, TIP1 and TIR1) is also cell cycle-regulated . TIP1 is expressed in G1 phase, CWP1, CWP2 and TIR1 are expressed in S/G2 phase, and SED1 in M phase . The data suggest that these proteins fulfil distinct functions in the cell wall.

Insect Biochem Mol Biol, 1997 Dec, 27(12), 999 - 1006
Functional expression of house fly (Musca domestica) cytochrome P450 CYP6D1 in yeast (Saccharomyces cerevisiae); Smith FF et al.; Cytochrome P450 CYP6D1 from the house fly is important in the detoxication of xenobiotics and in resistance to pyrethroid insecticides . In house fly microsomes CYP6D1 requires cytochrome b5 for the metabolism of some substrates, such as benzo{a}pyrene, but does not require cytochrome b5 for the metabolism of other substrates such as methoxyresorufin . To examine the molecular mechanisms involved in its metabolism of pyrethroids and other substrates, a system for the heterologous expression of CYP6D1 in the yeast Saccharomyces cerevisiae was developed . Heterologous CYP6D1 can be inducibly expressed by culture in media with galactose as the sole carbon source, and is successfully inserted into the yeast microsomes . CYP6D1 is enzymatically active, as measured by methoxyresorufin-O-demethylation, indicating that CYP6D1 is able to interact with yeast P450 reductase . However, CYP6D1 expression did not result in measurable benzo{a}pyrene hydroxylation, suggesting that CYP6D1 cannot interact with yeast cytochrome b5, or that there is insufficient cytochrome b5 in the yeast microsomes to support this CYP6D1-mediated activity . Some suggestions are made for improving the yeast microsomal oxidoreductase environment in order to optimize CYP6D1 function.

Biotechnol Appl Biochem, 1998 Apr, 27 ( Pt 2), 109 - 15
alpha-Factor pro-peptide N-linked oligosaccharides facilitate secretion of the insulin precursor in Saccharomyces cerevisiae; Kjeldsen T et al.; To evaluate the possible relationship between N-linked glycosylation of the Saccharomyces cerevisiae alpha-factor pro-peptide and transport of the alpha-factor pro-peptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway, we analysed secretion of insulin precursor facilitated by alpha-factor pro-peptides with one or more of the three N-linked glycosylation sites removed . Mutation of the three alpha-factor pro-peptide N-linked glycosylation sites drastically decreased insulin precursor secretion . The three alpha-factor pro-peptide N-linked glycosylation sites differ in their ability to facilitate secretion of the insulin precursor . The two alpha-factor pro-peptide N-linked glycosylation sites localized closest to the insulin precursor contributed significantly to secretion, whereas the most N-terminally linked glycosylation site did not appear to facilitate secretion . Only correctly folded insulin precursor was found in the culture supernatant, regardless of the pro-peptide used for secretion, indicating that alpha-factor pro-peptide N-linked oligosaccharide chains are not necessary for correct folding of the insulin precursor . Thus, N-linked glycosylation facilitates intracellular transport of the alpha-factor propeptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway and secretion of the insulin precursor . N-linked glycosylation per se is not sufficient to facilitate secretion of the insulin precursor; the position of the N-linked oligosaccharide chain on the alpha-factor pro-peptide is important for facilitating efficient secretion.

Biotechnol Appl Biochem, 1998 Apr, 27 ( Pt 2), 81 - 8
Expression and secretion of beta-galactosidase in Saccharomyces cerevisiae using the signal sequences of GgpI, the major yeast glycosylphosphatidylinositol-containing protein; Pignatelli R et al.; New secretory signals and strategies can be attempted to improve the secretion of heterologous proteins of biotechnological interest which encounter difficulties being exported in yeast . The GGPI gene of Saccharomyces cerevisiae codes for a 125 kDa glycoprotein transported through the secretory pathway and anchored to the plasma membrane by means of a glycosylphosphatidylinositol . The regions coding for the secretory signal or also for the first 46 amino acids were tested for efficiency in secretion by fusion to the lacZ gene of Escherichia coli resulting in the synthesis of the endoplasmic reticulum-targeted 1-22- and 1-68-GgpIp/beta-gal hybrids . A cytoplasmic form was also examined . The 1-22 beta gal is partially transported to the cell surface and in the medium in an unglycosylated form . The 1-68 beta gal is completely retained in the intracellular membranes and is N-glycosylated in the GgpIp moiety . The amount of hybrid protein produced is similar and independent from its targeted site, suggesting that translocation through endoplasmic reticulum is not a limiting step, whereas the amount of active enzyme is from 50 to 80% lower for the endoplasmic reticulum forms compared with the cytoplasmic form . BiP/Kar2p putative precursor is accumulated in cells expressing the endoplasmic reticulum-targeted forms but not in those producing the cytosolic beta-galactosidase or over-expressing an endogenous secretory protein . Thus, glycosylation and abnormal folding rather than over-expression are among the factors responsible for the decreased activity and exit of beta-galactosidase from the endoplasmic reticulum and for induction of BiP . The results obtained indicate that the sole secretory signal of GgpIp is suitable to drive secretion of foreign products with complex folding and point to the importance of the endoplasmic reticulum quality control in the secretion of heterologous proteins in yeast.

Mol Cell Biol, 1998 May, 18(5), 2884 - 91
Ash1, a daughter cell-specific protein, is required for pseudohyphal growth of Saccharomyces cerevisiae; Chandarlapaty S et al.; Ash1 (for asymmetric synthesis of HO) was first uncovered in genetic screens that revealed its role in mating-type switching . Ash1 prevents HO expression in daughter cells . Because Ash1 has a zinc finger-like domain related to that of the GATA family of transcription factors, it presumably acts by repressing HO transcription . Nonswitching diploid cells also express Ash1, suggesting it could have functions in addition to regulation of HO expression . We show here that Ash1 has an essential function for pseudohyphal growth . Our epistasis analyses are consistent with the deduction that Ash1 acts separately from the mitogen-activated protein kinase cascade and Ste12 . Similarly to the case in yeast form cells, Ash1 is asymmetrically localized to the nuclei of daughter cells during pseudohyphal growth . This asymmetric localization reveals that there is a previously unsuspected daughter cell-specific function necessary for pseudohyphal growth.

Mol Cell Biol, 1998 May, 18(5), 2738 - 47
Bub1p kinase activates the Saccharomyces cerevisiae spindle assembly checkpoint; Farr KA et al.; Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function . In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase . We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles . High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation . Delayed cells possessed a G2 DNA content and elevated Clb2p mitotic cyclin levels . Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate . Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3 . We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other . This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.

FEBS Lett, 1998 Apr 3, 425(3), 475 - 8
Hexokinase PII has a double cytosolic-nuclear localisation in Saccharomyces cerevisiae; Randez-Gil F et al.; We describe here that the HXK2 gene product, isoenzyme PII of hexokinase, is located in both the nucleus and the cytoplasm of Saccharomyces cerevisiae cells . This conclusion is supported by assays of hexokinase-specific activity in isolated nuclei from wild-type and hxk1lhxk2 double mutant strains, by immunoblot experiments using anti-Hxk2 antibodies and by observation of the fluorescence distribution of a Hxk2-GFP fusion protein in cells transformed with the HXK2::gfp gene.

Genetics, 1998 Apr, 148(4), 1787 - 98
Cdc1 and the vacuole coordinately regulate Mn2+ homeostasis in the yeast Saccharomyces cerevisiae; Paidhungat M et al.; The yeast CDC1 gene encodes an essential protein that has been implicated in the regulation of cytosolic {Mn2+} . To identify factors that impinge upon Cdc1 or the Cdc1-dependent process, we isolated second-site suppressors of the conditional cdc1-1(Ts) growth defect . Recessive suppressors define 15 COS (CdcOne Suppressor) genes . Seven of the fifteen COS genes are required for biogenesis of the vacuole, an organelle known to sequester intracellular Mn2+ . An eighth gene, COS16, encodes a vacuolar membrane protein that seems to be involved in Mn2+ homeostasis . These results suggest mutations that block vacuolar Mn2+ sequestration compensate for defects in Cdc1 function . Interestingly, Cdc1 is dispensable in a cos16delta deletion strain, and a cdc1delta cos16delta double mutant exhibits robust growth on medium supplemented with Mn2+ . Thus, the single, essential function of Cdc1 is to regulate intracellular, probably cytosolic, Mn2+.

Genetics, 1998 Apr, 148(4), 1777 - 86
Cdc1 is required for growth and Mn2+ regulation in Saccharomyces cerevisiae; Paidhungat M et al.; Cdc1 function was initially implicated in bud formation and nuclear division because cdc1(Ts) cells arrested with a small bud, duplicated DNA, and undivided nucleus . Our studies show that Cdc1 is necessary for cell growth at several stages of the cell cycle, as well as in pheromone-treated cells . Thus, Cdc1 depletion might affect bud formation and nuclear division, as well as other cellular processes, by blocking a process involved in general cell growth . Cells depleted of intracellular Mn2+ also exhibit a cdc1-like phenotype and recent results suggested Cdc1 might be a Mn2+-dependent protein . We show that all of the conditional Cdc1(Ts) alleles tested cause cells to become sensitive to Mn2+ depletion . In addition, Cdc1 overproduction alleviates the chelator sensitivity of several Mn2+ homeostasis mutants . These findings are compatible with a model in which Cdc1 regulates intracellular, and in particular cytosolic, Mn2+ levels which, in turn, are necessary for cell growth.

Genetics, 1998 Apr, 148(4), 1701 - 13
Cell cycle arrest in cdc20 mutants of Saccharomyces cerevisiae is independent of Ndc10p and kinetochore function but requires a subset of spindle checkpoint genes; Tavormina PA et al.; The spindle checkpoint ensures accurate chromosome segregation by inhibiting anaphase onset in response to altered microtubule function and impaired kinetochore function . In this study, we report that the ability of the anti-microtubule drug nocodazole to inhibit cell cycle progression in Saccharomyces cerevisiae depends on the function of the kinetochore protein encoded by NDC10 . We examined the role of the spindle checkpoint in the arrest in cdc20 mutants that arrest prior to anaphase with an aberrant spindle . The arrest in cdc20 defective cells is dependent on the BUB2 checkpoint and independent of the BUB1, BUB3, and MAD spindle checkpoint genes . We show that the lesion recognized by Bub2p is not excess microtubules, and the cdc20 arrest is independent of kinetochore function . We show that Cdc20p is not required for cyclin proteolysis at two points in the cell cycle, suggesting that CDC20 is distinct from genes encoding integral proteins of the anaphase promoting complex.

Genetics, 1998 Apr, 148(4), 1647 - 54
Topical reversion at the HIS1 locus of Saccharomyces cerevisiae . A tale of three mutants; von Borstel RC et al.; Mutants of the HIS1 locus of the yeast Saccharomyces cerevisiae are suitable reporters for spontaneous reversion events because most reversions are topical, that is, within the locus itself . Thirteen mutations of his1-1 now have been identified with respect to base sequence . Revertants of three mutants and their spontaneous reversion rates are presented: (1) a chain termination mutation (his1-208, nee his1-1) that does not revert by mutations of tRNA loci and reverts only by intracodonic suppression; (2) a missense mutation (his1-798, nee his1-7) that can revert by intragenic suppression by base substitutions of any sort, including a back mutation as well as one three-base deletion; and (3) a -1 frameshift mutation (his1-434, nee his1-19) that only reverts topically by +1 back mutation, +1 intragenic suppression, or a -2 deletion . Often the +1 insertion is accompanied by base substitution events at one or both ends of a run of A's . Missense suppressors of his1-798 are either feeders or nonfeeders, and at four different locations within the locus, a single base substitution encoding an amino acid alteration will suffice to turn the nonfeeder phenotype into a feeder phenotype . Late-appearing revertants of his1-798 were found to be slowly growing leaky mutants rather than a manifestation of adaptive mutagenesis . Spontaneous revertants of his1-208 and his1-434 produced no late-arising colonies.

Genetics, 1998 Apr, 148(4), 1525 - 33
The chromosome bias of misincorporations during double-strand break repair is not altered in mismatch repair-defective strains of Saccharomyces cerevisiae; McGill CB et al.; Recombinational repair of a site-specific, double-strand DNA break (DSB) results in increased reversion frequency for nearby mutations . Although some models for DSB repair predict that newly synthesized DNA will be inherited equally by both the originally broken chromosome and the chromosome that served as a template, the DNA synthesis errors are almost exclusively found on the chromosome that had the original DSB (introduced by the HO endonuclease) . To determine whether mismatch repair acts on the template chromosome in a directed fashion to restore mismatches to the initial sequence, these experiments were repeated in mismatch repair-defective (pms1, mlh1, and msh2) backgrounds . The results suggest that mismatch repair is not responsible for the observed bias.

Genetics, 1998 Apr, 148(4), 1507 - 24
Factors affecting inverted repeat stimulation of recombination and deletion in Saccharomyces cerevisiae; Lobachev KS et al.; Inverted DNA repeats are an at-risk motif for genetic instability that can induce both deletions and recombination in yeast . We investigated the role of the length of inverted repeats and size of the DNA separating the repeats for deletion and recombination . Stimulation of both deletion and recombination was directly related to the size of inverted repeats and inversely related to the size of intervening spacers . A perfect palindrome, formed by two 1.0-kb URA3-inverted repeats, increased intra- and interchromosomal recombination in the adjacent region 2,400-fold and 17,000-fold, respectively . The presence of a strong origin of replication in the spacer reduced both rates of deletion and recombination . These results support a model in which the stimulation of deletion and recombination by inverted repeats is initiated by a secondary structure formed between single-stranded DNA of inverted repeats during replication.

Genetics, 1998 Apr, 148(4), 1491 - 505
DNA sequence analysis of spontaneous mutagenesis in Saccharomyces cerevisiae; Kunz BA et al.; To help elucidate the mechanisms involved in spontaneous mutagenesis, DNA sequencing has been applied to characterize the types of mutation whose rates are increased or decreased in mutator or antimutator strains, respectively . Increased spontaneous mutation rates point to malfunctions in genes that normally act to reduce spontaneous mutation, whereas decreased rates are associated with defects in genes whose products are necessary for spontaneous mutagenesis . In this article, we survey and discuss the mutational specificities conferred by mutator and antimutator genes in the budding yeast Saccharomyces cerevisiae . The implications of selected aspects of the data are considered with respect to the mechanisms of spontaneous mutagenesis.

Yeast, 1998 Mar 30, 14(5), 471 - 7
The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae; Planta RJ et al.; Screening of the complete genome sequence from the yeast Saccharomyces cerevisiae has enabled us to compile a complete list of the genes encoding cytoplasmic ribosomal proteins in this organism . Putative ribosomal protein genes were selected primarily on the basis of the sequence similarity of their products with ribosomal proteins from other eukaryotic organisms, in particular the rat . These genes were subsequently screened for typical yeast rp-gene characteristics, viz . (1) a high codon adaptation index; (2) their promoter structure and (3) their responses to changes in growth conditions . The yeast genome appears to carry 78 different genes, of which 59 are duplicated, encoding 32 different small-subunit and 46 large-subunit proteins . A new nomenclature for these ribosomal proteins is proposed.

Yeast, 1998 Mar 15, 14(4), 383 - 90
Purification and properties of polyphosphatase from Saccharomyces cerevisiae cytosol; Andreeva N et al.; A homogenous polyphosphatase preparation was obtained from Saccharomyces cerevisiae cytosol with a 3.8% yield and 3540-fold purification . The enzyme hydrolysed polyphosphate (polyP) with various chain lengths, including polyP3, and split Pi off the end of the chain . It was inactive with respect to ATP, PPi, and p-nitrophenylphosphate . Its specific activity with polyP15 was 283 U/mg protein . The polyphosphatase was a monomeric protein with a molecular mass of 40 kDa . This enzyme was inactive without divalent cations and with Cu2+ and Ca2+ . The ability of other divalent cations to activate the enzyme decreased in the following order; Co2+ > Mn2+ > Mg2+ > Zn2+ . A kinetic model of the hydrolysis of polyP3 and action of Mg2+ is proposed.

Yeast, 1998 Mar 15, 14(4), 359 - 69
The SKS1 gene of Saccharomyces cerevisiae is required for long-term adaptation of snf3 null strains to low glucose; Vagnoli P et al.; The SKS1 gene was originally identified as a multicopy suppressor of the growth defect of snf3 null mutations on low glucose concentrations . Snf3p is required for the rapid induction of HXT2 during growth on low substrate concentrations . Loss of Snf3p leads to a dramatic delay in expression of HXT2 . Adaptation to low substrate concentrations does not occur in snf3 sks1 double null mutant strains, suggesting that SKS1 is required for the glucose-dependent expression of HXT2 in the absence of Snf3p activity . Over-expression of SKS1 leads to over-expression of Hxt2p, thus explaining the mechanism of suppression of the snf3 defect . SKS1 defines a novel, Snf3p-independent pathway for the expression of Hxt2p . Under certain growth conditions, over-expression of SKS1 itself leads to a growth defect which is diminished in snf3 hxt2 double mutants . This suggests that over-expression of Hxt2p at physiologically inappropriate times is detrimental to the cells.

Yeast, 1998 Mar 15, 14(4), 347 - 57
The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae; Larsson C et al.; Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism . Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions . Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle . We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2 delta mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P . To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction . The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle . However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol . Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g . the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane . By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1.2, whereas the shuttle gave low rates but a high P/O ratio of 1.7 . Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2 delta strain proved dependent on GPD1 but not on GPD2.

Yeast, 1998 Mar 15, 14(4), 311 - 21
Synthesis of monohydroxylated inositolphosphorylceramide (IPC-C) in Saccharomyces cerevisiae requires Scs7p, a protein with both a cytochrome b5-like domain and a hydroxylase/desaturase domain; Dunn TM et al.; Saccharomyces cerevisiae mutants lacking Scs7p fail to accumulate the inositolphosphorylceramide (IPC) species . IPC-C, which is the predominant form found in wild-type cells . Instead scs7 mutants accumulate an IPC-B species believed to be unhydroxylated on the amide-linked C26-fatty acid . Elimination of the SCS7 gene suppresses the Ca(2+)-sensitive phenotype of csg1 and csg2 mutants . The CSG1 and CSG2 genes are required for mannosylation of IPC-C and accumulation of IPC-C by the csg mutants renders them Ca(2+)-sensitive . The SCS7 gene encodes a protein that contains both a cytochrome b5-like domain and a domain that resembles the family of cytochrome b5-dependent enzymes that use iron and oxygen to catalyse desaturation or hydroxylation of fatty acids and sterols . Scs7p is therefore likely to be the enzyme that hydroxylates the C26-fatty acid of IPC-C.

Yeast, 1998 Mar 15, 14(4), 303 - 10
Mam33p, an oligomeric, acidic protein in the mitochondrial matrix of Saccharomyces cerevisiae is related to the human complement receptor gC1q-R; Seytter T et al.; Mam33p (mitochondrial acidic matrix protein) is a soluble protein, located in mitochondria of Saccharomyces cerevisiae . It is synthesized as a precursor with an N-terminal mitochondrial targeting sequence that is processed on import . Mam33p assembles to a homo-oligomeric complex in the mitochondrial matrix . It can bind to the sorting signal of cytochrome b2 that directs this protein into the intermembrane space . Mam33p is encoded by an 801 bp open reading frame . Gene disruption did not result in a significant growth defect . Mam33p exhibits sequence similarity to gC1q-R, a human protein that has been implicated in the binding of complement factor C1q and kininogen.

J Bacteriol, 1998 Apr, 180(8), 2253 - 6
Physiological regulation of the derepressible phosphate transporter in Saccharomyces cerevisiae; Martinez P et al.; The extracellular phosphate concentration permissive for the expression of different amounts of the active high-affinity Pho84 phosphate transporter in the plasma membrane as well as the PHO84 messenger RNA levels in low-phosphate-grown Saccharomyces cerevisiae cells is very narrow and essential for a tight regulation of the transporter . The Pho84 transporter undergoes a rapid degradation once the supply of phosphate and/or carbon source is exhausted.

Prog Cell Cycle Res, 1997, 3, 143 - 56
Cell cycle regulation of S phase entry in Saccharomyces cerevisiae; Piatti S; Eukaryotic DNA replication is restricted to a narrow window of the cell cycle called S phase, and occurs once and only once during each cell cycle . The combination of genetic and biochemical approaches in the budding yeast Saccharomyces cerevisiae has proven extremely helpful for studying the cell cycle regulation of S phase entry . This review will try to summarise the most recent discoveries which led to a new model to explain how entry into S phase is regulated in eukaryotic cells.

Mutat Res, 1998 Feb 2, 397(2), 271 - 8
Chloroform and carbon tetrachloride induce intrachromosomal recombination and oxidative free radicals in Saccharomyces cerevisiae; Brennan RJ et al.; Chlorination of drinking water results in the generation of low levels of numerous chlorinated hydrocarbons due to the reaction of chlorine with naturally occurring organic compounds in the water . Concern has been raised about the safety of these chlorinated contaminants as several of them, most notably chloroform (trichloromethane), have been shown to be carcinogenic in long-term rodent bioassays and weak correlations between trihalomethane levels in drinking water and an increased risk of bladder and colorectal cancer in humans have been found . Chloroform and carbon tetrachloride induce liver cancer in rats and mice only at doses where significant hepatotoxicity is observed and have been classed as non-genotoxic carcinogens . We have investigated the ability of chloroform, carbon tetrachloride and 1,1,1-trichloroethane to induce deletions via intrachromosomal recombination in the yeast Saccharomyces cerevisiae . Chloroform and carbon tetrachloride induced this genotoxic recombination event at similar doses, 1,1,1-Trichloroethane gave only a weak response in the DEL recombination assay and only at the highest dose . We further show that chloroform and carbon tetrachloride, but not trichloroethane, induced oxidative free radical species in our yeast strain . The free radical scavenger N-acetylcysteine reduced chloroform-induced toxicity and recombination, and both chloroform and carbon tetrachloride were able to oxidize the free radical-sensitive reporter compound dichlorofluorescein diacetate in vivo . The implications of these findings to the carcinogenic activities of the three compounds are discussed.

Biochemistry, 1998 Mar 31, 37(13), 4437 - 50
Mutation and modeling analysis of the Saccharomyces cerevisiae Swi6 ankyrin repeats; Ewaskow SP et al.; The Swi4-Swi6 family of transcription factors confers G1/S specific transcription in budding and fission yeast . These proteins contain four ankyrin repeats, which are present in a large number of functionally diverse proteins and have been shown to be important for protein-protein interaction . However, no specific sequence has been identified that is diagnostic of an ankyrin repeat-interacting protein . To determine the function of the ankyrin repeats of Swi6, we generated both random and site-directed mutations within the ankyrin repeat domain of Swi6 and assayed the transcriptional function of these mutant swi6 alleles . We found six single mutations, scattered within the first and the fourth repeats, that generate a temperature-sensitive Swi6 protein . In addition, we found that alanine substitutions for the most conserved residues in each repeat were highly deleterious and also confer temperature sensitivity . Most of these swi6 alleles are able to form ternary complexes with Swi4 and DNA, but these complexes display reduced mobility in band-shift gels, suggesting a dramatic conformational change . We have modeled the ankyrin repeats of Swi6 using the coordinates derived for 53BP2 and find that, despite its low level of sequence conservation, these modeling studies and our mutation data are consistent with Swi6 having a structure very similar to that of 53BP2 . Moreover, all but one of our single mutants and all of the site-directed mutants disrupt critical structural features of the predicted folding pattern of these repeats . We conclude that the ankyrin repeats play a major structural role in Swi6 . Ankyrin repeats are unlikely to have inherent protein or DNA binding properties . However, they form a characteristic and stable structure with surfaces that may be tailored for many different macromolecular interactions.

J Biol Chem, 1998 Mar 27, 273(13), 7628 - 36
Regulation of high affinity iron uptake in the yeast Saccharomyces cerevisiae . Role of dioxygen and Fe; Hassett RF et al.; High affinity iron uptake in Saccharomyces cerevisiae requires a metal reductase, a multicopper ferroxidase, and an iron permease . Fet3, the apparent ferroxidase, is proposed to facilitate iron uptake by catalyzing the oxidation of reductase-generated Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1 (Kaplan, J., and O'Halloran, T . V . (1996) Science 271, 1510-1512) . We show here that dioxygen also plays an essential role in the expression of these iron uptake activities . Cells grown anaerobically exhibited no Fe(III) reductase or high affinity iron uptake activity, even if assayed for these activities under air . Northern blot analysis showed that the amount of those mRNAs encoding proteins associated with this uptake was repressed in anaerobic cultures but was rapidly induced by exposure of the culture to dioxygen . The anaerobic repression was reduced in cells expressing an iron-independent form of the trans-activator, Aft1, a protein that regulates the expression of these proteins . Thus, the effect of oxygenation on this expression appeared due at least in part to the state or distribution of iron in the cells . In support of this hypothesis, the membrane-permeant Fe(II) chelator, 2, 2'-bipyridyl, in contrast to the impermeant chelator bathophenanthroline disulfonate, caused a strong and rapid induction of these transcripts under anaerobic conditions . An increase in the steady-state levels of iron-regulated transcripts upon oxygenation or 2,2'-bipyridyl addition occurred within 5 min, indicating that a relatively small, labile intracellular pool of Fe(II) regulates the expression of these activities . The strength of the anaerobic repression was dependent on the low affinity, Fe(II)-specific iron transporter, encoded by FET4, suggesting that this Fe(II) pool was linked in part to iron brought into the cell via Fet4 protein . The data suggest a model in which dioxygen directly or indirectly modulates the Fe(III)/Fe(II) ratio in an iron pool linked to Aft1 protein while bipyridyl increases this ratio by chelating Fe(II) . These results indicate that dioxygen both modulates the sensitivity to iron-dependent transcriptional regulation and acts as substrate for Fet3 in the ferroxidase reaction catalyzed by this ceruloplasmin homologue.

Mol Cell Biochem, 1998 Jan, 178(1-2), 33 - 40
Comparison of biochemical effects produced by calcium ions and by monomers of polyacrylamide (acrylamide and bisacrylamide) on strains of Saccharomyces cerevisiae used for production of chiral synthons; de Souza Pereira R; The biochemical behaviour of four commercial strains of Saccharomyces cerevisiae was studied in the presence of calcium ions, acrylamide and bisacrylamide . Calcium ions at a concentration of 300 microM induced an increase of NAD(P)+ reduction in commercial Turkish and American strains, while in Chilean and Brazilian commercial strains, it diminished NAD(P)+ reduction . On the other hand, polyacrylamide monomers (acrylamide and bisacrylamide) induced a decrease of NAD(P)+ reduction in all strains studied in this paper . When membrane potential (delta psi) and oxygen consumption were measured in the presence of polyacrylamide monomers, a decrease of both was observed in all strains studied.

