S. cerevisiae

Genetics, 1997 Dec, 147(4), 1545 - 56
Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TID1, in mitosis and meiosis; Shinohara M et al.; The RAD54 gene, which encodes a protein in the SWI2/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae . The yeast genome project revealed a homologue of RAD54, RDH54/TID1 . Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis . The rad54 mutant is sensitive to the alkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion . The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis . However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant . These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of R4D54 . In meiosis, both single mutants produce viable spores at slightly reduced frequency . However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants . Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type . These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins.

Genetics, 1997 Dec, 147(4), 1533 - 43
RDH54, a RAD54 homologue in Saccharomyces cerevisiae, is required for mitotic diploid-specific recombination and repair and for meiosis; Klein HL; Most mitotic recombination and repair genes of Saccharomyces cerevisiae show no specificity of action for the genome ploidy . We describe here a novel repair and recombination gene that is specific for recombination and repair between homologous chromosomes . The RDH54 gene is homologous to the RAD54 gene, but rdh54 mutants do not show sensitivity to methyl methanesulfonate at concentrations that sensitize a rad54 mutant . However, the rdh54 null mutation enhances the methyl methanesulfonate sensitivity of a rad54 mutant and single rdh54 mutants are sensitive to prolonged exposure at high concentrations of methyl methanesulfonate . The RDH54 gene is required for recombination, but only in a diploid . We present evidence showing that the RDH54 gene is required for interhomologue gene conversion but not intrachromosomal gene conversion . The rdh54 mutation confers diploid-specific lethalities and reduced growth in various mutant backgrounds . These phenotypes are due to attempted recombination . The RDH54 gene is also required for meiosis as homozygous mutant diploids show very poor sporulation and reduced spore viability . The role of the RDH54 gene in mitotic repair and in meiosis and the pathway in which it acts are discussed.

Gene, 1997 Nov 12, 201(1-2), 5 - 10
Suppression of the Saccharomyces cerevisiae hac1/ire15 mutation by yeast genes and human cDNAs; Nikawa J et al.; We previously reported that the Saccharomyces cerevisiae ire15 mutation results in an inositol-auxotrophic phenotype, and that human cDNAs can suppress the ire15 mutation (Nikawa, J., 1994 . A cDNA encoding the human transforming growth factor beta receptor suppresses the growth defect of a yeast mutant . Gene 149, 367 372; Nikawa, J., Nakano, H., Ohi, N., 1996b . Structural and functional conservation of human and yeast HAC1 genes which can suppress the growth defect of the Saccharomyces cerevisiae ire15 mutant . Gene 171, 107-111) . Herein, we present evidence that the gene responsible for the ire15 mutation is HAC1, which encodes a transcription factor for KAR2, obtained by isolating a yeast single-copy supressor gene and by performing complementation analysis . Sequencing analysis revealed that the mutant HAC1 gene obtained from the ire15 mutant contained an AAA codon at position 50 instead of the AGA codon observed in the wild-type gene, resulting in the alteration of the aa from Arg to Lys . All human cDNAs and yeast multicopy suppressors, which had been isolated as suppressors for the ire15 mutation, were able to suppress the inositol-auxotrophic phenotype but not the defect in KAR2 induction of the hac1-disrupted strain.

J Biol Chem, 1997 Nov 28, 272(48), 30061 - 6
The Saccharomyces cerevisiae ACR3 gene encodes a putative membrane protein involved in arsenite transport; Wysocki R et al.; The cluster of three genes, ACR1, ACR2, and ACR3, previously was shown to confer arsenical resistance in Saccharomyces cerevisiae . The overexpression of ACR3 induced high level arsenite resistance . The presence of ACR3 together with ACR2 on a multicopy plasmid was conducive to increased arsenate resistance . The function of ACR3 gene has now been investigated . Amino acid sequence analysis of Acr3p showed that this hypothetical protein has hydrophobic character with 10 putative transmembrane spans and is probably located in yeast plasma membrane . We constructed the acr3 null mutation . The resulting disruptants were 5-fold more sensitive to arsenate and arsenite than wild-type cells . The acr3 disruptants showed wild-type sensitivity to antimony, tellurite, cadmium, and phenylarsine oxide . The mechanism of arsenical resistance was assayed by transport experiments using radioactive arsenite . We did not observe any significant differences in the accumulation of 76AsO33- in wild-type cells, acr1 and acr3 disruptants . However, the high dosage of ACR3 gene resulted in loss of arsenite uptake . These results suggest that arsenite resistance in yeast is mediated by an arsenite transporter (Acr3p).

FEBS Lett, 1997 Oct 27, 416(3), 339 - 43
Mutational analysis of Yap1 protein, an AP-1-like transcriptional activator of Saccharomyces cerevisiae; Takeuchi T et al.; To define the essential amino acid residues of Yap1 in stress response, we generated yap1 mutations by in vitro mutagenesis, which cause defects in mediating resistance to the stress of H2O2, but not of CdCl2 . Sequence analysis of the mutant yap1 genes revealed three point mutations and two truncation mutations near the carboxy-terminus . The truncation mutations resulted in hyperresistance to cadmium . Northern blot analysis of stress-induced levels of TRX2 and GSH1 mRNAs indicated that the ability of the mutant Yap1 protein to induce transcriptional activation of target genes correlates well with its ability to confer stress resistance . The carboxy-terminal domain of Yap1 appears to act negatively in cadmium resistance.

Mol Cell Biol, 1997 Dec, 17(12), 7283 - 94
Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae; Kressler D et al.; A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A) . Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family . Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization . An HA epitope-tagged Fal1p is localized predominantly in the nucleolus . Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits . Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin . Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2 . Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits . Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.

Mol Cell Biol, 1997 Dec, 17(12), 7029 - 39
SSP1, a gene necessary for proper completion of meiotic divisions and spore formation in Saccharomyces cerevisiae; Nag DK et al.; During meiosis, a diploid cell undergoes two rounds of nuclear division following one round of DNA replication to produce four haploid gametes . In yeast, haploid meiotic products are packaged into spores . To gain new insights into meiotic development and spore formation, we followed differential expression of genes in meiotic versus vegetatively growing cells in the yeast Saccharomyces cerevisiae . Our results indicate that there are at least five different classes of transcripts representing genes expressed at different stages of the sporulation program . Here we describe one of these differentially expressed genes, SSP1, which plays an essential role in meiosis and spore formation . SSP1 is expressed midway through meiosis, and homozygous ssp1 diploid cells fail to sporulate . In the ssp1 mutant, meiotic recombination is normal but viability declines rapidly . Both meiotic divisions occur at the normal time; however, the fraction of cells completing meiosis is significantly reduced, and nuclei become fragmented soon after meiosis II . The ssp1 defect does not appear to be related to a microtubule-cytoskeletal-dependent event and is independent of two rounds of chromosome segregation . The data suggest that Ssp1 is likely to function in a pathway that controls meiotic nuclear divisions and coordinates meiosis and spore formation.

Mol Cell Biol, 1997 Dec, 17(12), 6982 - 93
Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions; Fernandes L et al.; Saccharomyces cerevisiae contains eight members of a novel and fungus-specific family of bZIP proteins that is defined by four atypical residues on the DNA-binding surface . Two of these proteins, Yap1 and Yap2, are transcriptional activators involved in pleiotropic drug resistance . Although initially described as AP-1 factors, at least four Yap proteins bind most efficiently to TTACTAA, a sequence that differs at position +/-2 from the optimal AP-1 site (TGACTCA); further, a Yap-like derivative of the AP-1 factor Gcn4 (A239Q S242F) binds efficiently to the Yap recognition sequence . Molecular modeling suggests that the Yap-specific residues make novel contacts and cause physical constraints at the +/-2 position that may account for the distinct DNA-binding specificities of Yap and AP-1 proteins . To various extents, Yap1, Yap2, Yap3, and Yap5 activate transcription from a promoter containing a Yap recognition site . Yap-dependent transcription is abolished in strains containing high levels of protein kinase A; in contrast, Gcn4 transcriptional activity is stimulated by protein kinase A . Interestingly, Yap1 transcriptional activity is stimulated by hydrogen peroxide, whereas Yap2 activity is stimulated by aminotriazole and cadmium . In addition, unlike other yap mutations tested, yap4 (cin5) mutations affect chromosome stability, and they suppress the cold-sensitive phenotype of yap1 mutant strains . Thus, members of the Yap family carry out overlapping but distinct biological functions.

J Biol Chem, 1997 Nov 21, 272(47), 29704 - 10
Hydroxylation of Saccharomyces cerevisiae ceramides requires Sur2p and Scs7p; Haak D et al.; The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids . Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid . Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p-dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast . Deletion of either gene suppresses the Ca2+-sensitive phenotype of csg2Delta mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation . Characterization of scs7 and sur2 mutants is expected to provide insight into the function of ceramide hydroxylation.

Genes Dev, 1997 Nov 15, 11(22), 3032 - 45
Rad53-dependent phosphorylation of Swi6 and down-regulation of CLN1 and CLN2 transcription occur in response to DNA damage in Saccharomyces cerevisiae; Sidorova JM et al.; Budding yeast possesses a checkpoint-dependent mechanism of delaying G1 progression in response to UV and ionizing radiation DNA damage . We have shown that after a pulse of DNA damage in G1 with the alkylating agent MMS, there is also a MEC1-, RAD53-, and RAD9-dependent delay in G1 . This delay occurs at or before Start, as the MMS-treated cells do not bud, remain sensitive to alpha-factor, and have low CLN1 and CLN2 transcript levels for a longer time than untreated cells . We further show that MMS directly and reversibly down-regulates CLN1 and CLN2 transcript levels . The initial drop in CLN transcript levels in MMS is not RAD53 dependent, but the kinetics of reaccumulation of CLN messages as cells recover from the damage is faster in rad53-11 cells than in wild type cells . This is not an indirect effect of faster progression through G1, because CLN transcripts reaccumulate faster in rad53-11 mutants arrested in G1 as well . In addition, the recovery of CLN mRNA levels can be also hastened by a SWI6 deletion or by overexpression of the truncated Swi4 (Swi4-t) that lacks the carboxy-terminal domain through which Swi4 associates with Swi6 . This indicates that both Rad53 and Swi6 are negative regulators of CLN expression after DNA damage . Finally, Swi6 undergoes an MMS-inducible, RAD53-dependent phosphorylation in G1 cells, and Rad53, immunoprecipitated from MMS-treated cells, phosphorylates Swi6 in vitro . On the basis of these observations, we suggest that the Rad53-dependent phosphorylation of Swi6 may delay the transition to S phase by inhibiting CLN transcription.

Genes Dev, 1997 Nov 15, 11(22), 2958 - 71
The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae; Chen GC et al.; BEM2 of Saccharomyces cerevisiae encodes a Rho-type GTPase-activating protein that is required for proper bud site selection at 26 degrees C and for bud emergence at elevated temperatures . We show here that the temperature-sensitive growth phenotype of bem2 mutant cells can be suppressed by increased dosage of the GIC1 gene . The Gic1 protein, together with its structural homolog Gic2, are required for cell size and shape control, bud site selection, bud emergence, actin cytoskeletal organization, mitotic spindle orientation/positioning, and mating projection formation in response to mating pheromone . Each protein contains a CRIB (Cdc42/Rac-interactive binding) motif and each interacts in the two-hybrid assay with the GTP-bound form of the Rho-type Cdc42 GTPase, a key regulator of polarized growth in yeast . The CRIB motif of Gic1 and the effector domain of Cdc42 are required for this association . Genetic experiments indicate that Gic1 and Gic2 play positive roles in the Cdc42 signal transduction pathway, probably as effectors of Cdc42 . Subcellular localization studies with a functional green fluorescent protein-Gic1 fusion protein indicate that this protein is concentrated at the incipient bud site of unbudded cells, at the bud tip and mother-bud neck of budded cells, and at cortical sites on large-budded cells that may delimit future bud sites in the two progeny cells . The ability of Gic1 to associate with Cdc42 is important for its function but is apparently not essential for its subcellular localization.

FEMS Microbiol Rev, 1997 Sep, 21(2), 113 - 34
Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae; Nelissen B et al.; On the basis of the complete genome sequence of the budding yeast Saccharomyces cerevisiae, a computer-aided analysis was carried out of all members of the major facilitator superfamily (MFS), which typically consists of permeases with 12 transmembrane spans . Analysis of all 5885 predicted open reading frames identified 186 potential MFS proteins . Binary sequence comparison made it possible to cluster 149 of them into 23 families . Putative permease functions could be assigned to 12 families, the largest including sugar, amino acid, and multidrug transport . Phylogenetic clustering of proteins allowed us to predict a possible permease function for a total of 119 proteins . Multiple sequence alignments were made for all families, and evolutionary trees were constructed for families with at least four members . The latter resulted in the identification of 21 subclusters with presumably tightly related permease function . No functional clues were predicted for a total of 41 clustered or unclustered proteins.

Mol Biol Cell, 1997 Oct, 8(10), 1829 - 44
The Rho-GEF Rom2p localizes to sites of polarized cell growth and participates in cytoskeletal functions in Saccharomyces cerevisiae; Manning BD et al.; Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements . ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1 delta, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae . A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections . Disruption of ROM2 results in temperature-sensitive growth defects at 11 degrees C and 37 degrees C . rom2 delta cells exhibit morphological defects . At permissive temperatures, rom2 delta cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate . High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1 delta and kar3 delta strains . KAR3 encodes a kinesin-related protein that interacts with Cik1p . Furthermore, rom2 delta strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl . These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.

Methods Cell Biol, 1998, 53, 545 - 57
In vivo nuclear transport kinetics in Saccharomyces cerevisiae; Roberts PM et al.; We have described a direct fluorescence assay to measure the relative rates of NLS-directed import and passive export of an NLS-GFP fusion protein in yeast . The design and construction of the reporter GFP fusion, its spectral qualities, size, use of inducible promoters, and the choice of NLS, are variables that could extend the method's utility . Future applications will almost certainly demand the quantification of transport rates in single cells using image analysis techniques . As is the case whenever cellular processes are studied in vivo, the in vivo nuclear trafficking properties of NLS-GFP are complicated and poorly understood . Some will be attracted to NLS-GFP kinetic assays simply because so little is known about the function and regulation of the transport apparatus in living cells . At the same time, the uncertainties that accompany in vivo work necessarily prevent the rigorous interpretation of data, which biochemists expect from experiments performed in vitro using highly purified enzymes.

J Mol Biol, 1997 Oct 31, 273(3), 714 - 28
The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism; Mathieu M et al.; The dimeric, peroxisomal 3-ketoacyl-CoA thiolase catalyses the conversion of 3-ketoacyl-CoA into acyl-CoA, which is shorter by two carbon atoms . This reaction is the last step of the beta-oxidation pathway . The crystal structure of unliganded peroxisomal thiolase of the yeast Saccharomyces cerevisiae has been refined at 1.8 A resolution . An unusual feature of this structure is the presence of two helices, completely buried in the dimer and sandwiched between two beta-sheets . The analysis of the structure shows that the sequences of these helices are not hydrophobic, but generate two amphipathic helices . The helix in the N-terminal domain exposes the polar side-chains to a cavity at the dimer interface, filled with structured water molecules . The central helix in the C-terminal domain exposes its polar residues to an interior polar pocket . The refined structure has also been used to predict the mode of binding of the substrate molecule acetoacetyl-CoA, as well as the reaction mechanism . From previous studies it is known that Cys125, His375 and Cys403 are important catalytic residues . In the proposed model the acetoacetyl group fits near the two catalytic cysteine residues, such that the oxygen atoms point towards the protein interior . The distance between SG(Cys125) and C3(acetoacetyl-CoA) is 3.7 A . The O2 atom of the docked acetoacetyl group makes a hydrogen bond to N(Gly405), which would favour the formation of the covalent bond between SG(Cys125) and C3(acetoacetyl-CoA) of the intermediate complex of the two-step reaction . The CoA moiety is proposed to bind in a groove on the surface of the protein molecule . Most of the interactions of the CoA molecule are with atoms of the loop domain . The three phosphate groups of the CoA moiety are predicted to interact with side-chains of lysine and arginine residues, which are conserved in the dimeric thiolases .

J Pept Res, 1997 Nov, 50(5), 319 - 28
Position one analogs of the Saccharomyces cerevisiae tridecapeptide pheromone; Zhang YL et al.; Analogs of the Saccharomyces cerevisiae alpha-mating factor {WHWLQLKPGQPMY}, in which a variety of residues replaced Trp1 were synthesized and assayed for biological activity and receptor affinity . Analogs containing Gly or Leu or many different aromatic residues in position 1 of the peptide exhibited bioactivity in a growth arrest assay slightly greater than, or equal to, that of the parent pheromone, whereas the Glu1 and Lys1 analogs exhibited significantly lower bioactivity . Analogs with an aromatic replacement at position 1 had 3- to 6-fold lower receptor affinity than the parent peptide, whereas analogs with a hydrophilic residue at the N-terminus exhibited large reductions in receptor affinity with the peptide with Glu in position 1 showing a 120-fold reduction . N alpha-Acetylation had little effect on bioactivity but lowered receptor affinity by 20- to 40-fold . Amidation of the carboxyl terminus resulted in a 10-fold decrease in activity and a 160-fold decrease in receptor affinity . These results indicate that the alpha-factor receptor has a large hydrophobic binding pocket, possibly containing a negatively charged side-chain, which interacts with the N-terminus of alpha-factor . The lack of correlation between activity and binding and several analogs suggests that small residues near the N-terminus of alpha-factor may be very efficient in triggering isomerization of the receptor to its activated state in the first step of the signal transduction pathway.

J Bacteriol, 1997 Dec, 179(24), 7644 - 52
The minimal transactivation region of Saccharomyces cerevisiae Gln3p is localized to 13 amino acids; Svetlov V et al.; Regulated nitrogen catabolic gene transcription in Saccharomyces cerevisiae is mediated by four positive (Gln3p and Gat1p/Nil1p) and negative (Dal80p/Uga43p and Deh1p/Nil2p/GZF3p) regulators which function in opposition to one another . All four proteins contain GATA-type zinc finger domains, and three of them (Gln3p, Dal80p, and Deh1p) have been shown to bind to GATA sequences situated upstream of genes whose expression is sensitive to nitrogen catabolite repression (NCR) . The positive regulators, Gln3p and Gat1p, are able to support transcriptional activation when tethered by LexAp to the promoter of a reporter gene whose upstream activation sequences have been replaced with one or more lexA operator sites . Existing data suggest that these four proteins regulate transcription by competing with one another for binding to the GATA sequences which mediate NCR-sensitive gene expression . We show that the minimal Gln3p domain mediating transcriptional activation consists of 13 amino acids with a predicted propensity to form an alpha-helix . Genetic analysis of this region (Gln3p residues 126 to 138, QQNGEIAQLWDFN) demonstrated that alanine may be substituted for the aromatic and acidic amino acids without destroying transcriptional activation potential . Similar substitution of alanine for the two hydrophobic amino acids, isoleucine and leucine, however, destroys activation, as does introduction of basic amino acids in place of the acidic residues or introduction of proline into the center of the sequence . A point mutation in the Gln3p activation region destroys its in vivo ability to support NCR-sensitive DAL5 expression . We find no convincing evidence that NCR regulates Gln3p function by modulating the functioning of its activation region.

J Bacteriol, 1997 Dec, 179(24), 7611 - 6
Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae; Athenstaedt K et al.; Lipid particles of the yeast Saccharomyces cerevisiae harbor two enzymes that stepwise acylate glycerol-3-phosphate to phosphatidic acid, a key intermediate in lipid biosynthesis . In lipid particles of the s1c1 disruptant YMN5 (M . M . Nagiec et al., J . Biol . Chem . 268:22156-22163, 1993) acylation stops after the first step, resulting in the accumulation of lysophosphatidic acid . Two-dimensional gel electrophoresis confirmed that S1c1p is a component of lipid particles . Lipid particles of a second mutant strain, TTA1 (T . S . Tillman and R . M . Bell, J . Biol . Chem . 261:9144-9149, 1986), which harbors a point mutation in the GAT gene, are essentially devoid of glycerol-3-phosphate acyltransferase activity in vitro . Synthesis of phosphatidic acid is reconstituted by combining lipid particles from YMN5 and TTA1 . These results indicate that two distinct enzymes are necessary for phosphatidic acid synthesis in lipid particles: the first step, acylation of glycerol-3-phosphate, is catalyzed by a putative Gat1p; the second step, acylation of lysophosphatidic acid, requires S1c1p . Surprisingly, YMN5 and TTA1 mutants grow like the corresponding wild types because the endoplasmic reticulum of both mutants has the capacity to form a reduced but significant amount of phosphatidic acid . As a consequence, an s1c1 gat1 double mutant is also viable . Lipid particles from this double mutant fail completely to acylate glycerol-3-phosphate, whereas endoplasmic reticulum membranes harbor residual enzyme activities to synthesize phosphatidic acid . Thus, yeast contains at least two independent systems of phosphatidic acid biosynthesis.

Mol Biol Cell, 1997 Dec, 8(12), 2575 - 90
A mutational analysis identifies three functional regions of the spindle pole component Spc110p in Saccharomyces cerevisiae; Sundberg HA et al.; The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB) . The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate . To dissect the functions of Spc110p, we created temperature-sensitive mutations in the amino and carboxy termini . Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p . Region I is located at the amino terminus . Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site . Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the gamma-tubulin complex composed of Spc97p, Spc98p, and Tub4p . Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque . Our results strongly argue that Spc110p links the gamma-tubulin complex to the central plaque of the SPB.

Mol Biol Cell, 1997 Dec, 8(12), 2519 - 37
Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase; Fiorentino DF et al.; The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like "lipid kinase" domain and are required for early G1 progression in eukaryotes . Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p) . This gene is identical to DNA2, encoding a helicase required for DNA replication . As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants . Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions . Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase . Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis . Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis . These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.

Biochemistry, 1997 Dec 2, 36(48), 14956 - 64
Role of mitochondrial and cytoplasmic serine hydroxymethyltransferase isozymes in de novo purine synthesis in Saccharomyces cerevisiae; Kastanos EK et al.; One-carbon units are essential to a variety of anabolic processes which yield necessary cellular components including purines, pyrimidines, amino acids, and lipids . Serine hydroxymethyltransferase (SHMT) is the major provider of one-carbon units in the cell . The other product of this reaction is glycine . Both of these metabolites are required in de novo purine biosynthesis . In Saccharomyces cerevisiae, mitochondrial and cytoplasmic SHMT isozymes are encoded by distinct nuclear genes (SHM1 and SHM2) . Molecular genetic analyses have begun to define the roles of these two isozymes in folate-mediated one-carbon metabolism {McNeil, J . B., et al . (1996) Genetics 142, 371-381} . In our study, the SHM1 and SHM2 genes were disrupted singly and in combination to investigate the contributions of the two SHMT isozymes to the production of glycine and one-carbon units required in purine biosynthesis . Cell subfractionation experiments indicated that while only 5% of total activity was localized in the mitochondria, the specific activity in that compartment was much higher than in the cytoplasm . Growth and 13C NMR experiments indicate that the two isozymes function in different directions, depending on the nutritional conditions of the cell . When yeast was grown on serine as the primary one-carbon source, the cytoplasmic isozyme was the main provider of glycine and one-carbon groups for purine synthesis . When grown on glycine, the mitochondrial SHMT was the predominant isozyme catalyzing the synthesis of serine from glycine and one-carbon units . However, when both serine and glycine were present, the mitochondrial SHMT made a significant contribution of one-carbon units, but not glycine, for purine synthesis . Finally, NMR data are presented that suggest the existence of at least two sites of de novo purine biosynthesis in growing yeast cells, each being fed by distinct pools of precursors.

Biochemistry, 1997 Nov 25, 36(47), 14439 - 46
Phosphorylation of the acidic ribosomal P proteins in Saccharomyces cerevisiae: a reappraisal; Zambrano R et al.; Previous reports had pointed to serines 62 and 71/79 as possible phosphorylation sites in the yeast acidic ribosomal proteins YP1 alpha and YP2 alpha, respectively . However, it has been found that mutation of these serine residues did not affect the phosphorylation level of the proteins . A detailed examination of the YP2 alpha tryptic digest from the in vivo labeled protein demonstrates the existence of a totally trypsin-insensitive site at lysine 88 that led to a misinterpretation of previous results . The unique YP2 alpha tryptic phosphopeptide obtained contains, in addition to serines 71 and 79, a serine at position 96 near the carboxyl end, which automatic Edman degradation confirmed as the phosphorylated residue . In addition, by using Staphyloccocus protease V8, it was possible to obtain phosphopeptides containing only serine 96, whose phosphorylation has likewise been confirmed by radioactive labeling as well as by chemical methods . A similar analysis of the other 12 kDa acidic proteins, YP1 alpha, YP1 beta, and YP2 beta, has shown the presence of equivalent phosphorylation sites in the four P proteins, which correspond to position 96 in proteins YP1 alpha, YP1 beta, and YP2 alpha and position 100 in YP2 beta . This conclusion has been confirmed by the fact that mutation of serine 96 in proteins YP1 alpha and YP2 alpha abolishes their capacity to be phosphorylated in vivo . The mutation of the phosphorylation site of the individual acidic proteins seems not to alter their interaction with the ribosome . However, it has been found that the level of phosphorylation of the stalk proteins has an effect on the response of the cells to some specific metabolic conditions, indicating that it may modulate the translation of specific proteins.

Biochim Biophys Acta, 1997 Nov 10, 1322(1), 8 - 18
Contribution to the physiological characterization of glycerol active uptake in Saccharomyces cerevisiae; Lages F et al.; Evidence is presented here that in Saccharomyces cerevisiae IGC 3507, grown either on glycerol, ethanol or acetate, glycerol is transported by a high affinity uptake system of the electrogenic proton symport type, with Km of 1.7 +/- 0.7 mM, Vmax 441 +/- 19 micromolh(-1) g(-1) dry weight and a stoichiometry of 1:1 proton per molecule of glycerol, at 30 degrees C and pH 5.0 . No competitors were found among other polyols and sugars . Glycerol maximum accumulation ratios followed p.m.f . with extracellular pH . CCCP prevented glycerol accumulation, and inhibited uptake . NaCl did not interfere with H+/glycerol kinetics and energetics . This transport system was shown to be under glucose repression and inactivation . Glucose-grown cells presented, instead, a lower affinity permease for glycerol, probably a facilitated diffusion . Growth on glucose in the presence of NaCl did not induce the high affinity carrier . The stringent control of cell physiological condition over induction suggests for glycerol proton symport rather a physiological role connected with growth under gluconeogenic conditions.

Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 13093 - 8
All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae; Dolinski K et al.; The cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown . Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during in vitro protein folding . We have addressed their functions by a genetic approach in the yeast Saccharomyces cerevisiae . Five cyclophilins and three FKBPs previously were identified in yeast . We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3 . None of the eight cyclophilins or four FKBPs were essential . Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation . We conclude that cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes . We propose that each cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified.

Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 12949 - 56
In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone; Nathan DF et al.; In the highly concentrated environment of the cell, polypeptide chains are prone to aggregation during synthesis (as nascent chains await the emergence of the remainder of their folding domain), translocation, assembly, and exposure to stresses that cause previously folded proteins to unfold . A large and diverse group of proteins, known as chaperones, transiently associate with such folding intermediates to prevent aggregation, but in many cases the specific functions of individual chaperones are still not clear . In vivo, Hsp90 (heat shock protein 90) plays a role in the maturation of components of signal transduction pathways but also exhibits chaperone activity with diverse proteins in vitro, suggesting a more general function . We used a unique temperature-sensitive mutant of Hsp90 in Saccharomyces cerevisiae, which rapidly and completely loses activity on shift to high temperatures, to examine the breadth of Hsp90 functions in vivo . The data suggest that Hsp90 is not required for the de novo folding of most proteins, but it is required for a specific subset of proteins that have greater difficulty reaching their native conformations . Under conditions of stress, Hsp90 does not generally protect proteins from thermal inactivation but does enhance the rate at which a heat-damaged protein is reactivated . Thus, although Hsp90 is one of the most abundant chaperones in the cell, its in vivo functions are highly restricted.

Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 12898 - 903
Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II; Yue Z et al.; 5'-Capping is an early mRNA modification that has important consequences for downstream events in gene expression . We have isolated mammalian cDNAs encoding capping enzyme . They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily . The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical . Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5'-triphosphatase activity and catalyzed formation of RNA 5'-terminal GpppG . A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA . Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site . The K294A mutant retained RNA 5'-triphosphatase activity, which was eliminated by N-terminal truncation . Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase . The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation . These results also explain the selective capping of RNA polymerase II transcripts.

J Bacteriol, 1997 Dec, 179(23), 7497 - 506
The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus; Ahn YT et al.; The efficient partitioning of the 2microm plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB) . By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir+ strain . Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir(0) host . GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background . Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells . This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle . Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex . Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins . Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well . The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.

Biochim Biophys Acta, 1997 Oct 20, 1321(3), 207 - 20
Patch clamp investigation into the phosphate carrier from Saccharomyces cerevisiae mitochondria; Herick K et al.; After heterologous expression in E . coli, functionally active phosphate carrier (PIC) from Saccharomyces cerevisiae mitochondria was purified and reconstituted into giant liposomes and used for patch clamp experiments . Single channel currents across excised patches revealed an anion channel function of the PIC protein . Besides the three transport modes known to date, namely phosphate/phosphate exchange, phosphate/OH exchange and mercurial-induced unidirectional transport, this channel activity represents the fourth transport mode of the PIC . The PIC channel activity was sensitive towards phosphate as its physiological substrate . Phosphate (10 mM) blocked in a specific but reversible manner the PIC channel, suggesting a phosphate-dependent conformational change of the protein into the carrier mode . Furthermore, the current through the channel and its gating activity were affected by divalent cations . In the presence of Ca2+ and Mg2+, the channel displayed a mean conductance of 25 +/- 5 pS whereas 40 +/- 10 pS was observed in the absence of divalent cations . Also, the dwell times in either the open or closed state of the PIC channel appeared to be prolonged in the presence of Ca2+ and Mg2+ . The observed PIC channel characteristics are discussed with respect to previously reported electrophysiological in situ measurements on anion channels of the inner mitochondrial membrane . Similarities of the PIC channel to the inner mitochondrial anion channel (IMAC) have been found.

Mol Gen Genet, 1997 Oct, 256(4), 376 - 86
Identification of the Saccharomyces cerevisiae genes STB1-STB5 encoding Sin3p binding proteins; Kasten MM et al.; The yeast SIN3 gene functions as a transcriptional repressor, despite the fact that Sin3p does not bind DNA directly . We have conducted a two-hybrid screen to look for proteins that interact with Sin3p, using the PAH2 domain of Sin3p as bait . Five new genes, STB1-STB5 were identified, as well as the STB6 gene, which is similar to STB2 . STB1, STB2, STB3, and STB6 are novel genes, and STB4 and STB5 encode C6 zinc cluster DNA-binding proteins . None of these genes is essential for viability, and several of these genes may encode transcriptional activators . Several special problems were encountered in using a transcriptional repressor in a two-hybrid screen . For example, the STB genes will interact with a LexA-Sin3(PAH2) fusion protein containing a region of Sin3p, but a LexA-Sin3p fusion protein containing full-length Sin3p, along with a STB clone, does not produce two-hybrid activation of a transcriptional reporter . In addition, a sin3 mutation reduces the transcriptional activation by two-hybrid partners, suggesting that a sin3 mutation reduces the transcriptional efficiency of the Gal4p and VP16 activation domains . We have shown previously that Sin3p is part of a large multiprotein complex, and we show here that Stb1p and Stb2p are present in this complex.

Yeast, 1997 Nov, 13(14), 1337 - 46
Lys80p of Saccharomyces cerevisiae, previously proposed as a specific repressor of LYS genes, is a pleiotropic regulatory factor identical to Mks1p; Feller A et al.; In Saccharomyces cerevisiae, an intermediate of the lysine pathway, alpha-aminoadipate semialdehyde (alpha AASA), acts as a coinducer for the transcriptional activation of LYS genes by Lys14p . The limitation of the production of this intermediate through feedback inhibition of the first step of the pathway results in apparent repression by lysine . Previously, the lys80 mutations, reducing the lysine repression and increasing the production of lysine, were interpreted as impairing a repressor of LYS genes expression . In order to understand the role of Lys80p in the control of the lysine pathway, we have analysed the effects of mutations epistatic to lys80 mutations . The effects of lys80 mutations on LYS genes expression were dependent on the integrity of the activation system (Lys14p and alpha AASA) . The increased production of lysine in lys80 mutants appeared to result from an improvement of the metabolic flux through the pathway and was correlated to an increase of the alpha-ketoglutarate pool and of the level of several enzymes of the tricarboxylic acid cycle . The LYS80 genes has been cloned and sequenced; it turned out to be identical to gene MKS1 cloned as a gene encoding a negative regulator of the RAS-cAMP pathway . We conclude that Lys80p is a pleiotropic regulatory factor rather than a specific repressor of LYS genes.

Biochem Biophys Res Commun, 1997 Nov 17, 240(2), 287 - 92
G-protein coupled receptor from yeast Saccharomyces cerevisiae; Yun CW et al.; The Saccharomyces cerevisiae GPR1 (G-protein coupled receptor) gene was isolated using two-hybrid system with a heterotrimeric GTP binding protein alpha subunit Gpa2p as a bait . The GPR1 gene encodes 961 amino acids with predicted seven transmembrane segments and two large cytosolic regions as third cytosolic loop with 350 amino acids where asparagine-rich region was found and the C-terminal region with 283 amino acids . The Gpr1p interacted with Gpa2p at C-terminal region with 131 amino acid residues as well as third cytosolic loop . Disruption of the GPR1 gene was not lethal and did not affect to the cell growth . The Gpr1p-GFP fusion protein localized at the cell surface . These results suggest that Gpr1p is a G-protein coupled receptor which localized at plasma membrane . It is likely that a Gpr1p monitors the extracellular signal such as nutrition and transduce it via Gpa2p a possible positive regulator of cAMP level.

Gene, 1997 Oct 1, 198(1-2), 165 - 9
Isocitrate lyase localisation in Saccharomyces cerevisiae cells; Chaves RS et al.; The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm . This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation . This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted.

