Yeast

Bellu, A. R.; van der Klei, I. J.; Rechinger, K. B.; Yavuz, M.; Veenhuis, M.; Kiel, J. A. K. W.

doi: 10.1002/(SICI)1097-0061(199902)15:3<181::AID-YEA355>3.0.CO;2-Ypmid: 10077185

We have isolated the Hansenula polymorpha CPY gene encoding carboxypeptidase Y (Hp‐CPY). The deduced amino acid sequence revealed that Hp‐CPY consists of 541 amino acids and has a calculated Mr of 60,793. The protein is highly similar to Saccharomyces cerevisiae CPY (61·8% identity). At the N‐terminus of Hp‐CPY signals for the entry into the secretory pathway and subsequent sorting to the vacuole were identified. Immunocytochemically, using monospecific antibodies raised against Hp‐CPY, the protein was localized to the vacuole. On Western blots, a diffuse protein band was observed in extracts of H. polymorpha cells, suggesting that the protein is glycosylated. This was confirmed by endoglycosidase H treatment, which resulted in a strong reduction of the apparent Mr of the protein. We have investigated the effect of CPY deletion on the degradation of peroxisomes, an autophagous process that occurs when the organelles become redundant for growth. In Δcpy cells peroxisomal proteins were degraded in the vacuole as efficiently as in wild‐type H. polymorpha cells, indicating that CPY is not a major proteinase in this pathway. Copyright © 1999 John Wiley & Sons, Ltd.

Parrou, Jean‐Luc; Enjalbert, Brice; Plourde, Lucile; Bauche, Anne; Gonzalez, Benjamin; François, Jean

doi: 10.1002/(SICI)1097-0061(199902)15:3<191::AID-YEA358>3.0.CO;2-Opmid: 10077186

The dynamic responses of reserve carbohydrates with respect to shortage of either carbon or nitrogen source was studied to obtain a sound basis for further investigations devoted to the characterization of mechanisms by which the yeast Saccharomyces cerevisiae can cope with nutrient limitation during growth. This study was carried out in well‐controlled bioreactors which allow accurate monitoring of growth and frequent sampling without disturbing the culture. Under glucose limitation, genes involved in glycogen and trehalose biosynthesis (GLG1, GSY1, GSY2, GAC1, GLC3, TPS1 ), in their degradation (GPH1, NTH1 ), and the typical stress‐responsive CTT1 gene were coordinately induced in parallel with glycogen, when the growth has left the pure exponential phase and while glucose was still plentiful in the medium. Trehalose accumulation was delayed until the diauxic shift, although TPS1 was induced much earlier, due to hydrolysis of trehalose by high trehalase activity. In contrast, under nitrogen limitation, both glycogen and trehalose began to accumulate at the precise time when the nitrogen source was exhausted from the medium, coincidentally with the transcriptional activation of genes involved in their metabolism. While this response to nitrogen starvation was likely mediated by the stress‐responsive elements (STREs) in the promoter of these genes, we found that these elements were not responsible for the co‐induction of genes involved in reserve carbohydrate metabolism during glucose limitation, since GLG1, which does not contain any STRE, was coordinately induced with GSY2 and TPS1. Copyright © 1999 John Wiley & Sons, Ltd.

Scott, Andrew D.; Neishabury, Maryam; Jones, D. Hugh; Reed, Simon H.; Boiteux, Serge; Waters, Raymond

doi: 10.1002/(SICI)1097-0061(199902)15:3<205::AID-YEA361>3.0.CO;2-1pmid: 10077187

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8‐dihydro‐8‐oxoguanine (8‐OxoG). When compared to wild‐type, ogg1 mutants show an increase in the frequency of GC to TA transversions, indicating a role for Ogg1 in the repair of 8‐OxoG. Here we report an increased frequency of forward mutation to canavanine resistance in mutants defective in the nucleotide excision repair (NER) gene RAD14. This was not increased further in strains additionally defective in OGG1. However, when compared to strains solely defective in OGG1, ogg1rad14 mutants displayed an increase in spontaneous GC to TA transversions. Intriguingly, reversion of the lys1‐1 ochre allele was not increased in rad14 mutants, suggesting that oxidative base damage may only represent a substrate for NER in certain regions of the genome. We also examined repair of oxidative DNA damage by transforming mutant strains with plasmid DNA treated with methylene blue plus visible light. Mutants defective in OGG1 showed no significant reduction in transformation efficiency compared with wild‐type strains. In contrast, disruption of RAD14 reduced the efficiency of transformation, yet there was no further decrease in an ogg1rad14 mutant. This strongly supports a role for NER in the repair of oxidative base damage in yeast, and differs from similar experiments carried out in E. coli, where transformation efficiency is only reduced in mutants defective in both fpg and uvrA. Finally, the repair of Fpg‐sensitive sites was examined at the MATα and HMLα mating type loci, and NER was found to play a role in their removal. Copyright © 1999 John Wiley & Sons, Ltd.