J Pediatr, 1998 Mar, 132(3 Pt 2), S24 - 9
Genetic analysis of iron uptake in the yeast Saccharomyces cerevisiae; Dancis A; OBJECTIVE: We used the methods of yeast genetics to identify genes involved in acquisition of iron by eukaryotic cells . METHODS: Mutants were identified with defects in cellular iron uptake . These were organized into an upstream group and a downstream group . The upstream group was involved in the delivery of copper to the multicopper oxidase FET3 . Mutants of this group were characterized by defective iron uptake that could be corrected by exposure of the cells to large amounts of copper . The downstream group was more directly involved in iron uptake . Mutant phenotypes from these genes could not be corrected by copper exposure . RESULTS: Genes in the upstream group encoded the regulator of copper transport, MAC1, and two copper transporters, CTR1 and CCC2 . Genes in the downstream group encoded the multicopper oxidase FET3 and its partner the iron permease FTR1 . In addition, the downstream genes encoded the surface reductases FRE1 and FRE2 and the iron regulatory protein AFT1 . CONCLUSIONS: The iron and copper uptake processes in yeast intersect because the FET3 gene encodes a multicopper oxidase that is required for iron transport . In human beings, an analogous function may be served by ceruloplasmin, a multicopper oxidase with a role in iron homeostasis.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 3312 - 7
In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae; Mouaheb N et al.; Whereas vertebrates possess only two thioredoxin genes, higher plants present a much greater diversity of thioredoxins . For example, Arabidopsis thaliana has five cytoplasmic thioredoxins (type h) and at least as many chloroplastic thioredoxins . The abundance of plant thioredoxins leads to the question whether the various plant thioredoxins play a similar role or have specific functions . Because most of these proteins display very similar activities on artificial or biological substrates in vitro, we developed an in vivo approach to answer this question . The disruption of both of the two Saccharomyces cerevisiae thioredoxin genes leads to pleiotropic effects including methionine auxotrophy, H2O2 hypersensitivity, altered cell cycle characteristics, and a limited ability to use methionine sulfoxide as source of methionine . We expressed eight plant thioredoxins (six cytoplasmic and two chloroplastic) in yeast trx1, trx2 double mutant cells and analyzed the different phenotypes . Arabidopsis type h thioredoxin 2 efficiently restored sulfate assimilation whereas Arabidopsis type h thioredoxin 3 conferred H2O2 tolerance . All thioredoxins tested could complement for reduction of methionine sulfoxide, whereas only type h thioredoxins were able to complement the cell cycle defect . These findings clearly indicate that specific interactions between plant thioredoxins and their targets occur in vivo.

EMBO J, 1998 Mar 2, 17(5), 1336 - 49
The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae; Shirayama M et al.; Proteolysis mediated by the anaphase promoting complex (APC) has a crucial role in regulating the passage of cells through anaphase . Destruction of the anaphase inhibitor Pds1p is necessary for separation of sister chromatids, whereas destruction of the mitotic cyclin Clb2p is important for disassembly of the mitotic spindle, cytokinesis and re-replication of the genome . Pds1p proteolysis precedes that of Clb2p by at least 15 min, which helps to ensure that cells never re-replicate their genome before they have separated sister chromatids at the previous mitosis . What triggers Pds1p proteolysis and why does it not also trigger that of Clb2p? Apart from sharing a dependence on the APC, these two proteolytic events differ in their dependence on other cofactors . Pds1p proteolysis depends on a WD-repeat protein called Cdc20p, whereas Clb2p proteolysis depends on another, related WD protein called Hct1/Cdh1p . On the other hand, destruction of Clb2p, but not that of Pds1p, depends on the Polo-like kinase, Cdc5p . Cdc20p is essential for separation of sister chromatids, whereas Cdc5p is not . We show that both Cdc5p and Cdc20p are unstable proteins whose proteolysis is regulated by the APC . Both proteins accumulate during late G2/M phase and disappear at a late stage of anaphase . Accumulation of Cdc20p contributes to activation of Pds1p proteolysis in metaphase, whereas accumulation of Cdc5p facilitates the activation of Clb2p proteolysis.

EMBO J, 1998 Aug 10, 17(5), 1236 - 47
The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae; Lorenz MC et al.; In response to nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form . This dimorphic transition is regulated by the Galpha protein GPA2, by RAS2, and by elements of the pheromone-responsive MAP kinase cascade, yet the mechanisms by which nitrogen starvation is sensed remain unclear . We have found that MEP2, a high affinity ammonium permease, is required for pseudohyphal differentiation in response to ammonium limitation . In contrast, MEP1 and MEP3, which are lower affinity ammonium permeases, are not required for filamentous growth . Deltamep2 mutant strains had no defects in growth rates or ammonium uptake, even at limiting ammonium concentrations . The pseudohyphal defect of Deltamep2/Deltamep2 strains was suppressed by dominant active GPA2 or RAS2 mutations and by addition of exogenous cAMP, but was not suppressed by activated alleles of the MAP kinase pathway . Analysis of MEP1/MEP2 hybrid proteins identified a small intracellular loop of MEP2 involved in the pseudohyphal regulatory function . In addition, mutations in GLN3, URE2 and NPR1, which abrogate MEP2 expression or stability, also conferred pseudohyphal growth defects . We propose that MEP2 is an ammonium sensor, generating a signal to regulate filamentous growth in response to ammonium starvation.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 611 - 4
Identification of a Caenorhabditis elegans Delta6-fatty-acid-desaturase by heterologous expression in Saccharomyces cerevisiae; Napier JA et al.; We identified a cDNA expressed sequence tag from an animal (the nematode worm Caenorhabditis elegans) that showed weak similarity to a higher-plant microsomal Delta6-desaturase . A full-length cDNA clone was isolated and expressed in the yeast Saccharomyces cerevisiae . This demonstrated that the protein encoded by the C . elegans cDNA was that of a fatty acid Delta6-desaturase, as determined by the accumulation of gamma-linolenic acid . The C . elegans Delta6-desaturase contained an N-terminalcytochrome b5 domain, indicating that it had a similar structure to that of the higher-plant Delta6-desaturase . The C . elegans Delta6-desaturase mapped to cosmid W08D2, a region of chromosome III . This is the first example of a Delta6-desaturase isolated from an animal and also the first example of an animal desaturase containing a cytochrome b5 domain.

Yeast, 1998 Feb, 14(3), 271 - 80
Generation of Saccharomyces cerevisiae deletants and basic phenotypic analysis of eight novel genes from the left arm of chromosome XIV; Maftahi M et al.; The disruption of eight novel genes was realized in two genetic backgrounds . Among these open reading frames, NO333, NO348 and NO364 presented homologies with other proteins of yeast or other organisms, whereas NO320, NO325, NO339, NO384 and NO388 showed no similarity with any protein . Tetrad analysis of heterozygous deletant strains revealed that NO348, NO364 and NO388 are essential genes for vegetative growth, whereas NO320, NO325, NO333, NO339 and NO384 are non-essential . Basic phenotypic analyses of the non-lethal deletant strains as suggested in the six-pack B0 programme did not reveal any significant differences between parental and mutant strains.

Yeast, 1998 Feb, 14(3), 239 - 53
Identification and kinetic analysis of a functional homolog of elongation factor 3, YEF3 in Saccharomyces cerevisiae; Sarthy AV et al.; Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis . EF-3 contains two ABC cassettes, and binds and hydrolyses ATP . We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database . This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A . YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains . However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters . The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified . Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nM) . K(m) values for ATP were similar, but the Kcat values differed significantly . Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the Kcat and Kcat/K(m) values for EF-3A were about two-fold higher; however, the difference in Kcat/K(m) values between the two factors was small for basal ATPase activity.

Yeast, 1998 Feb, 14(3), 215 - 24
Post-translational fate of CAN1 permease of Saccharomyces cerevisiae; Opekarova M et al.; To study the post-translational fate of arginine permease (Can1p), the gene coding for this transport protein was placed behind a constitutive promoter of plasma membrane ATPase (PMA1) and furnished with a Myc tag . In exponential-phase cells the amount of Can1p is constant, although turnover can be demonstrated . A rapid decrease in transport activity during the early stationary phase is paralleled by a corresponding net degradation of the protein . The amount of Can1p present in exponential cells grown on various nitrogen sources is the same, except in arginine-grown cells, in which the amount of the protein is markedly lower . This occurs solely when arginine serves as nitrogen source but not as an immediate consequence of, for example, arginine addition to cells growing on other nitrogen sources . it was demonstrated that Can1p is phosphorylated . Since Can1p expression under the PMA1 promoter is glucose-dependent, the amount of the permease expressed in high-glucose-grown cells is higher than in low-glucose-grown ones . Only a part of the Can1p overexpressed in high-glucose-grown cells is phosphorylated, while in low-glucose-grown cells the phosphorylated form probably represents the majority of Can1p . The permease phosphorylation or dephosphorylation is not related to transinhibition.

Genetics, 1998 Mar, 148(3), 1007 - 20
Identification of GCD14 and GCD15, novel genes required for translational repression of GCN4 mRNA in Saccharomyces cerevisiae; Cuesta R et al.; In Saccharomyces cerevisiae, expression of the transcriptional activator GCN4 increases at the translational level in response to starvation for an amino acid . The products of multiple GCD genes are required for efficient repression of GCN4 mRNA translation under nonstarvation conditions . The majority of the known GCD genes encode subunits of the general translation initiation factor eIF-2 or eIF-2B . To identify additional initiation factors in yeast, we characterized 65 spontaneously arising Gcd- mutants . In addition to the mutations that were complemented by known GCD genes or by GCN3, we isolated mutant alleles of two new genes named GCD14 and GCD15 . Recessive mutations in these two genes led to highly unregulated GCN4 expression and to derepressed transcription of genes in the histidine biosynthetic pathway under GCN4 control . The derepression of GCN4 expression in gcd14 and gcd15 mutants occurred with little or no increase in GCN4 mRNA levels, and it was dependent on upstream open reading frames (uORFs) in GCN4 mRNA that regulate its translation . We conclude that GCD14 and GCD15 are required for repression of GCN4 mRNA translation by the uORFs under conditions of amino acid sufficiency . The gcd14 and gcd15 mutations confer a slow-growth phenotype on nutrient-rich medium, and gcd15 mutations are lethal when combined with a mutation in gcd13 . Like other known GCD genes, GCD14 and GCD15 are therefore probably required for general translation initiation in addition to their roles in GCN4-specific translational control.

Genetics, 1998 Mar, 148(3), 975 - 88
Radiation-induced chromosome aberrations in Saccharomyces cerevisiae: influence of DNA repair pathways; Friedl AA et al.; Radiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks . The relationship between individual pathways of break repair and aberration formation is not clear . By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(= YKU70) gene, or combinations thereof . We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids . In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination . No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low . Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.

Genetics, 1998 Mar, 148(3), 947 - 61
Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment; Li E et al.; The pheromone-responsive Galpha protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal . In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp-) mutants were recovered . Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response . Eight of the dominant Adp- mutations showed tight linkage to the gene encoding the pheromone-responsive Gbeta, STE4 . Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response . Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gbeta dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone . Moreover, the signaling activity of the aberrant Gbetagamma subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gbetagamma, but not by E364K, a hyperadaptive form of Gpa1p . On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gbetagamma.

Genetics, 1998 Mar, 148(3), 937 - 45
The involvement of cellular recombination and repair genes in RNA-mediated recombination in Saccharomyces cerevisiae; Derr LK; We previously demonstrated that a reverse transcript of a cellular reporter gene (his3-AI) can serve as the donor for gene conversion of a chromosomal his3-deltaMscI target sequence, and that this process requires the yeast recombination gene RAD52 . In this study, we examine the involvement of other recombination and repair genes in RNA-mediated recombination, and gain insight into the nature of the recombination intermediate . We find that mutation of the mitotic RecA homologs RAD51, RAD55, and RAD57 increases the rate of RNA-mediated recombination relative to the wild type, and that these gene functions are not required for RNA-mediated gene conversion . Interestingly, RAD1 is required for RNA-mediated gene conversion of chromosomal his3-deltaMscI sequences, suggesting that the cDNA intermediate has a region of nonhomology that must be removed during recombination with target sequences . The observation that both RAD1 and RAD52 are required for RNA-mediated gene conversion of chromosomal but not plasmid sequences indicates a clear difference between these two pathways of homologous RNA-mediated recombination.

J Bacteriol, 1998 Apr, 180(7), 1962 - 4
Proton gradient-driven nickel uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae; Nishimura K et al.; A vacuolar H+-ATPase-negative mutant of Saccharomyces cerevisiae was highly sensitive to nickel ion . Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles . An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential . Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast.

J Bacteriol, 1998 Apr, 180(7), 1913 - 9
Sterol uptake in Saccharomyces cerevisiae heme auxotrophic mutants is affected by ergosterol and oleate but not by palmitoleate or by sterol esterification; Ness F et al.; The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed . To evaluate heme availability, a heterologous 17alpha-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo . Heme deficiency in G204 led to accumulation of squalene and lethality . The heterologous cytochrome P-450 was inactive in this strain . The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity . By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A . It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium . Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol . Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer . Sterol transfer was only slightly affected by a lack of esterification . It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.

J Bacteriol, 1998 Apr, 180(7), 1700 - 8
The Saccharomyces cerevisiae SCS2 gene product, a homolog of a synaptobrevin-associated protein, is an integral membrane protein of the endoplasmic reticulum and is required for inositol metabolism; Kagiwada S et al.; The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 and hac1/ire15 mutants (J . Nikawa, A . Murakami, E . Esumi, and K . Hosaka, J . Biochem . 118:39-45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from Aplysia californica (P . A . Skehel, K . C . Martin, E . R . Kandel, and D . Bartsch, Science 269:1580-1583, 1995) . In this study we have characterized an SCS2 gene product (Scs2p) . The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol . The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34 degrees C . Genetic studies reveal that the overexpression of the INO1 gene rescues the inositol auxotrophy of the SCS2 disruption strain . The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells . The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the SCS2 disruption strain . Therefore, the Scs2p function might be conserved among eukaryotic cells.

J Biol Chem, 1998 Mar 13, 273(11), 6271 - 6
Crystal structure of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Rad6 at 2.6 A resolution; Worthylake DK et al.; The Saccharomyces cerevisiae ubiquitin-conjugating enzyme (UBC) Rad6 is required for several functions, including the repair of UV damaged DNA, damage-induced mutagenesis, sporulation, and the degradation of cellular proteins that possess destabilizing N-terminal residues . Rad6 mediates its role in N-end rule-dependent protein degradation via interaction with the ubiquitin-protein ligase Ubr1 and in DNA repair via interactions with the DNA binding protein Rad18 . We report here the crystal structure of Rad6 refined at 2.6 A resolution to an R factor of 21.3% . The protein adopts an alpha/beta fold that is very similar to other UBC structures . An apparent difference at the functionally important first helix, however, has prompted a reassessment of previously reported structures . The active site cysteine lies in a cleft formed by a coil region that includes the 310 helix and a loop that is in different conformations for the three molecules in the asymmetric unit . Residues important for Rad6 interaction with Ubr1 and Rad18 are on the opposite side of the structure from the active site, indicating that this part of the UBC surface participates in protein-protein interactions that define Rad6 substrate specificity.

J Biol Chem, 1998 Mar 6, 273(10), 5431 - 4
Deletion of the Saccharomyces cerevisiae TRR1 gene encoding thioredoxin reductase inhibits p53-dependent reporter gene expression; Pearson GD et al.; The prevalence of p53 gene mutations in many human tumors implies that p53 protein plays an important role in preventing cancers . Central among the activities ascribed to p53 is its ability to stimulate transcription of other genes that inhibit cells from entering S phase with damaged DNA . Human p53 can be studied in yeast where genetic tools can be used to identify proteins that affect its ability to stimulate transcription . Although p53 strongly stimulated reporter gene expression in wild type yeast, it only weakly stimulated reporter gene expression in Deltatrr1 yeast that lacked the gene encoding thioredoxin reductase . Furthermore, ectoptic expression of TRR1 in Deltatrr1 yeast restored p53-dependent reporter gene activity to high levels . Immunoblot assays established that the Deltatrr1 mutation affected the activity and not the level of p53 protein . The results suggest that p53 can form disulfides and that these disulfides must be reduced in order for the protein to function as a transcription factor.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2384 - 9
Ectopic induction of Clb2 in early G1 phase is sufficient to block prereplicative complex formation in Saccharomyces cerevisiae; Detweiler CS et al.; Eukaryotic cells ensure the stable propagation of their genome by coupling each round of DNA replication (S phase) to passage through mitosis (M phase) . This control is exerted at the initiation of replication, which occurs at multiple origins throughout the genome . Once an origin has initiated, reinitiation is blocked until the completion of mitosis, ensuring that DNA is replicated at most once per cell cycle . Recent studies in several organisms have suggested a model in which S- and M-phase promoting cyclin-dependent kinases prevent reinitiation by blocking the repetition of an early step in the initiation reaction . In budding yeast, this regulation is thought to involve inhibition of prereplicative complex (pre-RC) formation at origins by S and M phase-promoting Clb kinases . To date, however, there has been no direct demonstration that these kinases can perform such an important function . In this report we provide such a confirmation by showing that ectopic induction in G1 phase of a mitotic Clb, Clb2, is sufficient to inhibit DNA replication and does so by preventing pre-RC formation . This inhibition requires that Clb2 be induced before Cdc6, an initiation protein required for pre-RC formation; once pre-RCs have formed, Clb2 can no longer inhibit initiation . These results support the notion that during the normal cell cycle reassembly of the pre-RC, and hence reinitiation at an origin, is directly inhibited by S and M phase-promoting cyclin-dependent kinases.

Mol Gen Genet, 1998 Feb, 257(4), 461 - 8
Suppressors of the temperature sensitivity of DNA polymerase alpha mutations in Saccharomyces cerevisiae; Formosa T et al.; We have isolated two high copy, allele-specific suppressors of the temperature sensitivity of mutations in POL1, the gene that encodes the catalytic subunit of DNA polymerase alpha in the yeast Saccharomyces cerevisiae . Both genes, PSP1 and PSP2, also partially suppressed a mutation in POL3 which encodes DNA polymerase delta, and both also affected a mutation in CDC6, which acts in initiation of DNA replication . Suppression was not general, since ts mutations in several genes unrelated to replication were not affected, PSP1 was partially effective on low-copy-number vectors, while PSP2 required high copy numbers . The presence of suppressing plasmids did not alter the steady-state level of Pol1 protein, so suppression does not appear to be due to an increase in production or stability of Pol1p . Deletion of either PSP gene or both in combination resulted in apparently normal viable cells . While neither gene is homologous to genes with known functions, PSP1 and PSP2 both have unusual amino acid compositions: PSP1 is rich in asparagine and glutamine, while PSP2 is rich in asparagine and contains "RGG" motifs that have been associated with RNA-binding proteins . We also describe a transposon-mediated strategy that should be generally effective for rapid characterization of multicopy suppressors.

Mol Gen Genet, 1998 Feb, 257(4), 404 - 11
The Aspergillus nidulans CCAAT-binding factor AnCP/AnCF is a heteromeric protein analogous to the HAP complex of Saccharomyces cerevisiae; Kato M et al.; The Aspergillus nidulans hapC gene was expressed as a fusion protein with MalE or glutathione-S-transferase (GST) in Escherichia coli, and used for the purification of HapC and the preparation of anti-HapC antiserum . The CCAAT-binding factor AnCP/AnCF contains a component with an approximate molecular mass of 32 kDa that cross-reacts with the antibody . The MalE-HapC fusion protein was able to replace authentic HapC in AnCP when incubated under appropriate conditions . Furthermore, reconstitution experiments with recombinant HapC, yHAP2 and yHAP5 polypeptides showed that all three polypeptides were required for the assembly of a complex capable of binding to CCAAT-containing taaG2 promoter DNA . The relationship between AnCP/AnCF and the Saccharomyces cerevisiae HAP complex is discussed.

Mol Cell Biol, 1998 Apr, 18(4), 1985 - 95
Multiple and distinct activation and repression sequences mediate the regulated transcription of IME1, a transcriptional activator of meiosis-specific genes in Saccharomyces cerevisiae; Sagee S et al.; IME1 encodes a transcriptional activator required for the transcription of meiosis-specific genes and initiation of meiosis in Saccharomyces cerevisiae . The transcription of IME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetate as the sole carbon source . Upon nitrogen depletion a transient induction in the transcription of IME1 is observed in MATa/MATalpha diploids but not in MAT-insufficient strains . In this study we demonstrate that the transcription of IME1 is controlled by an extremely unusual large 5' region, over 2,100 bp long . This area is divided into four different upstream controlling sequences (UCS) . UCS2 promotes the transcription of IME1 in the presence of a nonfermentable carbon source . UCS2 is flanked by three negative regions: UCS1, which exhibits URS activity in the presence of nitrogen, and UCS3 and UCS4, which repress the activity of UCS2 in MAT-insufficient cells . UCS2 consists of alternate positive and negative elements: three distinct constitutive URS elements that prevent the function of any upstream activating sequence (UAS) under all growth conditions, a constitutive UAS element that promotes expression under all growth conditions, a UAS element that is active only in vegetative media, and two discrete elements that function as UASs in the presence of acetate . Sequence analysis of IME1 revealed the presence of two almost identical 30- to 32-bp repeats . Surprisingly, one repeat, IREd, exhibits constitutive URS activity, whereas the other repeat, IREu, serves as a carbon-source-regulated UAS element . The RAS-cyclic AMP-dependent protein kinase cAPK pathway prevents the UAS activity of IREu in the presence of glucose as the sole carbon source, while the transcriptional activators Msn2p and Msn4p promote the UAS activity of this repeat in the presence of acetate . We suggest that the use of multiple negative and positive elements is essential to restrict transcription to the appropriate conditions and that the combinatorial effect of the entire region leads to the regulated transcription of IME1.

Mol Cell Biol, 1998 Apr, 18(4), 1891 - 902
Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA; Lewis LK et al.; RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae . Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells . Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase . Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains . In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining . Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed . Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination . These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.

Mol Cell Biol, 1998 Apr, 18(4), 1879 - 90
Identification and analysis of Mot3, a zinc finger protein that binds to the retrotransposon Ty long terminal repeat (delta) in Saccharomyces cerevisiae; Madison JM et al.; Spt3 and Mot1 are two transcription factors of Saccharomyces cerevisiae that are thought to act in a related fashion to control the function of TATA-binding protein (TBP) . Current models suggest that while Spt3 and Mot1 do not directly interact, they do function in a related fashion to stabilize the TBP-TATA interaction at particular promoters . Consistent with this model, certain combinations of spt3 and mot1 mutations are inviable . To identify additional proteins related to Spt3 and Mot1 functions, we screened for high-copy-number suppressors of the mot1 spt3 inviability . This screen identified a previously unstudied gene, MOT3, that encodes a zinc finger protein . We show that Mot3 binds in vitro to three sites within the retrotransposon Ty long terminal repeat (delta) sequence . One of these sites is immediately 5' of the delta TATA region . Although a mot3 null mutation causes no strong phenotypes, it does cause some mild phenotypes, including a very modest increase in Ty mRNA levels, partial suppression of transcriptional defects caused by a mot1 mutation, and partial suppression of an spt3 mutation . These results, in conjunction with those of an independent study of Mot3 (A . Grishin, M . Rothenberg, M . A . Downs, and K . J . Blumer, Genetics, in press), suggest that this protein plays a varied role in gene expression that may be largely redundant with other factors.

Mol Cell Biol, 1998 Apr, 18(4), 1855 - 65
Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae; Kressler D et al.; A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network . YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6) . Dbp6p is essential for cell viability . In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes . Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs . Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus . We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.

Biochim Biophys Acta, 1998 Feb 2, 1379(2), 245 - 56
Regulation of acid trehalase activity by association-dissociation in Saccharomyces cerevisiae; Biswas N et al.; Acid trehalase (AT) has always been reported to be copurified with invertase (I) and a 40 kDa additional protein . Glucose grown stationary phase cells of Saccharomyces cerevisiae contained least I activity . So, it was attempted to purify AT from these cells (I:AT = 10.83) . Studies on specific activity, percent recovery and I:AT ratio of different pools, collected during purification of AT, indicated that samples containing ratio I:AT < 2.2 were unstable . Purification methodology favouring association (DEAE-Sephadex chromatography) resulted in gaining total activity while methodology favouring dissociation (HPGPLC) resulted in tremendous loss in recovery . Active pool (Pool 1X) appeared to be electrophoretically homogeneous but dissociated into 175, 90, 68, 61, 57 (minor bands) and 37-41 (major band) molar mass (kDa) bands on SDS-PAGE . Inactive pools (Pools 1Y, 3X, 3Y) did not contain the 37-41 kDa major band . So, association of both I and a 37-41 kDa protein with AT appeared to be essential . Two bands of isoelectric pH (pI) 4.6 and 4.7 were present in pool 1X enzyme preparation . All SDS-PAGE-resolved bands of pool 1X, in an average, contained high aspartate/asparagine and low cysteine residues . AT activity appeared to be highly sensitive to the change in pH and also to agents affecting ionisation of protein, e.g., betaine, NaCl, acetate, etc . Association of AT components in presence of NaCl was demonstrated spectrophotometrically . Specific activity of AT decreased with dilution . Substrate mediated allosterism for this enzyme preparation suggested that AT existed as an equilibrium mixture of protomer-oligomer . It was suggested that reversible association-dissociation was a mechanism for the regulation of AT activity.

Curr Genet, 1998 Jan, 33(1), 10 - 5
Identification of a functional TATA element in the STA2 glucoamylase gene promoter from Saccharomyces cerevisiae; Vivier MA et al.; Accurate transcription by RNA polymerase II is usually dependent on the presence of a TATA element, and/or an initiator element, in the promoters of protein-encoding genes . The STA1-3 genes, encoding three glucoamylase isozymes (Sta1p, Sta2p and Sta3p, respectively) responsible for starch hydrolysis in the yeast Saccharomyces cerevisiae, have been shown to contain long and complex promoters with several regulatory regions . These promoters are also virtually identical to the yeast MUC1 gene promoter; this gene encodes a mucin-like protein and is evolutionary linked to, and transcriptionally co-regulated with, STA1-3 . The STA1-3 genes contain two putative TATA sequences; one conforming to the typical TATA box sequence, TATAAA, and another with the sequence of TATAAT . Here we present a study into the functional relevance of these putative TATA sequences and their effects on the transcription of the STA2 gene (as a representative model of the STA1-3 multigene family) and, by analogy, the MUC1 gene . We show that the TATAAA motif is the functional TATA box for STA2 and influences transcript levels, transcript initiation sites, and glucoamylase activities.

