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PubMed2004

Inactivation of endopolyphosphatase gene PPN1 results in inhibition of expression of exopolyphosphatase PPX1 and high-molecular-mass exopolyphosphatase not encoded by PPX1 in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses multiple forms of exopolyphosphatases, the enzymes involved in the metabolism of inorganic polyphosphates, which are important regulatory compounds. In S. cerevisiae, inactivation of endopolyphosphatase gene PPN1 leads to the inhibition of expression of both exopolyphosphatase PPX1 and high-molecular-mass exopolyphosphatase of approximately 1000 kDa not encoded by PPX1. In the single endopolyphosphatase mutant CRN, the expression of exopolyphosphatase PPX1 decreases 6.5-fold and 2.5-fold at the stationary and exponential growth phases, respectively, as compared with the parent strain CRY. In this mutant, the activity of the high-molecular-mass exopolyphosphatase of approximately 1000 kDa decreases approximately 10-fold as compared with that in strains with the PPN1 gene. In a double mutant of PPX1 and PPN1, no exopolyphosphatase activity is detected in the cytosol at the stationary growth phase. Thus, the exopolyPase activity in cell cytosol depends on the endopolyPase gene PPN1.

Acid Anhydride Hydrolases↗

PubMed2004

Enhanced peptide secretion by gene disruption of CYM1, a novel protease in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a widely used host in the production of therapeutic peptides and proteins. Here we report the identification of a novel endoprotease in S. cerevisiae. It is encoded by the CYM1 gene and is specific for the C-terminus of basic residues of heterologously expressed peptides. Gene disruption of CYM1 not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin. Cym1p resembles metalloendoproteases of the pitrilysin family with the HXXEH(X)E(71-77) catalytic domain as seen in insulysin, nardilysin and human metalloprotease 1. It is a nuclear encoded protease that localizes to mitochondria without a hydrophobic N-terminal signal sequence or a C-terminal tail-anchor. The protease does not require post-translational processing prior to activation and it contains cytosolic activity that processes peptides designated for the secretory pathway prior to translocation into the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1999

Interaction among the subunits of Golgi membrane mannosyltransferase complexes of the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae Mnn9 protein is a type II Golgi membrane protein which concerns in protein mannosylation. When solubilized by Triton X-100, it was recovered in two distinct complexes both having mannosyltransferase activity; one with Van1 protein (V-complex) and the other with Anp1, Hoc1, Mnn10, and Mnn11 proteins (A-complex). Characterization of the null mutants suggested that A-complex is also concerned in protein O-glycosylation. A-complex was more resistant than V-complex to dissociating conditions. Interaction between the lumenal domains of Van1 and Mnn9 was detected by a two-hybrid experiment. The anchor domain of Mnn9 protein could be replaced with other membrane anchors without losing the ability to form complexes similar to V- and A-complexes. Thus the lumenal domains are important to assemble these distinct complexes.

Fungal Proteins↗

PubMed1992

Purification, gene cloning, and gene disruption of the transcription elongation factor S-II in Saccharomyces cerevisiae.

Saccharomyces cerevisiae S-II was purified to near homogeneity as a protein stimulating RNA polymerase II. Four of seven lysyl endopeptidase-digested fragments of S-II were located in the PPR2 sequence reported previously. Analysis of a genomic clone of S-II revealed that S-II and PPR2 are the same protein consisting of 309 amino acid residues, and frame shifts were found in the sequence of PPR2 gene reported previously. Yeast S-II and mouse S-II showed high similarity in their amino acid sequences, especially in their amino-terminal and carboxyl-terminal regions. A gene disruption experiment showed that an S-II null mutant was not lethal under usual growth conditions, indicating that S-II is not essential for the growth of yeast.

Amino Acid Sequence↗

PubMed1996

Gat1p, a GATA family protein whose production is sensitive to nitrogen catabolite repression, participates in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.