FEBS Lett, 1997 Oct 13, 416(1), 1 - 5
Effect of temperature on the role of Hsp104 and trehalose in barotolerance of Saccharomyces cerevisiae; Iwahashi H et al.; We have studied the effect of temperature on the contribution of Hsp104 and trehalose to barotolerance using mutants deficient in Hsp104 and trehalose synthesis . When compared with a corresponding wild type strain, mutants of Hsp104 did not show temperature dependent barotolerance when the incubation temperature during the hydrostatic pressure treatment was increased . However, a mutant deficient in trehalose synthesis showed features similar to a wild type strain . Furthermore, the Hsp104 level was low in the insoluble fraction of the wild type strain after pressure treatment at 35 degrees C but not at 4 degrees C, and the protein profiles in the insoluble fraction were different between 35 degrees C and 4 degrees C . In contrast to the Hsp104 deficient mutants, the protein profile of the wild type after pressure treatment at 35 degrees C favors the role of Hsp104 as a disaggregator of proteins during hydrostatic pressure stress . These results suggest that the role of Hsp104 in barotolerance is temperature dependent in contrast to trehalose.

Cell Struct Funct, 1997 Aug, 22(4), 465 - 76
An altered nuclear migration into the daughter bud is induced by the cyclin A1-mediated Cdc28 kinase through an aberrant spindle movement in Saccharomyces cerevisiae; Sikder H et al.; A strain of Saccharomyces cerevisiae that contains an integrated copy of a Xenopus cyclin A1 gene under the control of the GAL1 promoter has been constructed . On inducing expression of cyclin A1, the nuclear migration that occurs prior to division becomes aberrant . Instead of migrating to the neck between the mother cell and daughter bud, the nucleus, the short mitotic spindle and its associated two spindle pole bodies entered the daughter bud . This phenotype was induced by expression of an indestructible cyclin mutant, but not by a mutated cyclin A1 unable to activate Cdc28 kinase . The nuclear abnormality induced by cyclin A1 was overcome by cdc28 mutations that abolish its ability to bind cyclin A1 . Both yeast cyclin Clb3 and Xenopus mitotic cyclin B produced the same phenotype, whereas G1 cyclin Cln2 did not . The results suggest that the proper movement of the nucleus through the spindle function during mitosis requires the appropriate activity of Cdc28 kinase mediated by specific cyclins.

FEMS Microbiol Lett, 1997 Nov 1, 156(1), 147 - 52
Cytochrome P-450 reductase is responsible for the ferrireductase activity associated with isolated plasma membranes of Saccharomyces cerevisiae; Lesuisse E et al.; Cytochrome P-450 reductase (encoded by the NCP1 gene) was found to catalyse all the NADPH-dependent ferrireductase activities associated with isolated plasma membranes of the yeast Saccharomyces cerevisiae . We therefore examined the contribution of this enzyme to the ferrireductase activity of cells in vivo . Cytochrome P-450 reductase was shown to be not essential for the cell ferrireductase activity, but it influenced this activity, with different effects on the Fre1- and the Fre2-dependent reductase systems . Overexpression of FRE1 did not lead to an increased ferrireductase activity of the cells when NCP1 was repressed . In contrast, cells that overexpressed FRE2 had maximal ferrireductase activity when NCP1 was repressed . The degree of NCP1 expression also affected the amount of iron and copper accumulated by the cells during growth . The biochemical implications and the physiological significance of these observations are discussed.

Genes Cells, 1997 Jul, 2(7), 443 - 55
Alteration of telomeric sequences and senescence caused by mutations in RAD50 of Saccharomyces cerevisiae; Kironmai KM et al.; BACKGROUND: Vegetatively dividing cells of Saccharomyces cerevisiae carrying a mutation in RAD50 grow significantly more slowly in rich medium and are sensitive to DNA damage inflicted by X-ray or chemical mutagens . RAD50 function is essential for the formation and repair of meiosis-specific double-strand breaks and chromosome stability . RESULTS: We present evidence for two new phenotypes associated with the rad50delta mutant; shortened telomeres and cell senescence . Comparison of TG1-3 telomeric sequences in an isogenic pair of RAD50 and rad50delta haploid strains showed that they were considerably shortened in the latter . Although rad50delta mutation conferred cell enlargement and slow growth, cell doubling was faster but caused an increase in the frequency of cell death . Telomeres were restored to the wild-type size in hemizygous RAD50/rad50delta and rad50S/rad50delta strains; however, they showed a significant increase in rad50S/rad50S diploid with a concomitant rise in cell viability . Telomeres were stabilized in hemizygous RAD50/rad50delta and rad50S/rad50delta diploids during prolonged growth, suggesting that even a half-dosage of RAD50 is sufficient to conserve the telomere size during successive cell divisions . Furthermore, cells bearing the rad50delta mutation revealed abnormalities in nuclear segregation and, in the presence of hydroxyurea, displayed phenotypes consistent with defects in S-phase checkpoint control . CONCLUSION: This report presents evidence of the involvement of a gene relevant to recombinational repair in the maintenance of telomeres . We conclude that the phenotypes displayed by yeast rad50delta cells have intriguing similarities among the human cell lines representing DNA repair-deficient chromosome instability syndromes.

Biochim Biophys Acta, 1997 Sep 26, 1342(1), 62 - 72
Purification and characterization of a novel poly(U), poly(C) ribonuclease from Saccharomyces cerevisiae; Lalioti VS et al.; A new ribonuclease from Saccharomyces cerevisiae, specific for poly(U) and poly(C) substrate, was purified near to homogeneity by successive fractionation with DEAE-Sepharose, Heparin-Sepharose and CM-Sepharose chromatography . The purified molecule detected by SDS/polyacrylimide gel electrophoresis has a molecular mass of 29 kDa . The optimum pH for the enzyme activity is 5.5-7 and its isoelectric point is 7.5 . The purified enzyme was able to degrade 26S, 18S and 5S rRNAs as well as mRNA obtained from in vitro transcription . No catalytic activity was observed when the RNase was incubated with tRNA and double stranded substrate . Our findings suggest that this novel RNase may play an important role in the processing of RNA in Saccharomyces cerevisiae.

Biochim Biophys Acta, 1997 Oct 11, 1358(3), 249 - 54
A novel dimeric oxovanadium (IV) species identified in Saccharomyces cerevisiae cells; Zoroddu MA et al.; Saccharomyces cerevisiae cells stored oxovanadium (IV) ions in a dimeric form . In the late stationary phase Saccharomyces cerevisiae cells grown in rich medium containing concentrations of oxovanadium (V), orthovanadate from 12 to 18 mM, causing growth stasis, a dimeric oxovanadium (IV) species was identified by EPR spectroscopy . The EPR spectrum exhibited at 110 K the low-field forbidden deltaMs = +/-2 transition at g around 4 and the half-field deltaMs = +/-1 15-lines feature at g around 2 out of the presence of a triplet state by the coupling of the oxovanadium (IV) ions in a dimeric form . Hyperfine splitting of 75.2 x 10(-4) cm(-1) and an interionic distance of about 4.4 angstroms was calculated . The dimeric species was localized in the cellular cytoplasmic space.

Yeast, 1997 Oct, 13(13), 1275 - 90
Expression profiles of transcripts from 126 open reading frames in the entire chromosome VI of Saccharomyces cerevisiae by systematic northern analyses; Naitou M et al.; Chromosome VI of Saccharomyces cerevisiae contains 126 open reading frames (ORFs), and the functions of proteins encoded by 80 ORFs are still unknown . In this report, we have systematically examined the expression profiles of all 126 ORFs on chromosome VI under five kinds of growth conditions by quantitative Northern hybridization . A series of Northern analyses and reverse transcription polymerase chain reactions have revealed that more than 64 novel ORFs are transcribed . Two ORFs (YFL059w and YFR011c) are specifically expressed in the presence of galactose . Two ORFs (YFL012w and YFR032c) are specifically transcribed in sporulation . Six ORFs (YFL049w, YFL035c, YFL010c, YFR006w, YFR010w and YFR017c) are abundantly expressed in many growth conditions.

Yeast, 1997 Oct, 13(13), 1265 - 74
Transcriptional regulation of SUP35 and SUP45 in Saccharomyces cerevisiae; Dagkessamanskaya A et al.; SUP35 and SUP45 encode translational release factors in the yeast Saccharomyces cerevisiae . In addition, Sup35p is related to the cytoplasmically inherited prion-like phenotype {PSI+} . The vital cellular role of Sup35p and Sup45p prompted us to study the regulation of transcription of the corresponding genes . Since the {PSI} state of the yeast strain affects the abundance of Sup35p and Sup45p, both {PSI+} and {psi-} variants were included in these analyses . It turned out that SUP35 and SUP45 transcript levels are regulated by nutritional changes and stress in a way strikingly similar to those of ribosomal protein genes . The {PSI} state did not influence the respective transcript levels nor their regulation, although HSP12 (as a monitor of general stress-responsive) gene expression appeared to differ in the two variant strains . The transcription activation sites of SUP35 and SUP45 were mapped using deletion analysis of the respective promoter-reporter fusion genes . The UAS in both cases was found to consist of an Abf1p-site and a T-rich element . Also in this respect SUP35 and SUP45 show a notable resemblance with ribosomal protein genes . Evidence was found that SUP35 in addition harbors a potential internal promoter element which became active after progressive 5'-deletion removing the first of the three in-frame ATGs.

Eur J Biochem, 1997 Oct 1, 249(1), 343 - 9
Identification and analysis of a static culture-specific cell wall protein, Tir1p/Srp1p in Saccharomyces cerevisiae; Kitagaki H et al.; A 100-kDa protein was found to be a major cell wall protein in Saccharomyces cerevisiae cells cultured without shaking, but was not present in cells cultured with shaking . The amino acid sequence of this protein was identical to the sequence of Tir1p/Srp1p . TIR1/SRP1 has previously been identified as a gene induced by glucose, cold shock or anaerobiosis and was believed to be a cell membrane protein but not a cell wall protein . However, we found that beta-1,3-glucanase solubilized Tir1p/Srp1p from the cell wall and the purified Tir1p/Srp1p reacted with antiserum to beta-1,6-glucan and contained glucose . These results suggest that Tir1p/Srp1p is a major structural cell wall protein in the static-cultured yeast cells and is bound to the cell wall through beta-1,6-glucan . TIR1/SRP1 mRNA was transcribed only in the static culture and its transcription was regulated by the ROX1 repressor.

Eur J Biochem, 1997 Oct 1, 249(1), 309 - 17
Purification and nucleic-acid-binding properties of a Saccharomyces cerevisiae protein involved in the control of ploidy; Weber V et al.; Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum . The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells {Wintersberger, U., Kuhne, C . & Karwan, A . (1995) Yeast 11, 929-944} . Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins . In this report, we demonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabditis elegans as well as with the vigilins, vertebrate RNA-binding proteins with a cellular location similar to that of Scp160p . Moreover, we found that Scp160p contains a potential nuclear-export signal (NES) near its N-terminus and a potential nuclear-localization signal (NLS) between KH domains 3 and 4 . To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA-cellulose and anti-Scp160p affinity chromatography . In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single-stranded and double-stranded DNA . Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes . Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role for Scp160p in nuclear RNA export or in RNA transport within the cytoplasm.

Int J Biochem Cell Biol, 1997 Apr, 29(4), 635 - 48
The role of the Src homology-2 domain in the lethal effect of Src expression in the yeast Saccharomyces cerevisiae; Trager JB et al.; Expression of the retroviral transforming gene v-src arrests the proliferation of the yeast Saccharomyces cerevisiae . A functional Src SH2 (Src homology 2) domain is required for this arrest . To examine the mechanism by which Src blocks yeast cell proliferation, and to determine the role of the Src SH2 domain in the growth arrest, src variants were expressed in yeast under the control of the galactose-inducible GAL1 promoter . Following galactose induction of Src expression, phosphotyrosyl-proteins were isolated by immunoprecipitation with beads coupled to either anti-phosphotyrosine antibody or to a recombinant fusion protein containing the Src SH2 domain . A group of SH2-binding phosphotyrosyl proteins was detected in cells expressing toxic forms of Src, but were not detected in cells expressing non-toxic variants . This group of phosphotyrosyl-proteins represents a minor subset of the proteins phosphorylated by v-Src . The lethality of v-Src and the phosphorylation of SH2-binding proteins were co-ordinately affected by alterations in phosphotyrosine-phosphatase activity . These observations indicate that the lethality of Src is correlated with the phosphorylation of proteins that bind to the Src SH2 domain . The role of the SH2 domain in determining the lethal effects of Src in yeast may be similar to its role in targeting Src to substrates necessary for its biological effects in vertebrate cells.

Mol Biol Cell, 1997 Nov, 8(11), 2119 - 32
Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes; Winey M et al.; The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells . Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs . Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined . NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle . However, accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells . Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance . However, NPCs were found in large clusters over regions of the nuclear envelope . Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.

Mol Biol Cell, 1997 Nov, 8(11), 2291 - 306
End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae; Wesp A et al.; end4-1 was isolated as a temperature-sensitive endocytosis mutant . We cloned and sequenced END4 and found that it is identical to SLA2/MOP2 . This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis . We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable . The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization . A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex . Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2 . The endocytic function of Abp1p depends on its SH3 domain . In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis . Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

Mol Biol Cell, 1997 Nov, 8(11), 2267 - 80
Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae; Dolinski K et al.; Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506 . The normal cellular functions of these proteins, however, are unknown . We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes . Surprisingly, most of these mutant enzymes were biologically active in vivo . In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay . However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading . In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases . Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo . One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast . Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

Acta Biochim Pol, 1997, 44(2), 191 - 200
The phosphorylation sites of ribosomal P proteins from Saccharomyces cerevisiae cells by endogenous CK-2, PK60S and RAP protein kinases; Boguszewska A et al.; The phosphorylation sites of ribosomal acidic proteins (P proteins) from Saccharomyces cerevisiae were studied in vivo and in vitro by using CK-2, PK60S and RAP protein kinases . The three enzymes phosphorylate the last serine residues located in a highly conserved carboxyl end of the polypeptide chains . This was established by two-dimensional analysis of tryptic phosphopeptides from 32P-labelled proteins YP1 alpha, YP1 beta, YP2 alpha and YP2 beta, and by kinetic studies of the protein kinases with synthetic peptides corresponding to the fragments of endogenous ribosomal acidic polypeptides . In experiments with both endogenous P proteins and synthetic peptides as substrates protein kinase PK60S demonstrated unusual substrate specificity . In contrast to CK-2 and RAP protein kinases, PK60S phosphorylates predominantly two of the four P proteins, YP1 alpha and YP2 beta, with kinetic constants dependent on the primary structure of the N-terminal region of the polypeptide containing the target residue . The neutral amino acid, alanine, at position 3 in the peptide AAEESDDD (polypeptide fragments of YP1 beta and YP2 alpha) decreases the K(m) value more than 10-fold by comparison with the basic lysine residue at the same position in the peptide AKEESDDD (polypeptide fragments of YP1 alpha and YP2 beta).

J Biochem (Tokyo), 1997 Jun, 121(6), 1182 - 9
The transcriptional activators of the PHO regulon, Pho4p and Pho2p, interact directly with each other and with components of the basal transcription machinery in Saccharomyces cerevisiae; Magbanua JP et al.; The transcriptional regulators Pho4p and Pho2p are involved in transcription of several genes in the PHO regulon of Saccharomyces cerevisiae . Genetic evidence with temperature-sensitive pho4 and pho2 mutants suggested that Pho4p and Pho2p interact with each other . Immunoprecipitation experiments showed that Pho4p and Pho2p form a complex on a 36-bp sequence bearing an upstream activation site (UAS) and protein binding assays indicated that these proteins interact directly . DNA-binding experiments with crude extracts prepared from yeast strains expressing T7-PHO4, encoding Pho4p tagged with the T7 epitope, indicated that Pho2p interacts with T7-Pho4p and enhances the binding affinity of T7-Pho4p to the UAS . Protein binding experiments also showed that both Pho4p and Pho2p could bind with the general transcription factors, TBP, TFIIB, and TFIIEbeta, suggesting that the Pho4p-Pho2p complex bound to the UAS activates transcription of the PHO genes by direct interaction with the general transcription factors.

Biometals, 1997 Oct, 10(4), 239 - 46
Reduction of vanadate to vanadyl by a strain of Saccharomyces cerevisiae; Bisconti L et al.; Three strains of Saccharomyces cerevisiae, SC-1, DBVPG 6173 and DBVPG 6037, were studied for vanadate resistance in complex Sabouraud medium since they did not thrive in different minimal media (yeast nitrogen base with and without amino acids) . The strain SC-1 was resistant up to 16 mM of vanadate, whereas the strains DBVPG 6173 and DBVPG 6037 were inhibited by 8 mM and 4 mM vanadate, respectively . The vanadate resistance in strain SC-1 was constitutive and due to the reduction of this oxyanion to vanadyl, which was detected by EPR spectroscopy and visible spectroscopy . The transformation of vanadate to vanadyl took place during the exponential growth phase; 10 mM of vanadate was reduced to vanadyl outside the cells since the oxyanion was not detected in the cell biomass and only a negligible concentration of vanadyl (25 nmoles mg-1 cells dry weight) was found in the biomass . The other two vanadate-sensitive yeast strains only accumulated vanadate and did not reduce the oxyanion to vanadyl.

J Biol Chem, 1997 Nov 7, 272(45), 28539 - 44
Purification and biochemical properties of Saccharomyces cerevisiae Mdj1p, the mitochondrial DnaJ homologue; Deloche O et al.; The DnaK/DnaJ/GrpE heat shock proteins of Escherichia coli constitute the prototype DnaK chaperone machine . Various studies have shown that these three proteins work synergistically in a diverse array of biological functions, including protein folding and disaggregation, proteolysis, and transport across biological membranes . We have overexpressed and purified the mitochondrial Saccharomyces cerevisiae DnaJ homologue, Mdj1pDelta55, which lacks the mitochondrial presequence, and studied its biochemical properties in well defined in vitro systems . We find that Mdj1pDelta55 interacts with DnaK as judged both by an enzyme-linked immunosorbent assay, as well as stimulation of DnaK's weak ATPase activity in the presence of GrpE . In addition, Mdj1pDelta55 not only interacts with denatured firefly luciferase on its own, but also enables DnaK to bind to it in an ATP-dependent mode . Using co-immunoprecipitation assays we can demonstrate the presence of a stable Mdj1pDelta55-luciferase-DnaK complex . However, in contrast to DnaJ, Mdj1pDelta55 does not appear to interact well with certain seemingly folded proteins, such as the sigma32 heat shock transcription factor or the lambdaP DNA replication protein . Finally, Mdj1pDelta55 can substitute perfectly well for DnaJ in the refolding of denatured firefly luciferase by the DnaK chaperone machine . These studies demonstrate that Mdj1pDelta55 has conserved most of DnaJ's known biological properties, thus supporting an analogous functional role in yeast mitochondria.

Genes Dev, 1997 Nov 1, 11(21), 2926 - 37
Rpp1, an essential protein subunit of nuclear RNase P required for processing of precursor tRNA and 35S precursor rRNA in Saccharomyces cerevisiae; Stolc V et al.; The gene for an essential protein subunit of nuclear RNase P from Saccharomyces cerevisiae has been cloned . The gene for this protein, RPP1, was identified by virtue of its homology with a human scleroderma autoimmune antigen, Rpp30, which copurifies with human RNase P . Epitope-tagged Rpp1 can be found in association with both RNase P RNA and a related endoribonuclease, RNase MRP RNA, in immunoprecipitates from crude extracts of cells . Depletion of Rpp1 in vivo leads to the accumulation of precursor tRNAs with unprocessed 5' and 3' termini and reveals rRNA processing defects that have not been described previously for proteins associated with RNase P or RNase MRP . Immunoprecipitated complexes cleave both yeast precursor tRNAs and precursor rRNAs.

J Bacteriol, 1997 Nov, 179(21), 6560 - 5
Effects of different carbon fluxes on G1 phase duration, cyclin expression, and reserve carbohydrate metabolism in Saccharomyces cerevisiae; Sillje HH et al.; By controlled addition of galactose to synchronized galactose-limited Saccharomyces cerevisiae cultures, the growth rate could be regulated while external conditions were kept constant . By using this method, the G1 phase duration was modulated and expression of cell cycle-regulated genes was investigated . The expression of the cyclin genes CLN1 and CLN2 was always induced just before bud emergence, indicating that this event marks the decision to pass Start . Thus, G1 phase elongation was not due to a slower accumulation of the CLN1 and CLN2 mRNA levels . Only small differences in CLN3 expression levels were observed . The maximal SWI4 expression preceded maximal CLN1 and CLN2 expression under all conditions, as expected for a transcriptional activator . But whereas SWI4 was expressed at about 10 to 20 min, before CLN1 and CLN2 expression at high growth rates, this time increased to about 300 min below a particular consumption rate at which the G1 phase strongly elongated . In the slower-growing cultures, also an increase in SWI6 expression was observed in the G1 phase . The increase in G1 phase duration below a particular consumption rate was accompanied by a strong increase in the reserve carbohydrate levels . These carbohydrates were metabolized again before bud emergence, indicating that below this consumption rate, a transient increase in ATP flux is required for progression through the cell cycle . Since Start occurred at different cell sizes under different growth conditions, it is not just a certain cell size that triggers passage through Start.

Appl Microbiol Biotechnol, 1997 Sep, 48(3), 339 - 45
Improved efficiency and stability of multiple cloned gene insertions at the delta sequences of Saccharomyces cerevisiae; Lee FW et al.; Two delta-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination . Cells containing integrations were selected by resistance to the aminoglycoside G418 . The first vector was a traditional construct containing only one delta sequence; with this vector, the transformation efficiency and the number of integrations per cell were quite low . The second carried two delta sequences flanking the desired insert, and the unneeded bacterial sequences were removed by restriction-enzyme digestion immediately before transformation . When this double delta vector was employed, the integrated copy number was more than doubled relative to the single delta system and final beta-galactosidase levels exceeded those obtained with the 2 mu-based plasmid . Furthermore, the integrations appeared more stable in long-term sequential culture (both with and without induction of the lacZ gene) than those obtained via the single delta vector.

J Biol Chem, 1997 Oct 31, 272(44), 27671 - 7
STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae; Cutler NS et al.; Wortmannin is a natural product that inhibits signal transduction . One target of wortmannin in mammalian cells is the 110-kDa catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase) . We show that wortmannin is toxic to the yeast Saccharomyces cerevisiae and present genetic and biochemical evidence that a phosphatidylinositol 4-kinase (PI 4-kinase), STT4, is a target of wortmannin in yeast . In a strain background in which stt4 mutants are rescued by osmotic support with sorbitol, the toxic effects of wortmannin are similarly prevented by sorbitol . In contrast, in a different strain background, STT4 is essential under all conditions and wortmannin toxicity is not mitigated by sorbitol . Overexpression of STT4 confers wortmannin resistance, but overexpression of PIK1, a related PI 4-kinase, does not . In vitro, the PI 4-kinase activity of STT4, but not of PIK1, was potently inhibited by wortmannin . Overexpression of the phosphatidylinositol 4-phosphate 5-kinase homolog MSS4 conferred wortmannin resistance, as did deletion of phospholipase C-1 . These observations support a model for a phosphatidylinositol metabolic cascade involving STT4, MSS4, and phospholipase C-1 and provide evidence that an essential product of this pathway is the lipid phosphatidylinositol 4,5-bisphosphate.

Arch Biochem Biophys, 1997 Nov 1, 347(1), 78 - 84
Purification and characterization of UBP6, a new ubiquitin-specific protease in Saccharomyces cerevisiae; Park KC et al.; Ubiquitin-specific protease-6 (UBP6) in Saccharomyces cerevisiae was expressed in Escherichia coli and purified from the cells using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a model substrate . The purified UBP6 behaved as a 58-kDa under both nondenaturing and denaturing conditions, indicating that the enzyme comprises a single polypeptide . It was maximally active at pH levels between 8.5 and 9, but showed little or no activity at pH below 7 and above 9.5 . As with other UBPs, its activity was strongly inhibited by sulfhydryl-blocking reagents, such as N-ethylmaleimide, and by ubiquitin-aldehyde . In addition to the model substrate, UBP6 hydrolyzed ubiquitin-alphaNH-protein extensions, such as the ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids and ubiquitin-alphaNH-dihydrofolate reductase, but not poly-His-tagged diubiquitin . It was also capable of releasing free ubiquitin from branched polyubiquitin chains that are ligated to proteins through epsilonNH-isopeptide bonds, although to a limited extent . These results suggest that UBP6 may play an important role in the generation of free ubiquitins and certain ribosomal proteins from ubiquitin-ribosomal fusion proteins as well as in deubiquitination of certain polyubiquitinated proteins targeted for degradation by the 26S proteasomes .

FEBS Lett, 1997 Sep 22, 415(1), 16 - 20
The acetyl-CoA synthetase gene ACS2 of the yeast Saccharomyces cerevisiae is coregulated with structural genes of fatty acid biosynthesis by the transcriptional activators Ino2p and Ino4p; Hiesinger M et al.; The yeast Saccharomyces cerevisiae contains two acetyl-CoA synthetase genes, ACS1 and ACS2 . While ACS1 transcription is glucose repressible, ACS2 shows coregulation with structural genes of fatty acid biosynthesis . The ACS2 upstream region contains an ICRE (inositol/choline-responsive element) as an activating sequence and requires the regulatory genes INO2 and INO4 for maximal expression . We demonstrate in vitro binding of the heterodimeric activator protein Ino2p/Ino4p to the ACS2 promoter . In addition, the pleiotropic transcription factor Abf1p also binds to the ACS2 control region . The identification of ACS2 activating elements also found upstream of ACC1, FAS1 and FAS2 suggests a role of this acetyl-CoA synthetase isoenzyme for the generation of the acetyl-CoA pool required for fatty acid biosynthesis.

J Biol Chem, 1997 Oct 10, 272(41), 25787 - 93
Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway; Yuan DS et al.; The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p . Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease . In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway . First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p . Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p . These findings were sufficient to explain the respiratory deficiency of these mutants . Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization . Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway.

Mol Gen Genet, 1997 Aug, 255(6), 561 - 9
Locus-specific suppression of ilv1 in Saccharomyces cerevisiae by deregulation of CHA1 transcription; Pedersen JO et al.; The ILV1 gene of Saccharomyces cerevisiae encodes the anabolic threonine deaminase, which catalyzes the first committed step in isoleucine biosynthesis . Strains devoid of a functional Ilv1p have a requirement for isoleucine . Threonine can also be deaminated by a second serine/threonine deaminase encoded by the CHA1 gene . CHA1 is regulated by transcriptional induction by serine and threonine, and enables yeast to utilize the hydroxyamino acids as sole nitrogen source . Phenotypic suppression of ilv1 can occur by inducer-mediated transcriptional activation of the CHA1 gene . To identify mutations in putative trnas-acting factors regulating CHA1 expression, we have isolated and characterized three extragenic suppressors of ilv1 . A dominant mutation, SIL4 (suppressor of ilv1), is allelic to HOM3 . It increases the size of the threonine pool, by 15- to 20-fold, which is sufficient to induce CHA1 transcription, thereby creating a metabolic bypass of ilv1 . A second dominant mutation, SIL3, and a recessive mutation, sil2, both suppress ilv1 by causing inducer-independent, constitutive transcription of CHA1 . Importantly, sil2 and SIL3 increase the expression of a CHA1p-lacZ translational gene fusion, demonstrating that they exert their action through the CHA1 promoter . Genetic analysis showed that both SIL3 and sil2 are alleles of CHA4, a positive regulator of CHA1, i.e., they convert Cha4p to a constitutive activator.

J Mol Biol, 1997 Oct 3, 272(4), 477 - 83
Mdj2p, a novel DnaJ homolog in the mitochondrial inner membrane of the yeast Saccharomyces cerevisiae; Westermann B et al.; Members of the heat shock protein 70 (Hsp70) family mediate import, folding, assembly and degradation of proteins in mitochondria . The function of Hsp70 proteins is dependent on their interaction with cofactors, including members of the DnaJ protein family . The mitochondrial DnaJ homolog, Mdj1p, has been shown to cooperate with the major mitochondrial Hsp70, mt-Hsp70 . We describe the identification of a second mitochondrial DnaJ homolog, Mdj2p, in the yeast Saccharomyces cerevisiae . The protein possesses an N-terminal transmembrane domain that anchors it in the mitochondrial inner membrane . The C-terminal J-domain shares 30% amino acid identity with the J-domain of Escherichia coli DnaJ and is exposed to the mitochondrial matrix . Mdj2p carries a putative internal mitochondrial targeting signal and is imported into mitochondria in a membrane potential-dependent manner . Deletion of the MDJ2 gene did not result in a detectable growth defect . Double mutants of mdj1 and mdj2 showed severe growth defects at elevated temperature, indicating a distinct overlap of the functions of Mdj1p and Mdj2p .

Mol Cell Biol, 1997 Nov, 17(11), 6765 - 71
Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae; Paques F et al.; During repair of a double-strand break (DSB) by gene conversion, one or both 3' ends of the DSB invade a homologous donor sequence and initiate new DNA synthesis . The use of the invading DNA strand as a primer for new DNA synthesis requires that any nonhomologous bases at the 3' end be removed . We have previously shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3 . We now report that these four proteins are needed only when the nonhomologous ends of recombining DNA are 30 nucleotides (nt) long or longer . An additional protein, the helicase Srs2, is required for the RAD1-dependent removal of long 3' tails . We suggest that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog . 3' tails shorter than 30 nt are removed by another mechanism that depends at least in part on the 3'-to-5' proofreading activity of DNA polymerase delta.

Mol Cell Biol, 1997 Nov, 17(11), 6693 - 9
The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae; Huang H et al.; It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres . This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci . Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains . Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2 . We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes . Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing . Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing . Telomeric silencing requires the amino terminus of Rad6 . Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.

Mol Cell Biol, 1997 Nov, 17(11), 6517 - 25
Coordination of the mating and cell integrity mitogen-activated protein kinase pathways in Saccharomyces cerevisiae; Buehrer BM et al.; Mating pheromone stimulates a mitogen-activated protein (MAP) kinase activation pathway in Saccharomyces cerevisiae that induces cells to differentiate and form projections oriented toward the gradient of pheromone secreted by a mating partner . The polarized growth of mating projections involves new cell wall synthesis, a process that relies on activation of the cell integrity MAP kinase, Mpk1 . In this report, we show that Mpk1 activation during pheromone induction requires the transcriptional output of the mating pathway and protein synthesis . Consequently, Mpk1 activation occurs subsequent to the activation of the mating pathway MAP kinase cascade . Additionally, Spa2 and Bni1, a formin family member, are two coil-coil-related proteins that are involved in the timing and other aspects of mating projection formation . Both proteins also affect the timing and extent of Mpk1 activation . This correlation suggests that projection formation comprises part of the pheromone-induced signal that coordinates Mpk1 activation with mating differentiation . Stimulation of Mpk1 activity occurs through the cell integrity phosphorylation cascade and depends on Pkc1 and the redundant MAP/Erk kinases (MEKs), Mkk1 and Mkk2 . Surprisingly, Mpk1 activation by pheromone was only partially impaired in cells lacking the MEK kinase Bck1 . This Bck1-independent mechanism reveals the existence of an alternative activator of Mkk1/Mkk2 in some strain backgrounds that at least functions under pheromone-induced conditions.

Mol Cell Biol, 1997 Nov, 17(11), 6491 - 501
Xbp1, a stress-induced transcriptional repressor of the Saccharomyces cerevisiae Swi4/Mbp1 family; Mai B et al.; We have identified Xbp1 (XhoI site-binding protein 1) as a new DNA-binding protein with homology to the DNA-binding domain of the Saccharomyces cerevisiae cell cycle regulating transcription factors Swi4 and Mbp1 . The DNA recognition sequence was determined by random oligonucleotide selection and confirmed by gel retardation and footprint analyses . The consensus binding site of Xbp1, GcCTCGA(G/A)G(C/A)g(a/g), is a palindromic sequence, with an XhoI restriction enzyme recognition site at its center . This Xbpl binding site is similar to Swi4/Swi6 and Mbp1/Swi6 binding sites but shows a clear difference from these elements in one of the central core bases . There are binding sites for Xbp1 in the G1 cyclin promoter (CLN1), but they are distinct from the Swi4/Swi6 binding sites in CLN1, and Xbp1 will not bind to Swi4/Swi6 or Mbp1/Swi6 binding sites . The XBP1 promoter contains several stress-regulated elements, and its expression is induced by heat shock, high osmolarity, oxidative stress, DNA damage, and glucose starvation . When fused to the LexA DNA-binding domain, Xbp1 acts as transcriptional repressor, defining it as the first repressor in the Swi4/Mbp1 family and the first potential negative regulator of transcription induced by stress . Overexpression of XBP1 results in a slow-growth phenotype, lengthening of G1, an increase in cell volume, and a repression of G1 cyclin expression . These observations suggest that Xbp1 may contribute to the repression of specific transcripts and cause a transient cell cycle delay under stress conditions.

Mol Cell Biol, 1997 Nov, 17(11), 6410 - 8
Transcriptional activation upon pheromone stimulation mediated by a small domain of Saccharomyces cerevisiae Ste12p; Pi H et al.; In the yeast Saccharomyces cerevisiae, Ste12p induces transcription of pheromone-responsive genes by binding to a DNA sequence designated the pheromone response element . We generated a series of hybrid proteins of Ste12p with the DNA-binding and activation domains of the transcriptional activator Gal4p to define a pheromone induction domain of Ste12p sufficient to mediate pheromone-induced transcription by these hybrid proteins . A minimal pheromone induction domain, delineated as residues 301 to 335 of Ste12p, is dependent on the pheromone mitogen-activated protein (MAP) kinase pathway for induction activity . Mutation of the three serine and threonine residues within the minimal pheromone induction domain did not affect transcriptional induction, indicating that the activity of this domain is not directly regulated by MAP kinase phosphorylation . By contrast, mutation of the two tyrosines or their preceding acidic residues led to a high level of transcriptional activity in the absence of pheromone and consequently to the loss of pheromone induction . This constitutively high activity was not affected by mutations in the MAP kinase cascade, suggesting that the function of the pheromone induction domain is normally repressed in the absence of pheromone . By two-hybrid analysis, this minimal domain interacts with two negative regulators, Dig1p and Dig2p (also designated Rst1p and Rst2p), and the interaction is abolished by mutation of the tyrosines . The pheromone induction domain itself has weak and inducible transcriptional activity, and its ability to potentiate transcription depends on the activity of an adjacent activation domain . These results suggest that the pheromone induction domain of Ste12p mediates transcriptional induction via a two-step process: the relief of repression and synergistic transcriptional activation with another activation domain.