Curr Genet, 1998 Jan, 33(1), 4 - 9
Low glutathione pools in the original pso3 mutant of Saccharomyces cerevisiae are responsible for its pleiotropic sensitivity phenotype; Brendel M et al.; The original pso3-1 mutant isolate of the yeast Saccharomyces cerevisiae exhibits a pleiotropic mutagen-sensitivity phenotype that includes sensitivity to UVA-activated 3-carbethoxypsoralen, to UVC-light, to mono- and bi-functional nitrogen mustard, to paraquat, and to cadmium; on the other hand, it shows hyper-resistance (HYR) to nitrosoguanidine when compared to established wild-type strains . Also, the original pso3-1 mutant exhibits a low UVC-induced mutability and mitotic gene conversion and a high rate of spontaneous and UVC-induced petite mutations . Since the HYR to the nitrosoguanidine (MNNG) phenotype resembles that of low glutathione-containing yeast cells, the original pso3-1 mutant was crossed to a gsh1 knock-out mutant that lacks the enzyme for the first step in glutathione biosynthesis and the resulting diploid was tested for complementation . While there was none for HYR to nitrosoguanidine, and other low glutathione-related phenotypes, some other phenotypic characteristics of pso3-1, e.g . UVC sensitivity and UVC-induced mutability were restored to a wild-type level . Tetrad analysis of a diploid derived from a cross of the original haploid pso3-1 isolate with a repair-proficient, normal glutathione-containing, PSO3 GSH1 wild-type led to the separation of a leaky gsh1 mutation phenotype from that of the repair-deficient pso3-1 phenotype . Linkage studies by tetrad and random spore analyses indicated no linkage of the two genes . This shows that the low glutathione content in the original pso3-1 isolate is due to a second, additional, mutation in the GSH1 locus and is unrelated to the pso3-1 mutation . Thus, the original pso3-1 isolate is a pso3-1 gsh1 double mutant with most of the particular characteristics of the pleiotropic sensitivity phenotype contributed by either the pso3-1 or the gsh1-leaky mutant allele . The expression of a few phenotypic characteristics of pso3, however, were most pronounced in pso3-1 mutants with a low glutathione pool.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1228 - 33
Substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae: excision of guanine lesions produced in DNA by ionizing radiation- or hydrogen peroxide/metal ion-generated free radicals; Karahalil B et al.; We have investigated the substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae (yOgg1 protein) for excision of modified DNA bases from oxidatively damaged DNA substrates using gas chromatography/isotope dilution mass spectrometry . Four DNA substrates prepared by treatment with H2O2/Fe(III)-EDTA/ascorbic acid, H2O2/Cu(II) and gamma-irradiation under N2O or air were used . The results showed that 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were efficiently excised from DNA exposed to ionizing radiation in the presence of N2O or air . On the other hand, 8-OH-Gua and FapyGua were not excised from H2O2/Fe(III)-EDTA/ascorbic acid-treated and H2O2/Cu(II)-treated DNA respectively . Fourteen other lesions, including the adenine lesions 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine, were not excised from any of the DNA substrates . Kinetics of excision significantly depended on the nature of the damaged DNA substrates . The findings suggest that, in addition to 8-OH-Gua, FapyGua may also be a primary substrate of yOgg1 in cells . The results also show significant differences between the substrate specificities of yOgg1 protein and its functional analog Fpg protein in Escherichia coli.

J Biol Chem, 1998 Apr 3, 273(14), 8099 - 105
Coenzyme Q6 and iron reduction are responsible for the extracellular ascorbate stabilization at the plasma membrane of Saccharomyces cerevisiae; Santos-Ocana C et al.; Yeast plasma membrane contains an electron transport system that maintains ascorbate in its reduced form in the apoplast . Reduction of ascorbate free radical by this system is comprised of two activities, one of them dependent on coenzyme Q6 (CoQ6) . Strains with defects in CoQ6 synthesis exhibit decreased capacity for ascorbate stabilization compared with wild type or with atp2 or cor1 respiratory-deficient mutant strains . Both CoQ6 content in plasma membranes and ascorbate stabilization were increased during log phase growth . The addition of exogenous CoQ6 to whole cells resulted in its incorporation in the plasma membrane, produced levels of CoQ6 in the coq3 mutant strain that were 2-fold higher than in the wild type, and increased ascorbate stabilization activity in both strains, although it was higher in the coq3 mutant than in wild type . Other antioxidants, such as benzoquinone or alpha-tocopherol, did not change ascorbate stabilization . The CoQ6-independent reduction of ascorbate free radical was not due to copper uptake, pH changes or to the presence of CoQ6 biosynthetic intermediates, but decreased to undetectable levels when coq3 mutant strains were cultured in media supplemented with ferric iron . Plasma membrane CoQ6 levels were unchanged by either the presence or absence of iron in wild type, atp2, or cor1 strains . Ascorbate stabilization appears to be a function of the yeast plasma membrane, which is partially based on an electron transfer chain in which CoQ6 is the central electron carrier, whereas the remainder is independent of CoQ6 and other antioxidants but is dependent on the iron-regulated ferric reductase complex.

Biochemistry, 1998 Mar 17, 37(11), 3858 - 64
Labeling and identification of the postulated acid/base catalyst in the alpha-glucosidase from Saccharomyces cerevisiae using a novel bromoketone C-glycoside; Howard S et al.; alpha-Glucosidase from Saccharomyces cerevisiae is a member of a sequence-related family of alpha-glycosidases (family 13) that includes digestive alpha-amylases and commercially important cyclodextrin glucanotransferases . These enzymes catalyze the hydrolysis of alpha-linked oligosaccharides by a two-step mechanism involving a glycosyl-enzyme intermediate . A novel bromoketone C-glycoside inactivator, 1'-bromo-3'-(alpha-D-mannopyranosyl)-2'-propanone, has been synthesized and used to label the putative acid/base catalyst (Glu-276) of yeast alpha-glucosidase . Electrospray ionization mass spectrometry was used to demonstrate stoichiometric labeling of the protein . The labeled residue was identified by comparative liquid chromatographic/mass spectrometric analysis of peptic digests of labeled and unlabeled enzyme samples, which confirmed the unique presence of two labeled peptides of m/z 745 and 694 . Subsequent tandem mass spectrometric analysis in the daughter-ion scan mode showed the two peptides to have an overlapping sequence in which Glu-276 was the labeled residue . Together with active-site-directed protection against inactivation with deoxynojirimycin, these results prove that Glu-276 is located within the active site of yeast alpha-glucosidase and, thus, provide further evidence for this residue playing an important role in catalysis.

Protein Sci, 1998 Feb, 7(2), 500 - 3
High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae a1 homeodomain expressed in Escherichia coli; Forman MD et al.; The Saccharomyces cerevisiae a1 homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium . Nuclear magnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances . Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site . Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E . coli genes . Lower level mistranslation at three other rare arginine codons also occurs . The percentage of lysine for arginine misincorporation in a1 homeodomain production is dependent on media composition . The dnaY gene, which encodes the rare 5'-AGA-3' tRNA(ARG), was co-expressed in E . coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain . Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression in minimal media, and homogeneous protein is produced.

Mol Gen Genet, 1998 Feb, 257(3), 362 - 7
Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae; Luhr B et al.; We have analysed the correction of defined mismatches in wild-type and msh2, msh3, msh6 and msh3 msh6 mutants of Saccharomyces cerevisiae in two different yeast strain backgrounds by transformation with plasmid heteroduplex DNA constructs . Ten different base/base mismatches, two single-nucleotide loops and a 38-nucleotide loop were tested . Repair of all types of mismatches was severely impaired in msh2 and msh3 msh6 mutants . In msh6 mutants, repair efficiency of most base/base mismatches was reduced to a similar extent as in msh3 msh6 double mutants . G/T and A/C mismatches, however, displayed residual repair in msh6 mutants in one strain background, implying a role for Msh3p in recognition of base/base mismatches . Furthermore, the efficiency of repair of base/base mismatches was considerably reduced in msh3 mutants in one strain background, indicating a requirement for MSH3 for fully efficient mismatch correction . Also the efficiency of repair of the 38-nucleotide loop was reduced in msh3 mutants, and to a lesser extent in msh6 mutants . The single-nucleotide loop with an unpaired A was less efficiently repaired in msh3 mutants and that with an unpaired T was less efficiently corrected in msh6 mutants, indicating non-redundant functions for the two proteins in the recognition of single-nucleotide loops.

Protein Expr Purif, 1998 Mar, 12(2), 173 - 84
Cloning, overexpression, and purification of cytosine deaminase from Saccharomyces cerevisiae; Hayden MS et al.; Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil . To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae . The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons . Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes . Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein . Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil . Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains . A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps . This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.

J Biol Chem, 1998 Feb 27, 273(9), 4950 - 6
Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae does not promote branch migration in vitro; Zhang Z et al.; It has been shown in vitro that Saccharomyces cerevisiae strand exchange protein 1 (Sep1) promotes the transfer of one strand of a linear duplex DNA to a homologous single-stranded DNA circle . Sep1 also has an exonuclease active on DNA and RNA . By using exonuclease III-treated linear duplex DNA with various lengths of single-stranded tail as well as Ca2+ to inhibit the exonuclease activity of Sep1, we show that the processivity of exonuclease activity of Sep1 is greater than previously reported . The results in this work also demonstrate that the joint molecule between the linear duplex and single-stranded circle observed from the Sep1-promoted strand transfer reaction is just the pairing between the long single-stranded tail of the linear duplex DNA (generated by the exonuclease activity of Sep1) and the single-stranded circular DNA . When a synthetic Holliday junction was used as substrate, branch migration facilitated by Sep1 could not be detected . Finally, using electron microscopy no alpha-structure, a joint molecule with displaced single-stranded DNA tail that indicates branch migration could be observed . The results imply that Sep1 cannot promote branch migration in vitro . Further investigation is needed to determine the role of Sep1 in recombination in vivo.

EMBO J, 1998 Feb 16, 17(4), 1128 - 40
Dob1p (Mtr4p) is a putative ATP-dependent RNA helicase required for the 3' end formation of 5.8S rRNA in Saccharomyces cerevisiae; de la Cruz J et al.; The temperature-sensitive mutation, dob1-1, was identified in a screen for dependence on overexpression of the yeast translation initiation factor eIF4B (Tif3p) . Dob1p is an essential putative ATP-dependent RNA helicase . Polysome analyses revealed an under accumulation of 60S ribosomal subunits in the dob1-1 mutant . Pulse-chase labelling of pre-rRNA showed that this was due to a defect in the synthesis of the 5.8S and 25S rRNAs . Northern and primer extension analyses in the dob1-1 mutant, or in a strain genetically depleted of Dob1p, revealed a specific inhibition of the 3' processing of the 5.8S rRNA from its 7S precursor . This processing recently has been attributed to the activity of the exosome, a complex of 3'-->5' exonucleases that includes Rrp4p . In vivo depletion of Dob1p also inhibits degradation of the 5' external transcribed spacer region of the pre-rRNA . A similar phenotype was observed in rrp4 mutant strains and, moreover, the dob1-1 and rrp4-1 mutations show a strong synergistic growth inhibition . We propose that Dob1p functions as a cofactor for the exosome complex that unwinds secondary structures in the pre-rRNA that otherwise block the progression of the 3'-->5' exonucleases.

J Biol Chem, 1998 Feb 13, 273(7), 4040 - 5
An essential domain within Cdc34p is required for binding to a complex containing Cdc4p and Cdc53p in Saccharomyces cerevisiae; Mathias N et al.; The CDC34 gene of the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating protein that transfers ubiquitin onto substrates to signal rapid degradation via the proteasome . Cdc34p has been implicated in signaling the destruction of a variety of substrates including the cyclin-dependent kinase inhibitor, Sic1p, which must be degraded for cells to enter S-phase . Mutants lacking CDC34 activity fail to degrade Sic1p and fail to enter S-phase, a phenotype that is also shared with cells lacking CDC4 and CDC53 activity . Here we demonstrate that Cdc4p, Cdc34p, and Cdc53p interact in vivo . We have mapped a Cdc4p/Cdc53p-binding region on Cdc34p; this region is essential for S-phase entry and thus the association of these three proteins is required for Sic1p degradation . All three proteins migrate in gel filtration to sizes that greatly exceed their actual size suggesting that they form stable associations with other proteins and we observe Cdc4p, Cdc34p, and Cdc53p fractionating into overlapping families of high molecular weight complexes . Finally, we demonstrate that Cdc4p, Cdc34p, and Cdc53p are stable throughout the cell cycle and that Cdc34p permanently resides as part of a complex throughout the cell cycle . This suggests that all Cdc34p substrates are ubiquitinated by a similar high molecular weight complex.

J Bacteriol, 1998 Mar, 180(6), 1418 - 24
Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity; Ram AF et al.; Deletion of GAS1/GGP1/CWH52 results in a lower beta-glucan content of the cell wall and swollen, more spherical cells (L . Popolo, M . Vai, E . Gatti, S . Porello, P . Bonfante, R . Balestrini, and L . Alberghina, J . Bacteriol . 175:1879-1885, 1993; A . F . J . Ram, S . S . C . Brekelmans, L . J . W . M . Oehlen, and F . M . Klis, FEBS Lett . 358:165-170, 1995) . We show here that gas1delta cells release beta1,3-glucan into the medium . Western analysis of the medium proteins with beta1,3-glucan- and beta1,6-glucan-specific antibodies showed further that at least some of the released beta1,3-glucan was linked to protein as part of a beta1,3-glucan-beta1,6-glucan-protein complex . These data indicate that Gas1p might play a role in the retention of beta1,3-glucan and/or beta-glucosylated proteins . Interestingly, the defective incorporation of beta1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in beta1,3-glucan synthase activity, probably caused by the induced expression of Fks2p . It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.

Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 239 - 42
The GTS1 gene product influences the ultradian oscillation of glycolysis in cell suspension of the yeast Saccharomyces cerevisiae; Wang J et al.; We tested the effect of the GTS1 gene of the yeast Saccharomyces cerevisiae on the cyanide-induced ultradian oscillation of the glycolytic metabolite NADH in cell suspension of strains with different copy numbers of the gene, that is, the wild-type, GTS1-disrupted and GTS1-overexpressing strains . The cells showed long-lasting oscillations when harvested between 1 and 2 hours after the diauxic shift from glucose to ethanol as a growth substrate . The frequencies of oscillation did not vary very much among the three strains tested . However, the amplitudes and durations of the oscillation were changed significantly as a function of the GTS1 gene-dosage . The effect of GTS1 on the amplitude was not caused by changing rates of glucose incorporation into cells as the rates were the same among the three strains during the macroscopic oscillation.

FEMS Microbiol Lett, 1997 Nov 15, 156(2), 245 - 9
Mutation thi81 causing a deficiency in the signal transduction of thiamine pyrophosphate in Saccharomyces cerevisiae; Nishimura H et al.; We isolated a strain carrying a recessive constitutive mutation (thi81) for the expression of thiamine metabolism in Saccharomyces cerevisiae . The thi81 mutant exhibits significant thiamine transport, thiamine-repressible acid phosphatase (T-rAPase) activities and significant activities of enzymes involved in thiamine biosynthesis which are repressed in the wild-type strain in medium supplemented with thiamine (2 x 10(-7) M) . The thi81 mutant exhibited the same level of thiamine pyrophosphokinase activity and intracellular thiamine pyrophosphate concentration as the wild-type strain in medium supplemented with exogenous thiamine . The mutant strain constitutively produced PHO3 mRNA encoding T-rAPase in medium supplemented with thiamine . These results suggest that the thi81 mutant lacks a negative factor involved in the regulation of the genes encoding proteins involved in yeast thiamine metabolism.

Biochim Biophys Acta, 1998 Feb 11, 1395(3), 315 - 20
Molecular identification of glutathione synthetase (GSH2) gene from Saccharomyces cerevisiae; Inoue Y et al.; The hypothetical protein YOL049w on the chromosome XV was identified to be the structural gene for glutathione synthetase (GSH2) of Saccharomyces cerevisiae . Translational initiation site was identified by making the GSH2-lacZ fusion . The GSH2 gene contained an open reading frame (1473 bp) with 491 amino acids, and molecular weight of the GSH2 gene product was calculated to be 55,812 . Glutathione synthetase activity in transformant carrying the GSH2 gene with multicopy plasmid increased approximately 4-fold . The GSH2 gene was not essential for growth of yeast cell, and glutathione was not detected from the gsh2 disrupter.

FEBS Lett, 1998 Feb 20, 423(2), 149 - 54
Son1p is a component of the 26S proteasome of the yeast Saccharomyces cerevisiae; Fujimuro M et al.; A son1 mutant was isolated as a mutant showing synthetic lethality with nin1-1 which is defective in the p31 component of the regulatory subunit of the yeast 26S proteasome . son1delta showed a synthetic effect with sen3delta and sun1delta, both components of the 26S proteasome, and with cdc28-1N . The 26S proteasome was partially purified from the wild type yeast . The FPLC fractions were analyzed by Western blotting using anti-Son1p antibody and antibodies against some authentic subunits of the 26S proteasome, and we found that Son1p co-migrated with components of the 26S proteasome . The 26S proteasome containing fraction was immunoprecipitated with anti-Son1p antibody . The resultant precipitate contained Nin1p, Sun1p, TBP1, and the 20S proteasome . Combining genetic and biochemical results together, we concluded that Son1p is a component of the yeast 26S proteasome.

Yeast, 1997 Dec, 13(16), 1535 - 45
Green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae; Walmsley RM et al.; The green fluorescent protein (GFP) of Aequorea victoria is now an established marker for gene expression and subcellular localization in budding yeast . Relatively high expression (greater than 2500 copies per cell) of GFP is required for direct microscopic visualization . This report provides a method for studying the expression of less highly expressed genes by the analysis of crude cell extracts--a simple and cheap alternative to the fluorescent activated cell sorter (FACS) . The utility of this marker is demonstrated in a study of the expression of the RAD54 gene . It is shown that the induction of the RAD54 promoter leads to the accumulation of Rad54p and of GFP and that the fluorescence induction is correctly regulated . This method should allow the screening of large numbers of novel gene disrupters for their effects on RAD54 expression and so identify trans-acting factors involved in the cellular response to DNA damage.

RNA, 1998 Mar, 4(3), 303 - 18
Transcription termination downstream of the Saccharomyces cerevisiae FBP1 {changed from FPB1} poly(A) site does not depend on efficient 3'end processing; Aranda A et al.; Efficient transcription termination downstream of poly(A) sites has been shown to correlate with the strength of an upstream polyadenylation signal and the presence of a polymerase pause site . To further investigate the mechanism linking termination with 3'-end processing, we analyzed the cis-acting elements that contribute to these events in the Saccharomyces cerevisiae FBP1 gene . FBP1 has a complex polyadenylation signal, and at least three efficiency elements must be present for efficient processing . However, not all combinations of these elements are equally effective . This gene also shows a novel organization of sequence elements . A strong positioning element is located upstream, rather than downstream, of the efficiency elements, and functions to select the cleavage site in vitro and in vivo . Transcription run-on analysis indicated that termination occurs within 61 nt past the poly(A) site . Deletion of two UAUAUA-type efficiency elements greatly reduces polyadenylation in vivo and in vitro, but transcription termination is still efficient, implying that FBP1 termination signals may be distinct from those for polyadenylation . Alternatively, assembly of a partial, but nonfunctional, polyadenylation complex on the nascent transcript may be sufficient to cause termination.

Appl Environ Microbiol, 1998 Mar, 64(3), 1139 - 42
Hydrostatic pressure enhances vital staining with carboxyfluorescein or carboxydichlorofluorescein in Saccharomyces cerevisiae: efficient detection of labeled yeasts by flow cytometry; Abe F; The extent of intracellular accumulation of the fluorescent dye carboxyfluorescein or carboxydichlorofluorescein (CDCF) in Saccharomyces cerevisiae was found to be increased 5- to 10-fold under a nonlethal hydrostatic pressure of 30 to 50 MPa . This observation was confirmed by analysis of individual labeled cells by flow cytometry . The pressure-induced enhancement of staining with CDCF required D-glucose and was markedly inhibited by 2-deoxy-D-glucose, suggesting that glucose metabolism has a role in the process.

J Biol Chem, 1998 Feb 20, 273(8), 4424 - 35
Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities . Role of the unique N terminus and putative intercalating methionine; Yen YM et al.; The non-histone proteins 6A/B (NHP6A/B) of Saccharomyces cerevisiae are high mobility group proteins that bind and severely bend DNA of mixed sequence . They exhibit high affinity for linear DNA and even higher affinity for microcircular DNA . The 16-amino acid basic segment located N-terminal to the high mobility group domain is required for stable complex formation on both linear and microcircular DNA . Although mutants lacking the N terminus are able to promote microcircle formation and Hin invertasome assembly at high protein concentrations, they are unable to form stable complexes with DNA, co-activate transcription, and complement the growth defect of Deltanhp6a/b mutants . A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch between residues 8 and 10 is required for the formation of monomeric complexes with linear DNA . Mutational analysis suggests that proline 18 may direct the path of the N-terminal arm to facilitate DNA binding, whereas the conserved proline at position 21, tyrosine 28, and phenylalanine 31 function to maintain the tertiary structure of the high mobility group domain . Methionine 29, which may intercalate into DNA, is essential for NHP6A-induced microcircle formation of 75-bp but not 98-bp fragments in vitro, and for full growth complementation of Deltanhp6a/b mutants in vivo.

Nucleic Acids Res, 1998 Feb 15, 26(4), 1002 - 9
Identification of cis-acting elements in the SUC2 promoter of Saccharomyces cerevisiae required for activation of transcription; Bu Y et al.; We analyzed the effects of site-directed mutations in the SUC2 promoter of Saccharomyces cerevisiae . Analyses were performed in wild-type as well as mig1 and tup1 mutant strains after the promoter mutants were reintroduced into the native SUC2 locus on the left arm of chromosome IX . Mutation of the two GC boxes revealed that these elements play two distinct roles: they are, as expected, required for Mig1-mediated repression but they are also necessary for activation of the SUC2 promoter in response to glucose limitation . The individual GC boxes are functionally redundant with regard to Mig1-mediated repression, however, only the upstream GC box is essential for high level expression of SUC2 . Microccocal nuclease sensitivity of the SUC2 promoter in derepressed cells was reduced in the GC box mutant promoters, particularly in the vicinity of the TATA box . The difference in nuclease sensitivity between wild-type and GC box mutant promoters was not evident in tup1- cells . The formation of nuclease-resistant chromatin does not require the GC boxes, indicating that other cis-acting elements can serve to recruit the Ssn6-Tup1 co-repressor complex to the SUC2 promoter.

Genetics, 1998 Feb, 148(2), 625 - 35
Afr1p regulates the Saccharomyces cerevisiae alpha-factor receptor by a mechanism that is distinct from receptor phosphorylation and endocytosis; Davis C et al.; The alpha-factor pheromone receptor activates a G protein signaling pathway that induces the conjugation of the yeast Saccharomyces cerevisiae . Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to alpha-factor . AFR1 also showed an interesting genetic relationship with the alpha-factor receptor gene, STE2, suggesting that the receptor is regulated by Afr1p . To investigate the mechanism of this regulation, we tested AFR1 for a role in the two processes that are known to regulate receptor signaling: phosphorylation and down-regulation of ligand-bound receptors by endocytosis . AFR1 overexpression diminished signaling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylation . The effects of AFR1 overexpression were weaker in strains that were defective in receptor endocytosis . However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein . Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to the number of receptors . These results indicate that AFR1 acts independently of endocytosis, and that the weaker effects of AFR1 in strains that are defective in receptor endocytosis were probably an indirect consequence of their increased receptor number caused by the failure of receptors to undergo ligand-stimulated endocytosis . Analysis of the ligand binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity for alpha-factor . Thus, Afr1p prevents alpha-factor receptors from activating G protein signaling by a mechanism that is distinct from other known pathways.

Genetics, 1998 Feb, 148(2), 599 - 610
Dominant alleles of Saccharomyces cerevisiae CDC20 reveal its role in promoting anaphase; Schott EJ et al.; We identified an allele of Saccharomyces cerevisiae CDC20 that exhibits a spindle-assembly checkpoint defect . Previous studies indicated that loss of CDC20 function caused cell cycle arrest prior to the onset of anaphase . In contrast, CDC20-50 caused inappropriate cell cycle progression through M phase in the absence of mitotic spindle function . This effect of CDC20-50 was dominant over wild type and was eliminated by a second mutation causing loss of function, suggesting that it encodes an overactive form of Cdc20p . Overexpression of CDC20 was found to cause a similar checkpoint defect, causing bypass of the preanaphase arrest produced by either microtubule-depolymerizing compounds or MPS1 overexpression . CDC20 overexpression was also able to overcome the anaphase delay caused by high levels of the anaphase inhibitor Pds1p, but not a mutant form immune to anaphase-promoting complex- (APC-)mediated proteolysis . CDC20 overexpression was unable to promote anaphase in cells deficient in APC function . These findings suggest that Cdc20p is a limiting factor that promotes anaphase entry by antagonizing Pds1p . Cdc20p may promote the APC-dependent proteolytic degradation of Pds1p and other factors that act to inhibit cell cycle progression through mitosis.

Genetics, 1998 Feb, 148(2), 571 - 9
Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae; Hata H et al.; The POP2 (Caf1) protein in Saccharomyces cerevisiae affects a variety of transcriptional processes and is a component of the Ccr4p complex . We have isolated five multicopy suppressor genes of a pop2 deletion mutation: CCR4, DHH1 (a putative RNA helicase), PKC1, STM1, and MPT5 (multicopy suppressor of pop two) . Overexpression of either the CCR4 or DHH1 genes effectively suppressed phenotypes associated with pop2 mutant cells; overexpression of PKC1, STM1, or MPT5 genes produced only partial suppression . Disruption of the CCR4 or DHH1 genes resulted in phenotypes similar to those observed for pop2 cells . In addition, overexpression of the DHH1 gene also suppressed the ccr4 mutation, suggesting a close relationship between the POP2, CCR4, and DHH1 genes . Two-hybrid analysis and coimmunoprecipitation experiments revealed that Pop2p and Dhh1p interact physically, and these and other data suggest that Dhh1p is also a component of the Ccr4p complex . Finally, we investigated the genetic interaction between factors associated with POP2 and the PKC1 pathway . The temperature-sensitive growth defect of dhh1 or mpt5 cells was suppressed by overexpression of PKC1, and the defect of mpk1 cells was suppressed by overexpression of MPT5 . These results and phenotypic analysis of double mutants from the POP2 and PKC1 pathways suggested that the POP2 and the PKC1 pathways are independent but have some overlapping functions.