Amino Acid Sequence↗

PubMed1998

Growth-independent regulation of CLN3 mRNA levels by nutrients in Saccharomyces cerevisiae.

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

Carbon↗

PubMed2002

Genome-wide expression analysis of genes affected by amino acid sensor Ssy1p in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Ssy1p is a membrane protein which senses extracellular amino acids and controls the expression of certain amino acid permease genes. Analysis by DNA micro-array newly identified DIP5 and MUP1 as the positive targets and CAN1, PUT4 and GAP1 as the negative targets under Ssy1p control. Interestingly, the effect of ssy1 deletion was not restricted to amino acid permease genes: the expression of nitrogen catabolite repression (NCR)-sensitive genes and methionine-biosynthesizing genes ( MET genes) was derepressed by the deletion of SSY1. Constitutive overexpression of the genes for glutamine permease ( GNP1) or methionine permease ( MUP1) enhanced the assimilation of glutamine or methionine in the ssy1Delta strain but could not fully suppress the derepression of the NCR-sensitive genes or MET genes. This result suggests that Ssy1p regulates not only the transcription of amino acid permease genes, but also transcription of many other nitrogen-metabolizing genes.

Amino Acid Transport Systems↗

PubMed1996

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach. We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo. Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis. These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function.

Binding Sites↗

PubMed2001

Multicopy suppressors of the sly1 temperature-sensitive mutation in the ER-Golgi vesicular transport in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Sly1 protein is a member of the Sec1/Munc18-family proteins, which are essential for vesicular trafficking, but their exact biological roles are yet to be determined. A temperature-sensitive sly1 mutant arrests the vesicular transport from the ER to Golgi compartments at 37 degrees C. We screened for multicopy suppressor genes that restore the colony formation of the sly1(ts) mutant to discover functionally interacting components. The suppressor genes obtained were classified as: (1) those that encode a multifunctional suppressor, SSD1; (2) heat shock proteins, SSB1 and SSB2; (3) cell surface proteins, WSC1, WSC2 and MID2; (4) ER-Golgi transport proteins, USO1 and BET1; and (5) an as-yet-uncharacterized protein, HSD1 (high-copy suppressor of SLY1 defect 1). By epitope tagging of the gene product, we found that Hsd1 protein is an ER-resident membrane protein. Its overproduction induced enlargement of ER-like membrane structures.

Amino Acid Sequence↗

PubMed2006

Polymerase eta is a short-lived, proteasomally degraded protein that is temporarily stabilized following UV irradiation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Rad30 is the homolog of human DNA polymerase eta whose inactivation leads to the cancer-prone syndrome xeroderma pigmentosum variant. Both human and yeast polymerase eta are responsible for error-free bypass of UV-induced cis-syn pyrimidine dimers and several other DNA lesions. Here we show, using yeast strains expressing TAP-tagged Rad30, that the level of this protein is post-translationally regulated via ubiquitination and proteasome-mediated degradation. The half-life of Rad30 is 20 min and it increases due to proteasomal defects. Mutations inactivating components of the Skp1/cullin/ F-box (SCF) ubiquitin ligase complex: Skp1 and the F-box protein Ufo1 stabilize Rad30. Our results indicate also that ultraviolet irradiation causes transient stabilization of Rad30, which leads, in turn, to temporary accumulation of this polymerase in the cell. We conclude that proteolysis plays an important role in regulating the cellular abundance of Rad30. These results are the first indication of a role for controlled proteasomal degradation in modulating cellular level of translesion DNA polymerase in eukaryotes.