Mol Cell Biol, 1997 Nov, 17(11), 6203 - 11
Genetic evidence for interaction between Cbp1 and specific nucleotides in the 5' untranslated region of mitochondrial cytochrome b mRNA in Saccharomyces cerevisiae; Chen W et al.; The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors . The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA . A previous deletion analysis identified an 11-nucleotide-long sequence within the 5' untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability . In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element . The CCG sequence within this region was shown to be necessary for stability . A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element . This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation.

Curr Genet, 1997 Oct, 32(4), 273 - 80
The Neurospora crassa cya-5 nuclear gene encodes a protein with a region of homology to the Saccharomyces cerevisiae PET309 protein and is required in a post-transcriptional step for the expression of the mitochondrially encoded COXI protein; Coffin JW et al.; The cya-5 nuclear mutant of Neurosopora crassa was previously shown to be deficient in cytochrome aa3, cytochrome c oxidase activity, and the immunologically detectable COXI protein . We have now demonstrated that the mitochondria of this mutant contain mRNA for the COXI protein and that COXI cannot be detected during pulse-chase labeling experiments of mitochondrial translation products . Cloning and analysis of the cya-5 gene reveal a long open reading frame capable of encoding a 1136 amino-acid protein . Sequence analysis suggests that the potential CYA-5 protein contains a mitochondrial targeting sequence at its amino-terminus . The long open reading frame also contains a 200 amino-acid region with homology to the PET309 protein, which is required for the production or stability of intron-containing coxI mRNAs, as well as the translation of mature coxI mRNAs, in the yeast Saccharomyces cerevisiae . These data suggest that the CYA-5 protein of N . crassa is required in a post-transcriptional step for COXI expression, most probably for the efficient translation of coxI mRNA.

Mol Gen Genet, 1997 Sep, 256(1), 88 - 91
Mitochondrial protein synthesis is not required for efficient excision of intron aI5 beta from COX1 pre-mRNA in Saccharomyces cerevisiae; Johnson CH et al.; Splicing of the group I intron aI5 beta from the yeast mitochondrial COX1 transcript requires at least four proteins, encoded by the nuclear genes PET54, MRS1/PET157, SUV3 and MSS18 . These proteins either act directly to facilitate intron aI5 beta excision, or indirectly in some manner . One possible indirect mode of action of these nuclear gene products is in stimulation of expression of a mitochondrial protein, such as a maturase, that is necessary for intron aI5 beta excision . To test this possibility, splicing of intron aI5 beta was examined in a rho-strain, which is incapable of mitochondrial protein synthesis . A quantitative RT-PCR assay was set up to compare levels of spliced COX1 mRNA present in three strains: a wild-type rho + strain; the rho-strain 7-49b-11, which retains the entire COX1 transcription unit; and a strain bearing a null mutation in the nuclear PET54 gene . The results showed that excision of aI5 beta occurs relatively efficiently in the rho-strain, and therefore does not require any mitochondrial-encoded proteins.

J Biol Chem, 1997 Oct 24, 272(43), 27091 - 8
Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3; Wendler F et al.; We have investigated the mechanisms underlying resistance to the drug diazaborine in Saccharomyces cerevisiae . We used UV mutagenesis to generate resistant mutants, which were divided into three different complementation groups . The resistant phenotype in these groups was found to be caused by allelic forms of the genes AFG2, PDR1, and PDR3 . The AFG2 gene encodes an AAA (ATPases associated to a variety of cellular activities) protein of unknown function, while PDR1 and PDR3 encode two transcriptional regulatory proteins involved in pleiotropic drug resistance development . The isolated PDR1-12 and PDR3-33 alleles carry mutations that lead to a L1044Q and a Y276H exchange, respectively . In addition, we report that overexpression of Yap1p, the yeast homologue of the transcription factor AP1, results in a diazaborine-resistant phenotype . The YAP1-mediated diazaborine resistance is dependent on the presence of functional PDR1 and PDR3 genes, although PDR3 had a more pronounced effect . These results provide the first evidence for a functional link between the Yap1p-dependent stress response pathway and Pdr1p/Pdr3p-dependent development of pleiotropic drug resistance.

J Biol Chem, 1997 Oct 24, 272(43), 26871 - 8
A 13C nuclear magnetic resonance investigation of the metabolism of leucine to isoamyl alcohol in Saccharomyces cerevisiae; Dickinson JR et al.; The metabolism of leucine to isoamyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy . The product of leucine transamination, alpha-ketoisocaproate had four potential routes to isoamyl alcohol . The first, via branched-chain alpha-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product . This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain alpha-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol . A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced . A third route utilizes alpha-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from alpha-hydroxyisocaproate because cell homogenates could not convert alpha-hydroxyisocaproate to isoamyl alcohol . The final possibility was that a pyruvate decarboxylase-like enzyme encoded by YDL080c appears to be the major route of decarboxylation of alpha-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent.

Mol Cells, 1997 Aug 31, 7(4), 532 - 6
Molecular cloning and characterization of the Saccharomyces cerevisiae SAB1 gene that suppresses a temperature-sensitive phenotype of the ARS-binding factor 1 mutant; So IS et al.; A high-copy number suppressor gene of the yeast temperature-sensitive lethal abf1 mutant was isolated and named SAB1 (suppressor of ABF1) . Chromoblot hybridization and grid-filter hybridization analyses showed that the SAB1 gene was located on chromosome IV . Deletion analyses of the SAB1 plasmid revealed that the suppressor activity was contained in a 1.1 kb DNA region . The nucleotide sequence of the 1.1 kb DNA fragment was determined and turned out to be identical to that of the yeast phosphoribosylanthranilate isomerase gene (TRP1) . A binding site for ARS-Binding Factor 1 was located in the coding sequence of the TRP1 gene, which has been known to be a part of the B domain of yeast autonomously replicating sequence 1 (ARS1) . Our results suggest that ABF1 might be important for the transcription of the yeast TRP1 gene in addition to having important roles in the stimulation of replication at the ARS1 locus.

J Bacteriol, 1997 Oct, 179(19), 6066 - 75
Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli; Deloche O et al.; The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine . We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E . coli homologs in vivo . We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E . coli, albeit only up to 40 degrees C . The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions . In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E . coli . This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p . Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature . A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ . In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E . coli . We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E . coli.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4257 - 63
Recovery of RNA polymerase II synthesis following DNA damage in mutants of Saccharomyces cerevisiae defective in nucleotide excision repair; Reagan MS et al.; We have measured the kinetics of the recovery of mRNA synthesis in the inducible GAL10 and RNR3 genes after exposure of yeast cells to ultraviolet (UV) radiation . Such recovery is abolished in mutant strains defective in nucleotide excision repair (NER) of DNA, including a rad23 mutant . Mutants defective in the RAD7 or RAD16 genes, which are required for the repair of the non-transcribed strand but not the transcribed strand of transcriptionally active genes, show slightly faster recovery of RNA synthesis than wild-type strains . A strain deleted of the RAD26 gene, which is known to be required for strand-specific NER in yeast, manifested delayed recovery of mRNA synthesis, whereas a rad28 mutant, which does not show defective strand-specific repair, showed normal kinetics of recovery . Measurement of the recovery of expression of selected individual yeast genes by Northern analysis following exposure of cells to UV radiation apparently correlates directly with the capacity of cells for strand-specific NER.

Genetics, 1997 Oct, 147(2), 557 - 66
Mutations in GSF1 and GSF2 alter glucose signaling in Saccharomyces cerevisiae; Sherwood PW et al.; One function of the Saccharomyces cerevisiae Snf1 protein kinase is to relieve glucose repression of SUC, GAL, and other genes in response to glucose depletion . To identify genes that regulate Snf1 kinase activity, we have selected mutants that inappropriately express a SUC2promoter::HIS3 gene fusion when grown in glucose and that require Snf1 function for this phenotype . Mutations representing two new complementation groups (gsf1 and gsf2) were isolated . gsf1 mutations affect two distinct responses to glucose: the Snf1-regulated glucose repression of SUC2 and GAL10 transcription and the Snf1-independent induction by glucose of HXT1 transcription . gsf2 mutations relieve glucose repression of SUC2 and GAL10 transcription and, in combination with snf1 delta, cause an extreme slow growth phenotype . The GSF2 gene was cloned by complementation of the gsf2-1 snf1 delta slow growth phenotype and encodes a previously uncharacterized 46kD protein.

Genetics, 1997 Oct, 147(2), 533 - 44
DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway; Dresser ME et al.; Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p . A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype . A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D . Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p . Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p . However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes . Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination . Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin . Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains.

Genetics, 1997 Oct, 147(2), 521 - 32
The role of Gcr1p in the transcriptional activation of glycolytic genes in yeast Saccharomyces cerevisiae; Uemura H et al.; To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and combinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-binding sites) . The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene . As observed previously, synthetic oligonucleotides containing both CT- and RPG-boxes conferred strong UAS activity . Likewise, a lone CT-box did not show any UAS activity . By contrast, oligonucleotides containing tandem Ct-boxes but no RPG-box conferred strong promoter activity . This UAS activity was not dependent on position or orientation of the oligonucleotides in the 5' noncoding region . However, it was dependent on both GCR1 and GCR2 . These results suggest that the ability of Gcr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent on Rap1p . Eleven independent mutants of GCR1 were isolated that conferred weak UAS activity to a single CT-box . Five mutants has single mutations in Gcr1p's DNA-binding domain and displayed slightly higher affinity for the CT-box . These results support the hypothesis that Gcr1p and Gcr2p play the central role in glycolytic gene expression and that the function of Rap1p is to facilitate the binding of Gcr1p to its target.

Genetics, 1997 Oct, 147(2), 507 - 19
Genetic and environmental factors affecting the de novo appearance of the {PSI+} prion in Saccharomyces cerevisiae; Derkatch IL et al.; It has previously been shown that yeast prion {PSI+} is cured by GuHCl, although reports on reversibility of curing were contradictory . Here we show that GuHCl treatment of both {PSI+} and {psi-} yeast strains results in two classes of {psi-} derivatives: Pin+, in which {PSI+} can be reinduced by Sup35p overproduction, and Pin-, in which overexpression of the complete SUP35 gene does not lead to the {PSI+} appearance . However, in both Pin+ and Pin- derivatives {PSI+} is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, {PSI+} curing remains reversible in both cases . Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin+ {psi-} derivatives are observed in Pin- {psi-} derivatives . Genetic analyses show that the Pin+ phenotype is determined by a non-Mendelian factor, which, unlike the {PSI+} prion, is independent of the Sup35p N-terminal domain . A Pin- {psi-} derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while {PSI+} curing by Hsp104 overproduction resulted exclusively in Pin+ {psi-} derivatives . We hypothesize that in addition to the {PSI+} prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin+ phenotype.

Genetics, 1997 Oct, 147(2), 479 - 92
Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions; Inouye C et al.; Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade . A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7) . Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction . Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein beta subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein . When expressed in a ste5 delta cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency . One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7 . Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

Genetics, 1997 Oct, 147(2), 467 - 78
Genetic interactions between a pep7 mutation and the PEP12 and VPS45 genes: evidence for a novel SNARE component in transport between the Saccharomyces cerevisiae Golgi complex and endosome; Webb GC et al.; The PEP7 gene from Saccharomyces cerevisiae encodes a 59-kD hydrophilic polypeptide that is required for transport of soluble vacuolar hydrolase precursors from the TGN to the endosome . This study presents the results of a high-copy suppression analysis of pep7-20 mutant phenotypes . This analysis demonstrated that both VPS45 and PEP12 are allele-specific high-copy suppressors of pep7-20 mutant phenotypes . Overexpression of VPS45 was able to completely suppress the Zn2+ sensitivity and partially suppress the carboxypeptidase Y deficiency . Overexpression of PEP12 was able to do the same, but to a lesser extent . Vps45p and Pep12p are Sec1p and syntaxin (t-SNARE) homologues, respectively, and are also thought to function in transport between the TGN and endosome . Two additional vacuole pathway SNARE complex homologues, Vps33p (Sec1p) and Pth1p (syntaxin), when overexpressed, were unable to suppress pep7-20 or any other pep7 allele, further supporting the specificity of the interactions of pep7-20 with PEP12 and VPS45 . Because several other vesicle docking/fusion reactions take place in the cell without discernible participation of Pep7p homologues, we suggest that Pep7p is a step-specific regulator of docking and/or fusion of TGN-derived transport vesicles onto the endosome.

Genetics, 1997 Oct, 147(2), 451 - 65
Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Srb/mediator complexes; Roberts SM et al.; The Saccharomyces cerevisiae transcription factor Spt20/Ada5 was originally identified by mutations that suppress Ty insertion alleles and by mutations that suppress the toxicity caused by Gal4-VP16 overexpression . Here we present evidence for physical associations between Spt20/Ada5 and three other Spt proteins, suggesting that they exist in a complex . A related study demonstrates that this complex also contains the histone acetyltransferase, Gcn5, and Ada2 . This complex has been named SAGA (Spt/Ada/Gcn5 acetyltransferase) . To identify functions that genetically interact with SAGA, we have screened for mutations that cause lethality in an spt20 delta/ada5 delta mutant . Our screen identified mutations in SNF2, SIN4, and GAL11 . These mutations affect two known transcription complexes: Snf/Swi, which functions in nucleosome remodeling, and Srb/mediator, which is required for regulated transcription by RNA polymerase II . Systematic analysis has demonstrated that spt20 delta/ada5 delta and spt7 delta mutations cause lethality with every snf/swi and srb/mediator mutation tested . Furthermore, a gcn5 delta mutation causes severe sickness with snf/swi mutations, but not with srb/mediator mutations . These findings suggest that SAGA has multiple activities and plays critical roles in transcription by RNA polymerase II.

Genetics, 1997 Oct, 147(2), 435 - 50
Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae; Lussier M et al.; The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes . We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes . The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database . Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor . Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

Genetics, 1997 Oct, 147(2), 421 - 34
Identification of genes controlling growth polarity in the budding yeast Saccharomyces cerevisiae: a possible role of N-glycosylation and involvement of the exocyst complex; Mondesert G et al.; The regulation of secretion polarity and cell surface growth during the cell cycle is critical for proper morphogenesis and viability of Saccharomyces cerevisiae . A shift from isotropic cell surface growth to polarized growth is necessary for bud emergence and a repolarization of secretion to the bud neck is necessary for cell separation . Although alterations in the actin cytoskeleton have been implicated in these changes in secretion polarity, clearly other cellular systems involved in secretion are likely to be targets of cell cycle regulation . To investigate mechanisms coupling cell cycle progression to changes in secretion polarity in parallel with and downstream of regulation of actin polarization, we implemented a screen for mutants defective specifically in polarized growth but with normal actin cytoskeleton structure . These mutants fell into three classes: those partially defective in N-glycosylation, those linked to specific defects in the exocyst, and a third class neither defective in glycosylation nor linked to the exocyst . These results raise the possibility that changes in N-linked glycosylation may be involved in a signal linking cell cycle progression and secretion polarity and that the exocyst may have regulatory functions in coupling the secretory machinery to the polarized actin cytoskeleton.

Genetics, 1997 Oct, 147(2), 409 - 20
Suppressors of the ndc10-2 mutation: a role for the ubiquitin system in Saccharomyces cerevisiae kinetochore function; Kopski KM et al.; We have isolated a new conditional-lethal mutation, ndc10-2, in the NDC10/CBF2/CTF14 gene that encodes the 110-kD subunit of the Saccharomyces cerevisiae CBF3 kinetochore complex . At the restrictive temperature of 37 degrees, ndc10-2 cells are able to assemble anaphase spindles, but fail to segregate their DNA, consistent with a defect in kinetochore function . To identify other factors that play a role in kinetochore assembly or function, we isolated both dosage and second site suppressors of the ndc10-2 mutation . These screens identified UBC6 as a dosage suppressor, and mutations in UBC6 and UBC7 as second-site suppressors of ndc10-2 heat sensitivity . Both UBC6 and UBC7 encode ubiquitin-conjugating enzymes that function in ubiquitin-mediated protein degradation . Furthermore, overexpression of a mutant ubiquitin suppresses the ndc10-2 mutation . These results implicate the ubiquitin system in the regulation of ndc10-2 function and suggest a role for the ubiquitin system in kinetochore function.

Genetics, 1997 Oct, 147(2), 383 - 97
The isolation and characterization of Saccharomyces cerevisiae mutants that constitutively express purine biosynthetic genes; Guetsova ML et al.; In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes . To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression . These mutants were named bra (for bypass of repression by adenine) . BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase . BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase . These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes . We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway . Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression . Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.

Genetics, 1997 Oct, 147(2), 371 - 82
"Break copy" duplication: a model for chromosome fragment formation in Saccharomyces cerevisiae; Morrow DM et al.; Introduction of a chromosome fragmentation vector (CFV) into the budding yeast Saccharomyces cerevisiae results in a targeted homologous recombination event that yields an independently segregating chromosome fragment (CF) and an alteration in the strain's karyotype . Fragmentation with an acentric CFV directed in a centromere-proximal orientation generates a CF that contains all sequences proximal to the targeting segment and results in loss of the endogenous targeted chromosome to yield a 2N-1 + CF karyotype . In contrast, fragmentation with a centric CFV directed in a centromere-distal orientation generates a CF that contains all sequences distal to the targeting segment and retention of the endogenous targeted chromosome to yield a 2N + CF karyotype . We have termed this phenomenon "break copy" duplication . Using yeast strains in which the centromere had been transposed to a new location, it was demonstrated that the centromere inhibited break copy duplication . These data suggest that CF formation is the product of an unscheduled DNA replication event initiated by the free end of the CFV and is analogous to a "half" double-strand break gap-repair reaction . We suggest that break copy duplication may have evolved as a mechanism for maintenance of ploidy following DNA breakage.

J Bacteriol, 1997 Oct, 179(20), 6279 - 84
Altered extent of cross-linking of beta1,6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall beta1,3-glucan content; Kapteyn JC et al.; The yeast cell wall contains beta1,3-glucanase-extractable and beta1,3-glucanase-resistant mannoproteins . The beta1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a beta1,6-glucan moiety, which in its turn is linked to beta1,3-glucan (J . C . Kapteyn, R . C . Montijn, E . Vink, J . De La Cruz, A . Llobell, J . E . Douwes, H . Shimoi, P . N . Lipke, and F . M . Klis, Glycobiology 6:337-345, 1996) . The beta1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of beta1,6-glucosylated proteins, including Cwp1p, as the B1,3-glucanase-extractable fraction . Chitin was linked to the proteins in the beta1,3-glucanase-resistant fraction through a beta1,6-glucan moiety . In wild-type cell walls, the beta1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less beta1,3-glucan but more chitin, beta1,3-glucanase-resistant proteins represented about 40% of the total . We propose that the increased cross-linking of cell wall proteins via beta1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening.

J Biol Chem, 1997 Oct 17, 272(42), 26087 - 90
The BST1 gene of Saccharomyces cerevisiae is the sphingosine-1-phosphate lyase; Saba JD et al.; Sphingolipids elicit a wide variety of eukaryotic cellular responses, most involving regulation of cell growth, differentiation, and apoptosis . Sphingosine 1-phosphate, a sphingolipid catabolite, is mitogenic in fibroblasts and inhibits the chemotactic mobility and invasiveness of human tumor cells . Sphingosine 1-phosphate degradation requires cleavage at the C2-3 carbon bond by sphingosine phosphate lyase . A yeast genetic approach was used to clone the first sphingosine phosphate lyase gene, BST1 . BST1 overexpression conferred resistance to sphingosine in yeast . BST1 deletion produced sensitivity to exogenous D-erythro-sphingosine and phytosphingosine and intracellular accumulation of sphingosine 1-phosphate upon exposure to exogenous sphingosine . This study confirms that sphingoid base metabolism is similar in all eukaryotes and suggests that yeast genetics may be useful in the isolation and identification of other genes involved in sphingolipid signaling and metabolism.

Biochemistry (Mosc), 1997 Jul, 62(7), 742 - 52
ADE6 gene of Saccharomyces cerevisiae yeast encoding formylglycinamidine-ribonucleotide synthetase . Cloning, sequencing, and analysis; Andreichuk YuV et al.; The ADE6 gene of Saccharomyces cerevisiae yeast encoding the enzyme formylglycinamidine-ribonucleotide (FGAM)-synthetase of de novo synthesis of purine nucleotides was cloned and sequenced . The gene encodes a protein consisting of 1358 amino acids . The flanking regions of 1208 (5') and 728 bp (3') were also sequenced . The nucleotide motif TGACTC inherent to the promotor regions of other purine genes of yeast was located (-276 bp) in the 5'-region of the gene . The amino acid sequence of the yeast FGAM-synthetase was found to contain repeats (Leu430-Ala620 and Pro810-Ile1000) . Repeats of similar patterns of conserved amino acids were also detected in the structure of all other FGAM-synthetases . A homology of FGAM-synthetases with certain proteins of viruses from the Herpesviridae family was found.

Nucleic Acids Res, 1997 Oct 15, 25(20), 3974 - 9
Saccharomyces cerevisiae mms19 mutants are deficient in transcription-coupled and global nucleotide excision repair; Lombaerts M et al.; The recently cloned Saccharomyces cerevisiae MMS19 gene appears to be involved in both nucleotide excision repair (NER) and transcription, which is also the case for components of the NER/transcription complex TFIIH . Unlike TFIIH however, the Mms19 protein does not affect NER in a highly purified in vitro system . In order to investigate the role of Mms19 in NER, we have analysed the repair capacity of the mms19 disruption mutant . We find that a cell-free extract of this mutant is deficient for NER in vitro . Since mms19 mutants are only moderately sensitive to irradiation with ultraviolet (UV) light, it is possible that such mutants are specifically deficient in one of the two modes of NER, i.e . transcription-coupled or global genome repair . To investigate this possibility, we have analysed the removal of cyclobutane-pyrimidine dimers (CPDs) at the nucleotide level in an mms19 mutant . Repair of CPDs was not detectable for both transcribed and non-transcribed sequences in this mutant, demonstrating a requirement for Mms19 in both transcription-coupled and global genome repair . Our data, combined with those obtained by others, suggest that Mms19 is required for NER in yeast, although it seems likely that the protein plays an indirect role in this process.

EMBO J, 1997 Oct 15, 16(20), 6272 - 80
The pheromone response pathway activates transcription of Ty5 retrotransposons located within silent chromatin of Saccharomyces cerevisiae; Ke N et al.; The Saccharomyces retrotransposon Ty5 integrates preferentially into transcriptionally inactive regions (silent chromatin) at the HM loci and telomeres . We found that silent chromatin represses basal Ty5 transcription, indicating that these elements are encompassed by silent chromatin in their native genomic context . Because transcription is a requirement for transposition, integration into silent chromatin would appear to prevent subsequent rounds of replication . Using plasmid-borne Ty5-lacZ constructs, we found that Ty5 expression is haploid specific and is repressed 10-fold in diploid strains . Ty5 transcription is also regulated by the pheromone response pathway and is induced approximately 20-fold upon pheromone treatment . Deletion analysis of the Ty5 LTR promoter revealed that a 33 bp region with three perfect matches to the pheromone response element is responsible for both mating pheromone and cell-type regulation . Transcriptional repression of Ty5 by silent chromatin can be reversed by pheromone treatment, which leads to transcription and transposition . Ty5 replication, therefore, is normally repressed by silent chromatin and appears to be induced during mating . This is the first example of transcriptional activation of a gene that naturally resides within silent chromatin.

J Immunol, 1997 Oct 1, 159(7), 3400 - 11
Complete protective immunity induced in mice by immunization with the 19-kilodalton carboxyl-terminal fragment of the merozoite surface protein-1 (MSP1{19}) of Plasmodium yoelii expressed in Saccharomyces cerevisiae: correlation of protection with antigen-specific antibody titer, but not with effector CD4+ T cells; Hirunpetcharat C et al.; The 19-kDa carboxyl-terminal fragment of the merozoite surface protein-1 (MSP1) is a leading malaria vaccine candidate but is unable to induce immunity in all monkeys or all strains of mice . The mechanism of immunity is unclear, although data show that cell-mediated immunity plays a critical role following immunization with the larger mature MSP1 protein . We optimized a vaccine protocol using the MSP1(19) fragment of Plasmodium yoelii expressed in Saccharomyces cerevisiae, such that following exposure of mice to parasites, they remained undetectable in peripheral blood, whereas control animals all died at very high parasitemia within 10 days . We then depleted the vaccinated mice of >99% of CD4+ T cells by anti-CD4 mAb treatment and could show that infections in most animals remained subpatent following challenge . Furthermore, mice in which the gene for the mu-chain of Ig had been disrupted could not be immunized with MSP1(19) . Immunity in normal mice did not depend on the presence of an intact spleen nor production of nitric oxide, persisting unabated when >70% of splenic macrophages were depleted . Thus, while effector CD4+ T cells may contribute to immunity, neither they nor factors associated with a Th1-type cell mediated immune response appeared to play the major role in MSP1(19)-induced protection in normal mice . Furthermore, T cells were not sufficient for immunity in mice lacking B cells . In normal mice, protection correlated with a very high titer of MSP1(19)-specific Abs (>6,400,000), predominantly G1 and G2b, which may function by merozoite neutralization.

Biochemistry, 1997 Oct 7, 36(40), 12036 - 44
Novel modifications to the farnesyl moiety of the a-factor lipopeptide pheromone from Saccharomyces cerevisiae: a role for isoprene modifications in ligand presentation; Dawe AL et al.; The a-factor of Saccharomyces cerevisiae is a dodecapeptide pheromone {YIIKGVFWDPAC(farnesyl)-OCH3} in which posttranslational modification with a farnesyl isoprenoid and carboxymethyl group is required for full biological activity . Utilizing novel synthetic techniques and a well-characterized array of biological assays, we prepared original modifications to the farnesyl moiety of the pheromone in order to assess the importance of this part of the lipopeptide for biological activity . Specifically, the 3-methyl group was replaced to create analogs containing the ethyl, vinyl, tert-butyl, and phenyl moieties at the 3-position of the farnesyl chain . Subsequent biological analyses demonstrated that all of these modifications render an active pheromone, with the vinyl and ethyl analogs exhibiting higher activity than the native a-factor . However, the level of activity varied with the modification; the bulkier and more hydrophobic groups (tert-butyl and phenyl) exhibited lower biological activity than the smaller moieties (ethyl and vinyl) . Furthermore, two analogs with phenyl substitutions that differ only in the presumed isomerization of the allylic double bond show up to an 8-fold difference in bioactivity . It has previously been surmised that the role of isoprenoid additions is solely to target the attached polypeptides to membranes by increasing their hydrophobicity . However, these studies demonstrate that even modest structural changes to the isoprenoid can significantly affect biological activity . These results are clearly inconsistent with a simple hydrophobic role for the isoprenoid and instead illustrate that it plays an active role in mediating optimal a-factor/receptor interaction.

Mol Cell Biol, 1997 Oct, 17(10), 6105 - 13
Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae; Huang M et al.; Ribonucleotide reductase (RNR), which catalyzes the rate-limiting step for deoxyribonucleotide production required for DNA synthesis, is an alpha2beta2 tetramer consisting of two large and two small subunits . RNR2 encodes a small subunit and is essential for mitotic viability in Saccharomyces cerevisiae . We have cloned a second essential gene encoding a homologous small subunit, RNR4 . RNR4 and RNR2 appear to have nonoverlapping functions and cannot substitute for each other even when overproduced . The lethality of RNR4 deletion mutations can be suppressed by overexpression of RNR1 and RNR3, two genes encoding the large subunit of the RNR enzyme, indicating genetic interactions among the RNR genes . RNR2 and RNR4 may be present in the same reductase complex in vivo, since they coimmunoprecipitate from cell extracts . Like the other RNR genes, RNR4 is inducible by DNA-damaging agents through the same signal transduction pathway involving MEC1, RAD53, and DUN1 kinase genes . Analysis of DNA damage inducibility of RNR2 and RNR4 revealed partial inducibility in dun1 mutants, indicating a DUN1-independent branch of the transcriptional response to DNA damage.

Mol Cell Biol, 1997 Oct, 17(10), 5968 - 75
FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae; Alarcon CM et al.; The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus) . Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown . Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12 . Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity . The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.

Mol Cell Biol, 1997 Oct, 17(10), 5867 - 75
Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit; Dalton S et al.; Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication . Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p . The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase . Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates . Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1 . We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions . The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro . This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.

Biochim Biophys Acta, 1997 Sep 4, 1328(2), 214 - 26
Overexpression of the sodium ATPase of Saccharomyces cerevisiae: conditions for phosphorylation from ATP and Pi; Benito B et al.; The ENA1 gene of Saccharomyces cerevisiae encodes a putative ATPase necessary for Na+ efflux . Plasma membranes and intracellular membranes of a yeast strain overexpressing the ENA1 gene contain significant amounts of ENA1 protein . Consequences of the overexpression with reference to the wild-type strain are: (1) a 5-fold higher content of the ENA1-protein in plasma membranes; (2) lower Na+ and Li+ effluxes; (3) slightly higher Na+ tolerance; and (4) much higher Li+ tolerance . The ENA1-specific ATPase activity in plasma membrane preparations of the overexpressing strain was low, but an ENA1 phosphoprotein was clearly detected when the plasma membranes were exposed to ATP in the presence of Na+ or to Pi in the absence of Na+ . The characteristics of this phosphoprotein, which correspond to the acyl phosphate intermediaries of P-type ATPases, the absolute requirement of Na+ or other alkali cations for phosphorylation, and the Na+ and pH dependence of phosphorylation from ATP and Pi suggest that the product of the ENA1 gene may be a Na,H-ATPase, which can also pump other alkali cations . The role of the intracellular membranes structures produced with the overexpression of ENA1 in Na+ and Li+ tolerances and the existence of a beta-subunit of the ENA1 ATPase are discussed.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10786 - 91
The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence; Theis JF et al.; Autonomously replicating sequence (ARS) elements, which function as the cis-acting chromosomal replicators in the yeast Saccharomyces cerevisiae, depend upon an essential copy of the 11-bp ARS consensus sequence (ACS) for activity . Analysis of the chromosome III replicator ARS309 unexpectedly revealed that its essential ACS differs from the canonical ACS at two positions . One of the changes observed in ARS309 inactivates other ARS elements . This atypical ACS binds the origin recognition complex efficiently and is required for chromosomal replication origin activity . Comparison of the essential ACS of ARS309 with the essential regions of other ARS elements revealed an expanded 17-bp conserved sequence that efficiently predicts the essential core of ARS elements.

J Dairy Sci, 1997 Sep, 80(9), 2035 - 44
Effects of a Saccharomyces cerevisiae culture on ruminal bacteria that utilize lactate and digest cellulose; Callaway ES et al.; The objective of this study was to determine the effects of a yeast (Saccharomyces cerevisiae) culture on lactate utilization and cellulose digestion by ruminal bacteria . Growth of Selenomonas ruminantium HD4 in medium that contained 5 g/L of DL-lactate, Trypticase, and yeast extract was stimulated 7 and 15% by 1 and 5% (vol/vol) yeast culture filtrate respectively . The 1 and 5% yeast culture filtrate stimulated growth of Selenomonas ruminantium H18 and Megasphaera elsdenii B159 and T81 on 5 g/L of DL-lactate in medium without Trypticase or yeast extract . Growth of Fibrobacter succinogenes S85 and Ruminococcus albus B199 on 6 g/L of cellobiose was stimulated by the addition of yeast culture filtrate to medium without Trypticase or yeast extract . The yeast culture filtrate increased the concentrations of acetate and total volatile fatty acids that were produced by Sel . ruminantium HD4 and increased the concentrations of propionate and total volatile fatty acids that were produced by Sel . ruminantium H18 but did not alter end-product formation of M . elsdenii or cellulolytic bacteria . Treatment with yeast culture increased the initial rate but not the extent of cellulose digestion by F . succinogenes S85 and Ruminococcus flavefaciens FD1 . Collectively, these results suggest that yeast culture provides soluble growth factors (i.e., organic acids, B vitamins, and amino acids) that stimulate growth of ruminal bacteria that utilize lactate and digest cellulose.

EMBO J, 1997 Sep 15, 16(18), 5629 - 38
Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1: coupling glucose sensing to gene expression and the cell cycle; Li FN et al.; Grr1 protein of the yeast Saccharomyces cerevisiae is a central component of a glucose signal transduction mechanism responsible for glucose-induced gene expression . It is required for glucose-stimulated regulation of Rgt1, a repressor of several glucose-induced HXT genes . Grr1 also plays a role in regulating the cell cycle, because it is required for degradation of the G1 cyclins Cln1 and Cln2 . We discovered that Grr1 physically interacts with Skp1, a protein that has been implicated in a ubiquitin-conjugating enzyme complex that targets for degradation the cell cycle regulators Cln1 and Cln2, and the cyclin-dependent kinase inhibitor Sic1 . Thus, Grr1 may regulate the cell cycle and glucose-induced gene expression via ubiquitin-mediated protein degradation . Consistent with this idea, Skp1, like Grr1, was found to be required for glucose-induced HXT gene expression . Two functional domains of Grr1 are required for its interaction with Skp1: 12 leucine-rich repeats (LRR) and an adjacent F-box . The Grr1-Skp1 interaction is enhanced by high levels of glucose . This could provide yeast with a mechanism for coupling nutrient availability to gene expression and cell cycle regulation.

J Virol, 1997 Oct, 71(10), 7781 - 90
In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae; Ishikawa M et al.; To facilitate manipulation of brome mosaic virus (BMV) RNA replicons in Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA replication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends . In galactose-induced yeast harboring such plasmids, expression of BMV RNA replication proteins 1a and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher than those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-encoded subgenomic mRNA for coat protein . Although the GAL1 promoter initiated transcription from multiple sites, 1a and 2a selectively amplified RNA3 with the authentic viral 5' end . As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be translated directly from DNA-derived RNA3 transcripts, so their expression depended on 1a- and 2a-directed subgenomic mRNA synthesis . In yeast in which DNA transcription of B3CAT, an RNA3 derivative with the chloramphenicol acetyltransferase (CAT) gene replacing the coat gene, was induced, CAT activity remained near background levels in the absence of 1a and 2a but increased over 500,000-fold when 1a and 2a were expressed . Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeast URA3 gene replacing the coat gene, conferred uracil-independent growth to ura3- yeast only after 1a and 2a expression and galactose induction . Once its 1a- and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after repression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid . These findings should be useful for many experimental purposes.