Genetics, 1998 Feb, 148(2), 559 - 69
The transcriptional regulator Hap1p (Cyp1p) is essential for anaerobic or heme-deficient growth of Saccharomyces cerevisiae: Genetic and molecular characterization of an extragenic suppressor that encodes a WD repeat protein; Chantrel Y et al.; We report here that Hap1p (originally named Cyp1p) has an essential function in anaerobic or heme-deficient growth . Analysis of intragenic revertants shows that this function depends on the amino acid preceding the first cysteine residue of the DNA-binding domain of Hap1p . Selection of recessive extragenic suppressors of a hap1-hem1- strain allowed the identification, cloning, and molecular analysis of ASC1 (Cyp1 Absence of growth Supressor) . The sequence of ASC1 reveals that its ORF is interrupted by an intron that shelters the U24 snoRNA . Deletion of the intron, inactivation of the ORF, and molecular localization of the mutations show unambiguously that it is the protein and not the snoRNA that is involved in the suppressor phenotype . ASC1, which is constitutively transcribed, encodes an abundant, cytoplasmically localized 35-kD protein that belongs to the WD repeat family, which is found in a large variety of eucaryotic organisms . Polysome profile analysis supports the involvement of this protein in translation . We propose that the absence of functional Asc1p allows the growth of hap1-hem1- cells by reducing the efficiency of translation . Based on sequence comparisons, we discuss the possibility that the protein intervenes in a kinase-dependent signal transduction pathway involved in this last function.

J Cell Biol, 1998 Feb 9, 140(3), 577 - 90
Retrieval of resident late-Golgi membrane proteins from the prevacuolar compartment of Saccharomyces cerevisiae is dependent on the function of Grd19p; Voos W et al.; The dynamic vesicle transport processes at the late-Golgi compartment of Saccharomyces cerevisiae (TGN) require dedicated mechanisms for correct localization of resident membrane proteins . In this study, we report the identification of a new gene, GRD19, involved in the localization of the model late-Golgi membrane protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A {DPAP A} fused to the transmembrane and lumenal domains of the alkaline phosphatase {ALP}), which localizes to the yeast TGN . A grd19 null mutation causes rapid mislocalization of the late-Golgi membrane proteins A-ALP and Kex2p to the vacuole . In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting . The recycling of the carboxypeptidase Y sorting receptor, Vps10p, between the TGN and the prevacuolar compartment is largely unaffected in grd19Delta cells . Kinetic assays of A-ALP trafficking indicate that GRD19 is involved in the process of retrieval of A-ALP from the prevacuolar compartment . GRD19 encodes a small hydrophilic protein with a predominantly cytosolic distribution . In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment . Using an in vitro binding assay, Grd19p was found to interact physically with the cytosolic domain of DPAP A . We conclude that Grd19p is a component of the retrieval machinery that functions by direct interaction with the cytosolic tails of certain TGN membrane proteins during the sorting/budding process at the prevacuolar compartment.

EMBO J, 1998 Feb 2, 17(3), 677 - 87
Peroxisomal beta-oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions; van Roermund CW et al.; The beta-oxidation of saturated fatty acids in Saccharomyces cerevisiae is confined exclusively to the peroxisomal compartment of the cell . Processing of mono- and polyunsaturated fatty acids with the double bond at an even position requires, in addition to the basic beta-oxidation machinery, the contribution of the NADPH-dependent enzyme 2,4-dienoyl-CoA reductase . Here we show by biochemical cell fractionation studies that this enzyme is a typical constituent of peroxisomes . As a consequence, the beta-oxidation of mono- and polyunsaturated fatty acids with double bonds at even positions requires stoichiometric amounts of intraperoxisomal NADPH . We suggest that NADP-dependent isocitrate dehydrogenase isoenzymes function in an NADP redox shuttle across the peroxisomal membrane to keep intraperoxisomal NADP reduced . This is based on the finding of a third NADP-dependent isocitrate dehydrogenase isoenzyme, Idp3p, next to the already known mitochondrial and cytosolic isoenzymes, which turned out to be present in the peroxisomal matrix . Our proposal is strongly supported by the observation that peroxisomal Idp3p is essential for growth on the unsaturated fatty acids arachidonic, linoleic and petroselinic acid, which require 2, 4-dienoyl-CoA reductase activity . On the other hand, growth on oleate which does not require 2,4-dienoyl-CoA reductase, and NADPH is completely normal in Deltaidp3 cells.

Nucleic Acids Res, 1998 Feb 1, 26(3), 860 - 1
PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae; Nikawa J et al.; We developed a novel method for synthesizing marker-disrupted alleles of yeast genes . The first step is PCR amplification of two sequences located upstream and downstream of the reading frame to be disrupted . Due to the addition of non-specific single A overhangs by Taq DNA polymerase, each PCR product can be ligated with a marker DNA which has T residues at its 3' ends . After amplification of individual ligation products through the second PCR, both products are mixed and annealed, and the single strand is converted to a double strand by an extension reaction . The final step is PCR amplification of the fragment composed of a selectable marker and two flanking sequences with the outermost primers . This method is rapid and needs only short oligonucleotides as primers.

Nucleic Acids Res, 1998 Feb 1, 26(3), 730 - 4
The small subunits of human and mouse DNA polymerase epsilon are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase epsilon; Jokela M et al.; Human DNA polymerase epsilon is composed of a 261 kDa catalytic polypeptide and a 55 kDa small subunit of unknown function . cDNAs encoding the small subunit of human and mouse DNA polymerase epsilon were cloned . The predicted polypeptides have molecular masses of 59.469 and 59.319 kDa respectively and they are 90% identical . The human and mouse polypeptides show 22% identity with the 80 kDa subunit of the five subunit DNA polymerase epsilon from the yeast Saccharomyces cerevisiae . The high degree of conservation suggests that the 55 kDa subunit shares an essential function with the yeast 80 kDa subunit, which was earlier suggested to be involved in S phase cell cycle control in a pathway that is able to sense and signal incomplete replication . The small subunits of human and mouse DNA polymerase epsilon also show homology to the C-terminal domain of the second largest subunit of DNA polymerase alpha . The gene for the small subunit of human DNA polymerase epsilon (POLE2) was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.

Mol Biol Cell, 1998 Jan, 9(1), 161 - 71
The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae; Lo WS et al.; Diploid yeast develop pseudohyphae in response to nitrogen starvation, while haploid yeast produce invasive filaments which penetrate the agar in rich medium . We have identified a gene, FLO11, that encodes a cell wall protein which is critically required for both invasion and pseudohyphae formation in response to nitrogen starvation . FLO11 encodes a cell surface flocculin with a structure similar to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins . Cells of the Saccharomyces cerevisiae strain Sigma1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids . In rich media, FLO11 is regulated by mating type; it is expressed in haploid cells but not in diploids . Upon transfer to nitrogen starvation media, however, FLO11 transcripts accumulate in diploid cells, but not in haploids . Overexpression of FLO11 in diploid cells, which are otherwise not invasive, enables them to invade agar . Thus, the mating type repression of FLO11 in diploids grown in rich media suffices to explain the inability of these cells to invade . The promoter of FLO11 contains a consensus binding sequence for Ste12p and Tec1p, proteins known to cooperatively activate transcription of Ty1 elements and the TEC1 gene during development of pseudohyphae . Yeast with a deletion of STE12 does not express FLO11 transcripts, indicating that STE12 is required for FLO11 expression . These ste12-deletion strains also do not invade agar . However, the ability to invade can be restored by overexpressing FLO11 . Activation of FLO11 may thus be the primary means by which Ste12p and Tec1p cause invasive growth.

J Bacteriol, 1998 Mar, 180(5), 1044 - 52
Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae; Boy-Marcotte E et al.; The multicopy suppressors of the snf1 defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive cis element (CCCCT) in response to various stresses . This cis element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway . We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of the msn2 msn4 double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition . Thirty-nine gene products (including those of ALD3, GDH3, GLK1, GPP2, HSP104, HXK1, PGM2, SOD2, SSA3, SSA4, TKL2, TPS1, and YBR149W) are dependent upon Msn2/4p for their induction at the diauxic transition . The expression of all these genes is repressed by cAMP . Thirty other genes identified during this study are still inducible in the mutant . A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including ACH1, ADH2, ALD6, ATP2, GPD1, ICL1, and KGD2) . We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response . In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift . Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway.

FEMS Microbiol Lett, 1998 Mar 1, 160(1), 31 - 6
The C-terminal domain of Snf3p mediates glucose-responsive signal transduction in Saccharomyces cerevisiae; Vagnoli P et al.; The SNF3 protein is composed of distinct cytoplasmic and integral-membrane domains and functions as a low glucose sensor required for the expression of hexose transporters (the HXT genes) in Saccharomyces . We report herein that the C-terminal domain, when expressed independently of the integral membrane domain, leads to glucose-independent expression of HXT2 on gluconeogenic carbon sources . The C-terminal-domain-induced expression of Hxt2p is reduced in a SNF3 wild-type strain, suggesting that Snf3p competes with this C-terminal peptide for interacting downstream elements . The probable active site for the signal transducing interaction was mapped to either of the redundant 17 of 23 amino acid sequences found in this C-terminal domain.

Genes Cells, 1997 Nov, 2(11), 667 - 78
Replication profile of Saccharomyces cerevisiae chromosome VI; Friedman KL et al.; BACKGROUND: An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA . As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae . RESULTS: Chromosome VI shows a wide variation in the percentage of cell cycles in which different replication origins are utilized . Most of the origins are activated in only a fraction of cells, suggesting that the pattern of origin usage on chromosome VI varies greatly within the cell population . The replication times of fragments containing chromosome VI origins show a temporal pattern that has been recognized on other chromosomes--the telomeres replicate late in S phase, while the central region of the chromosome replicates early . CONCLUSIONS: As demonstrated here for chromosome VI, analysis of the direction of replication fork movement along a chromosome and determination of replication time by measuring a period of hemimethylation may provide an efficient means of surveying origin activity over large regions of the genome.

Mol Gen Genet, 1998 Jan, 257(2), 230 - 7
Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination; Urrestarazu A et al.; This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 and aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9 affects aromatic aminotransferase II . Neither of the single mutants displays any nutritional requirement on minimal ammonia medium . In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, alpha-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor . Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known alpha-aminoadipate aminotransferases . Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways . Aromatic aminotransferase II also has a broader substrate specificity than initially described . In particular, it is responsible for all the measured kynurenine aminotransferase activity . Mutants lacking this activity grow very slowly on kynurenine medium.

Mol Gen Genet, 1998 Jan, 257(2), 143 - 8
Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1; Kishi T et al.; Cdc34, a ubiquitin-conjugating enzyme in Saccharomyces cerevisiae, is required for cell cycle progression . sic1, an S-phase cyclin-dependent kinase (CDK) inhibitor, is a critical target of Cdc34-mediated ubiquitination . Other essential target protein(s) could be defined since cdc34 sic1 double mutants still arrest in G2 phase . To identify proteins which function in the Cdc34-dependent ubiquitin pathway, a series of extragenic suppressors of the cdc34-1 sic1 double mutations was isolated . One of them was found to be defective in GRR1, which is involved not only in glucose repression but also in G1 cyclin destabilization . However, neither lack of glucose repression nor stabilization of G1 cyclin caused the suppression of cdc34-1 sic1 . Conversely, Grr1 overproduction in cdc34-1 sic1 cells impaired colony formation, even at the permissive temperature . A multicopy suppressor, MGO1, which rescued the growth defect associated with Grr1 overproduction was isolated, and found to be identical to SKP1 . Furthermore, Grr1 bound Skp1 directly in vitro . These results strongly suggest that Grr1 functions in the ubiquitin pathway through association with Skp1.

Mol Microbiol, 1998 Feb, 27(3), 643 - 50
The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae; Didion T et al.; Amino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity . We present evidence that a member of this family, encoded by the open reading frame (ORF) YDR160w (SSY1), is required for the expression of a set of transporter genes . Thus, deletion of the SSY1 gene causes loss of leucine-inducible transcription of the amino acid permease genes BAP2, TAT1 and BAP3 (ORF YDR046c) and the peptide transporter, PTR2 . D-leucine can generate the signal without entering the cell . We propose that Ssy1p is situated in the plasma membrane and is involved in sensing leucine in the medium.

Biochemistry, 1998 Feb 3, 37(5), 1264 - 73
Secretion efficiency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability; Kowalski JM et al.; Bovine pancreatic trypsin inhibitor (BPTI) has been widely used as a model protein to investigate protein structure and folding pathways . To study the role of its three disulfide bonds in folding, proofreading, and secretion of BPTI in an intact eucaryotic cell, BPTI was expressed and secreted from a synthetic gene in the yeast Saccharomyces cerevisiae . Site-directed mutagenesis was used to create all possible single and pairwise cysteine to alanine BPTI mutants, and the effect of these mutations on secretion efficiency was determined . The 5-55 disulfide bond is found to be essential for secretion-loss of either Cys5, Cys55, or both prevents secretion . Removal of the 14-38 disulfide bond results in a small reduction of secretion, but individual Cys14 or Cys38 replacements reduce secretion efficiency by 30% . Cys30 and Cys30-51 mutants are secreted at half the level of wild-type BPTI, while secretion of the Cys51 mutant is reduced by 90% . BPTI containing only a single disulfide bond (5-55) is not secreted . No relationship is observed between secretion efficiency and in vitro folding or unfolding rates, but mutant BPTI secretion is directly correlated with the in vitro unfolding temperature Tm and the free energy of stabilization provided by each of the three disulfides . These results indicate that structural fluctuations rather than the time-averaged structure observed by NMR or X-ray crystallography may determine recognition of a protein as misfolded and subsequent retention and degradation.

Mol Cell Biol, 1998 Mar, 18(3), 1736 - 45
Nonhomologous end joining during restriction enzyme-mediated DNA integration in Saccharomyces cerevisiae; Manivasakam P et al.; The BamHI restriction enzyme mediates integration of nonhomologous DNA into the Saccharomyces cerevisiae genome (R . H . Schiestl and T . D . Petes, Proc . Natl . Acad . Sci . USA 88:7585-7589, 1991) . The present study investigates the mechanism of such events: in particular, the mediating activity of various restriction enzymes and the processing of resultant fragment ends . Our results show that in addition to BamHI, BglII and KpnI increase DNA integration efficiencies severalfold, while Asp718, HindIII, EcoRI, SalI, SmaI, HpaI, MscI, and SnaBI do not . Secondly, the three active enzymes stimulated integrations only of fragments containing 5' or 3' overhangs but not of blunt-ended fragments . Thirdly, integrations mediated by one enzyme and utilizing a substrate created by another required at least 2 bp of homology . Furthermore, an Asp718 fragment possessing a 5' overhang integrated into a KpnI (isoschizomer) site possessing a 3' overhang, most likely by filling of the 5' overhang followed by 5' exonuclease digestion to produce a 3' end . We classified and analyzed the restriction enzyme-mediated integration events in the context of their genomic positions . The majority of events integrated into single sites . In the remaining 6 of 19 cases each end of the plasmid inserted into a different sequence, producing rearrangements such as duplications, deletions, and translocations.

Mol Cell Biol, 1998 Mar, 18(3), 1424 - 35
DNA-binding activities of Hop1 protein, a synaptonemal complex component from Saccharomyces cerevisiae; Kironmai KM et al.; The meiosis-specific HOP1 gene is important both for crossing over between homologs and for production of viable spores . hop1 diploids fail to assemble synaptonemal complex (SC), which normally provides the framework for meiotic synapsis . Immunochemical methods have shown that the 70-kDa HOP1 product is a component of the SC . To assess its molecular function, we have purified Hop1 protein to homogeneity and shown that it forms dimers and higher oligomers in solution . Consistent with the zinc-finger motif in its sequence, the purified protein contained about 1 mol equivalent of zinc whereas mutant protein lacking a conserved cysteine within this motif did not . Electrophoretic gel mobility shift assays with different forms of M13 DNA showed that Hop1 binds more readily to linear duplex DNA and negatively superhelical DNA than to nicked circular duplex DNA and even more weakly to single-stranded DNA . Linear duplex DNA binding was enhanced by the addition of Zn2+, was stronger for longer DNA fragments, and was saturable to about 55 bp/protein monomer . Competitive inhibition of this binding by added oligonucleotides suggests preferential affinity for G-rich sequences and weaker binding to poly(dA-dT) . Nuclear extracts of meiotic cells caused exonucleolytic degradation of linear duplex DNA if the extracts were prepared from hop1 mutants; addition of purified Hop1 conferred protection against this degradation . These findings suggest that Hop1 acts in meiotic synapsis by binding to sites of double-strand break formation and helping to mediate their processing in the pathway to meiotic recombination.

Mol Cell Biol, 1998 Mar, 18(3), 1296 - 302
Activation of chromosomal DNA replication in Saccharomyces cerevisiae by acidic transcriptional activation domains; Li R et al.; A large body of evidence from viral systems has established that transcription factors play an important and direct role in activating viral DNA replication . Among the transcriptional activation domains that can stimulate viral DNA replication are acidic domains such as those derived from herpes simplex virus VP16 and the tumor suppressor p53 . Here we show that acidic activation domains can also activate a cellular origin of replication in a chromosomal context . When tethered to the yeast ARS1 (autonomously replicating sequence 1) origin of replication, both VP16 and p53 activation domains can enhance origin function . In addition, the C-terminal acidic region of the yeast transcription factor ABF1, which normally activates the ARS1 origin, is sufficient for activating ARS1 function when tethered to the origin . Mutations at residues Trp-53 and Phe-54 of a 20-residue (41 to 60) activation region of p53 abolish the activation of both replication and transcription, suggesting that the same structural determinants may be employed to activate both processes in yeast . Furthermore, using a two-dimensional gel electrophoresis method, we demonstrate that the GAL4-p53 chimeric activator can activate initiation of chromosomal replication from an origin inserted at the native ARS1 locus . These findings strongly suggest functional conservation of the mechanisms used by the acidic activation domains to activate viral DNA replication in mammalian cells and chromosomal replication in yeast.

Mol Cell Biol, 1998 Mar, 18(3), 1190 - 200
The Saccharomyces cerevisiae RAD9 checkpoint reduces the DNA damage-associated stimulation of directed translocations; Fasullo M et al.; Genetic instability in the Saccharomyces cerevisiae rad9 mutant correlates with failure to arrest the cell cycle in response to DNA damage . We quantitated the DNA damage-associated stimulation of directed translocations in RAD9+ and rad9 mutants . Directed translocations were generated by selecting for His+ prototrophs that result from homologous, mitotic recombination between two truncated his3 genes, GAL1::his3-delta5' and trp1::his3-delta3'::HOcs . Compared to RAD9+ strains, the rad9 mutant exhibits a 5-fold higher rate of spontaneous, mitotic recombination and a greater than 10-fold increase in the number of UV- and X-ray-stimulated His+ recombinants that contain translocations . The higher level of recombination in rad9 mutants correlated with the appearance of nonreciprocal translocations and additional karyotypic changes, indicating that genomic instability also occurred among non-his3 sequences . Both enhanced spontaneous recombination and DNA damage-associated recombination are dependent on RAD1, a gene involved in DNA excision repair . The hyperrecombinational phenotype of the rad9 mutant was correlated with a deficiency in cell cycle arrest at the G2-M checkpoint by demonstrating that if rad9 mutants were arrested in G2 before irradiation, the numbers both of UV- and gamma-ray-stimulated recombinants were reduced . The importance of G2 arrest in DNA damage-induced sister chromatid exchange (SCE) was evident by a 10-fold reduction in HO endonuclease-induced SCE and no detectable X-ray stimulation of SCE in a rad9 mutant . We suggest that one mechanism by which the RAD9-mediated G2-M checkpoint may reduce the frequency of DNA damage-induced translocations is by channeling the repair of double-strand breaks into SCE.

Mol Cell Biol, 1998 Mar, 18(3), 1147 - 55
Regulation of transcription factor Pdr1p function by an Hsp70 protein in Saccharomyces cerevisiae; Hallstrom TC et al.; Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae requires the expression of several ATP binding cassette transporter-encoding genes under the control of the zinc finger-containing transcription factor Pdrlp . The ATP binding cassette transporter-encoding genes regulated by Pdrlp include PDR5 and YOR1, which are required for normal cycloheximide and oligomycin tolerances, respectively . We have isolated a new member of the PDR gene family that encodes a member of the Hsp70 family of proteins found in this organism . This gene has been designated PDR13 and is required for normal growth . Overexpression of Pdr13p leads to an increase in both the expression of PDR5 and YOR1 and a corresponding enhancement in drug resistance . Pdr13p requires the presence of both the PDR1 structural gene and the Pdr1p binding sites in target promoters to mediate its effect on drug resistance and gene expression . A dominant, gain-of-function mutant allele of PDR13 was isolated and shown to have the same phenotypic effects as when the gene is present on a 2microm plasmid . Genetic and Western blotting experiments indicated that Pdr13p exerts its effect on Pdr1p at a posttranslational step . These data support the view that Pdr13p influences pleiotropic drug resistance by enhancing the function of the transcriptional regulatory protein Pdr1p.

Cell Mol Life Sci, 1998 Jan, 54(1), 32 - 49
Telomere tales: chromatin, telomerase and telomere function in Saccharomyces cerevisiae; Lowell JE et al.; Unusual chromatin structures underlie epigenetic effects at the silent mating-type loci and telomeres in yeast . Many of the same genes appear to function in transcriptional silencing observed at both the silent mating-type loci and at telomeres . The observation that these loci are united by a requirement for shared factors suggests that the structure of chromatin at these regions is similar . Alteration of telomeric chromatin components affects regulation of transcription, telomeric length, recombination and chromosomal stability . Mutations in TLC1 and EST2, which both encode components of telomerase, cause identical phenotypes: progressive shortening of telomeric DNA, increased chromosome loss and eventually cell death . In this review, we examine the relationship between telomeric chromatin and telomere replication and discuss the possibility that telomerase itself is an integral part of telomeric chromatin structure.

FEMS Microbiol Lett, 1998 Feb 1, 159(1), 99 - 105
Protective roles of two aluminum (Al)-induced genes, HSP150 and SED1 of Saccharomyces cerevisiae, in Al and oxidative stresses; Ezaki B et al.; We isolated two yeast cDNA clones whose transcripts are induced by aluminum (Al) metal stress . Partial nucleotide sequencing showed that one is the HSP150 gene encoding a secreted heat shock protein, and the other corresponds to the SED1 gene encoding a putative membrane protein . To clarify the biological functions of these genes, we analyzed the sensitivity of gene-disrupted mutants to Al stress and to oxidative stresses . The Al tests indicated that the HSP150 protein served a basal protective role in Al stress, but SED1 did not; both of the genes had protective roles for oxidative stresses . The results for the HSP150 gene suggest that there is an overlap between Al ion stress, oxidative stress and heat shock stress in yeast.

FEMS Microbiol Lett, 1998 Feb 1, 159(1), 69 - 75
The ORF YBL042 of Saccharomyces cerevisiae encodes a uridine permease; Wagner R et al.; The purpose of this work was to identify the function of an open reading frame called YBL042, found during the systematic sequencing of Saccharomyces cerevisiae's chromosome II . The YBL042 gene product shows 70% similarity with the uracil permease and the allantoin permease encoded by FUR4 and DAL4, respectively . The mutation constructed by disruption of this ORF is allelic to the FUI1 gene previously described as encoding the uridine permease but not cloned yet . A strain carrying the disrupted allele and a fui1 mutant exhibit the same phenotype as they do not grow on a medium containing uridine as the sole source of pyrimidines and as they are resistant to 10(-3) M 5-fluorouridine (5FUI), a toxic analog of uridine . Even though the FUI1 gene has a multicopy suppressor effect on uracil transport, its product does not seem to be involved in this transport, in contrast to the FUR4 gene product which is involved in uridine transport . Moreover, the FUI1 gene product does not play any role in allantoin transport.

Yeast, 1998 Jan 30, 14(2), 161 - 9
Disruption of the Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin; Kerry-Williams SM et al.; Expression of recombinant human albumin (rHA) in Saccharomyces cerevisiae resulted in secretion of both mature albumin and a 45 kDa degradation product, comprising an N-terminal fragment of rHA with heterogeneous C-termini between residues 403 and 409 (Geisow et al., 1991) . Site-directed mutagenesis of the human albumin gene (HA) to change Arg410 to Ala (R410A) caused a significant reduction in the amount of fragment produced . Mutation of the adjacent dibasic site Lys413 Lys414 had little effect in isolation, but in combination with the R410A mutation resulted in a further reduction in the amount of rHA fragment produced . This reduction could be duplicated with nature-identical rHA by disruption of the gene for an aspartyl protease (YAP3), alone or in conjunction with disruption of the KEX2 gene . Disruption of KEX2 alone did not result in any improvement in the degree of degradation of the rHA . Reduced degradation was also observed when an rHA-human growth hormone fusion protein was secreted from a yap3 strain, suggesting that such strains may have a general utility for heterologous protein secretion.

Yeast, 1998 Jan 30, 14(2), 133 - 46
Mutations of the CDC28 gene and the radiation sensitivity of Saccharomyces cerevisiae; Koltovaya NA et al.; cdc28-srm, a non-temperature-sensitive (ts) mutation in the CDC28 gene of Saccharomyces cerevisiae that affects fidelity of mitotic transmission of both mitochondrial and nuclear genetic structures (Devin et al., 1990), also affected cell growth and sensitivity to lethal effects of ionizing radiation . At 30 degrees C cdc28-13, a ts mutation, was without appreciable effects on spontaneous mitochondrial rho(-)-mutagenesis, cell growth and radiation sensitivity, whereas all three cell characteristics mentioned were affected (although to a lesser degree than by cdc28-srm) by cdc28-1, another ts mutation . cdc28-srm was without any significant effect on the rates of spontaneous nuclear gene mutations and gamma-ray-induced mitotic recombination . An analysis of double mutants as regards their radiation sensitivity has revealed additive or even synergistic interactions between the cdc28-srm mutation and every one of the rad6-1 and rad52-1 mutations . The rad9 delta allele was found to be epistatic to cdc28-srm . These data suggest that the p34CDC28 protein is involved in the RAD9-dependent feedback control of DNA integrity operating at the cell cycle checkpoints.

Yeast, 1998 Jan 30, 14(2), 115 - 32
Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications; Brachmann CB et al.; A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed . These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression . Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications . We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.

