Polymorphism of the SUP35 gene and its product in the Saccharomyces cerevisiae yeasts

The product of the SUP35 gene of the saccharomycete yeast, the translation termination eRF3 factor, can be converted in prion, the heritable determinant of protein nature. The nucleotide sequence of this gene from the strain belonging to Peterhof genetic lines of the yeast Saccharomyces cerevisiae was determined. A comparison of the identified sequence with SUP35 sequences in the database of GenBank allowed the detection of polymorphic sites both in the SUP35 gene and its product. The location of polymorphic sites in the evolutionarily nonconserved N-terminal protein region confirmed that this eRF3 fragment lacks functions vital to life activity. Nevertheless, these sites are located in the vicinity of sites, whose role in the prion conversion of eRF3 has been established. Based on this, natural polymorphism of the primary eRF3 structure is assumed to be connected with the existence of different variants (strains) of its prion analog.     

Isolation and properties of an antisuppressor in Saccharomyces cerevisiae specific for an omnipotent suppressor.

A new Mendelian antisuppressor, ASU10, was isolated and shown to reduce the efficiency of the omnipotent yeast suppressor, sup35. ASU10 had no effect on the other omnipotent suppressor, sup45, or on several amber suppressors.     

Interaction of ATP17 gene with SUP45 and SUP35 genes in Saccharomyces cerevisiae yeast

Genes SUP35 and SUP45 have been identified in the saccharomycete yeast as genes controlling termination of translation in cytoplasmic ribosomes. However, many facts indicate that the control of translation termination is not the only function of these genes. This work is devoted to studying one of the pleiotropic effects of sup35 and sup45 mutations, a respiratory deficiency. The compensation for this deficiency in mutants for either gene can occur due to a mutation in the ATP17 gene encoding the f-subunit of mitochondrial F1F0 ATP synthase. It is assumed that the observed interaction can be related to the system of co-translational protein import into mitochondria.     

Yeast omnipotent supressor SUP1 (SUP45): nucleotide sequence of the wildtype and a mutant gene.

The primary structures of the yeast recessive omnipotent suppressor gene SUP1 (SUP45) and one of its mutant alleles (sup1-ts36) was determined. The gene codes for a protein of 49 kD. The mutant protein differs from the wildtype form in one amino acid residue (Ser instead of Leu) in the N-terminal part. The codon usage differs significantly from that of yeast ribosomal protein genes. However, an upstream element resembling a conserved oligonucleotide in the region 5' to ribosomal protein genes in S. cerevisiae has been found. A DNA probe internal to the SUP1 gene does not exhibit detectable homology to genomic DNA neither from higher eucaryotes nor from eu- or archaebacteria. The hypothetical function of this protein in control of translational fidelity is discussed.     

Prionization of the Pichia methanolica SUP35 gene product in the yeast Saccharomyces cerevisiae

Induction of the prionlike form of the SUP35 gene of Pichia methanolica, the [PSIP+] factor, was shown in the transgenic yeast Saccharomyces cerevisiae containing the P. methanolica SUP35 gene located in the chromosome instead of the indigenous SUP35 gene. Either the induction of the [PSIP+] factor in the transgenic yeast, unlike that of the classical [PSI+] factor, does not depend on the presence of the [PIN+] determinant in the cell or the substitution of the S. cerevisiae SUP35 gene for the P. methanolica SUP35 gene changes the PIN status of the strain. The [PSIP+] factor is unstable in mitosis and meiosis and is not effectively eliminated upon over-production of the chaperone protein Hsp104p of S. cerevisiae. The existence of an interspecific barrier during transmission of the prionlike state from S. cerevisiae Sup35p to P. methanolica Sup35p was shown.     

Deletion analysis of the SUP2 gene in Saccharomyces cerevisiae

The sup2 mutations of the yeast Saccharomyces cerevisiae or plasmid-mediated amplification of the wild type SUP2 gene lead to suppression of different types of nonsense mutations. The Sup2 protein includes a C-terminal region homologous to elongation factor EF-1 alpha and an unique N-terminal region. The SUP2 is an essential gene. The functional role of different regions of the SUP2 gene was investigated, by deleting them without disruption of the reading frame. Such constructs were maintained in yeast on episomal or centromeric plasmids. It was shown that the region, homologous to EF-1 alpha is necessary for viability, while the remaining N-terminal part is nonessential. The region of the first 154 amino acids is necessary and sufficient for the suppressor effect, caused by plasmid-mediated amplification of the SUP2 gene.     

Isolation and characterization of mutations in the HXK2 gene of Saccharomyces cerevisiae.

Several hundred new mutations in the gene (HXK2) encoding hexokinase II of Saccharomyces cerevisiae were isolated, and a subset of them was mapped, resulting in a fine-structure genetic map. Among the mutations that were sequenced, 35 were independent missense mutations. The mutations were obtained by mutagenesis of cloned HXK2 DNA carried on a low-copy-number plasmid vector and screened for a number of different phenotypes in yeast strains bearing chromosomal hxk1 and hxk2 null mutations. Some of these mutants were characterized both in vivo and in vitro; they displayed a wide spectrum of residual hexokinase activities, as indicated by three assays: in vitro enzyme activity, ability to grow on glucose and fructose, and ability to repress invertase production when growing on glucose. Of those that failed to support growth on fructose, only a small minority made normal-size, stable, and inactive protein. Analysis of the amino acid changes in these mutants in light of the crystallographically determined three-dimensional structure of hexokinase II suggests important roles in structure or catalysis for six amino acid residues, only two of which are near the active site.     

Characterization of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1

Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1-eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.     

Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase.

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate. The eki1Delta mutant did not exhibit a growth phenotype. Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p.     

Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae.

The DNA segments containing the ADR1 gene and a mutant allele, ADR1-5c, have been isolated by complementation of function in Saccharomyces cerevisiae. The ADR1 gene is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when S. cerevisiae cells are grown on a nonfermentable carbon source, whereas the ADR1-5c allele allows ADHII synthesis even during glucose repression. A plasmid pool consisting of yeast DNA fragments isolated from a strain carrying the ADR1-5c allele was used to transform a strain containing the adr1-1 allele, which prevents ADHII depression. Transformants were isolated which expressed ADHII during glucose repression. A plasmid isolated from one of these transformants was shown to carry the ADR1-5c allele by its ability to integrate at the chromosomal adr1-1 locus. The wild-type ADR1 gene was isolated by colony hybridization, using the cloned ADR1-5c gene as a probe. The ADR1-5c and ADR1 DNA segments were indistinguishable by restriction site mapping. A partial ADR1 phenotype could be conferred by a 1.9-kilobase region, but DNA outside of this region appeared to be necessary for normal activation of ADHII by the ADR1 gene.     

Viable nonsense mutants for the SUP45 gene in the yeast Saccharomyces cerevisiae are lethal at increased temperature

Nonlethal nonsense mutations obtained earlier in the essential gene SUP45 encoding the translation termination eRFI factor in the yeast Saccharomyces cerevisiae were further characterized. Strains carrying these mutations retain the viability, since the full-length eRF1 protein is present in these strains, although in decreased amounts as compared to wild-type cells, together with a truncated eRF1. All nonsense mutations are likely to be located in a weak termination context, because a change in the stop codon UGAA (in the case of mutation sup45-107) to UAGA (sup45-107.2) led to the alteration of the local context from a weak to strong and to the lethality of the strain carrying sup45-107.2. All nonsense mutations studied are characterized by thermosensitivity expressed as cell mortality after cultivation at 37 degrees C. When grown under nonpermissive conditions (37 degrees C), cells of nonsense mutants sup45-104, sup45-105. and sup45-107 display a decrease in the amount of the truncated eRF1 protein without reduction in the amount of the full-length eRF1 protein. The results of this study suggest that the N-terminal eRF1 fragment is indispensable for cell viability of nonsense mutants due to the involvement in termination of translation.     

Isolation and characterization of the gene encoding phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

The yeast PCK1 gene coding for phosphoenolpyruvate carboxykinase (PEPCK) was isolated by functional complementation of pck1 strains from S. cerevisiae. Only one copy of the gene was found per haploid yeast genome. An RNA of about 2 kb which hybridized with a DNA probe internal to the PCK1 gene was found only in cells growing in non-fermentable carbon sources. Yeast strains carrying multiple copies of the PCK1 gene showed normal catabolite repression of PEPCK except those carrying the shortest insertion complementing the mutation (2.2 kb) that presented an altered kinetics of derepression. Catabolite inactivation was decreased in strains transformed with multicopy plasmids carrying the PCK1 gene.     

The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.

The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon-specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction.     

Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae.

We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a 2.0-kilobase heat-inducible mRNA. This gene, which we have designated STI1, for stress inducible, was also induced by the amino acid analog canavanine and showed a slight increase in expression as cells moved into stationary phase. The STI1 gene encodes a 66-kilodalton protein, as determined from the sequence of the longest open reading frame. The putative STI1 protein, as identified by two-dimensional gel electrophoresis, migrated in the region of 73 to 75 kilodaltons as a series of four isoforms with different isoelectric points. STI1 is not homologous to the other conserved HSP70 family members in yeasts, despite similarities in size and regulation. Cells carrying a disruption mutation of the STI1 gene grew normally at 30 degrees C but showed impaired growth at higher and lower temperatures. Overexpression of the STI1 gene resulted in substantial trans-activation of SSA4 promoter-reporter gene fusions, indicating that STI1 may play a role in mediating the heat shock response of some HSP70 genes.     

Isolation and characterization of Saccharomyces cerevisiae glycolytic pathway mutants.

Yeast strains carrying recessive mutations representing four different loci that cause defects in pyruvate kinase, pyruvate decarboxylase, 3-phosphoglycerate kinase, and 3-phosphoglycerate mutase were isolated and partially characterized. Cells carrying these mutations were unable to use glucose as a carbon source as measured in turbidimetric growth experiments. Tetrad analysis indicated that these mutations were not linked to each other; one of the mutations, that affecting phosphoglycerate kinase, was located on chromosome III.     

Isolation and preliminary characterization of Saccharomyces cerevisiae proline auxotrophs.

Proline-requiring mutants of Saccharomyces cerevisiae were isolated. Each mutation is recessive and is inherited as expected for a single nuclear gene. Three complementation groups cold be defined which are believed to correspond to mutations in the three genes (pro1, pro2, and pro3) coding for the three enzymes of the pathway. Mutants defective in the pro1 and pro2 genes can be satisfied by arginine or ornithine as well as proline. This suggests that the blocks are in steps leading to glutamate semialdehyde, either in glutamyl kinase or glutamyl phosphate reductase. A pro3 mutant has been shown by enzyme assay to be deficient in delta 1-pyrroline-5-carboxylate reductase which converts pyrroline-5-carboxylate to proline. A unique feature of yeast proline auxotrophs is their failure to grown on the rich medium, yeast extract-peptone-glucose. This failure is not understood at present, although it accounts for the absence of proline auxotrophs in previous screening for amino acid auxotrophy.