DNA-Directed DNA Polymerase↗

PubMed2002

Rpf2p, an evolutionarily conserved protein, interacts with ribosomal protein L11 and is essential for the processing of 27 SB Pre-rRNA to 25 S rRNA and the 60 S ribosomal subunit assembly in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the maturation of 25 S rRNA and the 60 S ribosomal subunit assembly. In two-hybrid screening, using RRS1 as bait, we have cloned YKR081c/RPF2. Rpf2p is essential for growth and is mainly localized in the nucleolus. The amino acid sequence of Rpf2p is highly conserved in eukaryotes from yeast to human. Similar to Rrs1p, Rpf2p shows physical interaction with ribosomal protein L11 and appears to associate with preribosomal subunits fairly tightly. Northern, methionine pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a decrease of mature 25 S rRNA, and a shortage of 60 S subunits. An analysis of processing intermediates by primer extension shows that the Rpf2p depletion leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required for the processing of 27 SB into 25 S rRNA.

Alleles↗

PubMed1996

Identification of functional domains within the RAD1.RAD10 repair and recombination endonuclease of Saccharomyces cerevisiae.

Saccharomyces cerevisiae rad1 and rad10 mutants are unable to carry out nucleotide excision repair and are also defective in a mitotic intrachromosomal recombination pathway. The products of these genes are subunits of an endonuclease which recognizes DNA duplex/single-strand junctions and specifically cleaves the 3' single-strand extension at or near the junction. It has been suggested that such junctions arise as a consequence of DNA lesion processing during nucleotide excision repair and the processing of double-strand breaks during intrachromosomal recombination. In this study we show that the RAD1 RAD10 complex also cleaves a more complex junction structure consisting of a duplex with a protruding 3' single-strand branch that resembles putative recombination intermediates in the RAD1 RAD10-mediated single-strand annealing pathway of mitotic recombination. Using monoclonal antibodies, we have identified two regions of RAD1 that are required for the cleavage of duplex/single-strand junctions. These reagents also inhibit nucleotide excision repair in vitro, confirming the essential role of the RAD1 RAD10 endonuclease in this pathway.

Animals↗

PubMed2004

Eaf3 regulates the global pattern of histone acetylation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has a global pattern of histone acetylation in which histone H3 and H4 acetylation levels are lower at protein-coding sequences than at promoter regions. The loss of Eaf3, a subunit of the NuA4 histone acetylase and Rpd3 histone deacetylase complexes, greatly alters the genomic profile of histone acetylation, with the effects on H4 appearing to be more pronounced than those on H3. Specifically, the loss of Eaf3 causes increases in H3 and H4 acetylation at coding sequences and decreases at promoters, such that histone acetylation levels become evenly distributed across the genome. Eaf3 does not affect the overall level of H4 acetylation, the recruitment of the NuA4 catalytic subunit Esa1 to target promoters, or the level of transcription of the genes analyzed for histone acetylation. Whole-genome transcriptional profiling indicates that Eaf3 plays a positive, but quantitatively modest, role in the transcription of a small subset of genes, whereas it has a negative effect on very few genes. We suggest that Eaf3 regulates the genomic profile of histone H3 and H4 acetylation in a manner that does not involve targeted recruitment and is independent of transcriptional activity.

Acetylation↗

PubMed2002

A microarray-assisted screen for potential Hap1 and Rox1 target genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae adapts to altered oxygen availability by differentially expressing a number of genes. Under aerobic conditions oxygen control of gene expression is exerted through the activator Hap1 and the repressor Rox1. The Hap1 transcription factor senses cellular heme status and increases expression of aerobic genes in response to oxygen. The repression of hypoxic genes under normoxic conditions results from Hap1-mediated activation of ROX1 transcription. To allow the identification of additional Hap1 and Rox1 target genes, genome-wide expression was analysed in aerobically, chemostat-cultivated hap1 and rox1 null mutants. The microarray results show that deletion of HAP1 causes a lower transcript level of 51 genes. Transcription of 40 genes was increased in rox1 mutant cells compared to wild-type cells. Combining these results with our previously described transcriptome data of aerobically and anaerobically grown cells and with computational analysis of the promoters identified 24 genes that are potentially regulated by Hap1, and 38 genes satisfied the criteria of being direct targets of Rox1. In addition, this work provides further evidence that Rox1 controls transcription of anaerobic genes through repression under normoxic conditions.