Yeast, 1998 Jan 15, 14(1), 1 - 9
Sequences of Saccharomyces cerevisiae 2 microns DNA improving plasmid partitioning in Hansenula polymorpha; Bogdanova AI et al.; Insertion of the HindIII-PstI fragment of Saccharomyces cerevisiae 2 microns DNA into the Hansenula polymorpha replicative plasmids decreases plasmid copy number and ensures their distribution to daughter cells at both mitotic and meiotic cell divisions . This suggests that the stabilization effect is caused by the improvement of plasmid partitioning . Deletion analysis revealed that the region of 2 microns DNA sequence responsible for the increase of mitotic stability of H . polymorpha plasmids involves the 2 microns STB locus and adjoining region . Further analysis demonstrated that the stabilization effect may depend on the number of 24-28 bp imperfect repeats which were found in several copies in the STB locus and adjoining region.

Biochemistry, 1998 Feb 17, 37(7), 1754 - 61
Evolutionary conservation of enzymatic catalysis: quantitative comparison of the effects of mutation of aligned residues in Saccharomyces cerevisiae and Escherichia coli inorganic pyrophosphatases on enzymatic activity; Pohjanjoki P et al.; Soluble inorganic pyrophosphatase (PPase) is one of the better understood phosphoryl-transfer enzymes and is distinctive in having four divalent metal ions at the active site . Here we determine pH profiles for wild-type Saccharomyces cerevisiae PPase (Y-PPase) and for 14 of its active site variants and consider the effects of active site mutation on the pH-independent parameters and acid dissociation constants that characterize these profiles against the framework of the proposed structure of the activated complex . The results obtained (a) support the current mechanistic model in which a hydroxide ion, stabilized by binding to two metal ions at the active site and by an extended system of hydrogen bonds within the active site, is the nucleophile that attacks enzyme-bound inorganic pyrophosphate and (b) provide evidence that the acid group that is necessary for maximal activity is a water molecule coordinated to a third metal ion, as shown by the general rise in the pKa of this group that is a consequence of almost all of the mutations . We further compare the present results to those previously observed for the corresponding mutations in Escherichia coli PPase {E-PPase; Salminen et al . (1995) Biochemistry 34, 782-791} . Such comparison provides a measure of the extent to which different portions of the active site are conserved . We find that some corresponding mutations have different effects on catalytic function, demonstrating that even in the context of very similar active sites, interactions of the mutated site with less well conserved portions of the enzyme, in this case outside the active site, can lead to different outcomes . On the other hand, one region of the active site is highly conserved, suggesting that it may represent a common feature of phosphoryl-transfer enzymes or a vestige of a primitive ur-PPase active site.

Genes Dev, 1998 Feb 1, 12(3), 411 - 21
Nucleotide excision repair and photolyase preferentially repair the nontranscribed strand of RNA polymerase III-transcribed genes in Saccharomyces cerevisiae; Aboussekhra A et al.; A high-resolution primer extension technique was used to study the relationships between repair, transcription, and mutagenesis in RNA polymerase III transcribed genes in Saccharomyces cerevisiae . The in vivo repair of UV-induced DNA damage by nucleotide excision repair (NER) and by photoreactivation is shown to be preferential for the nontranscribed strand (NTS) of the SNR6 gene . This is in contrast to RNA polymerase II genes in which the NER is preferential for the transcribed strand (TS) . The repair-strand bias observed in SNR6 was abolished by inactivation of transcription in a snr6Delta2 mutant, showing a contribution of RNA polymerase III transcription in this phenomenon . The same strand bias for NER (slow in TS, fast in NTS) was discovered in the SUP4 gene, but only outside of the intragenic promoter element (box A) . Unexpectedly, the repair in the transcribed box A was similar on both strands . The strand specificity as well as the repair heterogeneity determined in the transcribed strand of the SUP4 gene, correlate well with the previously reported site- and strand-specific mutagenesis in this gene . These findings present a novel view regarding the relationships between DNA repair, mutagenesis, and transcription.

Genes Dev, 1998 Feb 1, 12(3), 357 - 69
Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae; Hartzog GA et al.; Previous characterization of the Saccharomyces cerevisiae Spt4, Spt5, and Spt6 proteins suggested that these proteins act as transcription factors that modify chromatin structure . In this work, we report new genetic and biochemical studies of Spt4, Spt5, and Spt6 that reveal a role for these factors in transcription elongation . We have isolated conditional mutations in SPT5 that can be suppressed in an allele-specific manner by mutations in the two largest subunits of RNA polymerase II (Pol II) . Strikingly, one of these RNA Pol II mutants is defective for transcription elongation and the others cause phenotypes consistent with an elongation defect . In addition, we show that spt4, spt5, and spt6 mutants themselves have phenotypes suggesting defects in transcription elongation in vivo . Consistent with these findings, we show that Spt5 is physically associated with RNA Pol II in vivo, and have identified a region of sequence similarity between Spt5 and NusG, an Escherichia coli transcription elongation factor that binds directly to RNA polymerase . Finally, we show that Spt4 and Spt5 are tightly associated in a complex that does not contain Spt6 . These results, taken together with the biochemical identification of a human Spt4-Spt5 complex as a transcription elongation factor (Wada et al . 1998), provide strong evidence that these factors are important for transcription elongation in vivo.

J Biol Chem, 1998 Jan 30, 273(5), 2829 - 34
Inhibition of amino acid transport by sphingoid long chain bases in Saccharomyces cerevisiae; Skrzypek MS et al.; Sphingoid long chain bases have many effects on cells including inhibition or stimulation of growth . The physiological significance of these effects is unknown in most cases . To begin to understand how these compounds inhibit growth, we have studied Saccharomyces cerevisiae cells . Growth of tryptophan (Trp-) auxotrophs was more strongly inhibited by phytosphingosine (PHS) than was growth of Trp+ strains, suggesting that PHS diminishes tryptophan uptake and starves cells for this amino acid . This hypothesis is supported by data showing that growth inhibition is relieved by increasing concentrations of tryptophan in the culture medium and by multiple copies of the TAT2 gene, encoding a high affinity tryptophan transporter . Measurement of tryptophan uptake shows that it is inhibited by PHS . Finally, PHS treatment induces the general control response, indicating starvation for amino acids . Multiple copies of TAT2 do not protect cells against two other cationic lipids, stearylamine, and sphingosine, indicating that the effect of PHS on tryptophan utilization is specific . Other data demonstrate that PHS reduces uptake of leucine, histidine, and proline by specific transporters . Our data suggest that PHS targets proteins in the amino acid transporter family but not other distantly related membrane transporters, including those necessary for uptake of adenine and uracil.

EMBO J, 1998 Jan 15, 17(2), 423 - 34
Multi-protein complexes in the cis Golgi of Saccharomyces cerevisiae with alpha-1,6-mannosyltransferase activity; Jungmann J et al.; Anp1p, Van1p and Mnn9p constitute a family of membrane proteins required for proper Golgi function in Saccharomyces cerevisiae . We demonstrate that these proteins colocalize within the cis Golgi, and that they are physically associated in two distinct complexes, both of which contain Mnn9p . Furthermore, we identify two new proteins in the Anp1p-Mnn9p-containing complex which have homology to known glycosyltransferases . Both protein complexes have alpha-1, 6-mannosyltransferase activity, forming a series of poly-mannose structures . These reaction products also contain some alpha-1, 2-linked mannose residues . Our data suggest that these two multi-protein complexes are responsible for the synthesis and initial branching of the long alpha-1,6-linked backbone of the hypermannose structure attached to many yeast glycoproteins.

Nucleic Acids Res, 1997 Dec 15, 25(24), 5057 - 64
Functional analysis of a replication origin from Saccharomyces cerevisiae: identification of a new replication enhancer; Raychaudhuri S et al.; Yeast replication origins have a modular arrangement of essential DNA sequences containing the ARS consensus sequence (ACS) flanked by auxiliary DNA elements which stimulate origin function . One of the auxiliary elements identified at several origins is a DNA replication enhancer that binds the Abf1p protein . We have isolated an ARS sequence from Saccharomyces cerevisiae based on its ability to bind Abf1p . Here we present a detailed molecular dissection of this ARS, designated ARS 1501, and we demonstrate that it functions as a genomic replication origin on chromosome XV . Mutagenesis of the Abf1p DNA-binding sites revealed that these sequences did not contribute significantly to ARS function . Instead, a new DNA element important for replication, designated REN1501, has been located 5' to the T-rich strand of the ACS . We show that REN1501 functions in either orientation and at variable distances from the ACS, defining this element as a DNA replication enhancer . Most significantly, point mutations within this element decreased the stability of plasmids bearing ARS 1501, suggesting that REN1501 binds a protein important for replication initiation . Only three elements found at origins are known to specifically bind proteins . These include the ARS essential sequences and the Abf1p and Rap1p DNA-binding sites . We show that the function of REN1501 at the origin cannot be replaced by a Rap1p DNA-binding site or a site that binds the transcriptional factor Gal4p and can only be partially substituted for by an Abf1p recognition sequence . This implies that the role of the REN1501 element at the ARS 1501 origin is specific, and suggest that the frequency of origin firing in eukaryotic cells may be regulated by origin-specific enhancers.

Nucleic Acids Res, 1997 Dec 15, 25(24), 4872 - 5
A new nomenclature for the cytoplasmic ribosomal proteins of Saccharomyces cerevisiae; Mager WH et al.; The availability of the complete sequence of the Saccharomyces cerevisiae genome has allowed a comprehensive analysis of the genes encoding cytoplasmic ribosomal proteins in this organism . On the basis of this complete inventory a new nomenclature for the yeast ribosomal proteins is presented.

Genetics, 1998 Jan, 148(1), 99 - 112
TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae; Helliwell SB et al.; The Saccharomyces cerevisiae genes TOR1 and TOR2 encode phosphatidylinositol kinase homologs . TOR2 has two essential functions . One function overlaps with TOR1 and mediates protein synthesis and cell cycle progression . The second essential function of TOR2 is unique to TOR2 and mediates the cell-cycle-dependent organization of the actin cytoskeleton . We have isolated temperature-sensitive mutants that are defective for either one or both of the two TOR2 functions . The three classes of mutants were as follows . Class A mutants, lacking only the TOR2-unique function, are defective in actin cytoskeleton organization and arrest within two to three generations as small-budded cells in the G2/M phase of the cell cycle . Class B mutants, lacking only the TOR-shared function, and class C mutants, lacking both functions, exhibit a rapid loss of protein synthesis and a G1 arrest within one generation . To define further the two functions of TOR2, we isolated multicopy suppressors that rescue the class A or B mutants . Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 suppressed the growth defect of a class A mutant . Surprisingly, overexpression of PLC1 and MSS4 also suppressed the growth defect of a class B mutant . These genes encode proteins that are involved in phosphoinositide metabolism and signaling . Thus, the two functions (readouts) of TOR2 appear to involve two related signaling pathways controlling cell growth.

Genetics, 1998 Jan, 148(1), 85 - 98
Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases; Srivastava A et al.; The PEP12 homolog Pth1p (Pep twelve homolog 1) is predicted to be similar in size to Pep12p, the endosomal syntaxin homolog that mediates docking of Golgi-derived transport vesicles and, like other members of the syntaxin family, is predicted to be a cytoplasmically oriented, integral membrane protein with a C-terminal transmembrane domain . Kinetic analyses indicate that deltapth1/vam3 mutants fail to process the soluble vacuolar hydrolase precursors and that PrA, PrB and most of CpY accumulate within the cell in their Golgi-modified P2 precursor forms . This is in contrast to a pep12 mutant in which P2CpY is secreted from the cell . Furthermore, pep12 is epistatic to pth1/vam3 with respect to the CpY secretion phenotype . Alkaline phosphatase, a vacuolar membrane hydrolase, accumulates in its precursor form in the deltapth1/vam3 mutant . Maturation of pro-aminopeptidase I, a hydrolase precursor delivered directly to the vacuole from the cytoplasm, is also blocked in the deltapth1/vam3 mutant . Subcellular fractionation localizes Pth1/Vam3p to vacuolar membranes . Based on these data, we propose that Pth1/Vam3p is the vacuolar syntaxin/t-SNARE homolog that participates in docking of transport vesicles at the vacuolar membrane and that the function of Pth1/Vam3p impinges on at least three routes of protein delivery to the yeast vacuole.

Genetics, 1998 Jan, 148(1), 71 - 83
Genetic interaction with vps8-200 allows partial suppression of the vestigial vacuole phenotype caused by a pep5 mutation in Saccharomyces cerevisiae; Woolford CA et al.; pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases . In addition, they show a vestigial vacuole morphology and a sensitivity to growth on media containing excess divalent cations . This pleiotropic phenotype observed for pep5::TRP1 mutants is partially suppressed by the vps8-200 allele . pep5::TRP1 vps8-200 mutants show near wild-type levels of mature-sized soluble vacuolar hydrolases, growth on zinc-containing medium, and a more "wild-type" vacuolar morphology; however, aminopeptidase I and alkaline phosphatase accumulate as precursors . These data suggest that Pep5p is a bifunctional protein and that the TRP1 insertion does not eliminate function, but results in a shorter peptide that can interact with Vps8-200p, allowing for partial function . vps8 deletion/disruption mutants contain a single enlarged vacuole . This genetic interaction was unexpected, since Pep5p was thought to interact more directly with the vacuole, and Vps8p is thought to play a role in transport between the Golgi complex and the prevacuolar compartment . The data are consistent with Pep5p functioning both at the site of Vps8p function and more closely proximal to the vacuole . They also provide evidence that the three transport pathways to the vacuole either converge or share gene products at late step(s) in the pathway(s).

Genetics, 1998 Jan, 148(1), 59 - 70
Evidence for independent mismatch repair processing on opposite sides of a double-strand break in Saccharomyces cerevisiae; Weng YS et al.; Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA) . We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT locus . Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape repair, and segregate to produce a sectored (Ura+/-) colony . If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored . For both a 14-bp palindrome, and a 37-bp near-palindrome, approximately 75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA . We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome . As seen in previous studies, these additional markers increased loop repair (likely reflecting corepair) . Among sectored products, few had additional segregating markers, indicating that the lack of repair at one marker is not associated with inefficient repair at nearby markers . Clear evidence was obtained for low levels of short tract mismatch repair . As seen with full gene conversions, donor alleles in sectored products were not altered . Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than nonsectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes . These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.

Genetics, 1998 Jan, 148(1), 33 - 47
An essential function of a phosphoinositide-specific phospholipase C is relieved by inhibition of a cyclin-dependent protein kinase in the yeast Saccharomyces cerevisiae; Flick JS et al.; The PLC1 gene product of Saccharomyces cerevisiae is a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C (PI-PLC) . We found that two genes (SPL1 and SPL2), when overexpressed, can bypass the temperature-sensitive growth defect of a plc1delta cell . SPL1 is identical to the PHO81 gene, which encodes an inhibitor of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk) . In addition to overproduction of Pho81p, two other conditions that inactivate this Cdk, a cyclin (pho80delta) mutation and growth on low-phosphate medium, also permitted growth of plc1delta cells at the restrictive temperature . Suppression of the temperature sensitivity of plc1delta cells by pho80delta does not depend upon the Pho4p transcriptional regulator, the only known substrate of the Pho80p/Pho85p Cdk . The second suppressor, SPL2, encodes a small (17-kD) protein that bears similarity to the ankyrin repeat regions present in Pho81p and in other known Cdk inhibitors . Both pho81delta and spl2delta show a synthetic phenotype in combination with plc1delta . Unlike single mutants, plc1delta pho81delta and plc1delta spl2delta double mutants were unable to grow on synthetic complete medium, but were able to grow on rich medium.

Indian J Exp Biol, 1997 Oct, 35(10), 1032 - 7
Hyperthermic modification of radiation induced gene conversion in Saccharomyces cerevisiae; Anjaria KB et al.; Effect of pre- and post-irradiation hyperthermia on the induction of gene conversion (non-reciprocal recombination) in diploid yeast cells was investigated . Post irradiation heat treatment does not significantly modify the frequency of gene conversion . Both low-level (51 degrees C-10 min) and lethal (51 degrees C, 40 min) pre irradiation heat treatments enhanced the gene conversion frequency by 17-49% . There was no quantitative correlation between the observed enhancement and the radiation dose . These results suggest that hyperthermia given prior to radiation not only sensitizes the cells to killing but also increases their convertogenic response . It has been suggested that recombination may be involved in carcinogenesis and tumour promotion . Based on these observations along with other reports, wherein hyperthermia was shown to modify the carcinogenic effects in animals as well as the genetic effects of radiation in vitro, it is possible to suggest that hyperthermia may have a potential to increase the second primary cancers, especially in long term survivors . Certain differences in the mechanism of interaction between radiation and heat in the pre- irradiation and post irradiation modalities appear likely.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 13 - 9
Functional analysis of the stress response element and its role in the multistress response of Saccharomyces cerevisiae; Treger JM et al.; The DDR2 gene of Saccharomyces cerevisiae is a multistress response gene whose transcription is rapidly and strongly induced by a diverse array of xenobiotic agents, and environmental and physiological conditions . The multistress response of this gene requires the pentanucleotide, 5' CCCCT, (C4T;STRE (STress Response Element)) and the zinc-finger transcription factors, Msn2p and Msn4p . A 51bp oligonucleotide (oligo 31/32) containing two STREs from the DDR2 promoter region was previously shown to direct heat shock activation of a lacZ reporter gene . In this work we demonstrate that the same element conferred a complete multistress response to an E . coli galK reporter gene introduced into yeast cells . A variant oligonucleotide in which both the STRE spacing and neighboring sequences were altered responded to the same spectrum of stresses, while substitution of nucleotides within the pentanucleotide completely abolished the multistress response . These results directly demonstrate that STREs are not only necessary but are sufficient for mediating a transcriptional response to a surprisingly diverse set of environmental and physiological conditions.

Nucleic Acids Res, 1998 Jan 1, 26(1), 68 - 72
The Yeast Protein Database (YPD): a curated proteome database for Saccharomyces cerevisiae; Hodges PE et al.; The Yeast Protein Database (YPD) is a curated database for the proteome of Saccharomyces cerevisiae . It consists of approximately 6000 Yeast Protein Reports, one for each of the known or predicted yeast proteins . Each Yeast Protein Report is a one-page presentation of protein properties, annotation lines that summarize findings from the literature, and references . In the past year, the number of annotation lines has grown from 25 000 to approximately 35 000, and the number of articles curated has grown from approximately 3500 to >5000 . Recently, new data types have been included in YPD: protein-protein interactions, genetic interactions, and regulators of gene expression . Finally, a new layer of information, the YPD Protein Minireviews, has recently been introduced . The Yeast Protein Database can be found on the Web at html

Acta Microbiol Immunol Hung, 1997, 44(3), 249 - 56
Effect of benomyl on Saccharomyces cerevisiae during continuous cultivation; Razmovski R et al.; The effect of Benomyl on Saccharomyces cerevisiae during continuous cultivation was investigated . The RNA, total protein and phosphorous contents decreased while the trehalose content increased with increasing Benomyl concentrations when an RD mutant and the parent strain were cultivated at different Benomyl concentrations under continuous conditions . The specific oxygen uptake rate of the respiratory deficient mutant (RD mutant) was approximately 5 times lower in comparison to the parent strain . The RQ value for the RD mutant was higher than 1 which clearly indicated the anaerobic metabolism of glucose degradation . The RD mutant lacked cytochrome aa3, while the cytochromes a and b contents were lower than those of the parent strain.

Oncogene, 1998 Jan 8, 16(1), 121 - 30
Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae; Asakura T et al.; We purified a novel actin filament (F-actin)-binding protein from the soluble fraction of Saccharomyces cerevisiae by successive column chromatographies by use of the 125I-labeled F-actin blot overlay method . The purified protein showed a minimum Mr of about 140 kDa on SDS-polyacrylamide gel electrophoresis and we named it ABP140 . A search with the partial amino acid sequences of ABP140 against the Saccharomyces Genome Database revealed that the open reading frame of the ABP140 gene (ABP140) corresponded to YOR239W fused with YOR240W by the +1 translational frame shift . The encoded protein consisted of 628 amino acids with a calculated Mr of 71,484 . The recombinant protein interacted with F-actin and showed the activity to crosslink F-actin into a bundle . Indirect immunofluorescence study demonstrated that ABP140 was colocalized with both cortical actin patches and cytoplasmic actin cables in intact cells . However, elimination of ABP140 by gene disruption did not show a deleterious effect on cell growth or affect the organization of F-actin . These results indicate that ABP140 is not required for cell growth but may be involved in the reorganization of F-actin in the budding yeast.

Microbiology, 1998 Jan, 144 ( Pt 1), 25 - 36
A mutation in the Rho1-GAP-encoding gene BEM2 of Saccharomyces cerevisiae affects morphogenesis and cell wall functionality; Cid VJ et al.; Saccharomyces cerevisiae strain V918 was previously isolated in a search for thermosensitive autolytic mutants and found to bear a recessive mutation that caused the development of multinucleate swollen cells undergoing cell lysis . The BEM2 gene has been isolated by complementation of the phenotype of a V918 segregant . BEM2 encodes a Rho-GTPase-activating protein (GAP) which is thought to act as a modulator of the Rho1 small GTPase . It is shown that the mutation causing the morphogenetic and autolytic phenotype in strain V918 and its segregants lies in the BEM2 gene, defining a new mutant allele, bem2-21 . Mutants in the BEM2 gene have been reported to display loss of cell polarity and depolarization of the actin cytoskeleton, causing a bud-emergence defect . Low resistance to sonication and to hydrolytic enzymes proved that the cell wall is less protective in bem2-21 mutants than in wild-type strains . Moreover, bem2-21 mutants are more sensitive than the wild-type to several antifungal drugs . Transmission electron microscopy revealed the development of abnormally thick and wide septa and the existence of thin areas in the cell wall which probably account for cell lysis . The depolarization of actin in bem2-21 mutants did not preclude morphogenetic events such as cell elongation in homozygous diploid strains during nitrogen starvation in solid media, hyperpolarization of growth in a background bearing a mutated septin, or sporulation . Multinucleate cells from bem2-21 homozygous diploids underwent sporulation giving rise to multispored asci ('polyads'), containing up to 36 spores . This phenomenon occurred only under osmotically stabilized conditions, suggesting that the integrity of the ascus wall is impaired in cells expressing the bem2-21 mutation . It is concluded that the function of the BEM2 gene product is essential for the maintenance of a functional cell wall.

Biochemistry (Mosc), 1997 Nov, 62(11), 1232 - 41
Saccharomyces cerevisiae telomeres . A review; Pryde FE et al.; Recent work has yielded considerable information concerning the structure and function of telomeres and their associated sequences in the budding yeast Saccharomyces cerevisiae . The structure and maintenance of telomeres depends not only on the RNA template and the catalytic subunit of telomerase, but on a number of other proteins . These include proteins involved in assessing DNA damage and cell cycle regulation . There are also non-telomerase mediated processes involved in the normal maintenance of telomeres . In addition to proteins involved in telomere maintenance, there are a number of other proteins involved in the chromatin structure of the region . Many of these proteins have roles in silencing, ageing, segregation and nuclear architecture . The structure of the subtelomeric regions has been well characterized and consists of a mosaic of repeats found in variable copy numbers and locations . Amidst the variable mosaic elements there are small conserved sequences found at all ends that may have functional roles . Recent work shows that the subtelomeric repeats can rescue chromosome ends when telomerase fails, buffer subtelomerically located genes against transcriptional silencing, and protect the genome from deleterious rearrangements due to ectopic recombination . Thus the telomeres of yeast have a variety of roles in the life of the yeast cell beyond the protection of the ends and overcoming the end replication problem associated with linear molecules.

Mol Microbiol, 1998 Jan, 27(1), 85 - 98
Pmt1 mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces cerevisiae; Bourdineaud JP et al.; We constructed hybrid proteins containing a plant alpha-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p . We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage . The cell wall-bound form was shown to be linked by means of a beta1,6-glucose-containing side-chain . Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins . We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the alpha-galactosidase-Srp1 fusion proteins, an alpha-galactosidase-Sed1 hybrid protein and an alpha-galactosidase-alpha-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions . However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an alpha-galactosidase-Cwp2 fusion protein was found linked to the cell wall but devoid of beta1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.

Appl Environ Microbiol, 1998 Feb, 64(2), 793 - 4
Construction of a sorbitol-based vector for expression of heterologous proteins in Saccharomyces cerevisiae; McGonigal T et al.; A new inducible yeast expression vector, pXS7, was constructed by using the promoter and terminator sequences from the Saccharomyces cerevisiae SOR1 gene, which codes for the sorbitol dehydrogenase protein . We cloned the coding sequence of the Saccharomyces YEF3 gene in this vector and demonstrated an increase in YEF3 protein levels when cells were grown in the presence of the sugar sorbitol.

Appl Environ Microbiol, 1998 Feb, 64(2), 779 - 83
The H(+)-ATPase in the plasma membrane of Saccharomyces cerevisiae is activated during growth latency in octanoic acid-supplemented medium accompanying the decrease in intracellular pH and cell viability; Viegas CA et al.; Saccharomyces cerevisiae plasma membrane H(+)-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth . The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pHi) . The cell population used as inoculum exhibited a significant heterogeneity of pHi, and the fall of pHi correlated with the loss of cell viability as determined by plate counts . When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.

Nature, 1998 Jan 29, 391(6666), 506 - 10
Crystal structure of the Saccharomyces cerevisiae phosphatidylinositol-transfer protein; Sha B et al.; The yeast phosphatidylinositol-transfer protein (Sec14) catalyses exchange of phosphatidylinositol and phosphatidylcholine between membrane bilayers in vitro . In vivo, Sec14 activity is essential for vesicle budding from the Golgi complex . Here we report a three-dimensional structure for Sec14 at 2.5 A resolution . Sec14 consists of twelve alpha-helices, six beta-strands, eight 3(10)-helices and has two distinct domains . The carboxy-terminal domain forms a hydrophobic pocket which, in the crystal structure, is occupied by two molecules of n-octyl-beta-D-glucopyranoside and represents the phospholipid-binding domain . This pocket is reinforced by a string motif whose disruption in a sec14 temperature-sensitive mutant results in destabilization of the phospholipid-binding domain . Finally, we have identified an unusual surface helix that may play a critical role in driving Sec14-mediated phospholipid exchange . From this structure, we derive the first molecular clues into how a phosphatidylinositol-transfer protein functions.

