S. G. Inge-Vechtomov

Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein

SUP35is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C‐terminal part similar to the elongation factor EF‐1α and a unique N‐terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF‐1α‐like C‐terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N‐terminal part of the Sup35 protein or the full‐length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi+] strains; (iii) expression of the C‐terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N‐ or C‐terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.

Multicopy SUP35 gene induces de-novo appearance of psi-like factors in the yeast Saccharomyces cerevisiae.

Previously, we have shown that plasmid-mediated multiplication of Saccharomyces cerevisiae wild-type SUP35 gene leads to omnipotent suppression and is incompatible with psi-factor, which is an endogenous extrachromosomal suppressor. Here, we describe a frequent de-novo appearance of psi-like factors in mitotic progeny of yeast transformants containing multicopy SUP35 gene.

Nucleotide sequence of the SUP2 (SUP35) gene of Saccharomyces cerevisiae

A nucleotide sequence of the yeast Saccharomyces cerevisiae omnipotent suppressor SUP2 (SUP35) gene is presented. The sequence contains a single open reading frame (ORF) of 2055 bp, which may encode a 76.5-kDa protein. A single transcript of 2.3 kb corresponding to a complete ORF is found. Analysis of codon bias suggests that the SUP2 gene is not highly expressed. The C-terminal part of the deduced amino acid sequence shows a high homology to yeast elongation factor EF-1 alpha, whereas the N-terminal part is unique for the SUP2 protein. The N terminus contains a number of short repeating elements and possesses an unusual amino acid composition. Analysis of the nucleotide and deduced amino acid sequences indicates that three additional proteins could possibly be expressed, two of which might be initiated on internal ATG codons and a third might be formed by alternative splicing. One of these proteins is supposed to be imported into mitochondria. Possible functions of the SUP2 gene product(s), especially its putative activity as a soluble factor controlling the fidelity of translation, are discussed.

Poly(A)-Binding Protein Acts in Translation Termination via Eukaryotic Release Factor 3 Interaction and Does Not Influence [PSI+] Propagation

ABSTRACT Recent studies of translational control suggest that translation termination may not be simply the end of synthesizing a protein but rather be involved in modulating both the translation efficiency and stability of a given transcript. Using recombinant eukaryotic release factor 3 (eRF3) and cellular extracts, we have shown for Saccharomyces cerevisiae that yeast eRF3 and Pab1p can interact. This interaction, mediated by the N+M domain of eRF3 and amino acids 473 to 577 of Pab1p, was demonstrated to be direct by the two-hybrid approach. We confirmed that a genetic interaction exists between eRF3 and Pab1p and showed that Pab1p overexpression enhances the efficiency of termination in SUP35 (eRF3) mutant and [PSI +] cells. This effect requires the interaction of Pab1p with eRF3. These data further strengthen the possibility that Pab1p has a role in coupling translation termination events with initiation of translation. Several lines of evidence indicate that Pab1p does not influence [PSI +] propagation. First, “[PSI +]-no-more” mutations do not affect eRF3-Pab1p two-hybrid interaction. Second, overexpression of PAB1 does not cure the [PSI +] phenotype or solubilize detectable amounts of eRF3. Third, prion-curing properties of overexpressed HSP104p, which is required for formation and maintenance of [PSI +], were not modified by excess Pab1p.

Yeast prion protein derivative defective in aggregate shearing and production of new 'seeds'.

According to the nucleated polymerization model, in vivo prion proliferation occurs via dissociation (shearing) of the huge prion polymers into smaller oligomeric ‘seeds’, initiating new rounds of prion replication. Here, we identify the deletion derivative of yeast prion protein Sup35 (Sup35‐Δ22/69) that is specifically defective in aggregate shearing and ‘seed’ production. This derivative, [PSI+], previously thought to be unable to turn into a prion state, in fact retains the ability to form a prion ([PSI+]Δ22/69) that can be maintained in selective conditions and transmitted by cytoplasmic infection (cytoduction), but which is mitotically unstable in non‐selective conditions. Moreover, the full‐size Sup35 prion ‘seeded’ by [PSI+]Δ22/69 retains its mitotic stability defect. The [PSI+]Δ22/69 cells contain more Sup35 protein in the insoluble fraction and form larger Sup35 aggregates compared with the conventional [PSI+] cells. Moderate excess of Hsp104 disaggregase increases transmission of the [PSI+]Δ22/69 prion, while excess Hsp70‐Ssa chaperone antagonizes it, opposite to their effects on conventional [PSI+]. Our results shed light on the mechanisms determining the differences between transmissible prions and non‐transmissible protein aggregates.