Biochimie, 1997 Jun, 79(6), 341 - 50
Mitochondrial DNA topoisomerase I of Saccharomyces cerevisiae; Tua A et al.; A mitochondrial DNA topoisomerase (type I, ATP-independent) can be biochemically distinguished from the nuclear enzyme DNA topoisomerase I . This conclusion is based on the subcellular localization of the mitochondrial enzyme, its optimal reaction conditions and sensitivity to enzyme inhibitors . Unlike its nuclear counterpart, the mitochondrial DNA topoisomerase exhibits an absolute requirement for a divalent cation (Mg2+ and Ca2+ work equally well in vitro) . In addition, it is slightly more sensitive to monovalent salts, with optimal activity obtained in 50-100 mM KCl . The mitochondrial enzyme is equally active at pH 7.5 or pH 9.5, but unlike its nuclear equivalent, is inactivated at higher pH values . The mitochondrial DNA topoisomerase is sensitive to coumermycin, berenil, camptothecin and 2,2,5,5-tetramethyl-4-imidazolidinone, a chemical that has no inhibitory effect on DNA topoisomerase I . Immunoblotting studies show that mitochondrial DNA topoisomerase activity is associated with a polypeptide (M(r) approximately 79,000) that cross-reacts with the antiserum against DNA topoisomerase I . Thus, the mitochondrial DNA topoisomerase may be derived by the differential expression of the DNA topoisomerase I gene or from the expression of a gene that is homologous to the DNA topoisomerase I gene.

Genes Dev, 1997 Sep 15, 11(18), 2414 - 25
Rpp1, an essential protein subunit of nuclear RNase P required for processing of precursor tRNA and 35S precursor rRNA in Saccharomyces cerevisiae; Stolc V et al.; The gene for an essential protein subunit of nuclear RNase P from Saccharomyces cerevisiae has been cloned . The gene for this protein, RPP1, was identified by virtue of its homology with a human scleroderma autoimmune antigen, Rpp30, which copurifies with human RNase P . Epitope-tagged Rpp1 can be found in association with both RNase P RNA and a related endoribonuclease, RNase MRP RNA, in immunoprecipitates from crude extracts of cells . Depletion of Rpp1 in vivo leads to the accumulation of precursor tRNAs with unprocessed 5' and 3' termini and reveals rRNA processing defects that have not been described previously for proteins associated with RNase P or RNase MRP . Immunoprecipitated complexes cleave both yeast precursor tRNAs and precursor rRNAs.

Genes Dev, 1997 Sep 15, 11(18), 2396 - 413
GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces cerevisiae; Huang HK et al.; We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae . Both suppressor genes are dominant in diploid cells and lethal in haploid cells . The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the gamma subunit of the eIF-2 complex and contains a mutation in the G2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein . The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex . Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon . Purified mutant eIF-2gamma complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis . Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the beta subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent . All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast . These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2 x GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.

Mol Biol Cell, 1997 Sep, 8(9), 1699 - 707
Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine; Grant CM et al.; Glutathione (GSH) synthetase (Gsh2) catalyzes the ATP-dependent synthesis of GSH from gamma-glutamylcysteine (gamma-Glu-Cys) and glycine . GSH2, encoding the Saccharomyces cerevisiae enzyme, was isolated and used to construct strains that either lack or overproduce Gsh2 . The identity of GSH2 was confirmed by the following criteria: 1) the predicted Gsh2 protein shared 37-39% identity and 58-60% similarity with GSH synthetases from other eukaryotes, 2) increased gene dosage of GSH2 resulted in elevated Gsh2 enzyme activity, 3) a strain deleted for GSH2 was dependent on exogenous GSH for wild-type growth rates, and 4) the gsh2 mutant lacked GSH and accumulated the dipeptide gamma-Glu-Cys intermediate in GSH biosynthesis . Overexpression of GSH2 had no effect on cellular GSH levels, whereas overexpression of GSH1, encoding the enzyme for the first step in GSH biosynthesis, lead to an approximately twofold increase in GSH levels, consistent with Gsh1 catalyzing the rate-limiting step in GSH biosynthesis . In contrast to a strain deleted for GSH1, which lacks both GSH and gamma-Glu-Cys, the strain deleted for GSH2 was found to be unaffected in mitochondrial function as well as resistance to oxidative stress induced by hydrogen peroxide, tert-butyl hydroperoxide, and the superoxide anion . Furthermore, gamma-Glu-Cys was at least as good as GSH in protecting yeast cells against an oxidant challenge, providing the first evidence that gamma-Glu-Cys can act as an antioxidant and substitute for GSH in a eukaryotic cell . However, the dipeptide could not fully substitute for the essential function of GSH in the cell as shown by the poor growth of the gsh2 mutant on minimal medium . We suggest that this function may be the detoxification of harmful intermediates that are generated during normal cellular metabolism.

Mol Biol Cell, 1997 Sep, 8(9), 1649 - 64
Evidence that mating by the Saccharomyces cerevisiae gpa1Val50 mutant occurs through the default mating pathway and a suggestion of a role for ubiquitin-mediated proteolysis; Xu BE et al.; The yeast G alpha subunit, Gpa1p, plays a negative role in the pheromone response pathway . The gpa1Val50 mutant was previously shown to have a growth defect, consistent with the GTPase defect predicted for this mutation, and greatly reduced mating . Various explanations for the mating defect have been proposed . One approach to analyze the gpa1Val50 mating defect involved epistasis analysis . The low mating of the gpa1Val50 mutant was independent of the pheromone receptor; therefore, it results from intracellular activation of the pathway, consistent with a GTPase defect . This result suggests that gpa1Val50 mating occurs through the default rather than the chemotropic pathway involved in pheromone response . We therefore tested the effect of a spa2 mutation on gpa1Val50 mating, because Spa2p has been implicated in the default pathway . The spa2 mutation greatly reduced the mating of the gpa1Val50 mutant, suggesting that gpa1Val50 mating occurs predominantly through the default pathway . In a second approach to investigate the gpa1Val50 phenotypes, suppressors of the gpa1Val50 mating defect were isolated . Two suppressor genes corresponded to SON1/UFD5 and SEN3, which are implicated in ubiquitin-mediated proteolysis . On the basis of these results, we suggest that a positive component of the default mating pathway is subject to ubiquitin-mediated degradation.

FEBS Lett, 1997 Sep 1, 414(1), 171 - 5
Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr-specific protein kinase; Stocchetto S et al.; Saccharomyces cerevisiae YGR262c gene, whose disruption causes severely defective growth, encodes a putative protein kinase shorter than any other protein kinase biochemically characterized to date and lacking some of the conserved features of these enzymes . Here we show that the product of the YGR262c gene, piD261, expressed in E . coli with a C-terminal (His)6 tag, is a bona fide Ser/Thr protein kinase as judged from its capability to autophosphorylate and to phosphorylate casein and osteopontin in the presence of {gamma-32P}ATP . In contrast, no phosphorylation of histones, myelin basic protein, phosvitin, bovine serum albumin and poly(Glu/Tyr)4:1 could be detected . Mn2+ or, less effectively, Co2+ are required for piD261 catalytic activity, which is conversely undetectable in the presence of Mg2+, a behaviour unique among Ser/Thr protein kinases.

EMBO J, 1997 Aug 15, 16(16), 4924 - 37
A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator; Gray JV et al.; The protein kinase C of Saccharomyces cerevisiae, Pkc1, regulates a MAP kinase, Mpk1, whose activity is stimulated at the G1-S transition of the cell cycle and by perturbations to the cell surface, e.g . induced by heat shock . The activity of the Pkc1 pathway is partially dependent on Cdc28 activity . Swi4 activates transcription of many genes at the G1-S transition, including CLN1 and CLN2 . We find that swi4 mutants are defective specifically in bud emergence . The growth and budding defects of swi4 mutants are suppressed by overexpression of PKC1 . This suppression requires CLN1 and CLN2 . Inhibition of the Pkc1 pathway exacerbates the growth and bud emergence defects of swi4 mutants . We find that another dose-dependent suppressor of swi4 mutants, the novel gene HCS77, encodes a putative integral membrane protein . Hcs77 may regulate the Pkc1 pathway; hcs77 mutants exhibit phenotypes like those of mpk1 mutants, are partially suppressed by overexpression of PKC1 and are defective in heat shock induction of Mpk1 activity . We propose that the Pkc1 pathway promotes bud emergence and organized surface growth and is activated by Cdc28-Cln1/Cln2 at the G1-S transition and by Hcs77 upon heat shock . Hcs77 may monitor the state of the cell surface.

Can J Microbiol, 1997 Aug, 43(8), 774 - 81
Mutants in the Saccharomyces cerevisiae RAS2 gene influence life span, cytoskeleton, and regulation of mitosis; Pichova A et al.; We investigated the phenotypic consequences in Saccharomyces cerevisiae of a disruption allele (ras2::LEU2) and of a dominant mutant form (RAS2ala18,val19) of RAS2 . In addition to the phenotypes described earlier for these mutants, we observed a small increase in the life span for the disruption allele and a drastic decrease of life span for the dominant mutant form, as compared with the isogenic wild type . This was found by analyzing these alleles in two different genetic backgrounds with nearly the same results . Life spans were determined by micromanipulating mother cells and counting generations until no further cell division occurred . A morphological analysis of the terminal phenotypes of very old mother cells was performed showing enlarged or rounded cells and in some cases elongated buds, some of which were difficult to separate from the mother cell . This was observed in wild-type cells, as well as mutant cells . However, the dominant RAS2 mutant (but not the wild-type or ras2::LEU2 mutant cells) after 2 days on complex media displayed phenotypes similar to the terminal phenotype of old mothers . A substantial fraction of the cells were enlarged and generated elongated buds, they lost Calcofluor staining of the bud scars, the cell surface appeared folded, the actin cytoskeleton was aberrant, and the mitotic spindle and the cytoplasmic microtubles were defective in their proper orientation, resulting in aberrant mitoses and empty buds . These phenotypic characteristics of the RAS2ala18,val19 mutation could be causative for the previously observed rapid loss of viability of these cells in stationary phase.

EMBO J, 1997 Aug 1, 16(15), 4788 - 95
Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair; Teo SH et al.; DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair . Mammalian cells have four DNA ligases, termed ligases I-IV . In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae . Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian DNA ligase IV . Unlike CDC9, LIG4 is not essential for DNA replication, RAD52-dependent homologous recombination nor the repair of UV light-induced DNA damage . Instead, it encodes a crucial component of the non-homologous end-joining (NHEJ) apparatus, which repairs DNA double-strand breaks that are generated by ionizing radiation or restriction enzyme digestion: a function which cannot be complemented by CDC9 . Lig4p acts in the same DNA repair pathway as the DNA end-binding protein Ku . However, unlike Ku, it does not function in telomere length homeostasis . These findings indicate diversification of function between different eukaryotic DNA ligases . Furthermore, they provide insights into mechanisms of DNA repair and suggest that the NHEJ pathway is highly conserved throughout the eukaryotic kingdom.

Yeast, 1997 Sep 30, 13(12), 1145 - 54
Specific labelling of cell wall proteins by biotinylation . Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae; Mrsa V et al.; Intact Saccharomyces cerevisiae cells were biotinylated with the non-permeable sulfosuccinimidyl-6-(biotinamido) hexanoate reagent . Twenty specifically labelled cell wall proteins would be extracted and visualized on SDS gels via streptavidin/horseradish peroxidase . Nine cell wall proteins were released by SDS extraction under reducing conditions and were designated Scw1-9p for (soluble cell wall proteins); five proteins were released from SDS-extracted cell walls by laminarinase (Ccw1-5p for covalently linked cell wall proteins) and six with mild (30 mM-NaOH, 4 degrees C, 14 h) alkali treatment (Ccw6-11p) . N-terminal sequences of the Ccw proteins 6, 7, 8 and 11 showed that these cell wall proteins are members of the PIR gene family (predicted proteins with internal repeats), CCW6 being identical to PIR1 and CCW8 to PIR3 . Single gene disruptions of all four genes did not yield a phenotype . In the CCW11 disruption the Ccw11p as well as the laminarinase-extracted Ccw5 protein was missing . The new cell wall proteins are O-mannosylated, contain a Kex2 processing site, but no C-terminal GPI anchor sequence.

Yeast, 1997 Sep 30, 13(12), 1135 - 44
Tandemly repeated 147 bp elements cause structural and functional variation in divergent MAL promoters of Saccharomyces cerevisiae; Bell PJ et al.; We have studied four novel MAL promoters isolated from a single strain of bakers' yeast . Within these promoters we have identified up to five tandem 147 bp repeats located between the MAL UAS region and the MALT TATA box . These repeats strongly reduce MALT (maltose permease) gene expression but only weakly reduce MALS (maltase) gene expression . Insertion of the 147 bp elements into the heterologous CYC1 promoter reduced expression when located between the CYC1 UAS and the TATA box, but not when located upstream of the UAS . We propose that these naturally occurring repeats have evolved as a mechanism to lower the level of MALT expression relative of MALS expression, thus avoiding possible toxic effects associated with over-expression from multiple copies of the permease gene.

Yeast, 1997 Sep 30, 13(12), 1099 - 133
A review of phenotypes in Saccharomyces cerevisiae; Hampsey M; A summary of previously defined phenotypes in the yeast Saccharomyces cerevisiae is presented . The purpose of this review is to provide a compendium of phenotypes that can be readily screened to identify pleiotropic phenotypes associated with primary or suppressor mutations . Many of these phenotypes provide a convenient alternative to the primary phenotype for following a gene, or as a marker for cloning a gene by genetic complementation . In many cases a particular phenotype or set of phenotypes can suggest a function for the product of the mutated gene.

Biochim Biophys Acta, 1997 Aug 15, 1341(1), 35 - 48
Identification of critical amino acid residues of Saccharomyces cerevisiae carbamoyl-phosphate synthetase: definition of the ATP site involved in carboxy-phosphate formation; Zheng W et al.; Carbamoyl-phosphate synthetases (CPSases) utilize two molecules of ATP at two homologous domains, B and C, with ATP(B) used to form the enzyme-bound intermediate carboxy-phosphate and ATP(C) used to phosphorylate the carbamate intermediate . To further define the role of one CPSase peptide suggested by affinity labeling studies to be near the ATP(B) site, we have carried out site-directed mutagenic analysis of peptide 234-242 of the Saccharomyces cerevisiae arginine-specific CPSase . Mutants E234A, E234D, E236A, E236D and E238A were unable to complement the CPSase-deficient yeast strain LPL26 whereas mutants Y237A, E238D, R241K, R241E and R241P supported LPL26 growth as well as wild-type CPSase . Kinetic analysis of E234A and Y237A indicated impaired utilization of ATP(B) but not of ATP(C) . D242A, a temperature-sensitive mutant, retained no detectable activity when assayed in vitro . These findings, together with the affinity labeling data and primary sequence analysis, strongly suggest that the yeast CPSase peptide 234-242 is located at the ATP(B) site and that some of its residues are important for functioning of the enzyme . D242 appears to occupy a critical structural position and E234, E236 and E238 appear to be critical for function, with the spatial arrangement of the carboxyl side chain also critical for E234 and E236.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 248 - 55
Overexpression of cytosolic malate dehydrogenase (MDH2) causes overproduction of specific organic acids in Saccharomyces cerevisiae; Pines O et al.; Saccharomyces cerevisiae accumulates L-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase . In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied . Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in L-malic acid accumulation in the production medium . The high apparent Km of MDH2 for L-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function in the enzyme and differs from the previously published Km of the mitochondrial enzyme (MDH1, 0.28 mM) . Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of L-malic acid production . The overexpression of MDH2 activity also causes an evaluation in the accumulation of fumaric acid and citric acid . Accumulation of fumaric acid is presumably caused by high intracellular L-malic acid concentrations and the activity of the cytosolic fumarase . The accumulation of citric acid may suggest the intriguing possibility that cytosolic L-malic acid is a direct precursor of citric acid in yeast.

Biochem Biophys Res Commun, 1997 Aug 28, 237(3), 562 - 5
Deregulation of the first N-glycosylation gene, ALG7, perturbs the expression of G1 cyclins and cell cycle arrest in Saccharomyces cerevisiae; Lennon K et al.; The evolutionarily conserved ALG7 gene encodes the dolichol-P-dependent N-acetylglucosamine-1-P transferase (GPT) and functions by initiating the dolichol pathway of protein N-glycosylation . In Saccharomyces cerevisiae, ALG7 has been shown to play a role in cell proliferation . The yeast alpha-factor-induced cell cycle arrest in G1 occurs, in part, by downregulation of CLN1 and CLN2 . The function of ALG7 in G1 arrest was examined in alg7 mutants containing diminished GPT activity . In wild type, CLN1 and CLN2 mRNAs were rapidly downregulated, while in alg7 mutants, these transcripts were only transiently repressed before becoming greatly augmented . Analyses of DNA contents and budding indices showed that alg7 mutants resumed cycling when wild type cells remained arrested . Thus, deregulation of ALG7 interferes with cell cycle arrest by preventing a sustained downregulation of CLN1 and CLN2 mRNAs . These results provide a molecular insight into the role of ALG7, and protein N-glycosylation in general, in proliferation.

J Mol Biol, 1997 Sep 12, 272(1), 1 - 8
The X-ray structure of the DNA-binding domain from the Saccharomyces cerevisiae cell-cycle transcription factor Mbp1 at 2.1 A resolution; Taylor IA et al.; The structure of the DNA-binding domain of the Saccharomyces cerevisiae cell-cycle transcription factor Mbp1 has been solved using the multiwavelength anomalous diffraction (MAD) technique on crystals of selenomethionyl protein and refined at 2.1 A resolution . The molecule is globular, consisting of a twisted, six-stranded beta-barrel that is packed against a loose bundle of four alpha-helices . Two of the beta-strands in combination with two of the helices form a structure characteristic of the DNA-binding motif found in the CAP family of helix-turn-helix transcription factors . In Mbp1, this beta2/alpha2 motif is associated with regions of both positive electrostatic potential and sequence conservation within the Mbp1/Swi4 family, suggesting a role in DNA-binding in these proteins . A combination of structural and biochemical data further indicate a similarity to HNF3gamma/fork head, a member of the family of "winged" helix-turn-helix proteins . We propose a model for DNA-binding involving a recognition helix in the major groove, phosphodiester backbone interactions through the beta-hairpin and further base and/or phosphate interactions mediated by a C-terminal, positively charged loop .

Electrophoresis, 1997 Aug, 18(8), 1429 - 40
Osmoresponsive proteins and functional assessment strategies in Saccharomyces cerevisiae; Blomberg A; Cells respond to increased external osmolarities by enhanced accumulation of compatible solutes . In yeast-cells, mainly exemplified by Saccharomyces cerevisiae, the premier compatible solute is the polyhydroxy-alcohol glycerol, the production of which is accompanied by overall metabolic changes . By applying two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled to computerized image quantification, a large body of valuable physiological information relating to this stress-adaptation has been gathered . One of the presumed key-enzymes in the production of glycerol in the cell is glycerol 3-phosphate dehydrogenase encoded by the GPD1 gene . The amount of this protein is enhanced during saline stress, and from 2-D analysis linked to microsequencing it became apparent that the osmo-regulated from contained a putative presequence . Sequence analysis of another salt-induced spot in the 2-D pattern revealed identity to a gene, YER062c, with previously unknown function . Biochemical characterization of this protein, including standard purification via chromatography and subsequent activity/specificity measurements, identified this salt-regulated protein as the missing protein/gene in glycerol production, namely the glycerol 3-phosphatase . The sequence of another salt regulated protein resolved in the 2-D gel revealed identity to a bacterial dihydroxyacetone kinase, thus indicating salt induced glycerol dissimilation . Comparing Northern data to the 2-D generated expression pattern revealed a strong correlation, indicating mainly regulation at the transcriptional level . In addition, altered expression during saline growth of some of the glycolytic enzymes was also apparent . Signalling mutants, either in the cAMP-dependent protein kinase A pathway or in a protein kinase cascade, have been analyzed during osmotic stress via 2-D PAGE, grouping proteins/genes apparently regulated via similar mechanismus . Proteome analysis has proven invaluable in the unravelling of the molecular physiology of yeast cells during adaptation and growth under osmotic stress, identifying vital components not selected by purely genetic approaches.

Electrophoresis, 1997 Aug, 18(8), 1361 - 72
Proteome analysis of Saccharomyces cerevisiae: a methodological outline; Fey SJ et al.; Proteome analysis offers a unique means of identifying important proteins, characterizing their modifications and beginning to describe their function . This is achieved through the combination of two technologies: protein separation and selection by two-dimensional gel electrophoresis, and protein identification and characterization by mass spectrometry . This methodological outline sketches the strengths and weaknesses of the two central technologies used, and provides both practical tips and the theoretical background for their utilization . One application of these technologies is illustrated by the characterization of genes, revealed by sequencing, but which have no--or only weak homology--to any other known genes . Other applications, for example the identification of protein markers for particular human diseases, are only referred to . The aim of the article is thus to provide the basis for a sound understanding of the full potential and limitations of proteome analysis.

J Biol Chem, 1997 Sep 19, 272(38), 24054 - 63
The activity of Cdc14p, an oligomeric dual specificity protein phosphatase from Saccharomyces cerevisiae, is required for cell cycle progression; Taylor GS et al.; The essential CDC14 gene of the budding yeast, Saccharomyces cerevisiae, encodes a 62-kDa protein containing a sequence that conforms to the active site motif found in all enzymes of the protein tyrosine phosphatase superfamily . Genetic studies suggest that Cdc14p may be involved in the initiation of DNA replication, but its precise cell cycle function is unknown . Recombinant Cdc14p was produced in bacteria, characterized, and shown to be a dual specificity protein phosphatase . Polyanions such as polyglutamate and double-stranded and single-stranded DNA bind to Cdc14p and affect its activity . Native molecular weights of 131,000 and 169,000 determined by two independent methods indicate that recombinant Cdc14p self-associates in vitro to form active oligomers . The catalytically inactive Cdc14p C283S/R289A mutant is not able to suppress the temperature sensitivity of a cdc14-1(ts) mutant nor replace the wild type gene in vivo, demonstrating that phosphatase activity is required for the cell cycle function of Cdc14p . A distinctive COOH-terminal segment (residues 375-551) is rich in Asn and Ser residues, carries a net positive charge, and contains two tandem 21-residue repeats . This COOH-terminal segment is not required for activity, for oligomerization, or for the critical cell cycle function of Cdc14p.

J Bacteriol, 1997 Sep, 179(18), 5971 - 4
SSU1 encodes a plasma membrane protein with a central role in a network of proteins conferring sulfite tolerance in Saccharomyces cerevisiae; Avram D et al.; The Saccharomyces cerevisiae SSU1 gene was isolated based on its ability to complement a mutation causing sensitivity to sulfite, a methionine intermediate . SSU1 encodes a deduced protein of 458 amino acids containing 9 or 10 membrane-spanning domains but has no significant similarity to other proteins in public databases . An Ssu1p-GEP fusion protein was localized to the plasma membrane . Multicopy suppression analysis, undertaken to explore relationships among genes previously implicated in sulfite metabolism, suggests a regulatory pathway in which SSU1 acts downstream of FZF1 and SSU3, which in turn act downstream of GRR1.

Curr Genet, 1997 Aug, 32(2), 93 - 9
Assessment of the essentiality of ERG genes late in ergosterol biosynthesis in Saccharomyces cerevisiae; Palermo LM et al.; Isogenic strains of yeast were constructed, differing only in insertionally inactivated genes for ergosterol biosynthesis . These and their allelic wild-types were grown in competition to ascertain growth differences and any selective advantage for organisms producing sterols with or without specific features of ergosterol . In every instance tested, the wild-type allele afforded a competitive advantage over the isogenic pair producing modified sterol structures instead of ergosterol . A general trend was seen in which the earlier in the biosynthetic pathway that a mutation occurred, the less able the strain producing the defective sterols could compete with the ergosterol-producing strains.

Mol Gen Genet, 1997 Aug, 255(5), 525 - 32
Cytoplasmic tyrosyl-tRNA synthetase rescues the defect in mitochondrial genome maintenance caused by the nuclear mutation mgm104-1 in the yeast Saccharomyces cerevisiae; Guan MX; The yeast nuclear mutation mgm104-1, which leads to slow growth on glucose medium and temperature-sensitive (ts) loss of mitochondrial DNA (mtDNA), has been identified by screening a collection of temperature-sensitive mutants on glycerol medium . A nuclear gene was isolated from a genomic DNA library by complementation of the mgm104-1 allele and was found to be identical to TTS1, which encodes the cytoplasmic tyrosyl-tRNA synthetase required for cytoplasmic protein synthesis . A gene disruption in a diploid strain demonstrated that the TTS1 gene is essential for cell viability . The lack of mutations in TTS1 in the mgm104-1 mutant indicates that TTS1 and MGM104 are different genes . The ability to rescue the mgm104-1 phenotype with a single additional copy of TTS1 suggests that TTS1 has an additional function that is directly or indirectly involved in the maintenance of the mitochondrial genome.

Appl Environ Microbiol, 1997 Sep, 63(9), 3341 - 4
Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae; Donald KA et al.; The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-R) is the major rate-limiting enzyme of the mevalonate pathway in many organisms, including yeasts . In the yeast Saccharomyces cerevisiae, there are two isoenzymes of HMG-R (Hmg1p and Hmg2p) . Both consist of an anchoring transmembrane domain and a catalytic domain . We have removed the known controlling features of HMG-R by overproducing the catalytic domain of Hmg1p . This overproduction leads to an enhancement of squalene production, implying that HMG-R has been deregulated . The enhancement is apparent under semianaerobic and aerobic conditions . Despite the increase in squalene production, the amount of ergosterol produced by the HMG-R-overproducing yeast was not increased . This result suggests the presence of another regulatory step between squalene and ergosterol formation . Squalene levels generated by cells overproducing the catalytic domain of HMG-R were estimated to be up to 10 times those produced by wild-type cells . The enhancement in squalene production coincided with a reduction in growth rate . This reduction may be a direct consequence of the buildup of high concentrations of squalene and presqualene intermediates of the pathway.

J Gen Virol, 1997 Sep, 78 ( Pt 9), 2315 - 8
A single dose immunization with rabbit haemorrhagic disease virus major capsid protein produced in Saccharomyces cerevisiae induces protection; Boga JA et al.; The gene coding for the major capsid protein (VP60) from rabbit haemorrhagic disease virus was expressed in Saccharomyces cerevisiae under the phosphoglycerate kinase promoter . The recombinant VP60 produced in yeast was antigenically similar to the viral polypeptide as determined with a polyclonal serum . Electron microscopic observation of the recombinant yeast-derived antigen revealed the presence of virus-like particles similar in size and appearance to native capsids . Subcutaneous vaccination of rabbits with a single dose of this antigen in the absence of commercial adjuvants conferred complete protection against the haemorrhagic disease.

Yeast, 1997 Sep 15, 13(11), 1053 - 8
Who's who among the Saccharomyces cerevisiae actin-related proteins? A classification and nomenclature proposal for a large family; Poch O et al.; Inspection of the complete Saccharomyces cerevisiae genome sequence and analysis of the actin-related proteins (ARPs) found therein revealed seven proteins, in addition to the previously designated actin-related proteins Arp1, Arp2 and Arp3, which contained substantial blocks of conservation relative to a chosen sub-set of actins . We have ordered the new ARPs relative to this group of actins and propose to name the more distantly related ARP members, according to their amino acid identity and similarity, Arp4-Arp10 . Most of these proteins appear to represent the first example of new classes of ARPs, each of which may have specific localization(s) and cellular function(s) . Recently reported ARPs from other species have also been included in the phylogenetic tree derived from the overall alignment of 29 actins and 28 ARPs.

Yeast, 1997 Sep 15, 13(11), 1029 - 42
Two new S-phase-specific genes from Saccharomyces cerevisiae; Le S et al.; Two new yeast genes, ASF1 (Anti-Silencing Function) and ASF2, as well as a C-terminal fragment of SIR3, were identified as genes that derepressed the silent mating type loci when overexpressed . ASF2 overexpression caused a greater derepression than did ASF1 . ASF1 overexpression also weakened repression of genes near telomeres, but, interestingly, ASF2 had no effect on telomeric silencing . Sequences of these two genes revealed open reading frames of 279 and 525 amino acids for ASF1 and ASF2, respectively . The ASF1 protein was evolutionarily conserved, MCB motifs, sequences commonly present upstream of genes transcribed specifically in S phase, were found in front of both genes, and, indeed, both genes were transcribed specifically in the S phase of the cell cycle . While an asf2 mutant was viable and had no obvious phenotypes, an asf1 mutant grew poorly . Neither mutant exhibited derepression of the silent mating type loci . The asf1 mutant was sensitive to methyl methane sulfonate, slightly UV-sensitive and somewhat deficient in minichromosome maintenance . It also lowered the restrictive temperature of a cdc13ts mutant . These phenotypes suggested a role for ASF1 in DNA repair and chromosome maintenance.

Yeast, 1997 Sep 15, 13(11), 1021 - 8
In vivo mutational analysis of highly conserved amino acid residues of the small subunit Cpa1p of the carbamylphosphate synthetase of Saccharomyces cerevisiae; Bernard A et al.; The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site-directed mutagenesis to change all residues to aspartic acid . Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo . The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate . These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site . Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS . C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mM) . The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mM, suggesting that this substitution is critical to NH3-dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme.

Yeast, 1997 Sep 15, 13(11), 999 - 1008
Vacuole segregation in the Saccharomyces cerevisiae vac2-1 mutant: structural and biochemical quantification of the segregation defect and formation of new vacuoles; Gomes De Mesquita DS et al.; The conditional vacuolar segregation mutant vac2-1 {Shaw and Wickner (1991) EMBO J . 10, 1741-1748} shifted to non-permissive temperature (37 degrees C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole . Some cells still contain normal segregation structures . Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37 degrees C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion . Thus, 10-15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell . To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping . The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37 degrees C . Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles . Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min . Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate . The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding.

Nucleic Acids Res, 1997 Sep 15, 25(18), 3570 - 9
Genetic evidence for selective degradation of RNA polymerase subunits by the 20S proteasome in Saccharomyces cerevisiae; Nouraini S et al.; scs32 was isolated as an extragenic suppressor of a temperature-sensitive (ts) mutation (rpo26-31) in the gene encoding Rpo26p, a subunit common to yeast nuclear RNA polymerases (RNAPs) . rpo26-31 also confers inositol auxotrophy, inhibits the assembly of RNAPI and RNAPII and reduces the steady-state level of Rpo26p and the largest subunit of RNAPI (Rpo11p or A190p) and RNAPII (Rpo21p) . rpo26-31p accumulated to wild-type levels in the scs32 strain; nevertheless, the amount of assembled RNAPII remained at a reduced level at high temperature . Hence, scs32 only partially suppressed the ts phenotype and was unable to suppress the Ino-phenotype of rpo26-31 . SCS32 is identical to PUP3, which encodes a subunit of the yeast proteasome . scs32 was able to suppress the phenotype of other ts alleles of RPO26, all of which reduce the steady-state level of this subunit . However, scs32 was unable to suppress the ts phenotype of mutant alleles of RPO21, or result in accumulation of the unstable rpo21-4p . These observations suggest that the stability of non-functional or unassembled forms of Rpo26p and Rpo21p are regulated independently.

J Biol Chem, 1997 Sep 12, 272(37), 23224 - 30
Saccharomyces cerevisiae basic region-leucine zipper protein regulatory networks converge at the ATR1 structural gene; Coleman ST et al.; Saccharomyces cerevisiae cells express a family of transcription factors belonging to the basic region-leucine zipper family . Two of these proteins, yAP-1 and Gcn4p, are known to be involved in oxidative stress tolerance and general control of amino acid biosynthesis, respectively . Strains lacking the YAP1 or GCN4 structural gene have very different phenotypes, which have been taken as evidence that these transcriptional regulatory proteins control separate batteries of target genes . In this study, we provide evidence that both yAP-1 and Gcn4p control the expression of a putative integral membrane protein, Atr1p . Both yAP-1 and Gcn4p can elevate resistance to 3-amino-1,2,4-triazole and 4-nitroquinoline-N-oxide but only if the ATR1 gene is intact . Expression of ATR1 is enhanced in the presence of constitutively active alleles of YAP1 and GCN4 . Regulation of ATR1 transcription by yAP-1 and Gcn4p occurs through a common DNA element related to the yAP-1 recognition element found upstream of other yAP-1-regulated genes . These data provide the first indication of overlap between the regulatory networks defined by yAP-1 and Gcn4p.

J Cell Biol, 1997 Sep 8, 138(5), 1023 - 40
Kinesin-related KIP3 of Saccharomyces cerevisiae is required for a distinct step in nuclear migration; DeZwaan TM et al.; Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes . Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis . The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells . Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase . Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position . The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant . The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase . Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement . This suggests that the lethality is due to the combination of sequential and possibly overlapping defects . Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus.

J Cell Biol, 1997 Sep 8, 138(5), 961 - 74
Osmotic balance regulates cell fusion during mating in Saccharomyces cerevisiae; Philips J et al.; Successful zygote formation during yeast mating requires cell fusion of the two haploid mating partners . To ensure that cells do not lyse as they remodel their cell wall, the fusion event is both temporally and spatially regulated: the cell wall is degraded only after cell-cell contact and only in the region of cell-cell contact . To understand how cell fusion is regulated, we identified mutants defective in cell fusion based upon their defect in mating to a fus1 fus2 strain (Chenevert, J., N . Valtz, and I . Herskowitz . 1994 . Genetics 136:1287-1297) . Two of these cell fusion mutants are defective in the FPS1 gene, which codes for a glycerol facilitator (Luyten, K., J . Albertyn, W.F . Skibbe, B.A . Prior, J . Ramos, J.M . Thevelein, and S . Hohmann . 1995 . EMBO {Eur . Mol . Biol . Organ.} J . 14:1360-1371) . To determine whether inability to maintain osmotic balance accounts for the defect in cell fusion in these mutants, we analyzed the behavior of an fps1Delta mutant with reduced intracellular glycerol levels because of a defect in the glycerol-3-phosphate dehydrogenase (GPD1) gene (Albertyn, J., S . Hohmann, J.M . Thevelein, and B.A . Prior . 1994 . Mol . Cell . Biol . 14:4135-4144): deletion of GPD1 partially suppressed the cell fusion defect of fps1 mutants . In contrast, overexpression of GPD1 exacerbated the defect . The fusion defect could also be partially suppressed by 1 M sorbitol . These observations indicate that the fusion defect of fps1 mutants results from inability to regulate osmotic balance and provide evidence that the osmotic state of the cell can regulate fusion . We have also observed that mutants expressing hyperactive protein kinase C exhibit a cell fusion defect similar to that of fps1 mutants . We propose that Pkc1p regulates cell fusion in response to osmotic disequilibrium . Unlike fps1 mutants, fus1 and fus2 mutants are not influenced by expression of GPD1 or by 1 M sorbitol . Their fusion defect is thus unlikely to result from altered osmotic balance.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9768 - 72
Changes of telomere length cause reciprocal changes in the lifespan of mother cells in Saccharomyces cerevisiae; Austriaco NR Jr et al.; Budding yeast cells divide asymmetrically, giving rise to a mother and its daughter . Mother cells have a limited division potential, called their lifespan, which ends in proliferation-arrest and lysis . In this report we mutate telomerase in Saccharomyces cerevisiae to shorten telomeres and show that, rather than shortening lifespan, this leads to a significant extension in lifespan . This extension requires the product of the SIR3 gene, an essential component of the silencing machinery which binds to telomeres . In contrast, longer telomeres in a genotypically wild-type strain lead to a decrease in lifespan . These findings suggest that the length of telomeres dictates the lifespan by regulating the amount of the silencing machinery available to nontelomeric locations in the yeast genome.