J Cell Biol, 1998 Jan 12, 140(1), 49 - 60
Pex17p of Saccharomyces cerevisiae is a novel peroxin and component of the peroxisomal protein translocation machinery; Huhse B et al.; The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis . pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways . The PEX17 gene (formerly PAS9; Albertini, M., P . Rehling, R . Erdmann, W . Girzalsky, J.A.K.W . Kiel, M . Veenhuis, and W.-H Kunau . 1997 . Cell . 89:83-92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures . However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes . Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants ("ghosts") . This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis . Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor . The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex . This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p . From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.

J Biol Chem, 1998 Jan 9, 273(2), 849 - 58
Analysis of the mechanisms of action of the Saccharomyces cerevisiae dominant lethal cdc42G12V and dominant negative cdc42D118A mutations; Davis CR et al.; The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity . The mechanisms of action of the dominant negative cdc42(D118A) mutant and the lethal, gain of function cdc42(G12V) mutant were examined . Cdc42(D118A,C188S)p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the two-hybrid system, which correlated with the temperature dependence of the cdc42(D118A) phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42(D118A) action . Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p . The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42(D118A, C188S)p-Cdc24p interactions and could suppress the cdc42(D118A) mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain . The cdc42(G12V) mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1Delta and cla4Delta cells but not ste20Delta cells, suggesting that the mechanism of cdc42(G12V) action is through the Skm1p and Cla4p protein kinases at the plasma membrane . Two intragenic suppressors of cdc42(G12V) were also identified that displayed a dominant negative phenotype at 16 degrees C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.

Biochem J, 1998 Jan 1, 329 ( Pt 1), 65 - 71
Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion; Yanez E et al.; The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 alpha-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229 . These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied . Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme . In contrast, substitutions of Asn-134 had little or no effect . The use of antibodies showed that alpha-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain . alpha-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F . These findings indicate that Asn-229 is the single glycosylated residue in SWA2 . Thermostability analysis of both purified wild-type (T50=50 degrees C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx . 3 degrees C in the T50 of the enzyme . The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km) . These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw . occidentalis SWA2 alpha-amylase in both its wild-type and mutant forms.

FEBS Lett, 1997 Dec 29, 420(2-3), 186 - 90
Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae; Odani T et al.; Mannosylphosphorylation is a major oligosaccharide modification that provides negative charge in the Saccharomyces cerevisiae cell wall . Although two genes, MNN6 and MNN4, which encode a mannosylphosphate transferase and its putative positive regulator, respectively, are involved in this modification, the amount of Mnn4p has been found to be a limiting factor for mannosylphosphorylation . The level of mannosylphosphorylation increased at late-logarithmic and stationary phases of cell growth, and this was correlated to the transcriptional enhancement of MNN4 . We also find that mannosylphosphate transfer to mannan is negatively regulated by the protein kinase A pathway, while the presence of 0.5 M potassium chloride enhanced MNN4 transcription . This type of transcriptional regulation is observed in many stress response genes, implying that mannosylphosphate transfer is involved in the cellular response to a variety of stresses.

FEBS Lett, 1997 Dec 29, 420(2-3), 147 - 50
Glucose exerts opposite effects on mRNA versus protein and activity levels of Pde1, the low-affinity cAMP phosphodiesterase from budding yeast, Saccharomyces cerevisiae; Wera S et al.; In budding yeast (Saccharomyces cerevisiae), a low-affinity phosphodiesterase, Pde1, and a high-affinity phosphodiesterase, Pde2, are responsible for the degradation of cAMP . Addition of glucose to glycerol-grown yeast cells is known to cause a transient increase in the cAMP level and recent work has indicated a specific involvement of Pde1 in this response . In this work we show that glucose addition induces the accumulation to high levels of mRNA encoding Pde1 . This increase continues for at least 8 hours and is due to enhanced transcription of the PDE1 gene, since glucose addition does not change the stability of the Pde1 mRNA . Surprisingly, using an assay method specific for Pde1, we observed that the activity of Pde1 remains constant and finally decreases several-fold during the same period . In addition, this activity profile closely follows the Pde1 protein level as judged from Western blotting with antibodies directed against Pde1 . Experiments using cycloheximide, a general inhibitor of translation, allow to exclude the possibility of a futile cycle of Pde1 synthesis and degradation . Hence, glucose addition appears to trigger an increase in PDE1 gene transcription together with a specific inhibition of the translation of Pde1 mRNA.

J Biol Chem, 1997 Dec 12, 272(50), 31908 - 14
The VRG4 gene is required for GDP-mannose transport into the lumen of the Golgi in the yeast, Saccharomyces cerevisiae; Dean N et al.; In the yeast Saccharomyces cerevisiae, glycoproteins and sphingolipids are modified in the Golgi by the addition of mannose residues . The critical mannosyl donor for these reactions is the nucleotide sugar, GDP-mannose, whose transport into the Golgi from the cytoplasm is required for mannosylation . This transport reaction has been well characterized, but the nucleotide sugar transporter has yet to be identified in yeast . VRG4 is an essential gene whose product is required for a number of Golgi-specific functions, including glycosylation and the organization of the endomembrane system . Here, data are presented that demonstrate that the primary role of Vrg4p is in the transport of GDP-mannose into the Golgi . The vrg4 mutation causes a general impairment in mannosylation, affecting N-linked and O-linked glycoprotein modifications as well as the mannosylation of sphingolipids . By using an in vitro assay, vrg4 mutants were shown to be specifically defective in the transport of GDP-mannose into Golgi vesicles . The Vrg4 protein localizes to the Golgi complex in a pattern that suggests a wide distribution throughout the Golgi . Vrg4p displays homology to other putative nucleotide sugar transporters, suggesting that the VRG4 gene encodes a Golgi GDP-mannose transporter . As Vrg4p is essential, these results suggest that a complete lack of mannosylation of glycoproteins in the Golgi leads to inviability . Alternatively, the essential function of Vrg4p in yeast involves its effect on sphingolipids, which would imply a critical role for mannosylinositol phosphorylceramides or mannosyl diphosphoinositol ceramides on growth and viability.

Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13804 - 9
A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae; Verna J et al.; The PKC1-MPK1 pathway in yeast functions in the maintenance of cell wall integrity and in the stress response . We have identified a family of genes that are putative regulators of this pathway . WSC1, WSC2, and WSC3 encode predicted integral membrane proteins with a conserved cysteine motif and a WSC1-green fluorescence protein fusion protein localizes to the plasma membrane . Deletion of WSC results in phenotypes similar to mutants in the PKC1-MPK1 pathway and an increase in the activity of MPK1 upon a mild heat treatment is impaired in a wscDelta mutant . Genetic analysis places the function of WSC upstream of PKC1, suggesting that they play a role in its activation . We also find a genetic interaction between WSC and the RAS-cAMP pathway . The RAS-cAMP pathway is required for cell cycle progression and for the heat shock response . Overexpression of WSC suppresses the heat shock sensitivity of a strain in which RAS is hyperactivated and the heat shock sensitivity of a wscDelta strain is rescued by deletion of RAS2 . The functional characteristics and cellular localization of WSC suggest that they may mediate intracellular responses to environmental stress in yeast.

Plant J, 1997 Oct, 12(4), 819 - 29
Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae; Hiraiwa N et al.; Vacuolar processing enzymes (VPEs) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants . A larger precursor to VPE synthesized on rough endoplasmic reticulum is converted to an active enzyme in the vacuoles . In this study, a precursor to castor bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE . Two VPE proteins of 59 and 46 kDa were detected in the vacuoles of the transformant . They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N-linked glycosylation sites . During the growth of the transformant, the level of the 59 kDa VPE increased slightly until a rapid decrease occurred after 9 h . By contrast, the 46 kDa VPE appeared simultaneously with the disappearance of the 59 kDa VPE . Vacuolar processing activity increased with the accumulation of the 46 kDa VPE, but not of the 59 kDa VPE . The specific activity of the 46 kDa VPE was at a similar level to that of VPE in plant cells . The 46 kDa VPE instead of proteinase A mediated the conversion of procarboxypeptidase Y to the mature form . This indicates that proteinase A responsible for maturation of yeast vacuolar proteins can be replaced functionally by plant VPE . These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self-catalytic proteolysis within the vacuoles.

Biol Chem, 1997 Oct, 378(10), 1187 - 91
Cloning and characterization of a dominant-negative vps1 allele of the yeast Saccharomyces cerevisiae; Finken-Eigen M et al.; The gene product of the yeast VPS1 gene is a member of a family of high-molecular-weight GTP-binding proteins that are involved in diverse cellular processes . The Vps1 protein (Vps1p) was shown to perform an essential function in the yeast secretory pathway . Here, we report the isolation and characterization of a mutant allele of the VPS1 gene, causing a dominant-negative vacuolar protein sorting (vps) defect, as demonstrated by the mislocalization of the vacuolar hydrolase carboxypeptidase Y (CPY) . DNA sequence analysis of the mutant vps1 allele (vps1d-293) revealed a single point mutation, resulting in an amino acid exchange at position 293 from Ala to Asp . The mutation is located downstream of the tripartite GTP-binding motif found in the amino-terminal half of the protein . The observation that expression of wild-type Vps1p partially suppressed the dominant-negative CPY sorting phenotype indicates competition of a non-functional mutant Vps1 protein and a functional wild-type VPS1p for a Vps1p-binding site of an as yet unknown vacuolar protein sorting factor.

J Bacteriol, 1998 Feb, 180(3), 483 - 90
Toxicity of linoleic acid hydroperoxide to Saccharomyces cerevisiae: involvement of a respiration-related process for maximal sensitivity and adaptive response; Evans MV et al.; Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity . LoaOOH is toxic to wild-type Saccharomyces cerevisiae at a very low concentration (0.2 mM) relative to other peroxides . By using isogenic mutant strains, the possible roles of glutathione (gsh1 and gsh2), glutathione reductase (glr1), respiratory competence ({rho0} petite), and yAP-1p-mediated expression (yap1) in conferring LoaOOH resistance have been examined . Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the {rho0} petite mutant was most resistant to LoaOOH but could not adapt . Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant . An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the yap1 and glr1 mutants were more sensitive than the wild type . In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme . Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 microg ml-1) . The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide . Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37 degrees C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.

Biochemistry (Mosc), 1997 Sep, 62(9), 1051 - 2
Adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate: new substrates of the cytosolic exopolyphosphatase of the yeast Saccharomyces cerevisiae; Kulakovskaya TV et al.; A cytosolic preparation of Saccharomyces cerevisiae is capable of hydrolyzing adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate with activities which are 1.5-2 times greater than that with polyP15 . The apparent K(m) values for hydrolysis of adenosine-5'-tetraphosphate and guanosine-5'-tetraphosphate are 100 and 80 microM, respectively . A comparative study of inhibitors shows that these activities are inherent characteristics of these exopolyphosphatases.

FEMS Microbiol Lett, 1998 Jan 1, 158(1), 121 - 6
Amino acid uptake is strongly affected during exponential growth of Saccharomyces cerevisiae in 0.7 M NaCl medium; Norbeck J et al.; Labelling of Saccharomyces cerevisiae grown in 0.7 M NaCl with 35S-methionine revealed a 5-6 fold lowering of the methionine incorporation into protein, which could not be attributed solely to the approximately 50% longer generation time of cells grown in 0.7 M NaCl . Subsequent studies of the high affinity methionine uptake system showed a strongly reduced uptake of methionine during growth in 0.7 M NaCl medium . This reduced uptake was shown to be strain-independent and caused mainly by an approximately 20-fold lowered maximum velocity (Vmax) of the transport system, while the substrate affinity (Km) displayed only a minor change . A salt-instigated reduction of uptake was furthermore demonstrated for the leucine and histidine high affinity uptake systems and also for a mixture of 15 different amino acids . We therefore suggest that the reduced amino acid uptake is a general phenomenon observed in salt-grown cells.

Genes Dev, 1997 Dec 15, 11(24), 3445 - 58
Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae; Matheos DP et al.; Ca2+ signals regulate gene expression in animal and yeast cells through mechanisms involving calcineurin, a protein phosphatase activated by binding Ca2+ and calmodulin . Tcn1p, also named Crz1p, was identified as a transcription factor in yeast required for the calcineurin-dependent induction of PMC1, PMR1, PMR2A, and FKS2 which confer tolerance to high Ca2+, Mn2+, Na+, and cell wall damage, respectively . Tcn1p was not required for other calcineurin-dependent processes, such as inhibition of a vacuolar H+/Ca2+ exchanger and inhibition of a pheromone-stimulated Ca2+ uptake system, suggesting that Tcn1p functions downstream of calcineurin on a branch of the calcium signaling pathway leading to gene expression . Tcn1p contains three zinc finger motifs at its carboxyl terminus resembling the DNA-binding domains of Zif268, Swi5p, and other transcription factors . When fused to the transcription activation domain of Gal4p, the carboxy terminal domain of Tcn1p directed strong calcineurin-independent expression of PMC1-lacZ and other target genes . The amino-terminal domain of Tcn1p was found to function as a calcineurin-dependent transcription activation domain when fused to the DNA-binding domain of Gal4p . This amino-terminal domain also formed Ca2+-dependent and FK506-sensitive interactions with calcineurin in the yeast two-hybrid assay . These findings suggest that Tcn1p functions as a calcineurin-dependent transcription factor . Interestingly, induction of Tcn1p-dependent genes was found to be differentially controlled in response to physiological Ca2+ signals generated by treatment with mating pheromone and high salt . We propose that different promoters are sensitive to variations in the strength of Ca2+ signals generated by these stimuli and to effects of other signaling pathways.

Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14300 - 5
An essential component of a C-terminal domain phosphatase that interacts with transcription factor IIF in Saccharomyces cerevisiae; Archambault J et al.; One of the essential components of a phosphatase that specifically dephosphorylates the Saccharomyces cerevisiae RNA polymerase II (RPII) large subunit C-terminal domain (CTD) is a novel polypeptide encoded by an essential gene termed FCP1 . The Fcp1 protein is localized to the nucleus, and it binds the largest subunit of the yeast general transcription factor IIF (Tfg1) . In vitro, transcription factor IIF stimulates phosphatase activity in the presence of Fcp1 and a second complementing fraction . Two distinct regions of Fcp1 are capable of binding to Tfg1, but the C-terminal Tfg1 binding domain is dispensable for activity in vivo and in vitro . Sequence comparison reveals that residues 173-357 of Fcp1 correspond to an amino acid motif present in proteins of unknown function predicted in many organisms.

Biochem Mol Biol Int, 1997 Nov, 43(4), 901 - 11
Nitrogen starvation in a Saccharomyces cerevisiae strain deleted in the trehalose-6-phosphate synthase complex; Fernandes PM et al.; Little is known about the mechanisms by which nutrient limitation leads to G1 arrest in Saccharomyces cerevisiae . We have shown that mutant cells deleted in the trehalose-6-phosphate synthase gene able to grow on glucose, when starved for nitrogen, did not arrest in the G1 phase of the cell cycle . We attribute this effect to an increase in the activity of cAMP-dependent protein kinase . When grown on maltose without nitrogen they arrested properly in G1 . Tests with a mutant cell deleted in the specific trehalose phosphatase proved that the presence of the trehalose precursor, trehalose-6-phosphate, was sufficient to trigger the negative sign during nitrogen starvation, leading the cells to arrest in the G1 phase of the cell cycle.

Genetics, 1997 Nov, 147(3), 1053 - 62
Separable functions of ORC5 in replication initiation and silencing in Saccharomyces cerevisiae; Dillin A et al.; Origin recognition complex (ORC) is a six subunit complex that functions as the replication initiator and is required for silencing the HML and HMR loci in the yeast Saccharomyces cerevisiae . The roles of ORC5 in replication initiation and silencing were investigated to determine whether the two roles were mechanistically coincident or separable . Some spontaneous revertants of orc5-1 were functional for replication initiation, but not silencing . Other alleles of ORC5 were obtained that were nonfunctional for replication initiation, but fully competent for silencing . The two types of alleles, when put in the same cell, complemented, establishing two separable functions for ORC5 . These data implied that replication initiation at HMR-E was not required for silencing . The data were consistent with a model in which different ORC species functioned at different origins within the genome and that only one Orc5p subunit functioned at any given origin.

Genetics, 1997 Nov, 147(3), 1017 - 24
A role for REV3 in mutagenesis during double-strand break repair in Saccharomyces cerevisiae; Holbeck SL et al.; Recombinational repair of double-strand breaks (DSBs), traditionally believed to be an error-free DNA repair pathway, was recently shown to increase the frequency of mutations in a nearby interval . The reversion rate of trp1 alleles (either nonsense or frameshift mutations) near an HO-endonuclease cleavage site is increased at least 100-fold among cells that have experienced an HO-mediated DSB . We report here that in strains deleted for rev3 this DSB-associated reversion of a nonsense mutation was greatly decreased . Thus REV3, which encodes a subunit of the translesion DNA polymerase zeta, was responsible for the majority of these base substitution errors near a DSB . However, rev3 strains showed no decrease in HO-stimulated recombination, implying that another DNA polymerase also functioned in recombinational repair of a DSB . Reversion of trp1 frameshift alleles near a DSB was not reduced in rev3 strains, indicating that another polymerase could act during DSB repair to make these frameshift errors . Analysis of spontaneous reversion in haploid strains suggested that Rev3p had a greater role in making point mutations than in frameshift mutations.

Genetics, 1997 Nov, 147(3), 1003 - 16
SRO9, a multicopy suppressor of the bud growth defect in the Saccharomyces cerevisiae rho3-deficient cells, shows strong genetic interactions with tropomyosin genes, suggesting its role in organization of the actin cytoskeleton; Kagami M et al.; RHO3 encodes a Rho-type small GTPase in the yeast Saccharomyces cerevisiae and is involved in the proper organization of the actin cytoskeleton required for bud growth . SRO9 (YCL37c) was isolated as a multicopy suppressor of a rho3delta mutation . An Sro9p domain required for function is similar to a domain in the La protein (an RNA-binding protein) . Disruption of SRO9 did not affect vegetative growth, even with the simultaneous disruption of an SRO9 homologue, SRO99 . However, sro9delta was synthetically lethal with a disruption of TPM1, which encodes tropomyosin; sro9delta tpm1delta cells did not distribute cortical actin patches properly and lysed . We isolated TPM2, the other gene for tropomyosin, as a multicopy suppressor of a tpm1delta sro9delta double mutant . Genetic analysis suggests that TPM2 is functionally related to TPM1 and that tropomyosin is important but not essential for cell growth . Overexpression of SRO9 suppressed the growth defect in tpm1delta tpm2delta cells, disappearance of cables of actin filaments in both rho3delta cells and tpm1delta cells, and temperature sensitivity of actin mutant cells (act1-1 cells), suggesting that Sro9p has a function that overlaps or is related to tropomyosin function . Unlike tropomyosin, Sro9p does not colocalize with actin cables but is diffusely cytoplasmic . These results suggest that Sro9p is a new cytoplasmic factor involved in the organization of actin filaments.

Curr Genet, 1997 Nov, 32(5), 315 - 22
A Saccharomyces cerevisiae mutant defective in the kinesin-like protein Kar3 is sensitive to NaCl-stress; Schoch CL et al.; Several mutants of Saccharomyces cerevisiae showing poor growth in the presence of elevated concentrations of NaCl were isolated to identify genes involved in the osmo-stress response . One of these mutants (WAY.5-4A-11; osr11) which showed a clear 2:2 segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain has been characterised . The mutation responsible for poor growth under salt-stress was recessive . The corresponding gene was cloned by complementation of the mutant phenotype and a 3.5-kb fragment was isolated . The sequence of this fragment matched that of KAR3, a gene previously identified to be involved in karyogamy and mitosis . Allelism of OSR11 to KAR3 was confirmed by tetrad analysis, and disruption mutants showed the same NaCl-phenotype as the original osr11 mutation . The disruption mutant was more sensitive to high sucrose concentrations than the original mutant was to high glucose concentrations . In a different genetic background (W303-1A), the kar3 disruptants were less sensitive to osmo-stress than the WAY.5-4A strain . Heat-stress, nitrogen-starvation and cultivation on ethanol failed to affect the growth of osr11 and kar3 mutants, pointing to a possible specific involvement of KAR3 in the osmotic-stress response . Microscopic studies showed that cell division of the kar3 mutants was impaired and NaCl-stress conditions aggravated the phenotype.

J Cell Sci, 1997 Dec, 110 ( Pt 23), 2987 - 99
A structure/function analysis of Rat7p/Nup159p, an essential nucleoporin of Saccharomyces cerevisiae; Del Priore V et al.; Rat7p/Nup159p is an essential nucleoporin of Sac-charomyces cerevisiae originally isolated in a genetic screen designed to identify yeast temperature-sensitive mutants defective in mRNA export . Here we describe a detailed structural-functional analysis of Rat7p/Nup159p . The mutation in the rat7-1 ts allele, isolated in the original genetic screen, was found to be a single base pair change that created a stop codon approximately 100 amino acids upstream of the actual stop codon of this 1,460 amino acid polypeptide, thus eliminating one of the two predicted coiled-coil regions located near the carboxyl terminus of the protein . These coiled-coil regions are essential since an allele lacking both coiled-coil regions was unable to support growth under any conditions . In contrast, no other region of the protein was absolutely required . The SAFG/PSFG repeat region in the central third of the protein was completely dispensable for growth at temperatures between 16 degrees C and 37 degrees C and cells expressing this mutant allele were indistinguishable from wild type . Deletion of the amino-terminal third of the protein, upstream from the repeat region, or the portion between the repeat region and the coiled-coils resulted in temperature-sensitivity, but the two alleles showed distinct phenotypes with respect to the behavior of nuclear pore complexes (NPCs) . Taken together, our data suggest that Rat7p/Nup159p is anchored within the NPC through its coiled-coil region and adjacent sequences . In addition, we postulate that the N-terminal third of Rat7p/Nup159p plays an important role in mRNA export.

Folia Microbiol (Praha), 1996, 41(3), 237 - 42
Three fluorescent probes for the flow-cytometric assessment of membrane potential in Saccharomyces cerevisiae; Denksteinova B et al.; Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3'-dipropylthiacarbocyanine iodide (diS-C3(3)) and 3,3'-dipropyloxacarbocyanine iodide (diO-C3(3)), were tested for their suitability as fluorescent indicators of membrane potential in Saccharomyces cerevisiae in studies performed by flow cytometry . For all these dyes the intensity of fluorescence of stained cells increased with probe concentration in the range of 60-3000 nmol/L . The optimum staining period was 15-20 min for diS-C3(3) . Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop in fluorescence intensity . In some yeast batches this depolarization was accompanied by a separation of subpopulations with different fluorescence properties.

Arch Biochem Biophys, 1998 Jan 15, 349(2), 397 - 401
In vivo and in vitro phosphorylation of the alpha 7/PRS1 subunit of Saccharomyces cerevisiae 20 S proteasome: in vitro phosphorylation by protein kinase CK2 is absolutely dependent on polylysine; Pardo PS et al.; In this paper, we show that the Saccharomyces cerevisiae 20 S proteasome subunit 1 (PRS1), recently renamed as alpha 7, is the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome component . In vitro phosphorylation occurs only in the presence of polylysine, a characteristic that distinguishes the yeast proteasome from mammalian ones which are phosphorylated by CK2 in the absence of polylysine . A peptide reproducing the long acidic C-terminal tail of alpha 7/PRS1, where consensus CK2 phosphorylation sites are located, was also phosphorylated by the CK2 holoenzyme in a polylysine-dependent manner, suggesting that this region contains structural features responsible for this particular behavior.

Mol Cell Biol, 1998 Feb, 18(2), 1049 - 54
Mutations in chromatin components suppress a defect of Gcn5 protein in Saccharomyces cerevisiae; Perez-Martin J et al.; The yeast GCN5 gene encodes the catalytic subunit of a nuclear histone acetyltransferase and is part of a high-molecular-weight complex involved in transcriptional regulation . In this paper we show that full activation of the HO promoter in vivo requires the Gcn5 protein and that defects in this protein can be suppressed by deletion of the RPD3 gene, which encodes a histone deacetylase . These results suggest an interplay between acetylation and deacetylation of histones in the regulation of the HO gene . We also show that mutations in either the H4 or the H3 histone gene, as well as mutations in the SIN1 gene, which encodes an HMG1-like protein, strongly suppress the defects produced by the gcn5 mutant . These results suggest a hierarchy of action in the process of chromatin remodeling.

Mol Cell Biol, 1998 Feb, 18(2), 960 - 6
Analysis of damage tolerance pathways in Saccharomyces cerevisiae: a requirement for Rev3 DNA polymerase in translesion synthesis; Baynton K et al.; The replication of double-stranded plasmids containing a single N-2-acetylaminofluorene (AAF) adduct located in a short, heteroduplex sequence was analyzed in Saccharomyces cerevisiae . The strains used were proficient or deficient for the activity of DNA polymerase zeta (REV3 and rev3delta, respectively) in a mismatch and nucleotide excision repair-defective background (msh2delta rad10delta) . The plasmid design enabled the determination of the frequency with which translesion synthesis (TLS) and mechanisms avoiding the adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to complete replication . To this end, a hybridization technique was implemented to probe plasmid DNA isolated from individual yeast transformants by using short, 32P-end-labeled oligonucleotides specific to each strand of the heteroduplex . In both the REV3 and rev3delta strains, the two strands of an unmodified heteroduplex plasmid were replicated in approximately 80% of the transformants, with the remaining 20% having possibly undergone prereplicative MSH2-independent mismatch repair . However, in the presence of the AAF adduct, TLS occurred in only 8% of the REV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation . All TLS observed in the REV3 strain was abolished in the rev3delta mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.

Mol Cell Biol, 1998 Feb, 18(2), 779 - 89
Role for the ubiquitin-proteasome system in the vacuolar degradation of Ste6p, the a-factor transporter in Saccharomyces cerevisiae; Loayza D et al.; Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains . Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast . Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside . Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis . In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p . We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4 and pre1,2) . Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant . One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion . Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p . This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate.

