The Hsp90/Cdc37p chaperone system is a determinant of molybdate resistance in Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks enzymes that contain the molybdopterin co‐factor and therefore any requirement for molybdenum as a trace mineral supplement. Instead, high molybdate levels are inhibitory to its growth. Low cellular levels of heat shock protein 90 (Hsp90), an essential chaperone, were found to enhance this sensitivity to molybdate. Certain Hsp90 point mutations and co‐chaperone protein defects that partially compromise the function of the Hsp90/Cdc37p chaperone system also rendered S. cerevisiae hypersensitive to high molybdate levels. Sensitivity was especially apparent with mutations close to the Hsp90 nucleotide binding site, with the loss of the non‐essential co‐chaperone Sti1p (the equivalent of mammalian Hop), and with the abolition of residue Ser14 phosphorylation on the essential co‐chaperone Cdc37p. While it remains to be proved that these effects reflect direct inhibition of the Hsp90 of the cell by the MoO₄²⁺ oxyanion in vivo; this possibility is suggested by molybdate sensitivity arising with a mutation in the Hsp90 nucleotide binding site that does not generate stress sensitivity or an impaired stress response. Molybdate sensitivity may therefore be a useful phenotype to score when studying mutations in this chaperone system. Copyright © 2009 John Wiley & Sons, Ltd.     

Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation

The Cdc48/p97 AAA-ATPase functions in membrane fusion and ubiquitin-dependent protein degradation. Here, we show that, in yeast, Cdc48p interacts with three novel proteins, Cuil-3p, which contain a conserved ubiquitin-related (UBX) domain. Cui2p and Cui3p are closely related, interact with each other, and are localized at the perinuclear membrane. Cdc48p binds directly the UBX domain of Cui3p in vitro. Multiple deletions of the CUI1, CUI2 and CUI3 genes confer deficiency in sporulation and degradation of model ubiquitin-protein fusions. The Cuil-3 proteins were also found to interact with Ufd3p, a WD repeat protein known to associate with Cdc48p. Together, these results indicate that the Cuil-3 proteins form complexes that are components of the ubiquitin-proteasome system.     

The role of two putative nitroreductases,Frm2p and Hbn1p,in the oxidative stress response in Saccharomyces cerevisiae

The nitroreductase family is comprised of a group of FMN‐ or FAD‐dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant‐enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4‐nitroquinoline‐oxide (4‐NQO) and N‐nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Δ and frm2Δ hbn1Δ. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H₂O₂ and t‐BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx. Copyright © 2009 John Wiley & Sons, Ltd.     

The S. cerevisiae nitrogen starvation-induced Yvh1p and Ptp2p phosphatases play a role in control of sporulation

Starvation for nitrogen in the absence of a fermentable carbon source causes diploid Saccharomyces cerevisiae cells to leave vegetative growth, enter meiosis, and sporulate; the former nutritional condition also induces expression of the YVH1 gene that encodes a protein phosphatase. This correlation prompted us to determine whether the Yvh1p phosphatase was a participant in the network that controls the onset of meiosis and sporulation. We found that expression of the IME2 gene, encoding a protein kinase homologue required for meiosis- and sporulation-specific gene expression, is decreased in a yvh1 disrupted strain. We also observed a decrease, albeit a smaller one, in the expression of IME1 which encodes an activator protein required for IME2 expression. Under identical experimental conditions, expression of the MCK1 and IME4 genes (which promote sporulation but do not require Ime1p for expression) was not affected. These results demonstrate the specificity of the yvh1 disruption phenotype. They suggest that decreased steady-state levels of IME1 and IME2 mRNA were not merely the result of non-specific adverse affects on nucleic acid metabolism caused by the yvh1 disruption. Sporulation of a homozygous yvh1 disruption mutant was delayed and less efficient overall compared to an isogenic wild-type strain, a result which correlates with decreased IME1 and IME2 gene expression. We also observed that expression of the PTP2 tyrosine phosphatase gene (a negative regulator of the osmosensing MAP kinase cascade), but not the PTP1 gene (also encoding a tyrosine phosphatase) was induced by nitrogen-starvation. Although disruption of PTP2 alone did not demonstrably affect sporulation or IME2 gene expression, sporulation was decreased more in a yvh1, ptp2 double mutant than in a yvh1 single mutant; it was nearly abolished in the double mutant. These data suggest that the YVH1 and PTP2 encoded phosphatases likely participate in the control network regulating meiosis and sporulation. Expression of YVH1 and PTP2 was not affected by nitrogen source quality (asparagine compared to proline) suggesting that nitrogen starvation-induced YVH1 and PTP2 expression and sensitivity to nitrogen catabolite repression are on two different branches of the nitrogen regulatory network.     