Anaerobiosis↗

PubMed2002

Ngl2p is a Ccr4p-like RNA nuclease essential for the final step in 3'-end processing of 5.8S rRNA in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains three nonessential genes (NGL1, NGL2, and NGL3) that encode proteins containing a domain with similarity to a Mg(2+)-dependent endonuclease motif present in the mRNA deadenylase Ccr4p. We have investigated a possible role of these proteins in rRNA processing, because for many of the pre-rRNA processing steps, the identity of the responsible nuclease remains elusive. Analysis of RNA isolated from cells in which the NGL2 gene has been inactivated (ngl2delta) demonstrates that correct 3'-end formation of 5.8S rRNA at site E is strictly dependent on Ngl2p. No role in pre-rRNA processing could be assigned to Ngl1p and Ngl3p. The 3'-extended 5.8S rRNA formed in the ngl2delta mutant is slightly shorter than the 6S precursor previously shown to accumulate upon combined deletion of the 3' --> 5' exonuclease-encoding REX1 and REX2 genes or upon depletion of the exosomal subunits Rrp40p or Rrp45p. Thus, our data add a further component to the set of nucleases required for correct 3'-end formation of yeast 5.8S rRNA.

Amino Acid Sequence↗

PubMed1995

A role of Sep1 (= Kem1, Xrn1) as a microtubule-associated protein in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells lacking the SEP1 (also known as XRN1, KEM1, DST2, RAR5) gene function exhibit a number of phenotypes in cellular processes related to microtubule function. Mutant cells show increased sensitivity to the microtubule-destabilizing drug benomyl, increased chromosome loss, a karyogamy defect, impaired spindle pole body separation, and defective nuclear migration towards the bud neck. Analysis of the arrest morphology and of the survival during arrest strongly suggests a structural defect accounting for the benomyl hypersensitivity, rather than a regulatory defect in a checkpoint. Biochemical analysis of the purified Sep1 protein demonstrates its ability to promote the polymerization of procine brain and authentic S.cerevisiae tubulin into flexible microtubules in vitro. Furthermore, Sep1 co-sediments with these microtubules in sucrose cushion centrifugation. Genetic analysis of double mutant strains containing a mutation in SEP1 and in one of the genes coding for alpha- or beta-tubulin further suggests interaction between Sep1 and microtubules. Taken together these three lines of evidence constitute compelling evidence for a role of Sep1 as an accessory protein in microtubule function in the yeast S.cerevisiae.

Animals↗

PubMed1995

Genetics of the synthesis of serine from glycine and the utilization of glycine as sole nitrogen source by Saccharomyces cerevisiae.

Saccharomyces cerevisiae can grow on glycine as sole nitrogen source and can convert glycine to serine via the reaction catalyzed by the glycine decarboxylase multienzyme complex (GDC). Yeast strains with mutations in the single gene for lipoamide dehydrogenase (lpd1) lack GDC activity, as well as the other three 2-oxoacid dehydrogenases dependent on this enzyme. The LPD1 gene product is also required for cells to utilize glycine as sole nitrogen source. The effect of mutations in LPD1 (L-subunit of GDC), SER1 (synthesis of serine from 3-phosphoglycerate), ADE3 (cytoplasmic synthesis of one-carbon units for the serine synthesis from glycine), and all combinations of each has been determined. The results were used to devise methods for isolating mutants affected either in the generation of one-carbon units from glycine (via GDC) or subsequent steps in serine biosynthesis. The mutants fell into six complementation groups (gsd1-6 for defects in conversion of glycine to serine). Representatives from three complementation groups were also unable to grow on glycine as sole nitrogen source (gsd1-3). Assays of the rate of glycine uptake and decarboxylation have provided insights into the nature of the mutations.

Amino Acid Oxidoreductases↗

PubMed2001

The rye mutants identify a role for Ssn/Srb proteins of the RNA polymerase II holoenzyme during stationary phase entry in Saccharomyces cerevisiae.

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

Cell Division↗