Mol Cell Biol, 1998 Feb, 18(2), 665 - 75
RNA polymerase I-promoted HIS4 expression yields uncapped, polyadenylated mRNA that is unstable and inefficiently translated in Saccharomyces cerevisiae; Lo HJ et al.; The HIS4 gene in Saccharomyces cerevisiae was put under the transcriptional control of RNA polymerase I to determine the in vivo consequences on mRNA processing and gene expression . This gene, referred to as rhis4, was substituted for the normal HIS4 gene on chromosome III . The rhis4 gene transcribes two mRNAs, of which each initiates at the polymerase (pol) I transcription initiation site . One transcript, rhis4s, is similar in size to the wild-type HIS4 mRNA . Its 3' end maps to the HIS4 3' noncoding region, and it is polyadenylated . The second transcript, rhis4l, is bicistronic . It encodes the HIS4 coding region and a second open reading frame, YCL184, that is located downstream of the HIS4 gene and is predicted to be transcribed in the same direction as HIS4 on chromosome III . The 3' end of rhis4l maps to the predicted 3' end of the YCL184 gene and is also polyadenylated . Based on in vivo labeling experiments, the rhis4 gene appears to be more actively transcribed than the wild-type HIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene . This finding indicated that rhis4 mRNAs are rapidly degraded, presumably due to the lack of a cap structure at the 5' end of the mRNA . Consistent with this interpretation, a mutant form of XRN1, which encodes a 5'-3' exonuclease, was identified as an extragenic suppressor that increases the half-life of rhis4 mRNA, leading to a 10-fold increase in steady-state mRNA levels compared to the wild-type HIS4 mRNA level . This increase is dependent on pol I transcription . Immunoprecipitation by anticap antiserum suggests that the majority of rhis4 mRNA produced is capless . In addition, we quantitated the level of His4 protein in a rhis4 xrn1delta genetic background . This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of capped HIS4 mRNA . Our data indicate that polyadenylation of mRNA in yeast occurs despite HIS4 being transcribed by RNA polymerase I, and the 5' cap confers stability to mRNA and affords the ability of mRNA to be translated efficiently in vivo.

Genetika, 1997 Sep, 33(9), 1221 - 8
{Role of the RAD57 gene in the repair of double-stranded DNA gaps in the yeast Saccharomyces cerevisiae}; Glazer VM et al.; The linearized plasmid with complementary (cohesive) ends was shown to restore the circular form in cells of the rad57 mutant with a lower efficiency than in Rad+ cells . This process proved to be cold-sensitive in mutant cells, in contrast to wild-type cells . When mutant cells were shifted from 23 up to 36 degrees C, the repair efficiency increased approximately 1.5 times . In most cases examined, the repair was not accompanied by the doublestrand gap repair within the break site and did not depend on temperature . Homology between chromosomal and plasmid DNA sequences in the break region and the presence of cohesive ends were shown to be essential for the repair of linearized plasmids with a double-strand gap in cells of the rad57 mutant . Degradation of cohesive ends of the linearized plasmid during its repair in rad57 cells is insignificant . Possible mechanisms of linearized plasmid repair in the rad57 mutant are proposed.

Curr Genet, 1997 Dec, 32(6), 389 - 98
A Saccharomyces cerevisiae mitochondrial DNA fragment activates Reg1p-dependent glucose-repressible transcription in the nucleus; Santangelo GM et al.; As part of an effort to identify random carbon-source-regulated promoters in the Saccharomyces cerevisiae genome, we discovered that a mitochondrial DNA fragment is capable of directing glucose-repressible expression of a reporter gene . This fragment (CR24) originated from the mitochondrial genome adjacent to a transcription initiation site . Mutational analyses identified a GC cluster within the fragment that is required for transcriptional induction . Repression of nuclear CR24-driven transcription required Reg1p, indicating that this mitochondrially derived promoter is a member of a large group of glucose-repressible nuclear promoters that are similarly regulated by Reg1p . In vivo and in vitro binding assays indicated the presence of factors, located within the nucleus and the mitochondria, that bind to the GC cluster . One or more of these factors may provide a regulatory link between the nucleus and mitochondria.

Curr Genet, 1997 Dec, 32(6), 384 - 8
Two genes of the putative mitochondrial fatty acid synthase in the genome of Saccharomyces cerevisiae; Schneider R et al.; In order to find further genes of the mitochondrial fatty acid synthase, we searched the genome of Saccharomyces cerevisiae for sequences that are homologous to conserved regions of bacterial fatty acid synthase genes . We found the gene products of ORF YKL055c (EMBL Accession No . X75781) and of YOR221C (EMBL Accession No . X92441) to be homologous to bacterial 3-oxoacyl-(acyl carrier protein) reductases and to malonyl-CoA:ACP-transferases, respectively . We disrupted these two genes which in both cases led to a respiratory deficient phenotype, as is the case for the genes encoding a mitochondrial acyl carrier protein and a beta-ketoacyl-ACP synthase . We propose to call the above mentioned genes OAR1 {3-oxo-acyl-(acyl carrier protein) reductase} and MCT1 (malonyl-CoA:ACP transferase) . They are presumed to be part of a type-II mitochondrial fatty acid synthase, a relic of the endosymbiontic origin of mitochondria, delivering substrates for phospholipid re-modelling and/or repair.

Curr Genet, 1997 Dec, 32(6), 377 - 83
REO1 and ROX1 are alleles of the same gene which encodes a transcriptional repressor of hypoxic genes in Saccharomyces cerevisiae; Kwast KE et al.; Both the REO1 and ROX1 genes are thought to encode heme-dependent, transcriptional repressors of hypoxic genes in Saccharomyces cerevisiae . However, genetic complementation studies have yielded conflicting results about whether these are the same or different genes . Because of the central importance of these repressors, which control the expression of nearly all known hypoxic genes in yeast, we have sought to resolve this confusion by comparing the phenotypes of reo1 and rox1 mutants using Northern-blot analyses, performing additional complementation studies, and sequencing the ROX1 gene in reo1 strains . Northern-blot analyses of a reo1 strain show wild-type expression of the aerobic genes examined, but de-repression of the Rox1-regulated, hypoxic genes . Aerobic transcript levels of these hypoxic genes were also de-repressed in a diploid strain created by mating a rox1 disrupted strain with a reo1 strain, indicating that genetic complementation did not occur between these two strains . Sequence analyses of ROX1 in reo1 strains reveal a frame-shift mutation in the 5'-end of its coding region, resulting in a nonsense codon in the sixth position . Taken together, these results provide compelling evidence that reo1 is an allele of ROX1.

J Virol, 1998 Feb, 72(2), 1036 - 42
The pokeweed antiviral protein specifically inhibits Ty1-directed +1 ribosomal frameshifting and retrotransposition in Saccharomyces cerevisiae; Tumer NE et al.; Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins . The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation . Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting . We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae . Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the alpha-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed +1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system . Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells . PAP does not affect programmed -1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M1-dependent killer virus phenotype, suggesting that -1 ribosomal frameshifting does not occur after the peptidyltransferase reaction . These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.

J Biol Chem, 1998 Jan 23, 273(4), 2109 - 17
The spermidine transport system is regulated by ligand inactivation, endocytosis, and by the Npr1p Ser/Thr protein kinase in Saccharomyces cerevisiae; Kaouass M et al.; We have characterized the regulation of spermidine transport in yeast and identified some of the genes involved in its control . Disruption of the SPE2 gene encoding S-adenosylmethionine decarboxylase, which catalyzes an essential step in polyamine biosynthesis, upregulated the initial velocity of spermidine uptake in wild-type cells as well as in the polyamine transport-deficient pcp1 mutants . Exogenous spermidine rapidly inactivated spermidine transport with a half-life of approximately 10-15 min via a process that did not require de novo protein synthesis but was accelerated by cycloheximide addition . Conversely, reactivation of spermidine influx upon polyamine deprivation required active protein synthesis . The stability of polyamine carrier activity was increased 2-fold in polyamine-depleted spe2 deletion mutants, indicating that endogenous polyamines also contribute to the down-regulation of spermidine transport . Ligand-mediated repression of spermidine transport was delayed in end3 and end4 mutants that are deficient in the initial steps of the endocytic pathway, and spermidine uptake activity was increased 4- to 5-fold in end3 mutants relative to parental cells, although the stability of the transport system was similar in both strains . Disruption of the NPR1 gene, which encodes a putative Ser/Thr protein kinase essential for the reactivation of several nitrogen permeases, resulted in a 3-fold decrease in spermidine transport in NH4(+)-rich media but did not prevent its down-regulation by spermidine . The defect in spermidine transport was more pronounced in NH4(+)- than proline-grown npr1 cells, suggesting that NPR1 protects against nitrogen catabolite repression of polyamine uptake activity . These results suggest that (a) the polyamine carrier is an unstable protein subject to down-regulation by spermidine via a process involving ligand inactivation followed by endocytosis and that (b) NPR1 expression fully prevents nitrogen catabolite repression of polyamine transport, unlike the role predicted for that gene by the inactivation/reactivation model proposed for other nitrogen permeases.

J Biol Chem, 1998 Jan 16, 273(3), 1298 - 302
Saccharomyces cerevisiae Cdc6 stimulates Abf1 DNA binding activity; Feng L et al.; In budding yeast Saccharomyces cerevisiae, an ARS binding factor 1 (Abf1) binds to the sequence-specific DNA element involved in DNA replication and transcription . We describe in this study how yeast Cdc6 protein stimulates Abf1 protein DNA binding activities . The Abf1 binding activity was reduced approximately 20-fold in a cdc6-1 mutant than in the wild-type strain . Introducing a copy of the wild-type CDC6 gene into the cdc6-1 mutant strain restored the Abf1 DNA binding activity . We demonstrated that recombinant Abf1 binds to ARS1 in vitro, and its DNA binding activity can be highly stimulated by the addition of a fusion glutathione S-transferase (GST)-Cdc6 protein . Deletion analysis revealed that the stimulating region is located at the amino terminus of the Cdc6 protein . However, we could not find the direct physical interaction between Cdc6 and Abf1 . Instead, we found that the GST-Cdc6 can compete with distamycin A for binding to the DNA molecule . As distamycin A is a specific reagent that binds noncovalently to DNA at (A + T)-rich tracks, the stimulation of Abf1 DNA binding activity may be mediated by the Cdc6/DNA interaction . Our results favor a hypothesis that Cdc6 may function as an architectural factor in the assembly of a functional initiation replication complex.

Genome Res, 1997 Dec, 7(12), 1169 - 73
The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form; Hudson JR Jr et al.; Nearly all of the open reading frames (ORFs) of the yeast Saccharomyces cerevisiae have been synthesized by PCR using a set of approximately 6000 primer pairs . Each of the forward primers has a common 22-base sequence at its 5' end, and each of the back primers has a common 20-base sequence at its 5' end . These common termini allow reamplification of the entire set of original PCR products using a single pair of longer primers-in our case, 70 bases . The resulting 70-base elements that flank each ORF can be used for rapid and efficient cloning into a linearized yeast vector that contains these same elements at its termini . This cloning by genetic recombination obviates the need for ligations or bacterial manipulations and should permit convenient global approaches to gene function that require the assay of each putative yeast gene.

J Bacteriol, 1998 Jan, 180(2), 225 - 30
Growth-independent regulation of CLN3 mRNA levels by nutrients in Saccharomyces cerevisiae; Parviz F et al.; Saccharomyces cerevisiae cells regulate progress through the G1 phase of the cell cycle in response to nutrients, moving quickly through G1 in rich medium and slowly in poor medium . Recent work has shown that the levels of Cln3 protein, a G1 cyclin, are low in cells growing in poor medium and high in cells growing rapidly in rich medium, consistent with the previously recognized role of Cln3 in promoting passage through Start . Cln3 protein levels appear to be regulated both transcriptionally and posttranscriptionally . We have worked to define the nutrient signals that regulate CLN3 mRNA levels . We find that CLN3 mRNA levels are high during log-phase growth in glucose medium, low in postdiauxic cells growing on ethanol, and slightly lower still in cells in stationary phase . CLN3 mRNA levels are induced by glucose in a process that involves transcriptional control, requires metabolism of the glucose, and is independent of the Ras-cyclic AMP pathway . CLN3 mRNA levels are also positively regulated by nitrogen sources, but phosphorus and sulfur limitation do not affect CLN3 message levels.

J Photochem Photobiol B, 1997 Nov, 41(1-2), 90 - 102
Selective photoaffinity labelling of one mitochondrial protein in living cells of Saccharomyces cerevisiae with the fluorescent probe APMC . Identification of the target protein as subunit I of cytochrome c oxidase; Haass-Mannle H et al.; The lipophilic, cationic fluorochrome azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living yeast cells (Saccharomyces cerevisiae WT X 2180) . It contains a photosensitive diazirine ring and is suitable for photoaffinity labelling . By combining photoaffinity labelling, micro-gel electrophoresis (SDS-PAGE), and detection of the APMC fluorescence with a microfluorimeter, we established a highly sensitive procedure for determining the apparent molecular weight of the APMC-labelled proteins in yeast cells . On vital staining at 0.1 microM APMC for 30 min, only one mitochondrial protein with an apparent molecular weight of 40 kDa is labelled with high intensity . At increased dye concentrations proteins of 47 and 49 kDa are labelled too, however not until all binding sites of the 40 kDa protein are occupied . Obviously, the APMC cations have a pronounced affinity for this protein . It was shown by fractional centrifugation that the labelled 40 kDa protein is a constituent of the inner mitochondrial membrane . One driving force for the accumulation of the APMC cations is the trans-membrane potential (TMP) across the inner mitochondrial membrane . Consequently, uncouplers like dinitrophenol (DNP) and carbonylcyanidechlorophenyl-hydrazone (CCCP), ionophores (valinomycin, gramicidin), and inhibitors of the respiratory chain (myxothiazol, KCN), which decrease the TMP, also diminish the APMC accumulation and labelling . And conversely, drugs, which hyperpolarize the inner membrane (nigericin, atractyloside), favour APMC labelling . Another driving force of APMC accumulation is the dye's lipophilicity, which facilitates dye accumulation by hydrophobic interaction with the very lipophilic proteins of the inner mitochondrial membranes . This was shown by competitive double staining experiments . Thiamine strongly inhibits APMC labelling . Obviously, the transport of the APMC cations is facilitated by the thiamine carrier, and thiamine competes for the same binding sites, which are occupied by the dye cations . Chloramphenicol is an inhibitor of the mitochondrial protein synthesis without affecting the TMP . On preincubation, chloramphenicol completely quenches the signal of the 40 kDa protein . Therefore, this protein must be encoded on the mtDNA . The only 40 kDa protein with adequate properties is the subunit I of cytochrome c oxidase . Obviously, it is the preferred target of the APMC cations on photoaffinity labelling . This assignment agrees with the strong hydrophobicity of the labelled 40 kDa protein, which was tested with various detergents . It also agrees with the solvatochromism of the protein-bound APMC label, and finally with the paralellism of the labelled protein with cytochrome c oxidase on fractional ammonium sulfate precipitation.

Mol Gen Genet, 1997 Dec, 257(1), 71 - 82
Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae; Selva EM et al.; The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences . In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition . In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains . When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains . The increases were greater in the msh2 msh3 double mutant than in either single mutant . When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions . However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type . The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation . Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination . In all strains, very few (< 10%) recombinants could be attributed to exceptional events . These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges.

J Biol Chem, 1997 Dec 19, 272(51), 32566 - 72
Involvement of yeast sphingolipids in the heat stress response of Saccharomyces cerevisiae; Jenkins GM et al.; A role for sphingolipids in the yeast heat stress response has been suggested by the isolation of suppressors of mutants lacking these lipids, which are unable to grow at elevated temperatures . The current study examines the possible role of sphingolipids in the heat adaptation of yeast cells as monitored by growth and viability studies . The suppressor of long chain base auxotrophy (SLC, strain 7R4) showed a heat-sensitive phenotype that was corrected by transformation with serine palmitoyltransferase . Thus, the deficiency in sphingolipids and not the suppressor mutation was the cause of the heat-sensitive phenotype of the SLC strain 7R4 . The ability of sphingolipids to rescue the heat-sensitive phenotype was examined, and two endogenous yeast sphingoid backbones, phytosphingosine and dihydrosphingosine, were found to be most potent in this effect . Next, the effect of heat stress on the levels of the three major classes of sphingolipids was determined . The inositol phosphoceramides showed no change over a 1.5-h time course . However, the four detected species of sphingoid bases increased after 15 min of heat stress from 1.4- to 10.8-fold . The largest increases were seen in two sphingoid bases, C20 phytosphingosine and C20 dihydrosphingosine, which increased 6.4- and 10.8-fold over baseline, respectively . At 60 min of heat stress two species of yeast ceramide increased by 9.2- and 10.6-fold over baseline . The increase seen in the ceramides was partially decreased by Fumonisin B1, a ceramide synthase inhibitor . Therefore, heat stress induces accumulation of sphingoid bases and of ceramides, probably through de novo synthesis . Taken together, these results demonstrate that sphingolipids are involved in the yeast heat stress adaptation.

Folia Microbiol (Praha), 1997, 42(6), 557 - 61
Uracilless death and papillae formation in rad6-1 polyauxotrophic strains of Saccharomyces cerevisiae; Rojas Gil AP et al.; Both uracilless death and papillae formation during uracil starvation are markedly more extensive in rad6-1 than in RAD6 strains . Osmotic stabilization with 1 mol/L glucitol improves the growth of rad6-1 polyauxotrophic strains in supplemented minimal medium and partially suppresses both the uracilless death and cannibalistic growth of papillae on colonies.

Folia Microbiol (Praha), 1997, 42(5), 451 - 6
Measurement of membrane potential in Saccharomyces cerevisiae by the electrochromic probe di-4-ANEPPS: effect of intracellular probe distribution; Chaloupka R et al.; Changes in the membrane potential of Saccharomyces cerevisiae were monitored by the electrochromic probe 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans- ethenyl)pyridinium)propanesulfonate (di-4-ANEPPS) that should incorporate into the plasma membrane . The probe had suitable spectral characteristics and exhibited an electrochromic shift upon a change in membrane potential but the magnitude of the response increased with time . The presence and properties of the cell wall affected the extent of cell staining . The time dependence of the fluorescent response indicated that the probe was not incorporated solely into the plasma membrane but spread gradually into the whole cell; this was confirmed by confocal microscopy . The probe is therefore suitable for assessing membrane potential changes only over time intervals up to 30 min . Longer monitoring will require either a modified staining protocol or a derivatization of the probe molecule . As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

Eur J Cell Biol, 1997 Dec, 74(4), 350 - 60
Disruption of three phosphatidylinositol-polyphosphate 5-phosphatase genes from Saccharomyces cerevisiae results in pleiotropic abnormalities of vacuole morphology, cell shape, and osmohomeostasis; Srinivasan S et al.; As a result of the genome sequencing project in Saccharomyces cerevisiae, three open reading frames were found in the yeast genome that contain sequences with strong homology to all the domains conserved among the four mammalian phosphatidylinositol-phosphate 5-phosphatases: inpp5bp, ocrl1p, synaptojanin, and ship . In addition, all three yeast gene products shared with synaptojanin regions of homology to the SAC1 gene of yeast . Disruption of each of these genes singly and in pairs produced mutant strains that were viable but demonstrated variable phenotypes of abnormal vacuolar and plasma membrane morphology as well as increased sensitivity to osmotic stress . Total phosphatidylinositol-(4,5)-bisphosphate 5-phosphatase activity was reduced to varying degrees in each of the strains . No defect in carboxypeptidase Y sorting was seen in a processing and targeting assay . Abnormal actin cytoskeleton morphology was present in some of the strains carrying mutations in two of the genes.

Yeast, 1997 Dec, 13(15), 1477 - 89
In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae; Caro LH et al.; Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway . On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal . Seven additional ORFs were found to belong to this group . Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine . In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall . It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site . Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins . For members of three plasma-membrane protein families, a function has been described . On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions . The GPI-attachment site of yeast slightly differs from mammalian cells . This might be of use in the development of anti-fungal drugs.

Yeast, 1997 Dec, 13(15), 1465 - 75
Identification of the gene encoding scHelI, a DNA helicase from Saccharomyces cerevisiae; Bean DW et al.; The gene encoding scHelI, a previously characterized DNA helicase from Saccharomyces cerevisiae, has been identified as YER176w, an open reading frame on chromosome V . The gene has been named HEL1 to indicate the DNA helicase activity of the gene product . HEL1 was identified by screening a lambda gt11 yeast protein expression library with antiserum to purified scHelI . Several independent immunopositive clones were isolated and shown to contain portions of HEL1 either by sequencing or by hybridization to a probe containing HEL1 sequences . The HEL1 open reading frame includes the seven conserved helicase motifs, consistent with the DNA helicase activity of scHelI, and the predicted size of the protein is in agreement with the size of purified scHelI . Partially purified cellular extracts from a hel1 deletion mutant strain did not contain scHelI activity . Homology searches revealed protein sequence homology between HEL1 and two previously identified and biochemically characterized yeast helicases, encoded by the DNA2 and UPF1 genes . Haploid hel1 deletion strains were constructed and shown to be viable with growth rates equivalent to those of parental strains . These strains did not differ from the parental strains in ultraviolet light sensitivity or the generation of petite colonies . Furthermore, these haploid deletion strains were capable for mating, the resultant diploid homozygous mutants were viable, capable of sporulation, and the spores displayed no reduction in viability.

Yeast, 1997 Dec, 13(15), 1423 - 35
Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription; Szczypka MS et al.; The metal ions, Cu2+/+ and Fe3+/2+, are essential co-factors for a wide variety of enzymatic reactions . However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins . Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu2+ for high affinity transport of Fe2+ into yeast cells has established a physiological link between these two trace metal ions . In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly for acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis . Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe2+ transport, also repressible by exogenous copper ions . The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution of redox states of Fe3+/2+ in the presence of elevated extracellular concentrations of copper ions . Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions.

Appl Environ Microbiol, 1998 Jan, 64(1), 316 - 24
Impaired secretion of a hydrophobic cutinase by Saccharomyces cerevisiae correlates with an increased association with immunoglobulin heavy-chain binding protein (BiP); Sagt CM et al.; This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028) . Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated . Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level . Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell . Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER) . The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase . Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP . These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.

Arch Biochem Biophys, 1997 Dec 15, 348(2), 383 - 90
Expression of four rat CYP2D isoforms in Saccharomyces cerevisiae and their catalytic specificity; Wan J et al.; We cloned four cDNAs belonging to the CYP2D subfamily to express these enzymes in yeast cells and to compare their catalytic activities simultaneously . Three are believed to be alleles of CYP2D1, 2D2, and 2D3, respectively, based on high nucleotide sequence similarity, while CYP2D4 had both sequences of CYP2D4 and CYP2D18 . Expression plasmids carrying CYP2D cDNAs were transformed into Saccharomyces cerevisiae . Typical P450 CO-difference spectra with absorbance maximum at 448 nm were recorded with microsomal preparations from the yeast cells expressing the four CYP2D forms . A catalytic study of these CYP2D forms was done with debrisoquine, bufuralol, and lidocaine . CYP2D2 had the highest debrisoquine 4-hydroxylation (2.2 nmol/min/nmol P450) activity, similar to that (2.2 nmol/min/nmol) of human CYP2D6 expressed in yeast cells . CYP2D3 had high lidocaine N-deethylation (43 nmol/min/nmol P450) activity, and both CYP2D3 and 2D2 exhibited high lidocaine 3-hydroxylation (2.4 and 1.6 nmol/min/nmol P450, respectively) activity . Bufuralol 1'-hydroxylation catalytic capabilities were comparable among the four isoforms . The activity of CYP2D1 was relatively low toward the three substrates (debrisoquine, 0.091; bufuralol, 1.5; lidocaine 3-hydroxylation, 0.019; lidocaine N-deethylation, 2.8 nmol/min/nmol P450) . These findings indicate that debrisoquine, a typical substrate for CYP2D forms, was mainly metabolized by CYP2D2 but not CYP2D1 in rat liver and that the CYP2D forms have different substrate specificity.

Biopolymers, 1998, 45(1), 21 - 34
Conformational analysis of cyclic analogues of the Saccharomyces cerevisiae alpha-factor pheromone; Antohi O et al.; Analogues of the alpha-factor mating pheromone (WHWLQLKPGQPMY) from Saccharomyces cerevisiae in which the side chains of residues 7 and 10 were joined by lactam bonds were studied by nmr and molecular modeling . These investigations were carried out to discern the effect of lactam ring size on conformation and to ascertain whether the side chain i to i + 3 cyclized tetramers {H . R . Marepalli et al . (1996) Journal of the American Chemical Society, Vol . 118, pp . 6531-6539} can be considered as conformation-constraining building blocks when introduced into a long peptide chain . Nuclear Overhauser effect constraints, temperature coefficients, and backbone torsional angles were derived from 1H-nmr spectra measured in DMSO-d6 . Modeling studies using the above constraints indicate that the lactam regions of the tridecapeptides assume various combinations of type II beta-turns, gamma-turns, and gamma 1-turns, but never type I beta-turns . These investigations provide evidence that the tetrapeptide building blocks retain their preferred conformations in larger molecules and can be used to control the architecture of regions of such peptides.

Gene, 1997 Dec 19, 204(1-2), 145 - 51
Identification of the regions of porcine VCP preventing its function in Saccharomyces cerevisiae; Madeo F et al.; Cdc48p is essential for homotypic endoplasmic reticular fusion in Saccharomyces cerevisiae . It is localized at the endoplasmic reticulum during most of the cell division cycle but concentrates in the nucleus at the G1/S-transition . Its mammalian homologue VCP alternates between the endoplasmic reticulum and the centrosome in dependence of the cell cycle . Though Cdc48p and porcine VCP show a high sequence conservation--almost 70% of their amino acid residues are identical the VCP gene fails to complement a disruption of CDC48 . Complementation studies with CDC48 and VCP gene hybrids show that an exchange of the central Cdc48p domain for the central VCP domain prevents a complementation of a CDC48 disruption, although this is the best conserved region between the two proteins . Protein chimeras containing the N-terminal part of VCP only complement a disruption of CDC48 when expressed at high levels . The respective yeast strain shows a nucleus devoid of Cdc48p . In contrast to VCP, Cdc48p contains an almost perfect nuclear targeting sequence in this region . Exchange of the C-terminal Cdc48p domain for the C-terminus of VCP leads to normal viability of the cell, even at low expression levels.

Biochim Biophys Acta, 1997 Dec 5, 1343(2), 187 - 92
Protein-protein interaction between the RVS161 and RVS167 gene products of Saccharomyces cerevisiae; Navarro P et al.; As previous studies indicated that the RVS161 and RVS167 gene products of Saccharomyces cerevisiae seem to be involved in the same cellular function, we considered the possibility of a complex between the proteins encoded by these two genes . Using the two hybrid system, we have shown that Rvs167p interacts with Rvs161p, through its N-terminal domain which contains predicted coiled-coil structures . Moreover, if a tagged Rvs 167p protein was immuno adsorbed under non-denaturing conditions, it brought along the Rvs161p protein . These results confirmed the hypothesis of an in vivo complex between the two proteins.