J Bacteriol, 1997 Sep, 179(17), 5609 - 13
Nitrogen GATA factors participate in transcriptional regulation of vacuolar protease genes in Saccharomyces cerevisiae; Coffman JA et al.; The expression of most nitrogen catabolic genes in Saccharomyces cerevisiae is regulated at the level of transcription in response to the quality of nitrogen source available . This regulation is accomplished through four GATA-family transcription factors: two positively acting factors capable of transcriptional activation (Gln3p and Gat1p) and two negatively acting factors capable of down-regulating Gln3p- and/or Gat1p-dependent transcription (Dal80p and Deh1p) . Current understanding of nitrogen-responsive transcriptional regulation is the result of extensive analysis of genes required for the catabolism of small molecules, e.g., amino acids, allantoin, or ammonia . However, cells contain another, equally important source of nitrogen, intracellular protein, which undergoes rapid turnover during special circumstances such as entry into stationary phase, and during sporulation . Here we show that the expression of some (CPS1, PEP4, PRB1, and LAP4) but not all (PRC1) vacuolar protease genes is nitrogen catabolite repression sensitive and is regulated by the GATA-family proteins Gln3p, Gat1p, and Dal80p . These observations extend the global participation of GATA-family transcription factors to include not only well-studied genes associated with the catabolism of small nitrogenous compounds but also genes whose products are responsible for the turnover of intracellular macromolecules . They also point to the usefulness of considering control of the nitrogen-responsive GATA factors when studying the regulation of the protein turnover machinery.

Genetics, 1997 Sep, 147(1), 33 - 42
Genetic interactions between HOP1, RED1 and MEK1 suggest that MEK1 regulates assembly of axial element components during meiosis in the yeast Saccharomyces cerevisiae; Hollingsworth NM et al.; During meiosis, axial elements are generated by the condensation of sister chromatids along a protein core as precursors to the formation of the synaptonemal complex (SC) . Functional axial elements are essential for wild-type levels of recombination and proper reductional segregation at meiosis I . Genetic and cytological data suggest that three meiosis-specific genes, HOP1, RED1 and MEK1, are involved in axial element formation in the yeast Saccharomyces cerevisiae . HOP1 and RED1 encode structural components of axial elements while MEK1 encodes a putative protein kinase . Using a partially functional allele of MEK1, new genetic interactions have been found between HOP1, RED1 and MEK1 . Overexpression of HOP1 partially suppresses the spore inviability and recombination defects of mek1-974; in contrast, overexpression of RED1 exacerbates the mek1-974 spore inviability . Co-overexpression of HOP1 and RED1 in mek1-974 diploids alleviates the negative effect of overexpressing RED1 alone . Red1p/Red1p as well as Hop1p/Red1p interactions have been reconstituted in two hybrid experiments . Our results suggest a model whereby Mek1 kinase activity controls axial element assembly by regulating the affinity with which Hop1p and Red1p interact with each other.

Genetics, 1997 Sep, 147(1), 19 - 32
Mating in Saccharomyces cerevisiae: the role of the pheromone signal transduction pathway in the chemotropic response to pheromone; Schrick K et al.; The mating process in yeast has two distinct aspects . One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell . To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation . Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism . In contrast, cells mutant for the receptor (ste2) or the beta or gamma subunit (ste4 and ste18) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.

Curr Biol, 1997 Sep 1, 7(9), 701 - 4
Imaging green fluorescent protein fusion proteins in Saccharomyces cerevisiae; Shaw SL et al.; Tagging expressed proteins with the green fluorescent protein (GFP) from Aequorea victoria {1} is a highly specific and sensitive technique for studying the intracellular dynamics of proteins and organelles . We have developed, as a probe, a fusion protein of the carboxyl terminus of dynein and GFP (dynein-GFP), which fluorescently labels the astral microtubules of the budding yeast Saccharomyces cerevisiae . This paper describes the modifications to our multimode microscope imaging system {2,3}, the acquisition of three-dimensional (3-D) data sets and the computer processing methods we have developed to obtain time-lapse recordings of fluorescent astral microtubule dynamics and nuclear movements over the complete duration of the 90-120 minute yeast cell cycle . This required low excitation light intensity to prevent GFP photobleaching and phototoxicity, efficient light collection by the microscope optics, a cooled charge-coupled device (CCD) camera with high quantum efficiency, and image reconstruction from serial optical sections through the 6 micron-wide yeast cell to see most or all of the astral molecules . Methods are also described for combining fluorescent images of the microtubules labeled with dynein-GFP with high resolution differential interference contrast (DIC) images of nuclear and cellular morphology {4}, and fluorescent images of the chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI) {5}.

Mol Cell Biol, 1997 Sep, 17(9), 5146 - 55
SQT1, which encodes an essential WD domain protein of Saccharomyces cerevisiae, suppresses dominant-negative mutations of the ribosomal protein gene QSR1; Eisinger DP et al.; QSR1 is an essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein required for joining of 40S and 60S subunits . Truncations of QSR1 predicted to encode C-terminally truncated forms of Qsr1p do not substitute for QSR1 but do act as dominant negative mutations, inhibiting the growth of yeast when expressed from an inducible promoter . The dominant negative mutants exhibit a polysome profile characterized by 'half-mer' polysomes, indicative of a subunit joining defect like that seen in other qsr1 mutants (D . P . Eisinger, F . A . Dick, and B . L . Trumpower, Mol . Cell . Biol . 17:5136-5145, 1997.) By screening a high-copy yeast genomic library, we isolated several clones containing overlapping inserts of a novel gene that rescues the slow-growth phenotype of the dominant negative qsr1 truncations . The suppressor of qsr1 truncation mutants, SQT1, is an essential gene, which encodes a 47.1-kDa protein containing multiple WD repeats and which interacts strongly with Qsr1p in a yeast two-hybrid system . SQT1 restores growth and the "half-mer" polysome profile of the dominant negative qsr1 mutants to normal, but it does not rescue temperature-sensitive qsr1 mutants or the original qsr1-1 missense allele . In yeast cell lysates, Sqt1p fractionates as part of an oligomeric protein complex that is loosely associated with ribosomes but is distinct from known eukaryotic initiation factor complexes . Loss of SQT1 function by down regulation from an inducible promoter results in formation of half-mer polyribosomes and decreased Qsr1p levels on free 60S subunits . Sqt1p thus appears to be involved in a late step of 60S subunit assembly or modification in the cytoplasm.

Mol Cell Biol, 1997 Sep, 17(9), 5067 - 76
Cla4p, a Saccharomyces cerevisiae Cdc42p-activated kinase involved in cytokinesis, is activated at mitosis; Benton BK et al.; Yeasts have three functionally redundant G1 cyclins required for cell cycle progression through G1 . Mutations in GIN4 and CLA4 were isolated in a screen for mutants that are inviable with deletions in the G1 cyclins CLN1 and CLN2 . cln1 cln2 cla4 and cln1 cln2 gin4 cells arrest with a cytokinesis defect; this defect was efficiently rescued by CLN1 or CLN2 expression . GIN4 encodes a protein with strong homology to the Snflp serine/threonine kinase . Cla4p is homologous to mammalian p21-activated kinases (PAKs) (kinases activated by the rho-class GTPase Rac or Cdc42) . We developed a kinase assay for Cla4p . Cla4p kinase was activated in vivo by the GTP-bound form of Cdc42p . The specific activity of Cla4p was cell cycle regulated, peaking near mitosis . Deletion of the Cla4p pleckstrin domain diminished kinase activity nearly threefold and eliminated in vivo activity . Deletion of the Cla4p Cdc42-binding domain increased kinase activity nearly threefold, but the mutant only weakly rescued cla4 function in vivo . This suggests that kinase activity alone is not sufficient for full function in vivo . Deletion of the Cdc42-binding domain also altered the cell cycle regulation of kinase activity . Instead of peaking at mitosis, the mutant kinase activity exhibited reduced cell cycle regulation and peaked at the G1/S border . Cla4p kinase activity was not reduced by mutational inactivation of gin4, suggesting that Gin4p may be downstream or parallel to Cla4p in the regulation of cytokinesis.

Mol Cell Biol, 1997 Sep, 17(9), 5044 - 52
Zap1p, a metalloregulatory protein involved in zinc-responsive transcriptional regulation in Saccharomyces cerevisiae; Zhao H et al.; Zinc ion homeostasis in Saccharomyces cerevisiae is controlled primarily through the transcriptional regulation of zinc uptake systems in response to intracellular zinc levels . A high-affinity uptake system is encoded by the ZRT1 gene, and its expression is induced more than 30-fold in zinc-limited cells . A low-affinity transporter is encoded by the ZRT2 gene, and this system is also regulated by zinc . We used a genetic approach to isolate mutants whose ZRT1 expression is no longer repressed in zinc-replete cells, and a new gene, ZAP1, was identified . ZAP1 encodes a 93-kDa protein with sequence similarity to transcriptional activators; the C-terminal 174 amino acids contains five C2H2 zinc finger domains, and the N terminus (residues 1 to 706) has two potential acidic activation domains . The N-terminal region also contains 12% histidine and cysteine residues . The mutant allele isolated, ZAP1-1up, is semidominant and caused high-level expression of ZRT1 and ZRT2 in both zinc-limited and zinc-replete cells . This phenotype is the result of a mutation that substitutes a serine for a cysteine residue in the N-terminal region . A zap1 deletion mutant grew well on zinc-replete media but poorly on zinc-limiting media . This mutant had low-level ZRT1 and ZRT2 expression in zinc-limited as well as zinc-replete cells . These data indicate that Zap1p plays a central role in zinc ion homeostasis by regulating transcription of the zinc uptake system genes in response to zinc . Finally, we present evidence that Zap1p regulates transcription of its own promoter in response to zinc through a positive autoregulatory mechanism.

Mol Cell Biol, 1997 Sep, 17(9), 5001 - 15
Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis; Zanchin NI et al.; The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes . The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect . NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved . Gene disruption analysis demonstrated that NIP7 is essential for growth . A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein . Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus . Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth . Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA . Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

J Biol Chem, 1997 Aug 29, 272(35), 22140 - 7
The Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is encoded by the oleate-inducible gene SPS19; Gurvitz A et al.; beta-Oxidation is compartmentalized in mammals into both mitochondria and peroxisomes . Fatty acids with double bonds at even-numbered positions require for their degradation the auxiliary enzyme 2,4-dienoyl-CoA reductase, and at least three isoforms, two mitochondrial and one peroxisomal, exist in the rat . The Saccharomyces cerevisiae Sps19p is 34% similar to the human and rat mitochondrial reductases, and an SPS19 deleted strain was unable to utilize petroselineate (cis-C18:1(6)) as the sole carbon source, but remained viable on oleate (cis-C18:1(9)) . Sps19p was purified to homogeneity from oleate-induced cells and the homodimeric enzyme (native molecular weight 69,000) converted 2,4-hexadienoyl-CoA into 3-hexenoyl-CoA in an NADPH-dependent manner and therefore contained 2,4-dienoyl-CoA reductase activity . Antibodies raised against Sps19p decorated the peroxisomal matrix of oleate-induced cells . SPS19 shares with the sporulation-specific SPS18 a common promoter region that contains an oleate response element . This element unidirectionally regulates transcription of the reductase and is sufficient for oleate induction of a promoterless CYC1-lacZ reporter gene . SPS19 is dispensable for growth and sporulation on solid acetate and oleate media, but is essential for these processes to occur on petroselineate.

Nature, 1997 Aug 28, 388(6645), 900 - 3
Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae; Tsukamoto Y et al.; DNA double-strand breaks are repaired by homologous recombination or DNA end-joining, but the latter process often causes legitimate recombination and chromosome rearrangements . One of the factors involved in the end-joining process is Hdf1, a yeast homologue of Ku protein . We used the yeast two-hybrid assay to show that Hdf1 interacts with Sir4, which is involved in transcriptional silencing at telomeres and HM loci . Analyses of sir4 mutants showed that Sir4 is required for deletion by illegitimate recombination and DNA end-joining in the pathway involving Hdf1 . Sir2 and Sir3, but not Sir1, were also found to participate in these processes . Furthermore, mutations of the SIR2, SIR3 and SIR4 genes conferred increased sensitivity to gamma-radiation in a genetic background with a mutation of the RAD52 gene, which is essential for double-strand break repair mediated by homologous recombination . These results indicate that Sir proteins are involved in double-strand break repair mediated by end-joining . We propose that Sir proteins act with Hdf1 to alter broken DNA ends to create an inactivated chromatin structure that is essential for the rejoining of DNA ends.

J Biol Chem, 1997 Aug 22, 272(34), 21509 - 19
Identification and phenotypic analysis of two glyoxalase II encoding genes from Saccharomyces cerevisiae, GLO2 and GLO4, and intracellular localization of the corresponding proteins; Bito A et al.; We have isolated and characterized two genes coding for the glyoxalase II enzyme from Saccharomyces cerevisiae . The coding region of the GLO2 gene corresponds to a protein with 274 amino acids and a molecular mass of 31,306 daltons . The open reading frame of the GLO4 gene could be translated into a protein with 285 amino acids and a molecular mass of 32,325 daltons . The amino acid sequences of the deduced proteins are 59.1% identical and show high similarities to the sequence of the human glyoxalase II . When grown on either glucose or glycerol as a carbon source, a glo2 glo4 double deletion strain contains no glyoxalase II activity at all and shows no obvious phenotype during vegetative growth . However, this strain showed a similar high sensitivity against exogenous methylglyoxal as compared with a glyoxalase I-deficient strain . Whereas the GLO2 gene is expressed on both glucose and glycerol, the GLO4 gene is only active on glycerol . The active Glo2p protein is localized in the cytoplasm and the active Glo4p in the mitochondrial matrix . Heterologous expression of the full-length GLO2 coding region in Escherichia coli resulted in an active protein . However, to get an active Glo4p protein in E . coli, the putative mitochondrial transit peptide at the N-terminal end had to be removed by shortening the 5' end of the GLO4 open reading frame.

J Biol Chem, 1997 Aug 22, 272(34), 21461 - 6
Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae; Hung IH et al.; Wilson disease is a disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration due to inherited mutations in a gene encoding a putative copper-transporting P-type ATPase . Polyclonal antisera generated against the amino terminus of the Wilson protein detected a specific 165-kDa protein in HepG2 and CaCo cell lysates . Further analysis revealed that this protein is synthesized as a single-chain polypeptide and localized to the trans-Golgi network under steady state conditions . An increase in the copper concentration resulted in the rapid movement of this protein to a cytoplasmic vesicular compartment . This copper-specific cellular redistribution of the Wilson protein is a reversible process that occurs independent of a new protein synthesis . Expression of the wild-type but not mutant Wilson protein in the ccc2Delta strain of Saccharomyces cerevisiae restored copper incorporation into the multicopper oxidase Fet3p, providing direct evidence of copper transport by the Wilson protein . Taken together these data reveal a remarkable evolutionary conservation in the cellular mechanisms of copper metabolism and provide a unique model for the regulation of copper transport into the secretory pathway of eucaryotic cells.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9214 - 9
Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination; Sugawara N et al.; When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated . In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp . These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over . A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles . When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required . Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required . We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3' tail.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9141 - 6
Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene; Middendorp S et al.; Among the numerous centrin isoforms identified by two-dimensional gel electrophoresis in human cells, an acidic and slow-migrating isoform is particularly enriched in a centrosome fraction . We report here that this isoform specifically reacts with antibodies raised against Saccharomyces cerevisiae Cdc31p and is present, as other centrin isoforms, in the distal lumen of centrioles . It is encoded by a new centrin gene, which we propose to name HsCEN3 (Homo sapiens centrin gene 3) . This gene is more closely related to the yeast CDC31 gene, and shares less identity with algae centrin than HsCEN1 and HsCEN2 . A murine CDC31-related gene was also found that shows 98% identity and 100% similarity with HsCEN3, demonstrating a higher interspecies conservation than the murine centrin gene MmCEN1 (Mus musculus centrin gene 1) with either HsCEN1, or HsCEN2 . Finally, immunological data suggest that a CDC31-related gene could exist in amphibians and echinoderms as well . All together, our data suggest the existence of two divergent protein subfamilies in the current centrin family, which might be involved in distinct centrosome-associated functions . The possible implication of this new mammalian centrin gene in centrosome duplication is discussed.

FEBS Lett, 1997 Aug 18, 413(2), 226 - 30
Isolation and characterization of mutations affecting expression of the delta9- fatty acid desaturase gene, OLE1, in Saccharomyces cerevisiae; Fujimori K et al.; Expression of the delta9- fatty acid desaturase gene, OLE1, of Saccharomyces cerevisiae is negatively regulated transcriptionally and post-transcriptionally by unsaturated fatty acids . In order to isolate mutants exhibiting irregulation of OLE1 expression, we constructed an OLE1p-PHO5 fusion gene as a reporter consisting of the PHO5 gene encoding repressible acid phosphatase (rAPase) under the control of the OLE1 promoter (OLE1p) . By EMS mutagenesis, we isolated three classes of mutants, pfo1, pfo2 and pfo3 positive regulatory factor for OLE1) mutants, which show decreased rAPase activity under derepression conditions (absence of oleic acid) . Analysis of the transcription of OLE1 in these pfo mutants revealed that pfo1 and pfo3 mutants have a defect in the regulation of OLE1 expression at the transcriptional level while pfo2 mutants were suggested to have a mutation affecting OLE1 expression at a post-transcriptional step . In addition, four other classes of mutants, nfo1, nfo2, nfo3 and nfo4 (negative factor for OLE1) mutants that have mutations causing strong expression of the OLE1p-PHO5 fusion gene under repression conditions (presence of oleic acid), were isolated . Results of Northern analysis of OLE1 as well as OLE1p-PHO5 transcripts in nfo mutants suggested that these mutations occurred in genes encoding global repressors . We also demonstrated that TUP1 and SSN6 gene products are required for full repression of OLE1 gene expression, by showing that either tup1 or ssn6 mutations greatly increase the level of the OLE1 transcript.

Nucleic Acids Res, 1997 Aug 15, 25(16), 3204 - 11
The Ogg1 protein of Saccharomyces cerevisiae: a 7,8-dihydro-8-oxoguanine DNA glycosylase/AP lyase whose lysine 241 is a critical residue for catalytic activity; Girard PM et al.; The OGG1 gene of Saccharomyces cerevisiae codes for a DNA glycosylase that excises 7,8-dihydro-8- oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Fapy) from damaged DNA . In this paper, we have analysed the substrate specificity and the catalytic mechanism of the Ogg1 protein acting on DNA subtrates containing 8-OxoG residues or apurinic/apyrimidinic (AP) sites . The Ogg1 protein displays a marked preference for DNA duplexes containing 8-OxoG placed opposite a cytosine, the rank order for excision of 8-OxoG and cleavage efficiencies being 8-OxoG/C >8-OxoG/T >>8-OxoG/G and 8-OxoG/A . The cleavage of the DNA strand implies the excision of 8-OxoG followed by abeta-elimination reaction at the 3'-side of the resulting AP site . The Ogg1 protein efficiently cleaves a DNA duplex where a preformed AP site is placed opposite a cytosine (AP/C) . In contrast, AP/T, AP/A or AP/G substrates are incised with a very low efficiency . Furthermore, cleavage of 8-OxoG/C or AP/C substrates implies the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre (NaBH4) . Therefore, the Ogg1 protein is a eukaryotic DNA glycosylase/AP lyase . Sequence homology searches reveal that Ogg1 probably shares a common ancestor gene with the endonuclease III of Escherichia coli . A consensus sequence indicates a highly conserved lysine residue, K120 of endonuclease III or K241 of Ogg1, respectively . Mutations of K241 to Gln (K241Q) and Arg (K241R) have been obtained after site directed mutagenesis of OGG1 . Mutation K241Q completely abolishes DNA glycosylase activity and covalent complex formation in the presence of NaBH4 . However, the K241Q mutant still binds DNA duplexes containing 8-OxoG/C . In contrast, K241R mutation results in a catalytically active form of Ogg1 . These results strongly suggest that the free amino group of Lys241 is involved in the catalytic mechanism of the Ogg1 protein.

FEBS Lett, 1997 Aug 4, 412(3), 615 - 20
Neutral trehalase Nth1p of Saccharomyces cerevisiae encoded by the NTH1 gene is a multiple stress responsive protein; Zahringer H et al.; We have shown previously that expression of the NTH1 gene is increased at heat stress (40 degrees C) both at the mRNA and enzymatic activity levels . This increased expression was correlated to the requirement of the NTH1 gene for recovery after heat shock at 50 degrees C and the presence of stress responsive elements STRE (CCCCT) 3 times in its promoter region {S . Nwaka et al., FEBS Lett . 360 (1995) 286-290; S . Nwaka et al., J . Biol . Chem . 270 (1995) 10193-10198} . We show here that expression of the NTH1 gene and its product, neutral trehalase (Nthlp), are also induced by other stressors such as H2O2, CuSO4, NaAsO2, and cycloheximide (CHX) . Heat-induced expression of the NTH1 gene is shown to be accompanied by accumulation of trehalose . In contrast, the chemical stressors which also induce the expression of NTH1 did not lead to accumulation of trehalose under similar conditions . Our data suggest that: (1) heat- and chemical stress-induced expression of neutral trehalase is largely due to de novo protein synthesis, and (2) different mechanisms may control the heat- and chemical stress-induced expression of NTH1 at the transcriptional level . Participation of neutral trehalase (Nth1p) in multiple stress response dependent and independent on trehalose is discussed.

FEBS Lett, 1997 Aug 4, 412(3), 547 - 50
Substrate specificity of alpha-1,6-mannosyltransferase that initiates N-linked mannose outer chain elongation in Saccharomyces cerevisiae; Nakayama K et al.; Yeast Saccharomyces cerevisiae OCH1 gene encodes the mannosyltransferase that is essential for the outer chain elongation of N-linked oligosaccharides . Mannosyltransferase activity of OCH1 gene product (Och1p) was measured on HPLC by using pyridylaminated Man8GlcNAc2 (Man8GlcNAc2-PA) as an acceptor and the reaction product was observed at the retention time corresponding to Man9GlcNAc2-PA . 1H-NMR and fast atom bombardment mass spectrometry (FAB-MS) fragmentation analysis of Man9GlcNAc2-PA showed that the additional mannose was attached with an alpha-1,6 linkage at the site where mannose outer chain elongation initiates . Substrate specificity of Och1p was investigated by using various high mannose-type oligosaccharides as acceptors . Man8GlcNAc2 was the best acceptor for Och1p . The loss of one or two alpha-1,2-mannoses from Man8GlcNAc2 reduced the mannosyltransferase activity and the Man5GlcNAc2 completely lacking alpha-1,2-mannose residues did not serve as an acceptor . Man8GlcNAcOH that involves an open sugar ring by reduction of reducing terminal GlcNAc residue did not serve as an acceptor for Och1p . The loss of three mannoses at the alpha-1,6-branch also reduced the Och1p activity . These results suggest that Och1p is an initiation specific alpha-1,6-mannosyltransferase that requires the intact structure of Man8GlcNAc for efficient mannose outer chain initiation.

FEBS Lett, 1997 Aug 4, 412(3), 531 - 4
Expression of PEP carboxylase from Escherichia coli complements the phenotypic effects of pyruvate carboxylase mutations in Saccharomyces cerevisiae; Flores CL et al.; We investigated the effects of the expression of the Escherichia coli ppc gene encoding PEP carboxylase in Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase . Functional expression of the ppc gene restored the ability of the yeast mutants to grow in glucose-ammonium medium . Growth yield in this medium was the same in the transformed yeast than in the wild type although the growth rate of the transformed yeast was slower . Growth in pyruvate was slowed down in the transformed strain, likely due to a futile cycle produced by the simultaneous action of PEP carboxykinase and PEP carboxylase.

Eur J Biochem, 1997 Aug 1, 247(3), 972 - 80
Cytosolic aspartate aminotransferase encoded by the AAT2 gene is targeted to the peroxisomes in oleate-grown Saccharomyces cerevisiae; Verleur N et al.; Fatty acid beta-oxidation in peroxisomes requires the continued uptake of fatty acids or their derivatives into peroxisomes and export of beta-oxidation products plus oxidation of NADH to NAD . In an earlier study we provided evidence for the existence of an NAD(H) redox shuttle in which peroxisomal malate dehydrogenase plays a pivotal role . In analogy to the NAD(H)-redox-shuttle systems in mitochondria we have investigated whether a malate/aspartate shuttle is operative in peroxisomes . The results described in this paper show that peroxisomes of oleate-grown Saccharomyces cerevisiae contain aspartate aminotransferase (AAT) activity . Whereas virtually all cellular AAT activity was peroxisomal in oleate-grown cells, we found that in glucose-grown cells most of the AAT activity resided in the cytosol . We demonstrate that the gene AAT2 codes for the cytosolic and peroxisomal AAT activities . Disruption of the AAT2 gene did not affect growth on oleate . Furthermore beta-oxidation of palmitate was normal . These results indicate that AAT2 is not essential for the peroxisomal NAD(H) redox shuttle.

Eur J Biochem, 1997 Aug 1, 247(3), 776 - 83
A heterodimer of the Zn2Cys6 transcription factors Pip2p and Oaf1p controls induction of genes encoding peroxisomal proteins in Saccharomyces cerevisiae; Rottensteiner H et al.; In the yeast Saccharomyces cerevisiae, two transcriptional activators belonging to the Zn2Cys6 protein family, Pip2p and Oaf1p, are involved in fatty-acid-dependent induction of genes encoding peroxisomal proteins . This induction is mediated via an upstream activation sequence called the oleate-response element (ORE) . DNA-bandshift experiments with ORE probes and epitope-tagged proteins showed that two binary complexes occurred: in wild-type cells the major complex consisted of a Pip2p x Oaf1p heterodimer, but in cells in which Oaf1p was overexpressed an Oaf1p homodimer was also observed . The genes encoding Oaf1p and Pip2p were controlled in different ways . The OAF1 gene was constitutively expressed, while the PIP2 gene was induced upon growth on oleate, giving rise to positive autoregulatory control . We have shown that the Pip2p x Oaf1p heterodimer is responsible for the strong expression of the genes encoding peroxisomal proteins upon growth on oleate . Pip2p and Oaf1p form an example of a heterodimere of yeast Zn2Cys6 zinc-finger proteins binding to DNA.

Genes Dev, 1997 Aug 1, 11(15), 1912 - 24
A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks; Schar P et al.; Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination . Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast . Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product . This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4 . Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro . Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells . In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation . Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I . Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores . The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis.

Yeast, 1997 Aug, 13(10), 891 - 902
The Saccharomyces cerevisiae MFS superfamily SGE1 gene confers resistance to cationic dyes; Jacquot C et al.; A gene from Saccharomyces cerevisiae whose overexpression confers resistance to 10-N-nonyl acridine orange (NAO) has been isolated . This cationic dye binds acidic phospholipids and more specifically cardiolipin (Petit, J . M., Maftah, A., Ratinaud, M . H . and Julien, R . Eur . J . Biochem . 209, 267-273, 1992) . The isolated gene was found to be identical to SGE1, a partial multicopy suppressor of the gal11 mutation (Amakasu, H., Suzuki, Y., Nishizawa, M . and Fukasawa, T . Genetics 134, 675-683, 1993), that also confers crystal violet resistance to a supersensitive strain (Ehrenhofer-Murray, A . E., Wurgler, F . E . and Sengstag, C . Mol . Gen . Genet . 244, 287-294, 1994) . The data presented in this paper show that the SGE1 gene product, a member of the major facilitator superfamily, confers a pleiotropic drug-resistance phenotype when present in high copy number . The results also demonstrate that Sge1p acts as an extrusion permease whose specificity seems restricted to dye molecules possessing a large unsaturated domain that stabilizes a permanent positive charge such as NAO, crystal violet, ethidium bromide or malachite green.

Protein Expr Purif, 1997 Aug, 10(3), 340 - 4
Purification of alcohol dehydrogenase from Entamoeba histolytica and Saccharomyces cerevisiae using zinc-affinity chromatography; Cabrera N et al.; We have developed a single-step method for the purification of NADP(+)-dependent alcohol dehydrogenase from Entamoeba histolytica and NAD(+)-dependent alcohol dehydrogenase from Saccharomyces cerevisiae . It is based on the affinity for zinc of both enzymes . The amebic enzyme was purified almost 800 times with a recovery of 54% and the yeast enzyme was purified 30 times with a recovery of 100% . The kinetic constants of the purified enzymes were similar to those reported for other purification methods . With mammalian alcohol dehydrogenase, we obtained a 40-kDa band suggestive of purified alcohol dehydrogenase, but we failed to retain enzymatic activity in this preparation . Our results suggest that the described method is more applicable to the purification of tetrameric alcohol dehydrogenases.

J Gen Virol, 1997 Aug, 78 ( Pt 8), 1829 - 32
Immunogenic, non-infectious polio subviral particles synthesized in Saccharomyces cerevisiae; Rombaut B et al.; Polioviral genes coding for P1, the precursor for the structural proteins, and 3CD, the viral protease, were cloned in a Saccharomyces cerevisiae inducible expression system . N-antigenic empty capsids could be isolated from the yeast cell extract provided that pirodavir, a capsid-binding compound and capsid stabilizer, was added during the induction period and during purification . Purification was by immunoaffinity chromatography . The purified empty capsids had the same immunogenicity as poliovirus virions . The techniques described might be useful for the production of new non-infectious vaccines.

Protein Sci, 1997 Aug, 6(8), 1774 - 6
Crystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X-ray characterization of the crystals; Erskine PT et al.; 5-Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid . Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo-octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions . The E . coli enzyme crystallized in space group I422 (unit cell dimensions a = b = 130.7 A, c = 142.4 A) . The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid . The yeast enzyme (expressed in E . coli) crystallized in the same space group (I422) but with a smaller unit cell volume (a = b = 103.7 A, c = 167.7 A) . High resolution synchrotron data sets were obtained from both E . coli and yeast ALAD crystals by cryocooling to 100 K.

Genetics, 1997 Aug, 146(4), 1287 - 98
Constitutive mutations of the Saccharomyces cerevisiae MAL-activator genes MAL23, MAL43, MAL63, and mal64; Gibson AW et al.; We report the sequence of several MAL-activator genes, including inducible, constitutive, and noninducible alleles of MAL23, MAL43, MAL63, and mal64 . Constitutive alleles of MAL23 and MAL43 vary considerably from inducible alleles in their C-terminal domain, with many of the alterations clustered and common to both alleles . The 27 alterations from residues 238-461 of Mal43-C protein are sufficient for constitutivity, but the minimal number of alterations needed for the constitutive phenotype could not be determined . The sequence of mal64, a nonfunctional homologue of MAL63, revealed that Mal64p is 85% identical to Mal63p . Two mutations that activate mal64 and cause constitutivity are nonsense mutations resulting in truncated proteins of 306 and 282 residues . We conclude that the C-terminal region of the MAL-activator, from residues 283-470, contains a maltose-responsive negative regulatory domain, and that extensive mutation or deletion of the entire region causes loss of the negative regulatory function . Additionally, certain sequence elements in the region appear to be necessary for efficient induction of the full-length Mal63 activator protein . These studies highlight the role of ectopic recombination as an important mechanism of mutagenesis of the telomere-associated family of MAL loci.

FEMS Microbiol Lett, 1997 Aug 1, 153(1), 233 - 6
Block of the cell cycle of the yeast Saccharomyces cerevisiae by tyrphostin, an inhibitor of protein tyrosine kinase; Fujimura H; Tyrphostins are inhibitors of the epidermal growth factor receptor tyrosine kinase . To elucidate the biological function of protein tyrosine kinases in yeast cells, a mutant hypersensitive to tyrphostin was isolated and investigated for its response to the drug . The mutation was recessive and was designated tpt1 for tyrphostin hypersensitive . A tpt1 strain cannot grow in the presence of tyrphostin, implying that a biological process sensitive to tyrphostin is essential for cell growth . Microscopic observation indicated that large-budded cells were accumulated in the presence of the inhibitor . The results suggest the involvement of protein tyrosine phosphorylation in the cell cycle progression of Saccharomyces cerevisiae.

FEMS Microbiol Lett, 1997 Aug 1, 153(1), 89 - 96
Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae; Daran JM et al.; Yeast cells lacking UDP-Glc pyrophosphorylase (UGPase) encoded by UGPI are not viable . Two strategies were developed to drastically reduce the intracellular concentration of UDP-Glc in order to study the consequences of this metabolic engineering on physiology and morphology . Firstly, UGP1 was placed under the strongly regulatable THI4 promoter . This resulted in a 95% reduction of UGPase activity in the presence of thiamine . The phenotypic effects of this reduction were slightly stronger than those of glucose on the GALI0/CYC1-UGP1 gene fusion {Daran et al . (1995) Eur . J . Biochem . 230, 520-530} . A further reduction of flux towards UDP-Glc was achieved by deletion of the two phosphoglucomutase genes in the ugp1 conditional strain . The growth of this new mutant strain was hardly affected, while it was extremely sensitive to cell wall interfering drugs . Surprisingly, UDP-Glc levels were reduced only by 5-fold, causing a proportional decrease in both glycogen and beta-glucans . Taken altogether, these results indicate that a few percent of enzymatic activities leading to the formation of UDP-Glc appears sufficient to provide the UDP-Glc demands required for cell viability, and that the loss of function of UGP1 is lethal mainly because of the inability of yeast cells to properly form the cell wall.