Shiratori, Akiko; Shibata, Takehiko; Arisawa, Mikio; Hanaoka, Fumio; Marakami, Yasufumi; Eki, Toshihiko

doi: 10.1002/(SICI)1097-0061(199902)15:3<219::AID-YEA349>3.0.CO;2-3pmid: 10077188

Helicase‐related proteins play important roles in various cellular processes incuding DNA replication, DNA repair, RNA processing and so on. It has been well known that the amino acid sequences of these proteins contain several conserved motifs, and that the open reading frames (ORFs) which encode helicase‐related proteins make up several gene families. In this study, we have identified 134 ORFs that encode helicase‐like proteins in the Saccharomyces genome, based on similarity with the ORFs of authentic helicase and helicase‐related proteins. Multiple alignment of the ORF sequences resulted in the 134 ORFs being classified to 11 clusters. Seven out of 21 previously uncharacterized ORFs (YDL031w, YDL070w, YDL084w, YGL150c, YKL078w, YLR276c, and YMR128w) were identified by systematic gene disruption, to be essential for vegetative growth. Three (YDR332w, YGL064c, and YOL095c) out of the remaining 14 dispensable ORFs exhibited the slow‐growth phenotype at 30°C and 37°C. Furthermore, the expression profiles of transcripts from 43 ORFs were examined under seven different growth conditions by Northern analysis and reverse transcription‐polymerase chain reaction, indicating that all of the 43 tested ORFs were transcribed. Interestingly, we found that the level of transcript from 34 helicase‐like genes was markedly increased by heat shock. This suggests that helicase‐like genes may be involved in the biosynthesis of nucleic acids and proteins, and that the genes can be transcriptionally activated by heat shock to compensate for the repressed synthesis of mRNA and protein. Copyright © 1999 John Wiley & Sons, Ltd.

Adachi, Nobuaki; Matsumoto, Miwa; Hasegawa, Satoshi; Yamamoto, Tohru; Horikoshi, Masami

doi: 10.1002/(SICI)1097-0061(199902)15:3<255::AID-YEA359>3.0.CO;2-Apmid: 10077189

We isolated a Schizosaccharomyces pombe (Sz. pombe ) gene encoding the counterpart of the TFIIH subunit Homo sapiens (H. sapiens ) p44 and Saccharomyces cerevisiae (S. cerevisiae ) SSL1, and we named this gene product p47. Contrary to the case of SSL1, which is an essential gene of S. cerevisiae, p47 is not essential for the viability of Sz. pombe. The deduced amino acid sequence revealed that this TFIIH subunit is highly conserved during evolution. Comparison of the primary structures revealed differences in the predicted positions of introns in the Caenorhabditis elegans (C. elegans ) gene encoding the p47 counterpart found during the genome project. A charged cluster in the N‐terminal region is present in the two yeasts. Two putative zinc‐binding motifs, an extended C2H2 zinc finger with a ‘C8 motif’ and a second putative zinc‐binding motif common to the two TFIIH subunits, were also found, the former being completely conserved. The latter motif consists of five cysteine residues and is also present in hp44, SSL1 and another TFIIH subunit, human p34 (hp34). Since one zinc atom can bind to four ligands in zinc‐binding motifs, the conservation of cysteine residues was given attention. This motif is completely conserved in p47 homologues derived from the four species. As one cysteine residue is not conserved among the homologues of hp34, the consensus of this motif is concluded to be Cys–X2–Cys–X(10,12)–Cys–X2–Cys. This nucleotide sequence has been deposited in the GenBank Data Library under Accession Number AF017646. Copyright © 1999 John Wiley & Sons, Ltd.