Eur J Biochem, 1997 Dec 1, 250(2), 232 - 41
Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity; Crabeel M et al.; We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis . While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase . Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine . Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein {Liu, Y-S., Van Heeswijck R., Hoj, P . & Hoogenraad, N . (1995) Eur . J . Biochem . 228, 291-296}; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable-type . The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence . Yeast Arg7p shares between 32-43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized . Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase . We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase) . Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay . The yeast enzyme is thus clearly, albeit modestly, bifunctional . As with several bacterial ornithine acetyltransferases, the activity of Arg7p was practically insensitive to arginine but strongly inhibited by ornithine, which behaved as a competitive inhibitor.

FEBS Lett, 1997 Dec 15, 419(2-3), 259 - 62
The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating; Fischer M et al.; The yeast Saccharomyces cerevisiae gene CCH1 (ORF YGR217w) shows high homology with animal calcium channel alpha1-subunit genes . Knock-out mutants were constructed of Cch1 and of Mid1 which is known to mediate Ca2+ influx in response to the alpha-mating pheromone . Cch1 is involved in calcium influx and the late stage of the mating process . The cch1 mutant sensitivity against the alpha-mating pheromone can be reduced by the addition of extra calcium . The product of this gene is likely to interact with the MID1 gene product in Ca influx or its control.

Mol Microbiol, 1997 Nov, 26(4), 809 - 20
The control of morphogenesis in Saccharomyces cerevisiae by Elm1 kinase is responsive to RAS/cAMP pathway activity and tryptophan availability; Garrett JM; Many fungi undergo a morphological transition to filamentous growth in response to limiting nutrient conditions . Constitutively elongated Saccharomyces cerevisiae mutants (elm) have been isolated; the ELM1 gene encodes a putative serine/threonine protein kinase . A novel allele, elm1-15, has been isolated in an S288C-derived strain, which causes a pleiotropic phenotype, including media-specific growth effects, abnormal morphology and altered stress response, in cells that are auxotrophic for tryptophan . elm1-15 trp1 cells cannot use many nitrogen sources, are sensitive to amino acid analogues, have very low general amino acid permease activity and do not accumulate trehalose . In contrast, haploid elm1-15 TRP1 cells grow well in budding form on all media, are stress resistant and overaccumulate trehalose . Several lines of evidence suggest that Elm1 acts on functions related to the RAS/cAMP pathway . Overexpression of Elm1 partially rescues the ts phenotype of cdc25 and cyr1 mutants . Deletion of ELM1 in low PKA activity mutants increased the severity of their phenotypes, and activation of Ras2 decreases the cell elongation phenotype of elm1 mutants . A 'signal integration' model for the complex relationship of Elm1 and the RAS/cAMP pathway in controlling morphogenesis in response to nutrients is proposed.

Mol Microbiol, 1997 Nov, 26(4), 675 - 85
Regulation of the yeast SPS19 gene encoding peroxisomal 2,4-dienoyl-CoA reductase by the transcription factors Pip2p and Oaf1p: beta-oxidation is dispensable for Saccharomyces cerevisiae sporulation in acetate medium; Gurvitz A et al.; The yeast SPS19 gene encoding the peroxisomally targeted 2,4-dienoyl-CoA reductase shares its promoter region (291 bp) with the sporulation-specific gene SPS18 . SPS19 is induced during sporulation in diploids but to a lesser extent than SPS18; under oleate induction conditions, SPS19, but not SPS18, is transcribed via an oleate response element (ORE) independently of ploidy or sporulation . The SPS19 ORE is the binding target of the Pip2p and Oaf1p transcription factors, and an SPS19-lacZ reporter gene, which is highly expressed in oleate-induced cells, is not activated in haploids devoid of either protein . We examined the expression of CYC1-lacZ reporter constructs carrying the SPS19 and CTA1 OREs in diploids propagated under sporulation conditions and have shown that OREs are not sufficient for heterologous expression during yeast development . In addition, diploids deleted at either PIP2 or OAF1 demonstrated abundant ascosporogenesis, indicating that these genes are not essential for sporulation . A deltapex6 strain lacking peroxisomal structures and one devoid of fatty acyl-CoA oxidase (deltapox1), the first step in fungal beta-oxidation, were both proficient for sporulation and, hence, beta-oxidation and the peroxisomal compartment containing it are dispensable for meiotic development.

Genes Cells, 1997 Oct, 2(10), 631 - 43
Nuclear accumulation of Saccharomyces cerevisiae Mcm3 is dependent on its nuclear localization sequence; Young MR et al.; BACKGROUND: The proteins of the Mcm2-7 family are required for the initiation of DNA replication . In Saccharomyces cerevisiae the nuclear envelope does not break down during the mitotic phase of the cell cycle . Large nuclear proteins, such as the Mcm proteins, which accumulate in the nucleus during specific portions of the cell cycle, must have regulated mechanisms to direct their entry into the nucleus . RESULTS: We have identified a nuclear localization sequence (NLS) in Mcm3, and demonstrated that it is necessary for the translocation of Mcm3 into the nucleus and sufficient for directing Escherichia coli beta-galactosidase to the nucleus . Immediately adjacent to the nuclear localization sequence are four potential sites for phosphorylation by Cdc28 . Mutagenesis of all four sites has no immediate phenotypic effect on cell growth or viability, nor does it affect nuclear accumulation of Mcm3, although two-dimensional protein gel analysis has shown that at least some of these sites are normally phosphorylated in vivo . Substitution of the Mcm3 NLS by the SV40 large T-antigen NLS also directs the nuclear accumulation of the Mcm3-T-antigen protein, although cell growth is compromised . Replication activity in cells bearing either the Mcm3-Cdc28 phosphorylation site mutations or the Mcm3 T-antigen NLS substitution, as measured by plasmid stability assays, is comparable to activity in wild-type cells . CONCLUSIONS: The Mcm3 protein is imported into the nucleus by a specific NLS . The cell cycle specific nuclear accumulation of Mcm3 appears to be a result of nuclear retention or nuclear targeting, rather than nuclear import regulated through the NLS.

Genes Cells, 1997 Oct, 2(10), 615 - 29
Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have both distinct and overlapping roles in meiotic recombination; Shinohara A et al.; BACKGROUND: Rad51 and Dmc1 are Saccharomyces cerevisiae homologues of the Escherichia coli recombination protein RecA . Mutant analysis has shown that both proteins are required for normal meiotic recombination, for timely and efficient formation of synaptonemal complex and for normal progression out from meiotic prophase . RESULTS: We have further characterized rad51 and dmc1 single mutants . A dmc1 mutation confers more severe defects in double strand break (DSB) resolution, crossover recombination and meiotic progression than does a rad51 mutant; in contrast, during return to growth, a rad51 mutation confers more severe defects in viability and intrachromosomal recombination than does a dmc1 mutation . Analysis of a rad51 dmc1 double mutant, in parallel with single mutants, shows that the double mutant is more defective with respect to the formation of crossovers during meiosis and, especially strikingly, with respect to interhomologue and intrachromosomal recombination during return to growth . Consistent with the observation of DMC1-dependent recombination in a rad51 mutant, subnuclear complexes of Dmc1 protein were detected for the first time in this mutant . In contrast to the effects on recombination, the effect of the double mutant on meiotic progression was similar to that of the rad51 single mutant . CONCLUSION: Rad51 and Dmc1 each make unique contributions to meiotic recombination . However, the two proteins are capable of substituting for one another under some circumstances, implying that they most likely share at least one recombination function . Recombination and cell cycle phenotypes are all consistent with the possibility that a dmc1 mutation causes an arrest of the post-DSB recombination complexes at a later, more stable stage than does a rad51 mutation.

Gene, 1997 Dec 12, 203(2), 189 - 97
Cloning of porcine neuron growth inhibitory factor (metallothionein III) cDNA and expression of the gene in Saccharomyces cerevisiae; Wang SH et al.; Growth inhibitory factor (GIF), a member of the metallothionein (MT) family, is also known as MTIII . This protein distinguishes itself from other MT isoforms by exerting an inhibitory effect on cortical neuron growth instead of metal ion buffering . In this work, we cloned MTIII genes from a porcine brain cDNA library . Two species of clones were isolated that vary with respect to one nt in the coding sequence . This discrepancy results in the translation of two MTIII primary structures having a different amino acid at residue 46 . Herein, both MTIII cDNAs were constructed into an expression vector and transformed into yeast cells, respectively . The yeast carrying either MTIII gene displayed a similar metal tolerance when cultured in a medium containing metal . The resistance to metal toxicity was attributed to the expression of MTIII gene which was confirmed by RNA and protein analyses . The characteristics of the protein stability, metal binding affinity and ultraviolet absorption spectrum of the yeast produced MTIII are also compared with those of MTII . The comparison reveals that both MTs have similar physical characteristics . Moreover, circular dichroism spectrum of Cd saturated MTIII was analyzed as well . Typical Cys-Cd bands for MTII appear in the spectrum, indicating similar metal-thiol interactions for MTIII to those for other MT isoforms.

Protein Expr Purif, 1997 Dec, 11(3), 284 - 8
Purification and functional reconstitution of the human CHIP28 water channel expressed in Saccharomyces cerevisiae; Laize V et al.; The yeast Saccharomyces cerevisiae was used for heterologous expression of the human CHIP28 water Aquaporin-1 channel (Aquaporin-1) . A nine-amino-acid epitope of the influenza hemagglutinin protein (HA epitope), recognized by the monoclonal antibody 12CA5, was chosen to tag CHIP28 at its N-terminus . Epitope-tagged CHIP28 was purified from yeast extracts by immunochromatography on protein A/ 12CA5-coupled beads, after KI extraction and detergent solubilization, then concentrated by anion exchange chromatography . Purified protein was reconstituted in proteoliposomes and was shown to function as a water channel by stopped-flow spectrophotometry . This study demonstrates that the yeast has the capacity to produce functional aquaporins at levels sufficient for biochemical and biophysical analyses.

Biochem Biophys Res Commun, 1997 Dec 18, 241(2), 334 - 40
Molecular cloning of DNAs encoding the regulatory subunits of elongin from Saccharomyces cerevisiae and Drosophila melanogaster; Aso T et al.; The Elongin complex strongly stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along the DNA . Elongin is composed of a transcriptionally active A subunit and two positive regulatory B and C subunits . The Elongin complex is a potential target for regulation by the von Hippel-Lindau (VHL) tumor suppressor protein, which is capable of binding stably to the Elongin BC complex and preventing it from activating Elongin A . Here, we report the molecular cloning of a Saccharomyces cerevisiae genomic DNA encoding Elongin C subunit and of Drosophila cDNAs encoding Elongin B and C subunits . The predicted amino acid sequence of each protein shows a high degree of similarity with the mammalian proteins . The recombinant yeast Elongin C protein interacts with both mammalian Elongin A and VHL tumor suppressor protein . Moreover, yeast Elongin C strongly induces the transcriptional elongation activity of mammalian Elongin A . The expression of yeast Elongin C mRNA is dramatically upregulated during sporulation; however, the gene is not essential for sporulation and viability in yeast cell.

Infect Immun, 1998 Jan, 66(1), 59 - 64
Saccharomyces cerevisiae-secreted fusion proteins Pfs25 and Pfs28 elicit potent Plasmodium falciparum transmission-blocking antibodies in mice; Gozar MM et al.; Transmission-blocking vaccines based on sexual-stage surface antigens of Plasmodium falciparum may assist in the control of this lethal form of human malaria . Two vaccine candidates, Pfs25 and Pfs28, were produced as single recombinant fusion proteins . The 39-kDa chimeric proteins, having a C-terminal His6 tag, were secreted by Saccharomyces cerevisiae, using the prepro-alpha-factor leader sequence . Pfs25-28 fusion proteins were significantly more potent than either Pfs25 or Pfs28 alone in eliciting antibodies in mice that blocked oocyst development in Anopheles freeborni mosquitoes: complete inhibition of oocyst development in the mosquito midgut was achieved with fewer vaccinations, at a lower dose, and for a longer duration than with either Pfs25 or Pfs28 alone . Increased antigen-specific immunoglobulin G titers and highly significant lymphoproliferative stimulation by Pfs28-containing antigens suggest the presence of an immunodominant helper T-cell epitope in the Pfs28 portion of the fusion proteins . This epitope may be responsible for the enhanced humoral response to both Pfs25 and Pfs28 antigens . Protein production of the fusion protein was improved 12-fold by converting Pfs28 codons to yeast-preferred codons (TBV28), using a modified ADH2 promoter and incorporating a (Glu-Ala)2 repeat after the Kex2 cleavage site.

J Biol Chem, 1997 Dec 5, 272(49), 30998 - 1005
Characterization of an ADP-ribosylation factor-like 1 protein in Saccharomyces cerevisiae; Lee FJ et al.; ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and are believed to participate in vesicular transport in both exocytic and endocytic pathways . Several ARF-like proteins (ARLs) have been cloned from Drosophila, rat, and human; however, the biological functions of ARLs are unknown . We have identified a yeast gene (ARL1) encoding a protein that is structurally related (>60% identical) to human, rat, and Drosophila ARL1 . Biochemical analyses of purified recombinant yeast ARL1 (yARL1) protein revealed properties similar to those ARF and ARL1 proteins, including the ability to bind and hydrolyze GTP . Like other ARLs, recombinant yARL1 protein did not stimulate cholera toxin-catalyzed auto-ADP-ribosylation . yARL1 was not recognized by antibodies against mammalian ARLs or yeast ARFs . Anti-yARL1 antibodies did not cross-react with yeast ARFs, but did react with human ARLs . On subcellular fractionation, yARL1, similar to yARF1, was localized to the soluble fraction . The amino terminus of yARL1, like that of ARF, was myristoylated . Unlike Drosophila Arl1, yeast ARL1 was not essential for cell viability . Like rat ARL1, yARL1 might be associated in part with the Golgi complex . However, yARL1 was not required for endoplasmic reticulum-to-Golgi protein transport, and it may offer an opportunity to define an ARL function in another kind of vesicular trafficking, such as the regulated secretory pathway.

J Biol Chem, 1997 Dec 5, 272(49), 30975 - 83
Biochemical characterization of mammalian translation initiation factor 3 (eIF3) . Molecular cloning reveals that p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1; Chaudhuri J et al.; Eukaryotic translation initiation factor 3 (eIF3), which plays an essential role in initiation of protein synthesis, was purified from rabbit reticulocyte lysates using an assay that specifically measures its ability to stimulate the binding of Met-tRNAf (as a Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits . Purified eIF3 consisted of six major polypeptides of molecular masses 110, 67, 42, 40, 36, and 35 kDa but lacked the 170-kDa polypeptide reported to be a constituent of other eIF3 preparations . Characterization of purified eIF3 lacking the 170-kDa polypeptide showed that the eIF3-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex . eIF3, which was originally bound to the 40 S initiation complex, was released from the 40 S subunit during the subunit joining reaction . Additionally, chicken antibodies raised against rabbit reticulocyte eIF3 were used to immunochemically characterize eIF3 subunits and to isolate a 3.1-kilobase pair human cDNA that encodes the p110 subunit of mammalian eIF3 . The derived amino acid sequence (calculated Mr 95,214) shows that the p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1p, a subunit of yeast eIF3.

Mol Cell Biol, 1998 Jan, 18(1), 433 - 41
Cln3-associated kinase activity in Saccharomyces cerevisiae is regulated by the mating factor pathway; Jeoung DI et al.; The Saccharomyces cerevisiae cell cycle is arrested in G1 phase by the mating factor pathway . Genetic evidence has suggested that the G1 cyclins Cln1, Cln2, and Cln3 are targets of this pathway whose inhibition results in G1 arrest . Inhibition of Cln1- and Cln2-associated kinase activity by the mating factor pathway acting through Far1 has been described . Here we report that Cln3-associated kinase activity is inhibited by mating factor treatment, with dose response and timing consistent with involvement in cell cycle arrest . No regulation of Cln3-associated kinase was observed in a fus3 kss1 strain deficient in mating factor pathway mitogen-activated protein (MAP) kinases . Inhibition occurs mainly at the level of specific activity of Cln3-Cdc28 complexes . Inhibition of the C-terminally truncated Cln3-1-associated kinase is not observed; such truncations were previously identified genetically as causing resistance to mating factor-induced cell cycle arrest . Regulation of Cln3-associated kinase specific activity by mating factor treatment requires Far1 . Overexpression of Far1 restores inhibition of C-terminally truncated Cln3-1-associated kinase activity . G2/M-arrested cells are unable to regulate Cln3-associated kinase, possibly because of cell cycle regulation of Far1 abundance . Inhibition of Cln3-associated kinase activity by the mating factor pathway may allow this pathway to block the earliest step in normal cell cycle initiation, since Cln3 functions as the most upstream G1-acting cyclin, activating transcription of the G1 cyclins CLN1 and CLN2 as well as of the S-phase cyclins CLB5 and CLB6.

Mol Cell Biol, 1998 Jan, 18(1), 420 - 32
A hydrophobic segment within the 81-amino-acid domain of TFIIIA from Saccharomyces cerevisiae is essential for its transcription factor activity; Rowland O et al.; Transcription factor IIIA (TFIIIA) binds to the internal control region of the 5S RNA gene as the first step in the in vitro assembly of a TFIIIB-TFIIIC-TFIIIA-DNA transcription complex . An 81-amino-acid domain that is present between zinc fingers 8 and 9 of TFIIIA from Saccharomyces cerevisiae is essential for the transcription factor activity of this protein (C . A . Milne and J . Segall, J . Biol . Chem . 268:11364-11371, 1993) . We have monitored the effect of mutations within this domain on the ability of TFIIIA to support transcription of the 5S RNA gene in vitro and to maintain cell viability . TFIIIA with internal deletions that removed residues 282 to 315, 316 to 334, 328 to 341, or 342 to 351 of the 81-amino-acid domain retained activity, whereas TFIIIA with a deletion of the short leucine-rich segment 352NGLNLLLN359 at the carboxyl-terminal end of this domain was devoid of activity . Analysis of the effects of double and quadruple mutations in the region extending from residue 336 to 364 confirmed that hydrophobic residues in this portion of the 81-amino-acid domain, particularly L343, L347, L354, L356, L357, and L358, and to a lesser extent F336 and L337, contributed to the ability of TFIIIA to promote transcription . We propose that these hydrophobic residues play a role in mediating an interaction between TFIIIA and another component of the transcriptional machinery . We also found that TFIIIA remained active if either zinc finger 8 or zinc finger 9 was disrupted by mutation but that TFIIIA containing a disruption of both zinc finger 8 and zinc finger 9 was inactive.

Mol Cell Biol, 1998 Jan, 18(1), 30 - 8
Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae; Lee DH et al.; An accumulation in cells of unfolded proteins is believed to be the common signal triggering the induction of heat shock proteins (hsps) . Accordingly, in Saccharomyces cerevisiae, inhibition of protein breakdown at 30 degrees C with the proteasome inhibitor MG132 caused a coordinate induction of many heat shock proteins within 1 to 2 h . Concomitantly, MG132, at concentrations that had little or no effect on growth rate, caused a dramatic increase in the cells' resistance to very high temperature . The magnitude of this effect depended on the extent and duration of the inhibition of proteolysis . A similar induction of hsps and thermotolerance was seen with another proteasome inhibitor, clasto-lactacystin beta-lactone, but not with an inhibitor of vacuolar proteases . Surprisingly, when the reversible inhibitor MG132 was removed, thermotolerance decreased rapidly, while synthesis of hsps continued to increase . In addition, exposure to MG132 and 37 degrees C together had synergistic effects in promoting thermotolerance but did not increase hsp expression beyond that seen with either stimulus alone . Although thermotolerance did not correlate with hsp content, another thermoprotectant trehalose accumulated upon exposure of cells to MG132, and the cellular content of this disaccharide, unlike that of hsps, quickly decreased upon removal of MG132 . Also, MG132 and 37 degrees C had additive effects in causing trehalose accumulation . Thus, the resistance to heat induced by proteasome inhibitors is not just due to induction of hsps but also requires a short-lived metabolite, probably trehalose, which accumulates when proteolysis is reduced.

Trends Biotechnol, 1997 Dec, 15(12), 487 - 94
G-protein-coupled receptors in Saccharomyces cerevisiae: high-throughput screening assays for drug discovery; Pausch MH; G-protein-coupled receptors are an important class of therapeutic drug targets by virtue of their roles in the regulation of diverse cellular functions . Recent advances in the expression of heterologous G-protein-coupled receptors in the yeast Saccharomyces cerevisiae have led to the development of sensitive and selective assays of their ligand-induced activation . Implementation of this new technology in the high-throughput screening of compound libraries has enabled the discovery of novel ligands for the G-protein-coupled somatostatin receptor . This article describes the broad applicability of the technology and its use in drug discovery.

Int J Biochem Cell Biol, 1997 Aug-Sep, 29(8-9), 1097 - 101
Carbon and nitrogen sources regulate delta-aminolevulinic acid and gamma-aminobutyric acid transport in Saccharomyces cerevisiae; Correa Garcia S et al.; Evidence has been obtained showing that transport of delta-aminolevulinic acid (ALA), a precursor of porphyrin biosynthesis in Saccharomyces cerevisiae, is mediated by the gamma-aminobutyric acid (GABA)-specific permease, UGA4 . In yeast GABA is also incorporated by the general amino acid permease (GAP1) and the specific proline permease (PUT4) . The aim of the present work was to carry out a comparative study on the regulation of ALA and GABA transport to confirm our proposal that both compounds share the UGA4 permease . ALA and GABA uptake were measured in cells grown on minimal media with different carbon and/or nitrogen sources . To study the effect of the carbon source on UGA4 permease, ALA and GABA incorporation were measured in D27 strain, lacking GAP1 permease, and grown in proline as the sole nitrogen source, so the activity of PUT4 permease was negligible . The effect of the nitrogen source on UGA4 permease was studied measuring ALA and GABA uptake rates in cells from media with ammonium, proline and urea as nitrogen sources . It was found that the regulation by the carbon source was similar on ALA and GABA transport; they depend equally on the energetic conditions of the cells . Moreover, regulation by the nitrogen source on ALA and GABA uptake was also similar, and identical to that described already for UGA4 permease . These results are further evidence that both compounds, ALA and GABA, share the GABA-specific permease, UGA4.

Nat Biotechnol, 1997 Dec, 15(13), 1359 - 67
Genome-wide expression monitoring in Saccharomyces cerevisiae; Wodicka L et al.; The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes . This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry . The measurements are quantitative, sensitive, specific, and reproducible . Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media . Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell . Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.

Mol Gen Genet, 1997 Nov, 256(5), 469 - 80
A defect in GTP synthesis affects mannose outer chain elongation in Saccharomyces cerevisiae; Shimma Y et al.; We have found that yeast mutants that are defective in mannose outer chain elongation of N-linked glycoproteins show higher cell wall porosity than normal cells, and are hypersensitive to antibiotics with a large molecular weight; such as neomycin and geneticin . Wild-type yeast cells also showed enhanced sensitivity to neomycin in the presence of tunicamycin, an inhibitor of N-glycosylation, suggesting that the extent of N-glycosylation may affect the sensitivity of yeast cells to drugs and that sensitivity to neomycin may be an effective method for screening for yeast mutants defective in N-glycosylation . Pursuing this logic, we isolated neomycin-sensitive yeast mutants and screened them for defects in N-glycosylation . The neomycin-sensitive, N-glycosylation-defective mutants fell into 15 complementation groups including alleles of the previously isolated temperature-sensitive nes mutants nes10, nes17, and nes25 . Gene cloning revealed that NES10 was identical to SEC20, which is involved in ER-Golgi protein transport, NES17 was identical to ALG1, which encodes a beta-1,4-mannosyltransferase present in the ER, MSN17, a multicopy suppressor of nes17/alg1, was also isolated and found to be an allele of PSA1, which is involved in GDP-mannose synthesis, NES25 was identical to GUK1, which encodes a GMP kinase . Overexpression of MSN17 increased the GDP-mannose level in a wild-type strain by about threefold, and guk1 decreased the GDP-mannose level to one-fourth, suggesting a close relationship between GTP metabolism and mannose outer chain elongation; the link is presumably provided by the process of GDP-mannose transport in the Golgi membranes.

Genetics, 1997 Dec, 147(4), 1609 - 33
Analysis of mutationally altered forms of the Cct6 subunit of the chaperonin from Saccharomyces cerevisiae; Lin P et al.; The Cct double-ring chaperonin complex of Saccharomyces cerevisiae is comprised of eight essential subunits, Cct1p-Cct8p, and assists the folding of substrates such as actins and tubulins . Single and multiple amino acid replacements of Cct6p were constructed by oligonucleotide-directed mutagenesis, including changes of charged to alanine residues and uncharged to charged residues . The replacements were targeted, in part, to residues corresponding to functionally critical regions identified in the published crystal structure of the Escherichia coli chaperonin, GroEL . Here, we report the critical hydrophobic residues and clusters of hydrophilic residues in regions corresponding to those from the apical domain of GroEL implicated in peptide binding and peptide release, and certain residues in the putative equatorial domain implicated in subunit-to-subunit interaction . In contrast to their homologous counterparts in Cct2p and Cct1p, the highly conserved putative ATP binding motifs of Cct6p were relatively amenable to mutations . Our data suggest that the entire Cct6p molecule might be essential for assembly of Cct complex and might participate in binding substrates . However, there appeared to exist a functional hierarchy in ATP binding/hydrolysis among Cct subunits, as suggested by the high tolerance of Cct6p to mutations within the putative ATP binding pocket.

Genetics, 1997 Dec, 147(4), 1569 - 84
Control of amino acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST8; Roberg KJ et al.; The SEC13 gene was originally identified by temperature-sensitive mutations that block all protein transport from the ER to the Golgi . We have found that at a permissive temperature for growth, the sec13-1 mutation selectively blocks transport of the nitrogen-regulated amino acid permease, Gap1p, from the Golgi to the plasma membrane, but does not affect the activity of constitutive permeases such as Hip1p, Can1p, or Lyp1p . Different alleles of SEC13 exhibit different relative effects on protein transport from the ER to the Golgi, or on Gap1p activity, indicating distinct requirements for SEC13 function at two different steps in the secretory pathway . Three new genes, LST4, LST7, and LST8, were identified that are also required for amino acid permease transport from the Golgi to the cell surface . Mutations in LST4 and LST7 reduce the activity of the nitrogen-regulated permeases Gap1p and Put4p, whereas mutations in LST8 impair the activities of a broader set of amino acid permeases . The LST8 gene encodes a protein composed of WD-repeats and has a close human homologue . The LST7 gene encodes a novel protein . Together, these data indicate that SEC13, LST4, LST7, and LST8 function in the regulated delivery of Gap1p to the cell surface, perhaps as components of a post-Golgi secretory-vesicle coat.

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