FEMS Microbiol Lett, 1997 Aug 1, 153(1), 17 - 23
Fluorescent measurement of the intracellular pH during sporulation of Saccharomyces cerevisiae; Aon JC et al.; This work reports the intracellular pH (pHi) dynamics of Saccharomyces cerevisiae cells in sporulation medium . Cells loaded with the pH-sensitive dye carboxy-seminaphthorhodafluor-1 (C.SNARF-1) exhibited an alkalization of the pHi following the extracellular pH during sporulation in the absence of buffer and almost no change in pHi or delta pH when sporulation was carried out in buffered medium . The results indicate that the pH gradient does not appear to be directly involved in the regulation of acetate uptake during sporulation . However, the alkalization of pHi by eliciting a decrease in metabolic fluxes could account for a lower demand for acetate.

Arch Biochem Biophys, 1997 Aug 1, 344(1), 1 - 10
Involvement of a 50-kDa mRNP protein from Saccharomyces cerevisiae in mRNA binding to ribosomes; Triana L et al.; A yeast 50-kDa mRNA-binding protein (50mRNP) is found selectively associated with the 48S and 80S initiation complexes . This protein is structurally related to the translational elongation factor EF-1alpha . The protein reacts with antibodies directed against EF-1alpha and, similarly, EF-1alpha recognizes antibodies against the 50mRNP protein . This is evidence that they share at least one epitope which allows a similar antigenic behavior . In addition, both proteins show similar cleavage patterns upon treatment with the endoproteinase Lys-C . A murine antibody raised against 50mRNP inhibits both 48S and 80S initiation complex formation . The inhibitory effect is relieved by preincubating anti-50mRNP with EF-1alpha . Antibody to EF-1alpha manifests a similar inhibitory pattern for the formation of 48S and 80S complexes . These data strongly suggest that 50mRNP is an EF-1alpha-like polypeptide essential for the formation of the above complexes.

J Bacteriol, 1997 Aug, 179(15), 4929 - 36
Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p; Bony M et al.; The Saccharomyces cerevisiae FLO1 gene encodes a large 1,536-amino-acid serine- and threonine-rich protein involved in flocculation . We have assessed the localization of Flo1p by immunoelectron microscopy, and in this study we show that this protein is located in the external mannoprotein layer of the cell wall, at the plasma membrane level and in the periplasm . The protein was also visualized in the endoplasmic reticulum and in the nuclear envelope, indicating that it was secreted through the secretory pathway . The protein was detected by Western blotting in cell wall extracts as a high-molecular-mass (>200 kDa) polydisperse material obviously as a result of extensive N and probably O glycosylation . Flo1p was extracted from cell walls in large amounts by boiling in sodium dodecyl sulfate, suggesting that it is noncovalently anchored to the cell wall network . The membranous forms of Flo1p were shown to be solubilized by phosphatidylinositol-phospholipase C treatment, suggesting that Flo1p is glycosyl phosphatidylinositol (GPI) anchored to this organelle . The expression of truncated forms with the hydrophobic C-terminal domain deleted led to the secretion of the protein in the culture medium . The hydrophobic C terminus, which is a putative GPI anchoring domain, is therefore necessary for the attachment of Flo1p in the cell wall . Deletion analysis also revealed that the N-terminal domain of Flo1p was essential for cellular aggregation . On the whole, our data indicate that Flo1p is a true cell wall protein which plays a direct role in cell-cell interaction.

J Bacteriol, 1997 Aug, 179(15), 4664 - 70
The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity; Berges T et al.; The mevalonate diphosphate decarboxylase is an enzyme which converts mevalonate diphosphate to isopentenyl diphosphate, the building block of isoprenoids . We used the Saccharomyces cerevisiae temperature-sensitive mutant defective for mevalonate diphosphate decarboxylase previously described (C . Chambon, V . Ladeveve, M . Servouse, L . Blanchard, C . Javelot, B . Vladescu, and F . Karst, Lipids 26:633-636, 1991) to characterize the mutated allele . We showed that a single change in a conserved amino acid accounts for the temperature-sensitive phenotype of the mutant . Complementation experiments were done both in the erg19-mutated background and in a strain in which the ERG19 gene, which was shown to be an essential gene for yeast, was disrupted . Epitope tagging of the wild-type mevalonate diphosphate decarboxylase allowed us to isolate the enzyme in an active form by a versatile one-step immunoprecipitation procedure . Furthermore, during the course of this study, we observed that a high level of expression of the wild-type ERG19 gene led to a lower sterol steady-state accumulation compared to that of a wild-type strain, suggesting that this enzyme may be a key enzyme in mevalonate pathway regulation.

J Biol Chem, 1997 Aug 1, 272(31), 19165 - 70
Isolation and characterization of a thiamin transport gene, THI10, from Saccharomyces cerevisiae; Enjo F et al.; We isolated a thiamin transporter gene, THI10, from a genomic library of Saccharomyces cerevisiae by the complementation of a yeast mutant defective in thiamin transport activity . The THI10 gene contained an open reading frame of 1,794 base pairs encoding a 598-amino acid polypeptide with a calculated molecular weight of 66, 903 . The nucleotide sequence of THI10 is completely identical to that of an anonymous DNA (open reading frame L8083.2) mapped to chromosome XII; two other genes (open reading frames YOR071c and YOR192c) in chromosome XV are extremely similar to THI10 . Moreover, the THI10 gene product showed significant sequence homology with yeast allantoin and uracil transporters . Hydropathy profile suggested that THI10 product is highly hydrophobic and contains many transmembrane regions . Gene disruption of the THI10 locus completely abolished the thiamin transport activity and thiamin binding activity in yeast plasma membrane fraction . Both the transport and thiamin binding activities were restored in the disrupted cells when the THI10 open reading frame was expressed by yeast GAL1 promoter, suggesting that the THI10 gene encodes for the thiamin transport carrier protein . Northern blot analysis demonstrated that THI10 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate and that it requires a positive regulatory factor encoded by THI3 gene.

Mol Cell Biol, 1997 Aug, 17(8), 4830 - 41
Characterization of the Wtm proteins, a novel family of Saccharomyces cerevisiae transcriptional modulators with roles in meiotic regulation and silencing; Pemberton LF et al.; Transcription is regulated by the complex interplay of repressors and activators . Much of this regulation is carried out by, in addition to gene-specific factors, complexes of more general transcriptional modulators . Here we present the characterization of a novel family of transcriptional regulators in yeast . Wtm1p (WD repeat-containing transcriptional modulator) was identified as a protein present in a large nuclear complex . This protein has two homologs, Wtm2p and Wtm3p, which probably arose by gene duplications . Deletion of these genes affects transcriptional repression at several loci, including derepression of IME2, a meiotic gene normally repressed in haploid cells . Targeting of these proteins to DNA resulted in a dramatic repression of activated transcription . In common with a mutation in the histone deacetylase RPD3, wtm mutants showed increased repression at the silent mating-type locus, HMR, and at telomeres . Although all three Wtm proteins could act as transcriptional repressors, Wtm3p, which is the least homologous, appeared to have functions separate from those of the other two . Wtm3p did not appear to be complexed with the other two proteins, was essential for IME2 repression, and could not efficiently repress transcription in the absence of the other Wtm proteins . These data suggested that Wtm1p and Wtm2p are repressors and that Wtm3p has different effects on transcription at different loci.

Mol Cell Biol, 1997 Aug, 17(8), 4654 - 66
Structure-function analysis of the Saccharomyces cerevisiae G1 cyclin Cln2; Huang KN et al.; We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis . With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A . Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules . Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function . A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function . Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis . All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions . Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function.

Mol Cell Biol, 1997 Aug, 17(8), 4490 - 500
Identification of RTF1, a novel gene important for TATA site selection by TATA box-binding protein in Saccharomyces cerevisiae; Stolinski LA et al.; Interaction of the TATA box-binding protein (TBP) with promoters of RNA polymerase II-transcribed genes is an early and essential step in mRNA synthesis . Previous studies have demonstrated that the rate-limiting binding of TBP to a TATA element can be influenced by transcriptional regulatory proteins . To identify additional factors that may regulate DNA binding by TBP in vivo, we performed a genetic selection for extragenic suppressors of a yeast TBP mutant that exhibits altered and relaxed DNA binding specificity . This analysis has led to the discovery of a previously unidentified gene, RTF1 . The original rtf1 suppressor mutation, which encodes a single amino acid change in Rtf1, and an rtf1 null allele suppress the effects of the TBP specificity mutant by altering transcription initiation . Differences in the patterns of transcription initiation in these strains strongly suggest that the rtf1 missense mutation is distinct from a simple loss-of-function allele . The results of genetic crosses indicate that suppression of TBP mutants by mutations in RTF1 occurs in an allele-specific fashion . In a strain containing wild-type TBP, the rtf1 null mutation suppresses the transcriptional effects of a Ty delta insertion mutation in the promoter of the HIS4 gene, a phenotype also conferred by the TBP altered-specificity mutant . Finally, as shown by indirect immunofluorescence experiments, Rtf1 is a nuclear protein . Taken together, our findings suggest that Rtf1 either directly or indirectly regulates the DNA binding properties of TBP and, consequently, the relative activities of different TATA elements in vivo.

Mol Cell Biol, 1997 Aug, 17(8), 4465 - 73
Functional domains of the Saccharomyces cerevisiae Mlh1p and Pms1p DNA mismatch repair proteins and their relevance to human hereditary nonpolyposis colorectal cancer-associated mutations; Pang Q et al.; The MutL protein is an essential component of the Escherichia coli methyl-directed mismatch repair system but has no known enzymatic function . In the yeast Saccharomyces cerevisiae, the MutL equivalent, an Mlh1p and Pms1p heterodimer, interacts with Msh2p bound to mismatch-containing DNA . Little is known of the functional domains of Mlh1p and Pms1p . In this report, we define the Mlh1p and Pms1p domains required for Mlh1p-Pms1p interaction . The Mlh1p-interactive domain of Pms1p is comprised of 260 amino acids near the carboxyl terminus while the Pms1p-interactive domain of Mlh1p resides in the final 212 residues . The two domains are sufficient for Mlh1p-Pms1p interaction, as determined by the two-hybrid assay and by in vitro protein affinity chromatography . Deletions within the domains completely eliminated Mlh1p-Pms1p interaction . Using site-directed mutagenesis, we altered a number of highly conserved residues in the Mlh1p and Pms1p proteins, including some alterations that mimic germline mutations observed for human hereditary nonpolyposis colorectal cancer . Alterations either in the consensus MutL box located in the amino-terminal portion of each protein or in the carboxyl-terminal homology motif of Mlh1p eliminated DNA mismatch repair function but had no effect on Mlh1p-Pms1p interaction . In addition, certain MLH1 and PMS1 mutant alleles caused a dominant negative mutator effect when overexpressed . We discuss the implications of these findings for the structural organization of the Mlh1p and Pms1p proteins and the importance of Mlh1p-Pms1p interaction.

Mol Cell Biol, 1997 Aug, 17(8), 4294 - 304
EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae; Tang HY et al.; Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15 . Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p . We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex . End3p has been shown by others to be an important factor in the process of endocytosis . End3p is also known to be required for the organization of the actin cytoskeleton . Here we report that Pan1p and End3p act as a complex in vivo . Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed . Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein . Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other . The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton . The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance . Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.

Mol Cell Biol, 1997 Aug, 17(8), 4282 - 93
A family of ammonium transporters in Saccharomyces cerevisiae; Marini AM et al.; Ammonium is a nitrogen source supporting growth of yeast cells at an optimal rate . We recently reported the first characterization of an NH4+ transport protein (Mep1p) in Saccharomyces cerevisiae . Here we describe the characterization of two additional NH4+ transporters, Mep2p and Mep3p, both of which are highly similar to Mep1p . The Mep2 protein displays the highest affinity for NH4+ (Km, 1 to 2 microM), followed closely by Mep1p (Km, 5 to 10 microM) and finally by Mep3p, whose affinity is much lower (Km, approximately 1.4 to 2.1 mM) . A strain lacking all three MEP genes cannot grow on media containing less than 5 mM NH4+ as the sole nitrogen source, while the presence of individual NH4+ transporters enables growth on these media . Yet, the three Mep proteins are not essential for growth on NH4+ at high concentrations (>20 mM) . Feeding experiments further indicate that the Mep transporters are also required to retain NH4+ inside cells during growth on at least some nitrogen sources other than NH4+ . The MEP genes are subject to nitrogen control . In the presence of a good nitrogen source, all three MEP genes are repressed . On a poor nitrogen source, MEP2 expression is much higher than MEP1 and MEP3 expression . High-level MEP2 transcription requires at least one of the two GATA family factors Gln3p and Nil1p, which are involved in transcriptional activation of many other nitrogen-regulated genes . In contrast, expression of either MEP1 or MEP3 requires only Gln3p and is unexpectedly down-regulated in a Nil1p-dependent manner . Analysis of databases suggests that families of NH4+ transporters exist in other organisms as well.

Mol Cell Biol, 1997 Aug, 17(8), 4199 - 207
Premature 3'-end formation of CBP1 mRNA results in the downregulation of cytochrome b mRNA during the induction of respiration in Saccharomyces cerevisiae; Sparks KA et al.; The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes . Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes . One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the nuclear gene CBP1 (cytochrome b processing) . CBP1 produces two types of transcripts with different 3' ends: full-length 2.2-kb transcripts and 1.2-kb transcripts truncated within the coding sequence of Cbp1 . Upon induction of respiration, the steady-state level of the long transcripts decreases while that of the short transcripts increases reciprocally, an unexpected result since the product of the long transcripts is required for COB mRNA stability and thus for respiration . Here we have tested the hypothesis that the short transcripts, or proteins translated from the short transcripts, are also required for respiration . A protein translated from the short transcripts was not detected by Western analysis, although polysome gradient fractions were shown to contain both long and short CBP1 transcripts . A mutant strain in which production of the short transcripts was abolished showed wild-type growth properties, indicating that the short transcripts are not required for respiration . Due to mutation of the carbon source-responsive element, the long transcript level in the mutant strain did not decrease during induction of respiration . The mutant strain had increased levels of COB RNA, suggestive that production of short CBP1 transcripts is a mechanism for downregulation of the levels of long CBP1 transcripts, Cbp1, and COB mRNA during the induction of respiration.

J Cell Biochem, 1997 Aug 1, 66(2), 184 - 96
Expression of the vitamin D and the retinoid X receptors in Saccharomyces cerevisiae: alternative in vivo models for ligand-induced transactivation; Berghofer-Hochheimer Y et al.; The transcription factors of the nuclear hormone receptor family regulate gene expression via a complex network of macromolecular interactions . The ligand dependent activity of the vitamin D receptor is of particular interest because it modulates gene expression by the heterodimeric interaction with retinoid X receptors . We report here that individual functions of the vitamin D receptor including DNA-binding, homo- and heterodimerization and transactivation can be reconstituted in the yeast Saccharomyces cerevisiae . Interestingly, the simultaneous expression of the native vitamin D receptor and the retinoid X receptor beta resulted in a ligand independent transactivation of the lacZ reporter gene coupled to a mouse osteopontin vitamin D response element . However, homodimerization of the vitamin D receptor and heterodimerization were strongly enhanced upon ligand binding, when the receptors were expressed as fusion proteins with the Gal4 transcription factor in a yeast two-hybrid system . Furthermore, transactivating activity of a Gal4-fused vitamin D receptor was induced by vitamin D in a one-hybrid system devoid of retinoid X receptors . In addition, both Gal4-based systems behaved similar with regard to their dose-dependent response to vitamin D and related compounds when compared to the transcriptional activity of the vitamin D receptor in transiently transfected MCF-7 cells . Our results point out that specific ligands strongly enhanced receptor dimerization and induced transactivation in yeast and in MCF-7 cells . The constitutive transactivation by vitamin D receptor-retinoid X receptor heterodimers in yeast, depending on DNA binding of the receptors, strongly argues for the existence of cofactors, which are absent in yeast, but play a fundamental role in gene regulation in higher eukaryotic organisms.

Biochim Biophys Acta, 1997 Jul 31, 1321(1), 79 - 92
Structural and functional analysis of deficient mutants in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae; Ortwein C et al.; Four point mutations in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae that had been selected for respiratory incompetence but still contained spectrally detectable haem aa3 were analysed . The isolated mutant enzymes exhibited minor band shifts in their optical spectra and contained all eleven subunits . However, steady state activities were only a few percent compared to wild type enzyme . Using a comprehensive experimental approach, we first checked the integrity of the enzyme preparations and then identified the specific functional defect . The results are discussed using information from the recently solved structures of cytochrome c oxidase at 2.8 A . Mutation 167N is positioned between haem a and a conserved glutamate residue (E243) . It caused a distortion of the EPR signal of haem a and shifted its midpoint potential by 54 mV to the negative . The high-resolution structure suggests that the primary reason for the low activity of the mutant enzyme could be that asparagine in position 67 might form a stable hydrogen bond to E243, which is part of a proposed proton channel . Cytochrome c oxidase isolated from mutant T316K did not meet our criteria for homogeneity and was therefore omitted from further analysis . Mutants G352V and V380M exhibited an impairment of electron transfer from haem a to a3 and ligand binding to the binuclear centre was affected . In mutant V380M also the midpoint potential of CuB was shifted by 65 mV to the positive . The results indicated for these two mutants changes primarily associated with the binuclear centre, possibly associated with an interference in the routes and/or sites of protonation which are required for stable formation of the catalytic intermediates . This interpretation is discussed in the light of the high resolution structure.

J Biol Chem, 1997 Jul 25, 272(30), 18790 - 800
Ras2 and Ras1 protein phosphorylation in Saccharomyces cerevisiae; Whistler JL et al.; This work describes the phosphorylation of Saccharomyces cerevisiae Ras proteins and explores the physiological role of the phosphorylation of Ras2 protein . Proteins expressed from activated alleles of RAS were less stable and less phosphorylated than proteins from cells expressing wild-type alleles of RAS . This difference in phosphorylation level did not result from increased signaling through the Ras-cAMP pathway or reflect the primarily GTP-bound nature of activated forms of Ras protein per se . In addition, phosphorylation of Ras protein was not dependent on proper localization of the Ras2 protein to the plasma membrane nor on the interaction of Ras2p with its exchange factor, Cdc25p . The preferred phosphorylation site on Ras2 protein was identified as serine 214 . This site, when mutated to alanine, led to promiscuous phosphorylation of Ras2 protein on nearby serine residues . A decrease in phosphorylation may lead to a decrease in signaling through the Ras-cAMP pathway.

Oncogene, 1997 Jul 24, 15(4), 417 - 22
Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae; Koch G et al.; The small GTP-binding proteins of the Rho family, consisting of the Rho, Rac, and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, cell motility and cytokinesis, through reorganization of actin cytoskeleton . Rho GDI is a general regulator which forms a complex with the GDP-bound inactive form of the Rho family members and inhibits their activation . We have purified Rho GDI from the yeast Saccharomyces cerevisiae, cloned its gene, and named it RDII (Rho GD) . In this study, we have further characterized yeast Rho GDI . Rho GDI was found in the cytosol by immunoblot and immunofluorescence microscopic analyses . Rho1p and Cdc42p were co-immunoprecipitated with Rho GDI from the cytosol . This immunoprecipitated Rho1p was mainly bound to GDP . In the disruption mutant of Rho GDI, which did not show any apparent phenotype, both Rho1p and Cdc42p were also present in the cytosol . These results indicate that yeast Rho GDI possesses properties similar to those of mammalian Rho GDI, and that there is a cytosolic factor which functionally substitutes for Rho GDI in yeast.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 8027 - 32
Transitions in the coupling of transcription and nucleotide excision repair within RNA polymerase II-transcribed genes of Saccharomyces cerevisiae; Tijsterman M et al.; The molecular mechanism of transcription-coupled nucleotide excision repair in eukaryotes is poorly understood . The identification of the dual role of basal transcription factor TFIIH in DNA repair and transcription provided a plausible link between both processes . However, TFIIH is not part of the elongating transcription complex, suggesting that additional components are required to recruit TFIIH when RNA polymerase II (RNAPII) stalls at the site of DNA damage . Previously, we have shown that the yeast Rad26 protein is involved in transcription-coupled DNA repair . This paper describes the differential contribution of the Rad26 protein to efficient removal of UV-induced cyclobutane pyrimidine dimers (CPDs) from transcribed DNA . Two distinct regions within the transcribed strand of RNAPII-transcribed genes are identified that differ in their requirement for the RAD26 gene product . Using high-resolution repair analysis, we determined the in vivo repair kinetics of cyclobutane pyrimidine dimers positioned around the transcription initiation site of RNAPII-transcribed genes RPB2 and URA3 . Although transcription-coupled repair is severely reduced in rad26 mutants, lesions positioned in a small region immediately downstream of transcription initiation are efficiently removed in the absence of Rad26 . The observed transition in repair characteristics is abrupt and in excellent agreement with the region where TFIIH dissociates from RNAPII in vitro, strongly suggesting an inverse correlation between TFIIH association and Rad26 requirement . These data suggest that a transcription repair coupling factor (Rad26/CSB) is required for efficient repair only during the elongating stages of RNAPII transcription.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 8010 - 5
Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae; Radicella JP et al.; The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase activity that is a functional analog of the Fpg protein from Escherichia coli and excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged DNA . The repair of this ubiquitous kind of oxidative damage is essential to prevent mutations both in bacteria and in yeast . A human cDNA clone carrying an ORF displaying homology to the yeast protein was identified . The predicted protein has 345 amino acids and a molecular mass of 39 kDa . This protein shares a 38% sequence identity with the yeast Ogg1 protein, adding this novel human gene product to the growing family of enzymes that the repair of oxidatively damaged bases and are related to the E . coli endonuclease III . Northern blot analysis indicates that this gene, localized to chromosome 3p25, is ubiquitously expressed in human tissues . The cloned coding sequence was expressed in an E . coli strain that carried a disrupted fpg gene, the bacterial functional analog of OGG1 . Cell-free extracts from these cultures displayed a specific lyase activity on duplex DNA that carried an 8-oxoG/C base pair . The products of the reaction are consistent with an enzymatic activity like the one displayed by the yeast Ogg1 . Analysis of the substrate specificity reveals a very strong preference for DNA fragments harboring 8-oxoG/C base pairs . The pattern of specificity correlates well with the one found for the yeast enzyme . Moreover, when the human coding sequence was expressed in a yeast strain mutant in OGG1 it was able to complement the spontaneous mutator phenotype . These results make this novel gene (hOGG1) a strong candidate for the human homolog of the yeast OGG1 and suggest an important role of its product in the protection of the genome from the mutagenic effects of the oxidatively damaged purines.

Biochemistry, 1997 Jul 22, 36(29), 9002 - 12
Crystal structure of Kex1deltap, a prohormone-processing carboxypeptidase from Saccharomyces cerevisiae,; Shilton BH et al.; Kex1p is a prohormone-processing serine carboxypeptidase found in Saccharomyces cerevisiae . In contrast to yeast serine carboxypeptidase (CPD-Y) and wheat serine carboxypeptidase II (CPDW-II), Kex1p displays a very narrow specificity for lysyl or arginyl residues at the C-terminus of the substrate . The structure of Kex1Deltap, an enzyme that lacks the acidic domain and membrane-spanning portion of Kex1p, has been solved by a combination of molecular replacement and multiple isomorphous replacement and refined to a resolution of 2.4 A . The S1' site of Kex1Deltap is sterically restricted compared to those from CPD-Y or CPDW-II; it also contains two acidic groups that are well positioned to interact with the basic group of a lysine or arginine side chain . The high specificity of Kex1p can therefore be explained by a combination of steric and electronic factors . The structure of the S1 site of Kex1Deltap is also well suited for binding of a lysine or arginine side chain, and the enzyme may therefore exhibit a preference for these residues at P1.

Biochemistry, 1997 Jul 22, 36(29), 8992 - 9001
Solution structure of oxidized Saccharomyces cerevisiae iso-1-cytochrome c; Banci L et al.; The solution structure of oxidized Saccharomycescerevisiae Cys102Ser iso-1-cytochromechas been determined using 1361 meaningful NOEs (of 1676 total) after extending the published proton assignment {Gao, Y., et al . (1990) Biochemistry 29, 6994-7003} to 77% of all proton resonances . The NOE patterns indicate that secondary structure elements are maintained upon oxidation in solution with respect to the solid state and solution structures of the reduced species . Constraints derived from the pseudocontact shifts {diamagnetic reference shift values are those of the reduced protein {Baistrocchi, P., et al . (1996) Biochemistry 35, 13788-13796}} were used in the final stages of structure calculations . After restrained energy minimization with constraints from NOEs and pseudocontact shifts, a family of 20 structures with rmsd values of 0.58 +/- 0.08 and 1.05 +/- 0.10 A (relative to the average structure) for the backbone and all heavy atoms, respectively, was obtained . The solution structure is compared with the crystal structure and the structures of related systems . Twenty-six amide protons were detected in the NMR spectrum 6 days after the oxidized lyophilized protein was dissolved in D2O (pH 7.0 and 303 K); in an analogous experiment, 47 protons were observed in the spectrum of the reduced protein . The decrease in the number of nonexchanging amide protons, which mainly are found in the loop regions 14-26 and 75-82, confirms the greater flexibility of the structure of oxidized cytochrome c in solution . Our finding of increased solvent accessibility in these loop regions is consistent with proposals that an early step in unfolding the oxidized protein is the opening of the 70-85 loop coupled with dissociation of the Met80-iron bond.

FEBS Lett, 1997 Jul 21, 412(1), 207 - 10
Fatty acids induced uncoupling of Saccharomyces cerevisiae mitochondria requires an intact ADP/ATP carrier; Polcic P et al.; Fatty acids stimulate the oxidation rate of mitochondria isolated from the wild-type Saccharomyces cerevisiae, but do not affect significantly the respiration of mitochondria isolated from mutants, in which the ADP/ATP carrier (AAC) was either modified (R96H) or deleted (delta aac2) . Similarly as in mammalian mitochondria, the transmembrane electrical potential difference (delta psi) in the wild-type yeast mitochondria was dissipated by low concentrations of free fatty acids, and this was partially inhibited by bongkrecate . In contrast to the wild-type mitochondria, the addition of increasing concentrations of fatty acids to the op1 (R96H) mutant mitochondria abolished only a small portion of delta psi, as compared to the change induced by classical uncouplers . The different effects of fatty acids on both, the respiration and the delta psi of mitochondria isolated from the wild-type and the aac mutants, respectively, demonstrates that the intact AAC is essential for the fatty acids induced H+ permeability of mitochondrial membrane.

J Biol Chem, 1997 Jul 18, 272(29), 18333 - 40
Characterization of translation initiation factor 5 (eIF5) from Saccharomyces cerevisiae . Functional homology with mammalian eIF5 and the effect of depletion of eIF5 on protein synthesis in vivo and in vitro; Maiti T et al.; Eukaryotic translation initiation factor 5 (eIF5) interacts in vitro with the 40 S initiation complex (40 S.AUG.Met-tRNAf.eIF2.GTP) to mediate the hydrolysis of ribosome-bound GTP . In Saccharomyces cerevisiae, eIF5 is encoded by a single copy essential gene, TIF5, that encodes a protein of 45,346 daltons . To understand the function of eIF5 in vivo, we constructed a conditional mutant yeast strain in which a functional but a rapidly degradable form of eIF5 fusion protein was synthesized from the repressible GAL promoter . Depletion of eIF5 from this mutant yeast strain resulted in inhibition of both cell growth and the rate of in vivo protein synthesis . Analysis of the polysome profiles of eIF5-depleted cells showed greatly diminished polysomes with simultaneous increase in free ribosomes . Furthermore, lysates of cells depleted of eIF5 were dependent on exogenously added yeast eIF5 for efficient translation of mRNAs in vitro . This is the first demonstration that the TIF5 gene encodes a protein involved in initiation of translation in eukaryotic cells . Additionally, we show that rat eIF5 can functionally substitute yeast eIF5 in translation of mRNAs in vitro as well as in complementing in vivo a genetic disruption in the chromosomal copy of TIF5.

J Biol Chem, 1997 Jul 18, 272(29), 18117 - 24
MNN6, a member of the KRE2/MNT1 family, is the gene for mannosylphosphate transfer in Saccharomyces cerevisiae; Wang XH et al.; In yeast Saccharomyces cerevisiae the N-linked sugar chain is modified at different positions by the addition of mannosylphosphate . The mnn6 mutant is deficient in the mannosylphosphate transferase activity toward mannotetraose (Karson, E . M., and Ballou, C . E . (1978) J . Biol . Chem . 253, 6484-6492) . We have cloned the MNN6 gene by complementation . It has encoded a 446-amino acid polypeptide with the characteristics of type II membrane protein . The deduced Mnn6p showed a significant similarity to Kre2p/Mnt1p, a Golgi alpha-1, 2-mannosyltransferase involved in O-glycosylation . The null mutant of MNN6 showed a normal cell growth, less binding to Alcian blue, hypersensitivity to Calcoflour White and hygromycin B, and diminished mannosylphosphate transferase activity toward the endoplasmic reticulum core oligosaccharide acceptors (Man8GlcNAc2-PA and Man5GlcNAc2-PA) in vitro, suggesting the involvement of the MNN6 gene in the endoplasmic reticulum core oligosaccharide phosphorylation . However, no differences were observed in N-linked mannoprotein oligosaccharides between Deltaoch1 Deltamnn1 cells and Deltaoch1Deltamnn1Deltamnn6 cells, indicating the existence of redundant genes required for the core oligosaccharide phosphorylation . Based on a dramatic decrease in polymannose outer chain phosphorylation by MNN6 gene disruption and a determination of the mannosylphosphorylation site in the acceptor, it is postulated that the MNN6 gene may be a structural gene encoding a mannosylphosphate transferase, which recognizes any oligosaccharides with at least one alpha-1,2-linked mannobiose unit.

Eur J Biochem, 1997 Jul 15, 247(2), 703 - 8
Dimerization of Cdc25p, the guanine-nucleotide exchange factor for Ras from Saccharomyces cerevisiae, and its interaction with Sdc25p; Camus C et al.; The oligomerization state of Cdc25p, the guanine nucleotide exchange factor for ras from yeast, was analyzed using different complementary approaches . The two-hybrid system showed that the C-terminal part of Cdc25p (Cdc25-Ct) can interact with itself but also with Sdc25p-Ct, the corresponding part of Sdc25p, the other guanine exchange factor from yeast . The homotropic interaction of Cdc25p-Ct has been confirmed in yeast using immunoprecipitation experiments with epitope-tagged and beta-galactosidase-fused polypeptides . No other component was required for this interaction, since dimerization was shown to occur with material synthesized in vitro . The size of Cdc25-Ct produced in Escherichia coli has been directly measured on gel filtration columns and corresponds to a dimer . The dimerization domain is localized in the same part of the molecule as the catalytic domain and the portion responsible for membrane localization . The biological relevance of dimerization is still an open question, however by allowing heterodimerization with Sdc25p it could permit a more complex combinatorial regulation of ras in yeast.

FEMS Microbiol Lett, 1997 Jul 15, 152(2), 293 - 8
Mutants of Saccharomyces cerevisiae defective in vacuolar function confirm a role for the vacuole in toxic metal ion detoxification; Ramsay LM et al.; To directly define vacuolar role(s) in metal detoxification, we have examined the responses of vacuole-deficient mutants of Saccharomyces cerevisiae to several potentially toxic metals known to be mainly detoxified in the cytosol (Cu, Cd) or the vacuole (Co, Mn, Ni, Zn) . Three mutants, deficient in targeting of vacuolar proteins, were used with JSR18 delta 1 being devoid of any vacuole-like structure while ScVatB and ScVatC were deficient in specific protein subunits of the V-ATPase . The results obtained show that the absence of a vacuole or a functional vacuolar H(+)-ATPase was associated with increased sensitivity and a largely decreased capacity of the vacuole-deficient strains to accumulate Zn, Mn, Co and Ni, confirming an essential role for the vacuole in detoxification of these metals . In addition, the lack of vacuolar involvement in detoxification of Cu and Cd was confirmed since these metals did not exhibit increased toxicity towards the vacuolar mutants nor were there significant differences in Cu or Cd accumulation between parental and mutant strains.

FEMS Microbiol Lett, 1997 Jul 15, 152(2), 261 - 7
On the unidirectionality of arginine uptake in the yeast Saccharomyces cerevisiae; Opekarova M et al.; The reversibility of arginine accumulation was followed in exponentially growing cells of Saccharomyces cerevisiae and in the same cells transferred to non-growing energized conditions . Under non-growing conditions the accumulated arginine is retained in the cells while in exponentially growing cells the accumulated radioactivity is released after the addition of high external concentrations of arginine . There are indications that the process is saturable . The accumulated arginine is not exchanged for other related amino acids (L-citrulline, L-histidine) . Only L-lysine (a low-affinity substrate of the specific arginine permease) provokes partial radioactivity efflux from the cells . The switch of the arginine-related radioactive label efflux to its complete retention in the cells after changing the growth conditions occurs within a few minutes and is tentatively attributed to two concomitantly occurring events: (1) the actual presence of radioactive arginine (not its metabolite(s)) in the cell and (2) a modification of the specific arginine permease . The specific exchange of arginine described in the present study contrasts with the currently widely accepted opinion of unidirectionality of amino acid fluxes in yeast . The reasons why this phenomenon has not been observed before are discussed.

J Biol Chem, 1997 Jul 11, 272(28), 17495 - 501
The N terminus of the Qcr7 protein of the cytochrome bc1 complex is not essential for import into mitochondria in Saccharomyces cerevisiae but is essential for assembly of the complex; Malaney S et al.; Subunit 7 of the yeast cytochrome bc1 complex is encoded by the nuclear QCR7 gene and is essential for respiration . This protein does not contain a cleavable N-terminal mitochondrial targeting sequence, and it is not understood how the Qcr7 protein is imported into mitochondria and assembled into the complex . To test the role of the N terminus of the Qcr7 protein in mitochondrial import, assembly of the complex, and proton translocation, we inactivated the endogenous QCR7 gene and expressed mutated qcr7 genes capable of synthesizing proteins truncated by 7, 10, 14, and 20 residues (Qcr7p-delta7, Qcr7p-delta10, Qcr7p-delta14, and Qcr7p-delta20, respectively) from the N terminus . In addition, we studied two mutants containing Qcr7 proteins with point mutations in addition to a delta7 truncation, Qcr7p-delta7(D13V) and Qcr7p-delta7(R10K) . All the mutant proteins with the exception of Qcr7p-delta10 were present in the mitochondria at 30 degrees C, although most at lower steady-state levels than the Qcr7p from the strain overexpressing wild type QCR7 . The absence of the Qcr7p-delta10 may be the result of an unstable protein or a decrease in the efficiency of mitochondrial import due to its compromised amphipathic alpha-helix and the presence of a negative charge exposed at the N terminus . Cytochrome c reductase activities and the amounts of ATP synthesized were comparable with the wild type in the strain expressing Qcr7p-delta7 . The strain expressing Qcr7p-delta7(R10K) had an identical phenotype to the one containing the Qcr7p-delta7, whereas strains expressing the Qcr7p-delta10, Qcr7p-delta14, Qcr7p-delta20, and Qcr7p-delta7(D13V) were all respiration-deficient . Examination of the steady-state levels of complex III subunits showed that core protein 2, cytochrome c1, the iron-sulfur protein, and the 11-kDa subunit are reduced in respiration-deficient mutant strains . Results from deletion analyses indicate that the N-terminal 20 residues (after Met-1) of the Qcr7 protein are not essential for import into mitochondria and that the N-terminal seven residues (after Met-1) are not involved in proton translocation . The results of this work show, however, that the N terminus of the Qcr7 protein is essential for the biosynthesis of ubiquinol-cytochrome c reductase.

