The yeast non-Mendelian factor [ETA+] is a variant of [PSI+], a prion-like form of release factor eRF3

The yeast non-Mendelian factor [ETA+] is lethal in the presence of certain mutations in the SUP35 and SUP45 genes, which code for the translational release factors eRF3 and eRF1, respectively. One such mutation, sup35-2, is now shown to contain a UAG stop codon prior to the essential region of the gene. The non-Mendelian inheritance of [ETA+] is reminiscent of the yeast [PSI+] element, which is due to a self-propagating conformation of Sup35p. Here we show that [ETA+] and [PSI+] share many characteristics. Indeed, like [PSI+], the maintenance of [ETA+] requires the N-terminal region of Sup35p and depends on an appropriate level of the chaperone protein Hsp104. Moreover, [ETA+] can be induced de novo by excess Sup35p, and [ETA+] cells have a weak nonsense suppressor phenotype characteristic of weak [PSI+]. We conclude that [ETA+] is actually a weak, unstable variant of [PSI+]. We find that although some Sup35p aggregates in [ETA+] cells, more Sup35p remains soluble in [ETA+] cells than in isogenic strong [PSI+] cells. Our data suggest that the amount of soluble Sup35p determines the strength of translational nonsense suppression associated with different [PSI+] variants.     

Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation.

The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.     

Propagation of the yeast prion-like [psi+] determinant is mediated by oligomerization of the SUP35-encoded polypeptide chain release factor.

The Sup35p protein of yeast Saccharomyces cerevisiae is a homologue of the polypeptide chain release factor 3 (eRF3) of higher eukaryotes. It has been suggested that this protein may adopt a specific self-propagating conformation, similar to mammalian prions, giving rise to the [psi+] nonsense suppressor determinant, inherited in a non-Mendelian fashion. Here we present data confirming the prion-like nature of [psi+]. We show that Sup35p molecules interact with each other through their N-terminal domains in [psi+], but not [psi-] cells. This interaction is critical for [psi+] propagation, since its disruption leads to a loss of [psi+]. Similarly to mammalian prions, in [psi+] cells Sup35p forms high molecular weight aggregates, accumulating most of this protein. The aggregation inhibits Sup35p activity leading to a [psi+] nonsense-suppressor phenotype. N-terminally altered Sup35p molecules are unable to interact with the [psi+] Sup35p isoform, remain soluble and improve the translation termination in [psi+] strains, thus causing an antisuppressor phenotype. The overexpression of Hsp104p chaperone protein partially solubilizes Sup35P aggregates in the [psi+] strain, also causing an antisuppressor phenotype. We propose that Hsp104p plays a role in establishing stable [psi+] inheritance by splitting up Sup35p aggregates and thus ensuring equidistribution of the prion-like Sup35p isoform to daughter cells at cell divisions.     

Yeast polypeptide chain release factors eRF1 and eRF3 are involved in cytoskeleton organization and cell cycle regulation

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. eRF1 recognizes nonsense codons UAA, UAG, and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRF1-dependent manner. In the yeast Saccharomyces cerevisiae, eRF1 and eRF3 are encoded by the SUP45 and SUP35 genes, respectively. Here we show that in yeast shortage of any one of the release factors was accompanied by a reduction in the levels of the other release factor and resulted in a substantial increase of nonsense codon readthrough. Besides, repression of the genes encoding these factors caused different effects on cell morphology. Repression of the SUP35 gene caused accumulation of cells of increased size with large buds. This was accompanied by the disappearance of actin cytoskeletal structures, impairment of the mitotic spindle structure, and defects in nuclei division and segregation in mitosis. The evolutionary conserved C-terminal domain of eRF3 similar to the elongation factor EF-1alpha was responsible for these effects. Repression of the SUP45 gene caused accumulation of unbudded cells with 2C and higher DNA content, indicating that DNA replication is uncoupled from budding. The data obtained suggest that eRF1 and eRF3 play additional, nontranslational roles in the yeast cell.     

Prion-Dependent Lethality of sup45 Mutants in Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI⁺]. It is known that combination of [PSI⁺] with some mutant alleles of the SUP35 or SUP45 genes in one and the same haploid yeast cell causes synthetic lethality. In this study, we perform detailed analysis of synthetic lethality between various sup45 nonsense and missense mutations on one hand, and different variants of [PSI⁺] on the other hand. Synthetic lethality with sup45 mutations was detected for [PSI⁺] variants of different stringencies. Moreover, we demonstrate for the first time that in some combinations, synthetic lethality is dominant and occurs at the postzygotic stage after only a few cell divisions. The tRNA suppressor SUQ5 counteracts the prion-dependent lethality of the nonsense alleles but not of the missense alleles of SUP45, indicating that the lethal effect is due to the depletion of Sup45. Synthetic lethality is also suppressed in the presence of the C-proximal fragment of Sup35 (Sup35C) that lacks the prion domain and cannot be included into the prion aggregates. Remarkably, the production of Sup35C in a sup45 mutant strain is also accompanied by an increase in the Sup45 levels, suggesting that translationally active Sup35 up-regulates Sup45 or protects it from degradation.Key Words: Sup45, Sup35, eRF1, eRF3, amyloid, [PSI⁺], translation termination, Saccharomyces cerevisiae     

Overexpression of gene PPZ1 in the yeast Saccharomyces cerevisiae affects the efficiency of nonsense suppression

The phenomenon of nonsense suppression, which leads to the reading of stop codons as sense codons, may be related to disturbances in the operation of various components of the translation apparatus and the proteins interacting with them. The phosphatase Ppzlp is one of the factors affecting the nonsense suppression efficiency in the saccharomycete yeast. In this work, the impact of the overexpression of gene PPZ1 and its mutant allele PPZ1-R451L on the phenotypic expression of various mutant alleles of genes SUP35 and SUP45 or the yeast prion [PSI+] was analyzed. On the basis of the data obtained, a suggestion about the possible role of proteins Sup35p and Sup45p in the processes mediating the influence of gene PPZ1 overexpression on the efficiency of nonsense suppression is made.     