XIV. Yeast sequencing reports. Sequence of MKT1, needed for propagation of M2 satellite dsRNA of the L-A virus of Saccharomyces cerevisiae

MKT1 is required for m aintenance of K₂ above 30°C in strains with the L-A-HN variant of the L-A double-stranded RNA virus of Saccharomyces cerevisiae. We report that MKT1 encodes a 92 979 Da protein with serine-rich regions and the retroviral protease signature, DTG, but with no substantial homology to proteins presently in the databases. This sequence is available from GenBank under Accession Number U09129.     

Mam33p,an oligomeric,acidic protein in the mitochondrial matrix of Saccharomyces cerevisiae is related to the human complement receptor gC1q-R

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

The Genes Encoding the Transcription Factor yTAFII60, the G4p1 Protein and a Putative Glucose Transporter are Contained in a 12·3 kb DNA Fragment on the Left Arm of Saccharomyces cerevisiae Chromosome VII

We report the nucleotide sequence of a DNA fragment of 12 325 base pairs from the left arm of the Saccharomyces cerevisiae chromosome VII. Inspection of the coding capacity revealed 11 open reading frames (ORFs) longer than 100 amino acids. Five ORFs are significantly homologous to known proteins. The region encoding ORF G2985 corresponds (100%) to the gene encoding the yeast TATA binding protein-associated factor TAF_II60. The G3075 ORF is 47·8% identical to the hypothetical yeast protein YB88. G3080 shows 36·7% identity to the eel calmodulin. G3085 shows 94·9% identity with the published sequence of the quadruplex DNA binding protein G4p1. G3090 reveals 46·7% identity with the probable glucose transport protein yBR1625. The DNA sequence has been submitted to the EMBL data library under Accession Number X97644. © 1997 by John Wiley & Sons, Ltd.     

Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1

Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni(2+) affinity chromatography and gel filtration. N-terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD(+) concentration, consistent with the loss of NAMase activity in the null mutant. In wild-type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a P(PNC1)::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5'non-coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD-dependent biological processes.     

The S1, S2 and SGA1 ancestral genes for the STA glucoamylase genes all map to chromosome IX in Saccharomyces cerevisiae

The polymorphic extracellular glucoamylase-encoding genes STA1 (chr. IV), STA2 (chr. II) and STA3 (chr. XIV), from Saccharomyces cerevisiae var. diastaticus probably evolved by genomic rearrangement of DNA regions (S1, S2 and SGA1) present in S. cerevisiae, and subsequent translocation to unlinked regions of chromosomal regions. S1, encoding a homologue to the threonine/serine-rich domain of STA glucoamylases (GAI-III), mapped to the right arm of chromosome IX. S2, encoding the hydrophobic leader peptide of GAI-III, was also mapped on the right arm of chromosome IX, next to S1, close to DAL81. The SGA1 sporulation-specific, intracellular glucoamylase-encoding gene is located on the left arm of chromosome IX, 32 kb proximal of HIS5.     

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae

Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.     

IIX. Yeast mapping reports. ATP1 and ATP2, the F1F0-ATPase α and β subunit genes of Saccharomyces cerevisiae,are respectively located on chromosomes II and X

Southern blot analysis showed that ATP1 and ATP2 map on chromosomes II and X, respectively. Physical mapping of ATP1 and ATP2 by chromosome fragmentation showed that ATP1 is at the left end of chromosome II and ATP2 is at the right end of chromosome X. Both are located close to telomere sequences of each chromosome; ATP1 and ATP2 being approximately 30 kb and 85 kb from the respective telomeres.