J Biol Chem, 1997 Jul 11, 272(28), 17376 - 84
ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation; Oh CS et al.; ELO2 and ELO3 were identified from the Saccharomyces cerevisiae genome data base as homologues of ELO1, a gene involved in the elongation of the fatty acid 14:0 to 16:0 . Mutations in these genes have previously been shown to produce pleiotropic effects involving a number of membrane functions . The simultaneous disruption of ELO2 and ELO3 has also been shown to produce synthetic lethality, indicating that they have related and/or overlapping functions . Gas chromatography and gas chromatography/mass spectroscopy analyses reveal that null mutations of ELO2 and ELO3 produce defects in the formation of very long chain fatty acids . Analysis of the null mutants indicates that these genes encode components of the membrane-bound fatty acid elongation systems that produce the 26-carbon very long chain fatty acids that are precursors for ceramide and sphingolipids . Elo2p appears to be involved in the elongation of fatty acids up to 24 carbons . It appears to have the highest affinity for substrates with chain lengths less than 22 carbons . Elo3p apparently has a broader substrate specificity and is essential for the conversion of 24-carbon acids to 26-carbon species . Disruption of either gene reduces cellular sphingolipid levels and results in the accumulation of the long chain base, phytosphingosine . Null mutations in ELO3 result in accumulation of labeled precursors into inositol phosphoceramide, with little labeling in the more complex mannosylated sphingolipids, whereas disruption of ELO2 results in reduced levels of all sphingolipids.

Biochim Biophys Acta, 1997 Jul 5, 1327(1), 107 - 18
Purification of functional human P-glycoprotein expressed in Saccharomyces cerevisiae; Mao Q et al.; A system for expression and facile purification of the human P-glycoprotein (Pgp) from the yeast Saccharomyces cerevisiae is described . The wild-type human mdr1 cDNA was cloned into a high copy number yeast expression vector under the control of the constitutive promoter of the yeast plasma membrane H+-ATPase . Western blots of membranes from the stable transformants confirmed that the Pgp is expressed in yeast cells in amounts approximately 0.4% of the total yeast membrane protein . Density gradient sedimentation analysis of the yeast membranes indicated that the expressed Pgp is localized in the plasma membrane . Yeast cells transformed with the Pgp expression plasmid acquire increased resistance to valinomycin, suggesting that the expressed Pgp is properly folded and functional . The expressed Pgp can be solubilized from the yeast membranes with lysophosphatidylcholine, and when tagged with ten histidines at its C-terminus, can be readily purified to about 90% homogeneity by Ni2+ affinity chromatography . About 50 microg of the Pgp can be purified from 20 mg of crude yeast membranes . The purified human Pgp exhibits a verapamil-stimulated ATPase activity and the maximal activity is 2.5 +/- 0.5 micromol/min per mg of Pgp, suggesting that the purified Pgp from yeast is highly functional . The Pgp expressed in yeast has the same electrophoretic mobility (ca . 130 kDa) as the Pgp produced in Sf9 insect cells and is unaffected by N-glycosidase treatment, suggesting that it is not glycosylated . Because of the relative ease of growing yeast in massive quantities this expression system appears to be excellent for producing this membrane transporter at levels sufficient for further biochemical and biophysical studies, and for site-directed mutagenesis studies as well.

J Biol Chem, 1997 Jul 4, 272(27), 17045 - 54
Thioredoxin reductase-dependent inhibition of MCB cell cycle box activity in Saccharomyces cerevisiae; Machado AK et al.; Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S . Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Deltaswi6 yeast . Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6 . The mutations identified a single complementation group . Functional cloning revealed the mutations were alleles of the TRR1 gene encoding thioredoxin reductase . Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity . The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements . The trr1 mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, and SWI4) . Deletion of thioredoxin genes TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations on MCB reporter gene activity . Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression . The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.

Mol Gen Genet, 1997 Jul, 255(3), 322 - 31
Heat stress transcription factors from tomato can functionally replace HSF1 in the yeast Saccharomyces cerevisiae; Boscheinen O et al.; The fact that yeast HSF1 is essential for survival under nonstress conditions can be used to test heterologous Hsfs for the ability to substitute for the endogenous protein . Our results demonstrate that like Hsf of Drosophila, tomato Hsfs A1 and A2 can functionally replace the corresponding yeast protein, but Hsf B1 cannot . In addition to survival at 28 degrees C, we checked the transformed yeast strains for temperature sensitivity of growth, induced thermotolerance and activator function using two different lacZ reporter constructs . Tests with full-length Hsfs were supplemented by assays using mutant Hsfs lacking parts of their C-terminal activator region or oligomerization domain, or containing amino acid substitutions in the DNA-binding domain . Remarkably, results with the yeast system are basically similar to those obtained by the analysis of the same Hsfs as transcriptional activators in a tobacco protoplast assay . Most surprising is the failure of HsfB1 to substitute for the yeast Hsf . The defect can be overcome by addition to HsfB1 of a short C-terminal peptide motif from HsfA2 (34 amino acid residues), which represents a type of minimal activator necessary for interaction with the yeast transcription apparatus . Deletion of the oligomerization domain (HR-A/B) does not interfere with Hsf function for survival or growth at higher temperatures . But monomeric Hsf has a markedly reduced affinity for DNA, as shown by lacZ reporter and band-shift assays.

Mol Gen Genet, 1997 Jul, 255(4), 400 - 9
The MBR1 gene from Saccharomyces cerevisiae is activated by and required for growth under sub-optimal conditions; Reisdorf P et al.; The MBR1 gene was isolated as a multicopy suppressor of the phenotype on glycerol medium of a Saccharomyces cerevisiae strain mutant for the Hap2/3/4/5 transactivator complex . In this paper, we show that Mbr1p is a limiting factor for growth on glycerol medium under the following sub-optimal culture conditions: in late growth phase, at low temperature, at high external pH or in the presence of 1,10-phenanthroline . Moreover, deletion of MBR1 protects cells against stress, whilst overexpression of this gene has the opposite effect . MBR1 expression is induced in the late growth phase and is negatively controlled by the cAMP-dependent protein kinase A (PKA) . Both activation of PKA or overexpression of SOK1 or SCH9-two genes isolated as multicopy suppressors of a PKA null mutant-suppress the mbr1 growth defect . Our results indicate that Mbr1p is not an essential element of any one of these pathways . Deletion of SAC1, a gene implicated in vesicular transport, in association with MBR1 deletion, causes synthetic lethality . A possible role of Mbr1p in intracellular trafficking is discussed.

Biotechnol Prog, 1997 Jul-Aug, 13(4), 368 - 73
Sequential delta-integration for the regulated insertion of cloned genes in Saccharomyces cerevisiae; Lee FW et al.; A novel delta-integration vector was developed to allow the sequential insertion of multiple cloned genes in the yeast Saccharomyces cerevisiae . To allow repetitive integrations, the reusable URA3 Blaster selection cassette was employed; the insertions (of CUP1p-lacZ in this study) were selected using the URA3 marker which was subsequently "popped" out by recombination between flanking direct repeats . Transformants contained only one new integrated copy after the loss of the URA3 marker, and subsequent transformations were effective for the sequential insertion of a series of genes (one at a time) into dispersed chromosomal delta sequences . The structural stability of the integrations was location-dependent (ranging from 75% to 100% after 50 generations in complex medium with or without gene expression), and the integrations (at least up to five) had no significant effects on the growth of the cells . In addition, beta-galactosidase specific activity levels varied linearly with integrated copy number . The repetitive, regulated nature of integration with this vector is not possible with traditional delta-integration or other homologous recombination methods, and is promising for fine-tuning cloned gene copy number and for the insertion of metabolic pathway genes.

Biosci Biotechnol Biochem, 1997 Jul, 61(7), 1172 - 8
Isolation and characterization of kar2-404 mutation in Saccharomyces cerevisiae; Kawamura-Watabe A et al.; We have devised a direct screening method to isolate mutations in the KAR2 gene, and have isolated a BiP/KAR2 mutant, kar2-404, from Saccharomyces cerevisiae as a small halo-forming mutant of secreted mouse alpha-amylase . The mutation site was identified as a point mutation at t1337 to c1337 resulting in the Ile-404Thr mutation of mature Kar2-404p, located at the most NH2-terminal first beta-sheet structure (beta 1) of the putative peptide-binding domain . This isoleucine is highly conserved in the Hsp70 family . By pulse-chase experiments, no obvious difference was detected in the intracellular secretion rate of MF alpha 1-prepro-signal-mouse-alpha-amylase between the wild type and the kar2-404 mutant . However, only about half the amount of secreted heterologous protein, mouse alpha-amylase, was detected in the mutant culture medium compared with wild type . A smaller amount of homologous protein, alpha-factor, was also detected and decreased faster in the mutant culture medium than in wild type . Kar2-404p was expressed about 3-fold more than wild type Kar2p, probably to cover its defective functions, and the turnover rates of Kar2p and Kar2-404p were about the same in vivo . The purified Kar2-404p was slightly more sensitive to chymotryptic digestion than Kar2p in vitro.

J Photochem Photobiol B, 1997 Jul, 39(3), 185 - 96
Role of PSO genes in the repair of photoinduced interstrand cross-links and photooxidative damage in the DNA of the yeast Saccharomyces cerevisiae; Henriques JA et al.; Recent progress in elucidating the molecular structure of the PSSO genes PSO2 to PSO7 is presented . Their role in DNA repair and mutagenesis is discussed in the light of the putative proteins encoded in the respective ORFs and with the knowledge of recent progress in biological and biochemical experimentation . The role of the RecA protein in some steps of DNA repair in Saccharomyces cerevisiae is presented and discussed.

Eur J Biochem, 1997 Jul 1, 247(1), 142 - 7
Periplasmic Bar1 protease of Saccharomyces cerevisiae is active before reaching its extracellular destination; Ballensiefen W et al.; Saccharomyces cerevisiae MATa and MAT alpha cells secrete a-factor and alpha-factor pheromones . These peptides act on cells of the opposite mating type . They induce physiological changes which allow the formation of diploid cells . MATa strains produce an extracellular protease which cleaves, and thus inactivates the MAT alpha cell-specific alpha-factor pheromone . This pepsin-like enzyme is encoded by the BAR1(SST1) gene and is secreted into the periplasmic space of MATa cells . We found that the Bar1p protease is already active in early compartments of the secretory pathway . Our results indicate that Bar1 protease tolerates large N-terminal extensions of its substrate and does not require Golgi-specific modifications such as outer-chain glycosylation for activity.

Lett Appl Microbiol, 1997 Jul, 25(1), 43 - 7
Barotolerance is dependent on both trehalose and heat shock protein 104 but is essentially different from thermotolerance in Saccharomyces cerevisiae; Iwahashi H et al.; The contribution of trehalose and hsp104 to barotolerance in Saccharomyces cerevisiae has been investigated . Mutant strains, which lacked the ability to accumulate trehalose and/or hsp104, were examined for barotolerance and thermotolerance . All the mutants showed lower barotolerance and thermotolerance than their control strains . Trehalose had a greater protective effect towards high pressure than high temperature . Thus, trehalose and hsp104 are important factors for barotolerance and thermotolerance, but trehalose is more important for barotolerance than for thermotolerance.

Microbiology, 1997 Jul, 143 ( Pt 7), 2465 - 71
Reduction of ATPase activity accompanied by photodecomposition of ergosterol by near-UV irradiation in plasma membranes prepared from Saccharomyces cerevisiae; Arami S et al.; When plasma membranes prepared from the yeast Saccharomyces cerevisiae were exposed to near-UV radiation, photodecomposition of ergosterol and reduction of ATPase-activity occurred simultaneously . The Vmax for ATPase activity decreased markedly with increasing near-UV dosage while the Km value remained constant . When ATPase solubilized from the plasma membrane was exposed to near-UV, the activity remained constant irrespective of dosage, indicating that the ATPase molecule itself was not damaged by near-UV irradiation . The relationship between content of ergosterol and ATPase activity was examined using liposomes constructed with lipids extracted from the membrane . Maximum activity of ATPase was seen at 5% ergosterol in liposomes; this activity was 2.5 times greater than that in liposomes without ergosterol . Activity of ATPase bound to liposomes with 5% ergosterol was reduced after near-UV irradiation, while the activity remained unchanged in the case of the liposomes without ergosterol . Fluidity of the liposomes with 5% ergosterol also decreased with increasing near-UV dosage . Dosage-response curves for reduction of ATPase activity and for decrease in fluidity were similar to that for photodecomposition of ergosterol . These results suggested that the reduction of ATPase activity in the membrane by near-UV irradiation was not caused by photochemical degradation of the primary structure of the ATPase molecule, but was attributable to conformational change resulting from an alteration in the higher-order structure of the membrane due to photochemical decomposition of ergosterol.

Mol Biol Cell, 1997 Jul, 8(7), 1273 - 91
A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism; Chen P et al.; Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms . The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids . We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p . In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself . We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications . The formation and export of AFRP displays three striking features . First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production . Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor . Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues . Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity . Third, a-factor and AFRP use distinct cellular machinery for their secretion . Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant . Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion . Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor . Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.

Mol Biol Cell, 1997 Jul, 8(7), 1233 - 42
Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA; Nunnari J et al.; To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells . To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells . Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker . Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events . Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating . In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell . This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission . We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.

Yeast, 1997 Jul, 13(9), 861 - 9
The characterization of two new clusters of duplicated genes suggests a 'Lego' organization of the yeast Saccharomyces cerevisiae chromosomes; Feuermann M et al.; The systematic sequencing of 42,485 bp of yeast chromosome VII (nucleotides 377948 to 420432) has revealed the presence of 27 putative open reading frames (ORFs) coding for proteins of at least 100 amino acids . The degree of redundancy observed is elevated since five of the 27 ORFs are duplications of a previously identified gene . These duplicated copies may be classified in two types of cluster organization . The first type includes genes sharing a significant level of identity in the amino acid sequences of their predicted protein product . They are recovered on two different chromosomes, transcribed in the same orientation and the distance between them is conserved . The second type of cluster is based on one gene unit tandemly repeated . This duplication is itself repeated elsewhere in the genome . The level of nucleic acid identity is high within the coding sequence and the non-coding region between the two repeats . In addition, the basic gene unit is recovered many times in the genome and is a component of a multigene family of unknown function . These organizations in clusters of genes suggest a 'Lego organization' of the yeast chromosomes, as recently proposed for the genome of plants (Moore, 1995) . The sequence is deposited in the Yeast Genome Databank under Accession Number from Z72562 to Z72586.

Yeast, 1997 Jul, 13(9), 849 - 60
Sequence analysis of the 33 kb long region between ORC5 and SUI1 from the left arm of chromosome XIV from Saccharomyces cerevisiae; Sen-Gupta M et al.; We have determined the nucleotide sequence of a chromosomal region of 33,016 bp located on the left arm of chromosome XIV from budding yeast between the ORC5 and the SUI1 gene . Subsequent sequence analysis revealed the presence of 18 non-overlapping open reading frames (ORFs) including eight previously identified and sequenced genes (ORC5, ATX1, SIP3, NRD1, RAD50, MPA43, RPA49 and SUI1) . Three other ORFs (YNL256w, YNL255c and YNL247w) code for putative proteins with significant homology to proteins from other organisms, while 4 ORFs exhibit only weak homology to known proteins . Three ORFs have no homology with sequences in the databases.

Yeast, 1997 Jul, 13(9), 829 - 36
Cloning, sequencing and expression of a full-length cDNA copy of the M1 double-stranded RNA virus from the yeast, Saccharomyces cerevisiae; Russell PJ et al.; Strains of the budding yeast, Saccharomyces cerevisiae, may contain one or more cytoplasmic viruses with double-stranded RNA (dsRNA) genomes . The killer phenomenon in yeast, in which one cell secretes a killer toxin that is lethal to another cell, is dependent upon the presence of the L-A and M1 dsRNA viruses . The L-A viral genome encodes proteins for the viral capsid, and for synthesis and encapsidation of single-stranded RNA replication cycle intermediates . The M1 virus depends upon the L-A-encoded proteins for its capsid and for the replication of its killer-toxin-encoding genome . A full-length cDNA clone of an M genome has been made from a single dsRNA molecule and shown to encode functional killer and killer-immunity functions . The sequence of the clone indicates minor differences from previously published sequences of parts of the M1 genome and of the complete genome of S14 (an internal deletion derivative of M1) but no unreported amino acid variants and no changes in putative secondary structures of the single-stranded RNA . A 118-nucleotide contiguous segment of the M1 genome has not previously been reported; 92 of those nucleotides comprise a segment of A nucleotides in the AU-rich bubble that follows the toxin-encoding reading frame.

Yeast, 1997 Jul, 13(9), 795 - 807
Characterization of CHS4 (CAL2), a gene of Saccharomyces cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4; Trilla JA et al.; We have cloned CHS4, a gene that complements the resistance to Calcofluor of the Saccharomyces cerevisiae cal2 mutant . We show that CHS4 is allelic to the previously described SKT5 and CSD4 genes . CHS4 encodes a 696 amino acids protein with no potential transmembrane domain . chs4-null mutants are resistant to Calcofluor white and exhibit a considerable reduction in cell wall chitin and in chitin synthase III (CSIII) activity . Biochemical characterization of chitin synthase III from these null mutants indicates that the defect is due to a reduced V(max) of the enzyme . This defect can be overcome in vitro by trypsin treatment of the membrane preparations . Chs4p does not act as a transcriptional or translational regulator of CHS3, the gene coding for the catalytic subunit of CSIII activity, and we therefore propose that Chs4p would be an essential component of the CSIII complex, acting as a post-translational regulator of this activity . In addition to the chitin defect, the chs4 mutant shows a severe defect in mating.

FEMS Microbiol Lett, 1997 Jul 1, 152(1), 17 - 21
Effect of hydrostatic pressure of a mutant of Saccharomyces cerevisiae deleted in the trehalose-6-phosphate synthase gene; Fernandes PM et al.; Mutants Saccharomyces cerevisiae deleted on the trehalose-6-phosphate synthase gene (tps1) and their parental wild-type cells were submitted to hydrostatic pressure in the range of 0-200 MPa . Experimental evidence showed that viability for both strains decreased with increasing pressure and that tps1 mutants, unable to accumulate trehalose, were more sensitive to hydrostatic pressure than the wild-type cells . Additionally, both tps1 and wild-type cells in the stationary phase, when there is an accumulation of endogenous trehalose, were more resistant to pressure than proliferating cells . Under these conditions, mutant cells were also more sensitive to pressure treatment than the wild type . The present work also showed that mild pressure pretreatment did not induce hydrostatic pressure resistance (barotolerance) in yeast cells.

FEMS Microbiol Lett, 1997 Jul 1, 152(1), 11 - 5
A possible role of trehalose in osmotolerance and ethanol tolerance in Saccharomyces cerevisiae; Sharma SC; The effect of salt stress on ethanol endurance of yeast cells was studied . Cells grown under increased NaCl concentrations were more ethanol tolerant than controls . The increase in trehalose content under hyper-saline conditions has been suggested to allow cells to withstand higher ethanolic conditions . There seems to be an overlap between osmotolerance and ethanol endurance in Saccharomyces cerevisiae.

Protein Expr Purif, 1997 Jul, 10(2), 209 - 13
Overexpression and purification of elongation factor 3 from Saccharomyces cerevisiae; Kambampati R et al.; The translational elongation factor 3 (EF-3) from Saccharomyces cerevisiae was overexpressed and purified to near homogeneity from the post-ribosomal supernatant fraction (S-100) . A detailed protocol for the isolation of overexpressed EF-3 is presented . The procedure involves ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose and affinity chromatography on ATP-agarose . A protein purity of > or = 96% was established by quantitating the silver-stained SDS/polyacrylamide gel . The present method facilitates isolation of EF-3 in large amounts in high yield.

Genes Dev, 1997 Jul 1, 11(13), 1690 - 702
Differential regulation of FUS3 MAP kinase by tyrosine-specific phosphatases PTP2/PTP3 and dual-specificity phosphatase MSG5 in Saccharomyces cerevisiae; Zhan XL et al.; The Saccharomyces cerevisiae mating pheromone response is mediated by activation of a MAP kinase (Fus3p and Kss1p) signaling pathway . Pheromone stimulation causes cell cycle arrest . Therefore, inactivation of the Fus3p and Kss1p MAP kinases is required during recovery phase for the resumption of cell growth . We have isolated a novel protein tyrosine phosphatase gene, PTP3, as a negative regulator of this pathway . Ptp3p directly dephosphorylates and inactivates Fus3p MAP kinase in vitro . Multicopy PTP3 represses pheromone-induced transcription and promotes recovery . In contrast, disruption of PTP3 in combination with its homolog PTP2 results in constitutive tyrosine phosphorylation, enhanced kinase activity of Fus3p MAP kinase on stimulation, and delayed recovery from the cell cycle arrest . Both tyrosine phosphorylation and kinase activity of Fus3p are further increased by disruption of PTP3 and PTP2 in combination with MSG5, which encodes a dual-specificity phosphatase . Cells deleted for all three of the phosphatases (ptp2delta ptp3delta msg5delta) are hypersensitive to pheromone and exhibit a severe defect in recovery from pheromone-induced growth arrest . Our data indicate that Ptp3p is the major phosphatase responsible for tyrosine dephosphorylation of Fus3p to maintain a low basal activity; it also has important roles, along with Msg5p, in inactivation of Fus3p following pheromone stimulation . These data present the first evidence for a coordinated regulation of MAP kinase function through differential actions of protein tyrosine phosphatases and a dual-specificity phosphatase.

Genetics, 1997 Jul, 146(3), 849 - 57
Saccharomyces cerevisiae PAC2 functions with CIN1, 2 and 4 in a pathway leading to normal microtubule stability; Hoyt MA et al.; The products of the Saccharomyces cerevisiae CIN1, CIN2 and CIN4 genes participate in a nonessential pathway required for normal microtubule function . In this article, we demonstrate that the product of PAC2 also functions in this pathway . PAC2 deletion mutants displayed phenotypes and genetic interactions similar to those caused by cin1 delta, cin2 delta and cin4 delta . These include cold-sensitive microtubule structures and sensitivity to the microtubule depolymerizing agent benomyl . Involvement in a common functional pathway is indicated by the observation that all double mutant recombinations are viable and no more affected than any single mutant . In addition, extra copies of CIN1 were found to suppress the benomyl sensitivity of pac2 delta, cin2 delta and cin4 delta, but not that caused by other mutations that affect microtubule function . Cin1p and Pac2p were found to be related in sequence to mammalian proteins that aid in the folding of beta-tubulin into an assembly-competent state . Alleles of CIN1 were identified that could suppress the benomyl sensitivity of cin4-4 in a highly specific fashion . Our findings suggest that the guanine nucleotide-binding Cin4p interacts with Cin1p and regulates its tubulin folding activity.

Genetics, 1997 Jul, 146(3), 817 - 34
Mutations in Saccharomyces cerevisiae that block meiotic prophase chromosome metabolism and confer cell cycle arrest at pachytene identify two new meiosis-specific genes SAE1 and SAE3; McKee AH et al.; Two new meiosis-specific genes, SAE1 and SAE3, have been identified in a screen for mutations that confer an intermediate block in meiotic prophase . Such mutations confer a block to spore formation that is circumvented by addition of a mutation that eliminates meiotic recombination initiation and other aspects of chromosome metabolism, i.e., spo11 . We show that sae1-1 and sae3-1 mutations each confer a distinct defect in meiotic recombination . sae1-1 produces recombinants but very slowly and ultimately to less than half the wild-type level; sae3-1 makes persistent hyper-resected meiotic double-strand breaks and has a severe defect in formation of recombinants . Both mutants arrest at the pachytene stage of meiotic prophase, sae1-1 temporarily and sae3-1 permanently . The phenotypes conferred by sae3-1 are similar to those conferred by mutation of the yeast RecA homologue DMC1, suggesting that SAE3 and DMC1 act at the same step(s) of chromosome metabolism . These results provide further evidence that intermediate blocks to prophase chromosome metabolism cause cell-cycle arrest . SAE1 encodes a 208-residue protein homologous to vertebrate mRNA cap-binding protein 20 . SAE3 corresponds to a meiosis-specific RNA encoding an unusually short open reading frame of 50 codons.

Genetics, 1997 Jul, 146(3), 797 - 816
A general method for identifying recessive diploid-specific mutations in Saccharomyces cerevisiae, its application to the isolation of mutants blocked at intermediate stages of meiotic prophase and characterization of a new gene SAE2; McKee AH et al.; We describe a general new approach for identifying recessive mutations that affect diploid strains of yeast Saccharomyces cerevisiae and the application of this method to the identification of mutations that confer an intermediate block in meiotic prophase chromosome metabolism . The method uses a temperature-sensitive conjugation mutation ste7-1 in combination with homothallism . The mutations of interest confer a defect in spore formation that is dependent upon a gene required for initiation of meiotic recombination and development of meiosis-specific chromosome structure (SPO11) . Identified in this screen were null mutations of the DMC1 gene, nonnull mutations of RAD50 (rad50S), and mutations in three new genes designed SAE1, SAE2 and SAE3 (Sporulation in the Absence of Spo Eleven) . Molecular characterization of the SAE2 gene and characterization of meiotic and mitotic phenotypes of sae2 mutants are also presented . The phenotypes conferred by a sae2 null mutation are virtually indistinguishable from those conferred by the previously identified nonnull mutations of RAD50 (rad50S) . Most notably, both mutations confer only weak sensitivity to the radiomimetic agent methyl methane sulfonate (MMS) but completely block resection and turnover of meiosis-specific double-strand breaks . These observations provide further evidence that this constellation of phenotypes identifies a specific molecular function.

Genetics, 1997 Jul, 146(3), 781 - 95
Isolation of COM1, a new gene required to complete meiotic double-strand break-induced recombination in Saccharomyces cerevisiae; Prinz S et al.; We have designed a screen to isolate mutants defective during a specific part of meiotic prophase I of the yeast Saccharomyces cerevisiae . Genes required for the repair of meiotic double-strand breaks or for the separation of recombined chromosomes are targets of this mutant hunt . The specificity is achieved by selecting for mutants that produce viable spores when recombination and reductional segregation are prevented by mutations in SPO11 and SPO13 genes, but fail to yield viable spores during a normal Rec+ meiosis . We have identified and characterized a mutation com1-1, which blocks processing of meiotic double-strand breaks and which interferes with synaptonemal complex formation, homologous pairing and, as a consequence, spore viability after induction of meiotic recombination . The COM1/SAE2 gene was cloned by complementation, and the deletion mutant has a phenotype similar to com1-1, com1/sae2 mutants closely resemble the phenotype of rad50S, as assayed by phase-contrast microscopy for spore formation, physical and genetic analysis of recombination, fluorescence in situ hybridization to quantify homologous pairing and immunofluorescence and electron microscopy to determine the capability to synapse axial elements.

Appl Environ Microbiol, 1997 Jul, 63(7), 2857 - 62
Thiram and dimethyldithiocarbamic acid interconversion in Saccharomyces cerevisiae: a possible metabolic pathway under the control of the glutathione redox cycle; Elskens MT et al.; A rapid decrease of intracellular glutathione (GSH) was observed when exponentially growing cells of Saccharomyces cerevisiae were treated with sublethal concentrations of either dimethyldithiocarbamic acid or thiram {bis(dimethylthiocarbamoyl) disulfide} . The underlying mechanism of this effect possibly involves the intracellular oxidation of dimethyldithiocarbamate anions to thiram, which in turn oxidizes GSH . Overall, a linear relationship was found between thiram concentrations up to 21 microM and production of oxidized GSH (GSSG) . Cytochrome c can serve as the final electron acceptor for dimethyldithiocarbamate reoxidation, and it was demonstrated in vitro that NADPH handles the final electron transfer from GSSG to the fungicide by glutathione reductase . These cycling reactions induce transient alterations in the intracellular redox state of several electron carriers and interfere with the respiration of the yeast . Thiram and dimethyldithiocarbamic acid also inactivate yeast glutathione reductase when the fungicide is present within the cells as the disulfide . Hence, whenever the GSH regeneration rate falls below its oxidation rate, the GSH:GSSG molar ratio drops from 45 to 1 . Inhibition of glutathione reductase may be responsible for the saturation kinetics observed in rates of thiram elimination and uptake by the yeast . The data suggest also a leading role for the GSH redox cycle in the control of thiram and dimethyldithiocarbamic acid fungitoxicity . Possible pathways for the handling of thiram and dimethyldithiocarbamic acid by yeast are considered with respect to the physiological status, the GSH content, and the activity of glutathione reductase of the cells.

J Bacteriol, 1997 Jul, 179(13), 4415 - 8
Mck1, a member of the glycogen synthase kinase 3 family of protein kinases, is a negative regulator of pyruvate kinase in the yeast Saccharomyces cerevisiae; Brazill DT et al.; An interaction between the Saccharomyces cerevisiae protein kinase Mck1 and pyruvate kinase (Pyk1) was detected by using the two-hybrid method . Purified Mck1 was able to phosphorylate purified Pyk1 on Ser in vitro . Pyruvate kinase activity was elevated in mck1 delta cells . Several of the phenotypes of mck1 delta mutants are similar to those observed in cells overexpressing PYK1 . Co-overexpression of MCK1 suppressed all of the phenotypes associated with PYK1 overexpression . These results indicate that Mck1 negatively regulates pyruvate kinase activity, possibly by direct phosphorylation.

Mol Cell Biol, 1997 Jul, 17(7), 4178 - 90
The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein; Wittmeyer J et al.; We have used DNA polymerase alpha affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomyces cerevisiae . Two proteins that bound to the catalytic subunit of DNA polymerase alpha (Pol1 protein) are encoded by the essential genes CDC68/SPT16 and POB3 . The binding of both proteins was enhanced when extracts lacking a previously characterized polymerase binding protein, Ctf4, were used . This finding suggests that Cdc68 and Pob3 may compete with Ctf4 for binding to Pol1 . Pol1 and Pob3 were coimmunoprecipitated from whole-cell extracts with antiserum directed against Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum directed against the amino terminus of Pob3, suggesting that these proteins may form a complex in vivo . CDC68 also interacted genetically with POL1 and CTF4 mutations; the maximum permissive temperature of double mutants was lower than for any single mutant . Overexpression of Cdc68 in a pol1 mutant strain dramatically decreased cell viability, consistent with the formation or modulation of an essential complex by these proteins in vivo . A mutation in CDC68/SPT16 had previously been shown to cause pleiotropic effects on the regulation of transcription (J . A . Prendergrast et al., Genetics 124:81-90, 1990; E . A . Malone et al., Mol . Cell . Biol . 11:5710-5717, 1991; A . Rowley et al., Mol . Cell . Biol . 11:5718-5726, 1991), with a spectrum of phenotypes similar to those caused by mutations in the genes encoding histone proteins H2A and H2B (Malone et al., Mol . Cell . Biol . 11:5710-5717, 1991) . We show that at the nonpermissive temperature, cdc68-1 mutants arrest as unbudded cells with a 1C DNA content, consistent with a possible role for Cdc68 in the prereplicative stage of the cell cycle . The cdc68-1 mutation caused elevated rates of chromosome fragment loss, a phenotype characteristic of genes whose native products are required for normal DNA metabolism . However, this mutation did not affect the rate of loss or recombination for two intact chromosomes, nor did it affect the retention of a low-copy-number plasmid . The previously uncharacterized Pob3 sequence has significant amino acid sequence similarity with an HMG1-like protein from vertebrates . Based on these results and because Cdc68 has been implicated as a regulator of chromatin structure, we postulate that polymerase alpha may interact with these proteins to gain access to its template or to origins of replication in vivo.

Mol Cell Biol, 1997 Jul, 17(7), 3580 - 8
Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae; Weidenhammer EM et al.; The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae . Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions . Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex . Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs . These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.

FEBS Lett, 1997 Jun 30, 410(2-3), 219 - 22
Mitochondrial function is required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae; Grant CM et al.; Yeast strains that lack mitochondrial function are sensitive to oxidative stress caused by reactive oxygen species (ROS) . Specifically, rho0 mutants that lack mitochondrial DNA, and strains deleted for the nuclear genes COX6 and COQ3 that are required for function of the respiratory electron transport chain, were sensitive to H2O2 . In addition, treatment with mitochondrial inhibitors including antimycin A, oligomycin, potassium cyanide and sodium azide increased sensitivity to H2O2 . The mechanism does not appear to depend on the antioxidant status of the cell since respiratory-deficient strains were able to mount an inducible adaptive response to H2O2 . We suggest that the oxidant sensitivity is due to a defect in an energy-requiring process that is needed for detoxification of ROS or for the repair of oxidatively damaged molecules.

Yeast, 1997 Jun 30, 13(8), 735 - 45
Characterization of the checkpoint gene RAD53/MEC2 in Saccharomyces cerevisiae; Kim S et al.; Saccharomyces cerevisiae cells carrying mutations in RAD53/MEC2 fail to arrest in the S phase when DNA replication is blocked (the S/M checkpoint) or in the G2 phase when DNA is damaged (the G2/M checkpoint) . We isolated and determined the DNA sequence of RAD53 and found that it is identical to the SPK1 gene previously identified by Stern et al . (1991) . In addition to its checkpoint functions, we show here that RAD53 is essential for cell viability because null mutants are inviable . Weak genomic suppressors of the essential function do arise frequently, though they do not suppress the checkpoint defects of the null mutant . This genetically separates the essential and checkpoint functions . We show genetically that the protein kinase domain is essential for all RAD53-dependent functions tested because a site-specific mutation that inactivates the protein kinase activity results in a mutant phenotype indistinguishable from that of a null mutant . Overexpression of RAD53, or its kinase domain alone, resulted in a delay in cell-cycle progression that required the intact kinase function . The cell-cycle delay did not require any of the checkpoint genes tested (e.g . rad9 or mecl), indicating that the cell-cycle delay is either unrelated to the checkpoint responses, or that it occurs constitutively because RAD53 acts further downstream of the checkpoint genes tested . Finally, elimination of sequences in the promoter region of RAD53 revealed complex regulatory elements.