Prion variant maintained only at high levels of the Hsp104 disaggregase

The yeast prion [PSI+] is a self-perpetuating aggregated isoform of the translation termination factor Sup35. [PSI+] propagation is promoted by moderate levels and antagonized by high levels of the chaperone Hsp104. In agreement with the model postulating that excess Hsp104 acts on [PSI+] by disaggregating prion polymers, we show that an increase in Sup35 levels, accompanied by an increase in size of prion aggregates, also partially protects [PSI+] from elimination by excess Hsp104. Despite retention of [PSI+], excess Hsp104 decreases toxicity of overproduced Sup35 in [PSI+] strains. A heritable variant of [PSI+], which has been isolated and is maintained only in the presence of increased levels of Hsp104, is characterized by an abnormally large aggregate size, and exhibits an altered response to overproduction of the Hsp70 chaperone Ssa1. These features resemble the previously described prion generated by a deletion derivative of Sup35, but are not associated with any sequence alteration and are controlled exclusively at the protein level. Our data provide a proof of the existence of conditionally stable prion variants maintained only at altered levels of Hsps, that could in principle be beneficial if the normal cellular function of a prion protein becomes detrimental to the cell in such conditions.

Recessive mutations in SUP35 and SUP45 genes coding for translation release factors affect chromosome stability in Saccharomyces cerevisiae.

Abstract Chromosome stability in suppressor mutants for SUP35 and SUP45 genes coding for translation release factors was studied. We obtained spontaneous and UV-induced sup35 or sup45 mutants in a haploid strain disomic for chromosome III and tested the stability of an extra copy of this chromosome. The majority of the mutants showed increased chromosome instability. This phenotype was correlated with an increased sensitivity to the microtubule-poisoning drug benomyl which affects chromosome segregation at anaphase. Our data suggest that termination-translation factors eRF3 and eRF1 control chromosome transmission at mitotic anaphase in Saccharomyces cerevisiae.

Nonsense mutations in the essential gene SUP35 of Saccharomyces cerevisiae are non-lethal

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.

Sensitivity of sup35 and sup45 suppressor mutants in Saccharomyces cerevisiae to the anti-microtubule drug benomyl.

Abstract SUP35 and SUP45 genes determine the accuracy of translation at the stage of termination. We present indirect evidence indicating that these genes may also control some cellular process mediated by microtubules. A majority of sup35 and sup45 suppressor mutations confer supersensitivity to benomyl, the drug which de-polymerizes microtubules. In addition, data correlating phenotypic manifestations of sup45 suppressor mutations, involving sensitivity to benomyl, respiratory deficiency and a suppressor effect, are also presented.

[NSI+]: a novel non-Mendelian nonsense suppressor determinant in Saccharomyces cerevisiae

Non-Mendelian determinants that control heritable traits in yeast are subdivided into two major groups—one that includes DNA- or RNA-based elements and another that comprises protein-based factors that are analogous to mammalian prion. All yeast non-Mendelian determinants show dominant inheritance, and some of them demonstrate cytoplasmic infectivity. Only prions, however, harbor-specific features, such as high frequency of induction following overproduction of prion-encoding protein, loss of the protein’s normal function, and reversible curability. Here, we describe a novel nonchromosomal determinant that, in addition to [PSI+] and [ISP+], is involved in epigenetic control of nonsense suppression. This determinant, which we have designated [NSI+], causes nonsense suppression in the strains bearing the N-terminal-deleted or -modified SUP35 gene, but has no manifestation in the strains with the intact copy of SUP35. [NSI+] shows dominant non-Mendelian inheritance, reversible curability and may be transmitted by cytoduction, albeit with low frequency. Similar to yeast prions, this determinant can be cured by deletion or mutational inactivation of Hsp104. We have shown that [NSI+] does not correspond to the already identified yeast prions. Based on the data obtained, we hypothesize that [NSI+] is a novel prion factor involved in epigenetic control of nonsense suppression.