Yeast, 1997 Jun 30, 13(8), 717 - 25
Constitutive flocculation in Saccharomyces cerevisiae through overexpression of the GTS1 gene, coding for a 'Glo'-type Zn-finger-containing protein; Bossier P et al.; The product of the cloned GTS1 gene is characterized by structural features found in transcription factors . It contains one Zn-finger motif (CXXCX16CXXC) situated in the N-terminal end with a high degree of homology to the newly identified 'Glo' family of Zn-finger proteins (Ireland et al., 1994, EMBO J . 13, 3812-3821) . The C-terminal end of the protein is characterized by poly (Ala-Gln) and poly-Gln stretches . Poly-Gln are part of trans-acting motifs in known transcription factors . Overexpression of the GTS1 gene results in constitutive flocculation . Whole cell electrophoretic mobility and hydrophobicity of GTS1 overexpressing cells was respectively lower and higher relative to control cells . GTS1-induced flocculation is hardly sensitive to mannose in contrast to FLO1-determined flocculation . Overexpression of the GTS1 gene in a flo1 background does not abolish flocculation, suggesting that the FLO1 gene is not linked with the GTS1 gene in a 'flocculation pathway'.

Yeast, 1997 Jun 30, 13(8), 707 - 15
Deregulation of CLN1 and CLN2 in the Saccharomyces cerevisiae whi2 mutant; Radcliffe P et al.; Wild-type cells of the budding yeast Saccharbmyces cerevisiae arrest in G1 upon nutrient exhaustion . Cell cycle arrest requires the WHI2 gene since whi2 mutants continue to divide and become abnormally small as nutrients are depleted . Here we show that CLN1 and CLN2 transcript levels in a whi2 strain are higher during exponential growth, and persist longer upon starvation, than in an isogenic wild-type strain . In contrast to CLN1 and CLN2, CLN3 levels declined only at very high cell density and were unaffected by the whi2 mutation . Elevated CLN expression is sufficient to explain the whi2 phenotype since ectopic expression of CLN1 in a nutrient-depleted culture caused cells to continue dividing and interfered with the acquisition of heat resistance . These observations show that, either directly or indirectly, Whi2 negatively regulates G1 cyclin expression . Interestingly extremely high levels of Cln1 induced filamentous growth upon nutrient deprivation, suggesting a direct connection between G1 cyclin activity and morphological responses to poor nutrient conditions.

J Cell Biol, 1997 Jun 30, 137(7), 1483 - 93
The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon in Saccharomyces cerevisiae; Corsi AK et al.; We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae . A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP . This domain, purified as a fusion protein (63Jp) with glutathione S-transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP . The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein . GST alone was inactive in these assays . Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP . Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein . We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.

J Cell Biol, 1997 Jun 30, 137(7), 1469 - 82
Physiological regulation of membrane protein sorting late in the secretory pathway of Saccharomyces cerevisiae; Roberg KJ et al.; In mammalian cells, extracellular signals can regulate the delivery of particular proteins to the plasma membrane . We have discovered a novel example of regulated protein sorting in the late secretory pathway of Saccharomyces cerevisiae . In yeast cells grown on either ammonia or urea medium, the general amino acid permease (Gap1p) is transported from the Golgi complex to the plasma membrane, whereas, in cells grown on glutamate medium, Gap1p is transported from the Golgi to the vacuole . We have also found that sorting of Gap1p in the Golgi is controlled by SEC13, a gene previously shown to encode a component of the COPII vesicle coat . In sec13 mutants grown on ammonia, Gap1p is transported from the Golgi to the vacuole, instead of to the plasma membrane . Deletion of PEP12, a gene required for vesicular transport from the Golgi to the prevacuolar compartment, counteracts the effect of the sec13 mutation and partially restores Gap1p transport to the plasma membrane . Together, these studies demonstrate that both a nitrogen-sensing mechanism and Sec13p control Gap1p transport from the Golgi to the plasma membrane.

J Mol Biol, 1997 Jun 27, 269(5), 676 - 83
In vivo analyses of upstream promoter sequence elements in the 5 S rRNA gene from Saccharomyces cerevisiae; Lee Y et al.; Upstream promoter elements of the Saccharomyces cerevisiae 5 S rRNA gene have been characterized by genomic DNase I "footprinting" and by in vivo mutational analyses using base substitutions and deletions . A high copy shuttle-vector was used to efficiently express the mutant 5 S rRNA genes in vivo and a structural mutation in the 5 S rRNA, which was previously shown to be functionally neutral but easily detected by gel electrophoresis, allowed for an accurate measure of gene expression . The results provide direct evidence for upstream regulatory elements which confirms a start site element (sse) from -1 to -8 and identifies a new independent upstream promoter element (upe) centered from about -17 to -20 . In contrast to previous reports with reconstituted systems, both elements dramatically affect the efficiency of gene expression and suggest that the saturated conditions which are used in reconstituted studies mask sequence dependence; a dependency that could be physiologically significant and play a role in the regulation of 5 S rRNA expression . The footprint analyses support an extended region of protein interaction as recently observed in reconstituted systems but again provide evidence of significant structural rearrangements when the upstream sequence is changed.

Biochem Biophys Res Commun, 1997 Jun 27, 235(3), 799 - 805
Structural modifications of RNA influence the 5' exoribonucleolytic hydrolysis by XRN1 and HKE1 of Saccharomyces cerevisiae; Poole TL et al.; Two 5' exoribonucleases, XRN1 and HKE1, of Saccharomyces cerevisiae have been found to have very important cellular roles, XRN1 playing a key role in mRNA turnover and HKE1 in pre-rRNA processing . Here, an analysis of strong secondary structures in RNA that cause blocks or stalls (accumulation of RNA fragments that are shortened from the 5' end to the site of the secondary structure insertion) in the processive exoribonucleolytic hydrolysis reactions is reported . With both enzymes, oligo(G) tracts of lengths 18, 16, and 9 stall quite effectively, and the stalls are close to the start of the oligo(G) stretch . Two strong stem-loop structures cause measurable but low-level stalls with both enzymes . If the stem-loop structure is placed close to the 5' end of the RNA, substantial inhibition of overall RNA hydrolysis occurs with HKE1 and less, but measurable, inhibition with XRN1 . RNA structural modification caused by protein complexing has been investigated by using poly(A) binding protein . The hydrolysis of poly(A) by XRN1 is inhibited by poly(A) binding protein, while HKE1 activity is not affected.

Biochem Biophys Res Commun, 1997 Jun 27, 235(3), 669 - 74
Evaluation of clinical and environmental anti-estrogens with human estrogen receptor expressed in Saccharomyces cerevisiae: a novel role for ABC-cassette transporters in mediating anti-estrogenic activity; Tran DQ et al.; The effectiveness of anti-estrogens in treating estrogen-dependent diseases is limited by the acquired resistance of some diseases to anti-estrogens . This effect could occur by the export of anti-estrogens by cell membrane transport proteins . To study this phenomenon we have expressed human estrogen receptor (hER) and an estrogen-sensitive reporter in wild-type yeast and two transport-defective strains . In the wild-type strain, the most effective anti-estrogen was nafoxidine . 4-Hydroxy tamoxifen and clomiphene were inactive whereas tamoxifen had significant inhibitory activity in the wild-type strain . Using a strain missing the ABC-cassette transporter Snq2, clomiphene had anti-estrogenic activity . 4-Hydroxy tamoxifen had anti-estrogenic activity only in yeast lacking the transporter Pdr5 . Whole cell binding assays indicated that 4-hydroxy tamoxifen is exported by Pdr5 . Environmental chemicals such as polychlorinated biphenyls function as partial estrogens and anti-estrogens in yeast . In the absence of Pdr5 or Snq2, the estrogenic activity of 4-hydroxy, 2',4',6'-trichloro biphenyl (3-PCB) was substantially reduced in comparison to its activity in the wild-type strain . Interestingly, the antiestrogenic activity of 3-PCB was equivalent in the wild-type and transporter-defective strains . Our results suggest a novel role for ABC-cassette transporters in regulating the activity of clinical and environmental anti-estrogens.

J Biol Chem, 1997 Jun 27, 272(26), 16103 - 9
Ssd1p of Saccharomyces cerevisiae associates with RNA; Uesono Y et al.; The SSD1 gene has been isolated as a single copy suppressor of many mutants, such as sit4, slk1/bck1, pde2, and rpc31, in the yeast Saccharomyces cerevisiae . Ssd1p has domains showing weak but significant homology with RNase II-related proteins, Cyt4p, Dss1p, VacB, and RNase II, which are involved in the modification of RNA . We found that Ssd1p had the ability to bind RNA, preferably poly(rA), as well as single-stranded DNA . Interestingly, the most conserved domain among the RNase II-related proteins was not necessary for interaction with RNA . Indirect immunofluorescence staining with anti-Ssd1p antibody revealed that Ssd1p was detected mainly in the cytoplasm . Furthermore, sucrose gradient sedimentation analysis demonstrated that Ssd1p was not cofractionated with polyribosomes, suggesting that Ssd1p is not particularly bound to a translationally active subpopulation of mRNA in the cytoplasm.

Proc Natl Acad Sci U S A, 1997 Jun 24, 94(13), 6730 - 4
Similar processes mediate glycopeptide export from the endoplasmic reticulum in mammalian cells and Saccharomyces cerevisiae; Romisch K et al.; Glycopeptides are transported from the lumen of the yeast endoplasmic reticulum (ER) to the cytosol and in contrast to secretory proteins do not enter ER-to-Golgi transport vesicles . In a cell-free system, this process is ATP- and cytosol-dependent . While yeast cytosol promotes the export of glycopeptides from mammalian ER in vitro, glycopeptide release cannot be detected in the presence of mammalian cytosol . We demonstrate that this is due to an N-glycanase activity in mammalian cytosol rather than lack of glycopeptide transport activity in mammalian microsomes . Monitoring the amount of glycopeptide enclosed in ER membranes we show the cytosol- and ATP-dependent release of glycopeptide from mammalian microsomes . The fact that glycopeptide export can be achieved with ER and cytosol derived from heterologous sources indicates that glycopeptide export from the ER is an important process conserved during evolution.

J Biol Chem, 1997 Jun 20, 272(25), 15936 - 42
Promoter structure-dependent functioning of the general transcription factor IIE in Saccharomyces cerevisiae; Sakurai H et al.; General transcription factor (TF) IIE is an essential component of the basal transcription complex for protein-encoding genes, which is widely conserved in eukaryotes . Here we analyzed requirement for TFIIE for transcription in vivo by using yeast Saccharomyces cerevisiae cells harboring mutations in the TFA1 gene encoding the larger one of the two subunits of TFIIE . Deletion analysis indicated that the N-terminal half of Tfa1 protein has an essential function to support the cell growth . In a temperature-sensitive tfa1 mutant cell, the steady-state level of bulk poly(A)+ RNA decreased rapidly at the restrictive temperature . Surprisingly, levels of several mRNAs, whose transcription is directed by the promoters lacking the typical TATA sequence, were not affected in the mutant cells at that temperature . This promoter-specific functioning of TFIIE was reproduced in a cell-free system composed of TFIIE-depleted nuclear extracts . These results strongly suggest that requirement for TFIIE varies in each gene depending on the promoter structures in vivo.

J Biol Chem, 1997 Jun 20, 272(25), 15587 - 90
Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs; Chong S et al.; The protein splicing element (intein) of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae catalyzes both protein splicing and site-specific DNA cleavage . It has been demonstrated that the conserved splice junction residues are directly involved in protein splicing and the central dodecapeptide motifs are required for DNA cleavage . To examine whether the splicing activity of the intein can be structurally separated from the endonuclease motifs, we made large in-frame deletions at the central region of the intein . We demonstrate for the first time that protein splicing can proceed efficiently after the removal of the central region of the intein including the endonuclease motifs . Our results suggest that the N- and C-terminal regions of the Sce VMA intein may form a separate domain that is not only catalytically sufficient for protein splicing but also structurally independent from the endonuclease domain.

Gene, 1997 Jun 19, 192(2), 245 - 50
Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae; Matsuura A et al.; Autophagic protein degradation includes bulk protein turnover with dynamic membrane reorganization, in which formation of novel organelles autophagosomes play key roles . We have shown that Saccharomyces cerevisiae performs the autophagy in the vacuole, a lytic compartment of yeast, in response to various kinds of nutrient starvation . Here we show that the APG1 gene, involved in the autophagic process in yeast, encodes a novel type of Ser/Thr protein kinase . Our results provide direct evidence for involvement of protein phosphorylation in regulation of the autophagic process . We found overall homology of Apglp with C . elegans Unc-51 protein, suggesting that homologous molecular mechanisms, conserved from unicellular to multicellular organisms, are involved in dynamic membrane flow.

Gene, 1997 Jun 19, 192(2), 207 - 13
Analyses of APG13 gene involved in autophagy in yeast, Saccharomyces cerevisiae; Funakoshi T et al.; We have isolated 14 apg mutants defective in autophagy in yeast Saccharomyces cerevisiae (Tsukada and Ohsumi, 1993) . Among them, APG1 encodes a novel Ser/Thr protein kinase whose kinase activity is essential for autophagy . In the course of searching for genes that genetically interact with APG1, we found that overexpression of APG1 under control of the GAL1 promoter suppressed the autophagy-defective phenotype of apg13-1 mutant . Cloning and sequencing analysis showed that the APG13 gene encodes a novel hydrophilic protein of 738 amino acid residues . APG13 gene is constitutively expressed bot not starvation-inducible . Though dispensable for cell proliferation, APG13 is important for maintenance of cell viability under starvation conditions . apg13 disruptants were defective in autophagy like apg13-1 mutants . Morphological and biochemical investigation showed that a defect in autophagy of delta apg13 was also suppressed by APG1 overexpression . These results imply genetic interaction between APG1 and APG13.

FEMS Microbiol Lett, 1997 Jun 15, 151(2), 131 - 3
GAP1 activity is dependent on cAMP in Saccharomyces cerevisiae; Amitrano AA et al.; General amino acid permease (GAP1) activity was evaluated in adenylate cyclase-deficient Saccharomyces cerevisiae to determine the effect of cAMP levels on GAP1 activity . Lowering cAMP concentrations in the culture media led to a decrease in the initial rates of L-citrulline uptake . Kinetics of the amino acid transport system showed a partial loss of transport capacity, with no apparent modifications in permease affinity.

Yeast, 1997 Jun 15, 13(7), 621 - 37
The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae; Casas C et al.; High-affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases . Ferrous ions are then transported across the plasma membrane through the FET3 oxidase-FTR1 permease complex . Expression of the high-affinity iron uptake genes is induced upon iron deprivation . We demonstrate that AFT1 is differentially involved in such regulation . Aft1 protein is required for maintaining detectable non-induced level of FET3 expression and for induction of FRE2 in iron starvation conditions . On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi-Iwai et al., EMBO J . 14: 1231-1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way . The alterations in the basal levels of expression of the high-affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources . Overexpression of AFT1 leads to growth arrest of the G1 stage of the cell cycle . Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high-affinity iron uptake genes . Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirement for cell growth.

Yeast, 1997 Jun 15, 13(7), 599 - 612
Reconstitution of the NF-kappa B system in Saccharomyces cerevisiae for isolation of effectors by phenotype modulation; Epinat JC et al.; NF-kappa B is a ubiquitous transcription factor that contributes to the induction of many genes playing a central role in immune and inflammatory responses . The NF-kappa B proteins are subject to multiple regulatory influences including post-translational modifications such as phosphorylation and proteolytic processing . A very important component of this regulation is the control of their subcellular localization: cytoplasmic retention of NF-kappa B is achieved through interaction with I kappa B molecules . In response to extracellular signals, these molecules undergo degradation, NF-kappa B translocates to the nucleus and activates its target genes . To investigate novel proteins involved in this dynamic response, we have reconstituted the NF-kappa B/I kappa Beta system in the yeast Saccharomyces cerevisiae . We have successively introduced p65, the main transcriptional activator of the NF-kappa B family, which leads to the activation of two reporter genes controlled by kappa B sites, and the I kappa B alpha inhibitory protein, which abolishes this activation . By transforming such a yeast strain with a cDNA library we have performed a genetic screen for cDNAs encoding proteins capable of either dissociating the p65/I kappa B alpha complex or directly transactivating the expression of the reporter genes . The efficiency of our screen was demonstrated by the isolation of a cDNA encoding the p105 precursor of the p50 subunit of NF-kappa B . We also used this system to test stimuli known to activate signalling pathways in yeast, in order to investigate whether the related mammalian cascades might be involved in NF-kappa B activation . We showed that yeast endogenous kinase cascades activated by pheromone, hypo- or hyperosmotic shock cannot modulate NF-kappa B activity in our system, and that the p38 human MAP kinase does not act directly on the p65/I kappa B alpha complex.

J Biol Chem, 1997 Jun 13, 272(24), 15553 - 61
Expression and purification of the Saccharomyces cerevisiae alpha-factor receptor (Ste2p), a 7-transmembrane-segment G protein-coupled receptor; David NE et al.; A plasmid vector was developed that permitted high-level expression of a functional form of the Saccharomyces cerevisiae alpha-factor receptor (the STE2 gene product) tagged at its C-terminal end with an epitope (FLAG) and a His6 tract . When expressed in yeast from this plasmid, Ste2p was produced at a level at least 3-fold higher than that reported previously for any other 7-transmembrane-segment receptor expressed in the same cells . For purification, isolated cell membranes containing the overexpressed receptor were solubilized with detergent under specific conditions and subjected to immobilized metal affinity chromatography . Yields as high as 1 mg of nearly homogeneous (95%) receptor were routinely obtained even from relatively small scale preparations (60 g of frozen cell paste) . The purified receptor was reconstituted into artificial phospholipid vesicles . Radioligand binding studies demonstrated that the purified receptor, in the reconstituted vesicles, bound its tridecapeptide ligand (alpha-factor) with a KD (155 nM) consistent with the affinity expected for this receptor in the absence of its associated G protein . Efficient restoration of ligand binding activity upon reconstitution required the addition of solubilized membranes prepared from a yeast strain lacking the receptor . Sufficient amounts of active material can be obtained by this procedure to allow physical studies of this receptor and other 7-transmembrane-segment receptors expressed in this system.

J Biol Chem, 1997 Jun 13, 272(24), 15206 - 12
Ribosomal 5 S rRNA maturation in Saccharomyces cerevisiae; Lee Y et al.; The maturation of the ribosomal 5 S RNA in Saccharomyces cerevisiae is examined based on the expression of mutant 5 S rRNA genes, in vivo, and a parallel analysis of RNA processing, in vitro . Both types of analysis indicate that 5 S rRNA processing is not dependent on the nucleotide sequence of either the external transcribed spacer or the mature 5 S rRNA . The results further indicate the RNA is processed by an exonuclease activity which is limited primarily or entirely by helix I, the secondary structure formed between the mature and interacting termini . The 5 S RNA binding protein (YL3) also appears not to influence directly the maturation process, but rather to play a role in protecting the rRNA from further degradation by "housekeeping" nucleases . Taken together, the results continue to support a "quality control" function which helps to ensure that during maturation only normal precursors are processed and assembled into active ribosomes.

Mutat Res, 1997 Jun 9, 384(1), 33 - 44
Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae; Kupiec M et al.; Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms . We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events . We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced . We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents . We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes . We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction . Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination . We discuss the mechanistic and evolutionary aspects of these results.

J Biol Chem, 1997 Jun 6, 272(23), 14683 - 9
Structure-function analysis of the mRNA cap methyltransferase of Saccharomyces cerevisiae; Wang SP et al.; The Saccharomyces cerevisiae mRNA cap methylating enzyme is a 436-amino acid protein encoded by the essential ABD1 gene . To identify structural features of ABD1 required for enzyme function, we introduced alanine mutations at 19 positions within a 205-amino acid region of similarity to the methyltransferase domain of the vaccinia capping enzyme . Three new recessive lethal mutations, E170A, D194A, and R206A, were identified . Structure-function relationships were clarified by introducing conservative substitutions at Glu-170, Asp-194, and Arg-206, and at Tyr-254 (an essential residue identified previously) . Alleles E170D and D194E were viable, whereas E170Q and D194N were lethal; hence, acidic side chains were critical at both positions . R206K was viable, suggesting that a basic residue sufficed . Y254S was lethal, whereas Y254F was viable, albeit slow growing; thus, an aromatic side chain was important . The ABD1 mutations that were deleterious in vivo elicited catalytic defects in vitro . By studying the effects of amino- and carboxyl-terminal deletions, we defined a fully active catalytic domain of ABD1 from residues 130 to 426 . Residues 110-129 were dispensable for methyltransferase activity in vitro, but essential for function in vivo . This analysis allowed us to delineate a subfamily of ABD1-like proteins within the superfamily of AdoMet-dependent methyltransferases . In addition, we identify a candidate Caenorhabditis elegans gene encoding a putative cap methyltransferase . All residues essential for ABD1 activity are conserved in the C . elegans homologue.

FEBS Lett, 1997 Jun 2, 409(1), 96 - 100
The transcription of NAM7/UPF1 is enhanced in the absence of Cyp1p/Hap1p concomitant with the appearance of an ISF1-NAM7 cotranscript in Saccharomyces cerevisiae; Altamura N et al.; The two adjacent nuclear genes ISF1 and NAM7 cooperatively participate in mitochondrial functions . It is well known that Cyp1p(Hap1p) activates a number of genes involved in these same functions . We show in this paper that Cyp1p influences the transcriptional regulation of NAM7 . In addition, a significant amount of ISF1-NAM7 cotranscript is observed in a cyp1 mutant context . An extensive analysis of the intergenic region which separates the two genes revealed 5' starts of the NAM7 transcripts, additional to those previously mapped . These new 5' starts overlap the 3' ends of ISF1 . We propose that NAM7 is under the control of a negative Cyp1p-dependent regulator and that its absence favours a transcriptional read-through which results in the ISF1-NAM7 cotranscript we have identified.

Genetika, 1997 Jun, 33(6), 858 - 61
{A simple method of selecting Saccharomyces cerevisiae ade1 and ade2 mutants}; Alenin VV et al.; A simple technique of selection for spontaneous red mutants of yeast Saccharomyces cerevisiae is proposed . It is based on incubation of yeast cells carrying a deletion in the GCN4 gene on a solid selective medium containing 3-amino-1, 2, 4-triazole (3AT) . After incubation for three weeks at 30 degrees C, the 3 AT-resistant mutants ade2 and ade1 were easily visualized due to the pink or red colony color; they were then selected and analyzed by genetic and biochemical methods . The frequency of these colored mutants was approximately 10-9 . The method proposed is shown to be suitable for selection and analysis of both spontaneous mutants ade2 and ade1 and those induced by various mutagens . The possible molecular mechanism of 3AT-resistance of red mutants is discussed . This mechanism involves GCN4-independent activation of expression of genes of purine and histidine metabolism . This activation results from purine starvation caused by arrest of purine biosynthesis the ade2 and ade2 mutations.

Mol Gen Genet, 1997 Jun, 255(2), 209 - 18
The Uba2 and Ufd1 proteins of Saccharomyces cerevisiae interact with poly(A) polymerase and affect the polyadenylation activity of cell extracts; del Olmo M et al.; Poly(A) polymerase is responsible for the addition of the adenylate tail to the 3' ends of mRNA . Using the two-hybrid system we have identified two proteins which interact specifically with the Saccharomyces cerevisiae poly(A) polymerase, Pap1 . Uba2 is a homolog of ubiquitin-activating (E1) enzymes and Ufd1 is a protein whose function is probably also linked to the ubiquitin-mediated protein degradation pathway . These two proteins interact with Pap1 and with each other, but not with eight other target proteins which were tested in the two-hybrid system . The last 115 amino acids of Uba2, which contains an 82-amino acid region not present in previously characterized E1 enzymes, is sufficient for the interaction with Pap1 . Both Uba2 and Ufd1 can be co-immunoprecipitated from extracts with Pap1, confirming in vitro the interaction identified by two-hybrid analysis . Depletion of Uba2 from cells produces extracts which polyadenylate precursor RNA with increased efficiency compared to extracts from nondepleted cells, while depletion of Ufd1 yields extracts which are defective in processing . These two proteins are not components of polyadenylation factors, and instead may have a role in regulating poly(A) polymerase activity.

Mol Gen Genet, 1997 Jun, 255(2), 166 - 71
The role of cysteine residues in the homeodomain protein Mat alpha 2 in mating-type control of Saccharomyces cerevisiae; Mukai Y et al.; The Mat alpha 2 homeodomain protein plays a pivotal role in the control of cell type in Saccharomyces cerevisiae . The homeodomain in the C-terminal region of Mat alpha 2 functions as a DNA-binding domain and the N-terminal region, containing two cysteine residues at positions 33 and 34, is thought to be involved in formation of Mat alpha 2 homodimers via disulfide bonds . mat alpha 2 mutants, isolated in a previous study, in which haploid-specific genes cannot be repressed by the Matal-Mat alpha 2 heterodimer but a-specific genes can be repressed by the Mat alpha 2 homodimer, were found to produce mutant Mat alpha 2 with a substitution of tyrosine or phenylalanine for Cys33 . To clarify the role of Cys33 and Cys34 in the Mat alpha 2 protein, we generated several mat alpha 2 mutants by site-directed mutagenesis which had serine residues in place of these Cys residues . Transforming MATa cells with plasmids carrying these mat alpha 2 (MAT alpha 1+) mutations rendered transformants unable to mate . Northern blot analysis revealed that transcription of the a-specific gene STE2 and the haploid-specific locus RME1 in these transformants is repressed to the same level as in wild-type MATa/MAT alpha cells . We concluded that neither Cys33 nor Cys34 is required for repression of a-specific genes by the Mat alpha 2 homodimer or of haploid-specific genes by the Matal-Mat alpha 2 heterodimer, and therefore suggest that Mat alpha 2 homodimer formation in vivo is not mediated by disulfide linkage.

Can J Microbiol, 1997 Jun, 43(6), 569 - 76
Increased cellular fatty acid desaturation as a possible key factor in thermotolerance in Saccharomyces cerevisiae; Guerzoni ME et al.; An increase of the unsaturation level of the cellular fatty acids was observed at sublethal or superoptimal temperatures in Saccharomyces cerevisiae . The hypothesis of this paper is that a high unsaturated fatty acids relative content "per se" is not a prerequisite for withstanding sublethal temperature stress in yeast but is the result of oxygen-consuming desaturase activation, with consequent reduction of oxygen and the oxygen free radicals as they form during thermal stress . In the thermotolerant strains, no increase of cellular thiobarbituric acid reactive substances (TBARSs) was observed when temperature approached the maximal growth temperature, suggesting prevention of oxidative damage . On the other hand, the values of TBARSs tripled at 42 degrees C in nonthermotolerant strains . When a sublethal hydrogen peroxide treatment preceded a rapid temperature rise, a selected thermotolerant strain responded with a relative increase of saturated fatty acids . This response, associated with an insignificant viability loss due to the double stress, suggests the induction an alternative oxygen consumption mechanism preventing excessive fatty acid unsaturation, which could be detrimental to the cells in the presence of hydrogen peroxide at sublethal temperatures.

EMBO J, 1997 Jun, 16(12), 3506 - 18
Lipid remodeling leads to the introduction and exchange of defined ceramides on GPI proteins in the ER and Golgi of Saccharomyces cerevisiae; Reggiori F et al.; Previous experiments with Saccharomyces cerevisiae had suggested that diacylglycerol-containing glycosylphosphatidylinositols (GPIs) are added to newly synthesized proteins in the endoplasmic reticulum (ER) and that ceramides subsequently are incorporated into GPI proteins by lipid remodeling . Here we prove this hypothesis by labeling yeast cells with {3H}dihydrosphingosine ({3H}DHS) and showing that this tracer is incorporated into many GPI proteins even when protein synthesis and, hence, anchor addition, is blocked by cycloheximide . {3H}DHS incorporation is greatly enhanced if endogenous synthesis of DHS is inhibited by myriocin . Labeled GPI anchors contain three types of ceramides which, based on previous and present results, are identified as DHS-C26:0, phytosphingosine-C26:0 and phytosphingosine-C26:0-OH, the latter being found only on proteins which have reached the Golgi . Lipid remodeling can occur both in the ER and in a later secretory compartment . In addition, ceramide is incorporated into GPI proteins a long time after their initial synthesis by a process in which one ceramide gets replaced by another ceramide . Remodeling outside the ER requires vesicular flow from the ER to the Golgi, possibly to supply the remodeling enzymes with ceramides.

EMBO J, 1997 Jun, 16(12), 3494 - 505
Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae; Sipos G et al.; Glycosylphosphatidylinositol (GPI)-anchored membrane proteins of Saccharomyces cerevisiae exist with two types of lipid moiety--diacylglycerol or ceramide--both of which contain 26:0 fatty acids . To understand at which stage of biosynthesis these long-chain fatty acids become incorporated into diacylglycerol anchors, we compared the phosphatidylinositol moieties isolated from myo-{2-(3)H}inositol-labelled protein anchors and from GPI intermediates . There is no evidence for the presence of long-chain fatty acids in any intermediate of GPI biosynthesis . However, GPI-anchored proteins contain either the phosphatidylinositol moiety characteristic of the precursor lipids or a version with a long-chain fatty acid in the sn-2 position of glycerol . The introduction of long-chain fatty acids into sn-2 occurs in the endoplasmic reticulum (ER) and is independent of the sn-2-specific acyltransferase SLC1 . Analysis of ceramide anchors revealed the presence of two types of ceramide, one added in the ER and another more polar molecule which is found only on proteins which have reached the mid Golgi . In summary, the lipid of GPI-anchored proteins can be exchanged by at least three different remodelling pathways: (i) remodelling from diacylglycerol to ceramide in the ER as proposed previously; (ii) remodelling from diacylglycerol to a more hydrophobic diacylglycerol with a long-chain fatty acid in sn-2 in the ER; and (iii) remodelling to a more polar ceramide in the Golgi.

Chromosoma, 1997 Jun, 105(7-8), 523 - 31
Variable region V1 of Saccharomyces cerevisiae 18S rRNA participates in biogenesis and function of the small ribosomal subunit; van Nues RW et al.; The role of helix 6, which forms the major portion of the most 5'-located expansion segment of Saccharomyces cerevisiae 18S rRNA, was studied by in vivo mutational analysis . Mutations that increased the size of the helical part and/or the loop, even to a relatively small extent, abolished 18S rRNA formation almost completely . Concomitantly, 35S pre-rRNA and an abnormal 23S precursor species accumulated . rDNA units containing these mutations did not support cell growth . A deletion removing helix 6 almost completely, on the other hand, had a much less severe effect on the formation of 18S rRNA, and cells expressing only the mutant rRNA remained able to grow, albeit at a much reduced rate . Disruption of the apical A.U base pair by a single point mutation did not cause a noticeable reduction in the level of 18S rRNA but did result in a twofold lower growth rate of the cells . This effect could not be reversed by introduction of a second point mutation that restores base pairing . We conclude that both the primary and the secondary structure of helix 6 play an important role in the formation and the biological function of the 40S subunit.

Curr Genet, 1997 Jun, 31(6), 462 - 8
Influence of gene dosage and autoregulation of the regulatory genes INO2 and INO4 on inositol/choline-repressible gene transcription in the yeast Saccharomyces cerevisiae; Schwank S et al.; Expression of structural genes of phospholipid biosynthesis in yeast is mediated by the inositol/choline-responsive element (ICRE) . ICRE-dependent gene activation, requiring the regulatory genes INO2 and INO4, is repressed in the presence of the phospholipid precursors inositol and choline . INO2 and, to a less extent, INO4 are positively autoregulated by functional ICRE sequences in the respective upstream regions . However, an INO2 allele devoid of its ICRE functionally complemented an ino2 mutation and completely restored inositol/choline regulation of Ino2p-dependent reporter genes . Low-level expression of INO2 and INO4 genes, each under control of the heterologous MET25 promoter, did not alter the regulatory pattern of target genes . Thus, upstream regions of INO2 and INO4 are not crucial for transcriptional control of ICRE-dependent genes by inositol and choline . Interestingly, over-expression of INO2, but not of INO4, counteracted repression by phospholipid precursors . Possibly, a functional antagonism between INO2 and a negative regulator is the key event responsible for repression or de-repression.

Microbiology, 1997 Jun, 143 ( Pt 6), 1901 - 8
Glucose-dependent, cAMP-mediated ATP efflux from Saccharomyces cerevisiae; Boyum R et al.; Extracellular ATP plays an important role in the physiology of multicellular organisms; however, it is unknown whether unicellular organisms such as yeast also release ATP extracellularly . Experiments are described here which show that Saccharomyces cerevisiae releases ATP to the extracellular fluid . This efflux required glucose and the rate was increased dramatically by the proton ionophores nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; ATP efflux was also increased by the plasma membrane proton pump inhibitor diethylstilbestrol . The increase in the concentration of extracellular ATP was not due to cell lysis or general disruption of plasma membrane integrity as measured by colony-forming and methylene-blue-staining assays . ATP efflux was strictly correlated with a rise in intracellular cAMP; therefore, the cAMP pathway is likely to be involved in triggering ATP efflux . These results demonstrate that yeast cells release ATP in a regulated manner.

Microbiology, 1997 Jun, 143 ( Pt 6), 1885 - 9
An important role for glutathione and gamma-glutamyltranspeptidase in the supply of growth requirements during nitrogen starvation of the yeast Saccharomyces cerevisiae; Mehdi K et al.; When the yeast Saccharomyces cerevisiae sigma 1278b was starved for nitrogen, the total glutathione (GSH) pool increased from 7 to 17 nmol (mg dry wt)-1 during the first 2 h and then declined . More than 90% of the total GSH shifted towards the central vacuole during this time . This transient stimulation was not observed in the presence of buthionine-(S,R)-sulphoximine (BSO), a specific transition-state-analogue inhibitor of gamma-glutamylcysteine synthase (gamma-GCS), nor in a mutant strain deficient in this enzyme- gamma-Glutamyltranspeptidase (gamma-GT), a vacuolar enzyme responsible for the initial step of GSH degradation, was derepressed during nitrogen starvation . This mechanism can apparently enable the starved yeast cell to use the constituent amino acids from GSH which accumulate in the vacuole to satisfy its growth requirements for nitrogen.

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