Suppression of nonsense and frameshift mutations obtained by different methods for inactivating the translation termination factor eRF3 in yeast Saccharomyces cerevisiae

Mutations in genes of omnipotent nonsense suppressors SUP35 and SUP45 in yeast Saccharomyces cerevisiae encoding translation termination factors eRF3 and eRF1, respectively, and prionization of the eRF3 protein may lead to the suppression of some frameshift mutations (CPC mutations). Partial inactivation of the translation termination factor eRF3 was studied in strains with unstable genetically modified prions and also in transgenic yeast S. cerevisiae strains with the substitution of the indigenous SUP35 gene for its homolog from Pichia methanolica or for a recombinant S. cerevisiae SUP35 gene. It was shown that this partial inactivation leads not only to nonsense suppression, but also to suppression of the frameshift lys2-90 mutation. Possible reasons for the correlation between nonsense suppression and suppression of the CPC lys2-90 mutation and mechanisms responsible for the suppression of CPC mutations during inactivation of translation termination factors are discussed.     

Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.

Termination of translation in higher organisms is a GTP-dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.     

Overexpression of gene PPZ1 in the yeast Saccharomyces cerevisiae affects the efficiency of nonsense suppression

The phenomenon of nonsense suppression, which leads to the stop codons reading-through, may be related to disturbances in the operation of various components of the translation apparatus and the proteins interacting with them. The phosphatase Ppzlp is one of the factors affecting the nonsense suppression efficiency in Saccharomyces yeast. In this work, the impact of the overexpression of gene PPZ1 and its mutant allele PPZ1-R451L on the phenotypic expression of various mutant alleles of genes SUP35 and SUP45 or the yeast prion [PSI ⁺] was analyzed. On the basis of the data obtained, a suggestion about the possible role of proteins Sup35p and Sup45p in the processes mediating the influence of gene PPZ1 overexpression on the efficiency of nonsense suppression is made.     

The product of the mammalian orthologue of the Saccharomyces cerevisiae HBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3) but does not carry eRF3-like activity

We describe here the cloning and sequencing of human and mouse cDNAs encoding a putative GTP binding protein. Sequence comparison shows that these cDNAs (named eRFS) are likely to represent the orthologues of the yeast Saccharomyces cerevisiae HBS1 gene and that the C-terminal domains of the encoded proteins share structural features with eukaryotic elongation factor eEF-1A and release factor 3 (eRF3) families. The phylogenetic analysis suggests that eRFS proteins and Hbs1p form a cluster of orthologous sequences branching with the eRF3 family. Nevertheless, in yeast, the human eRFS protein and Hbs1p do not complement eRF3/Sup35p thermosensitive mutation and do not interact with eRF1.     

Polyglutamine toxicity is controlled by prion composition and gene dosage in yeast

Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.     

Eukaryotic release factors (eRFs) history

In the present review, we describe the history of the identification of the eukaryotic translation termination factors eRF1 and eRF3. As in the case of several proteins involved in general and essential processes in all cells (e.g., DNA replication, gene expression regulation.) the strategies and methodologies used to identify these release factors were first established in prokaryotes. The genetic investigations in Saccharomyces cerevisiae have made a major contribution in the field. A large amount of data have been produced, from which it was concluded that the SUP45 and SUP35 genes were controlling translation termination but were also involved in other functions important for the cell organization and the cell cycle accomplishment. This does not seem to be restricted to yeast but is also probably the case in eukaryotes in general. The biochemical studies of the proteins encoded by the higher eukaryote homologs of SUP45 and SUP35 were efficient and permitted the identification of eRF1 as being the key protein in the termination process, eRF3 having a stimulating role. Around 25 years were needed after the identification of sup45 and sup35 mutants for the characterization of their gene products as eRF1 and eRF3, respectively. It also has to be pointed out that if the results came first from bacteria, the identification of RF3 and eRF3 was made practically at the same time. Moreover, eRF1 was the first crystal structure obtained for a class-1 release factor, the bacterial RF2 structure came later. The goal is now to understand at the molecular level the roles of both eRF1 and eRF3 in addition to their translation termination functions.     

The paradox of viable <Emphasis Type="Italic">sup45</Emphasis> STOP mutations: a necessary equilibrium between translational readthrough,activity and stability of the protein

The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q → TAA, 242R → TGA, 317L → TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.     

Mechanism of inhibition of Psi+ prion determinant propagation by a mutation of the N-terminus of the yeast Sup35 protein.

The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3. This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited Psi+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58-->Asp change in the Sup35p N-terminal domain eliminates Psi+. Here we observed that the mutant Sup35p can be converted to the prion-like form in vitro, but such conversion proceeds slower than that of wild-type Sup35p. The overexpression of mutant Sup35p induced the de novo appearance of Psi+ cells containing the prion-like form of mutant Sup35p, which was able to transmit its properties to wild-type Sup35p both in vitro and in vivo. Our data indicate that this Psi+-eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Psi+-specific aggregates, but rather inhibits its subsequent prion-like rearrangement and/or binding of the next Sup35p molecule to the growing prion-like Sup35p aggregate.     

Interactions of [NSI +] prion-like determinant with SUP35 and VTS1 genes in Saccharomyces cerevisiae

Previously we characterized [NSI ⁺], determinant, that possesses the features of a yeast prion. This determinant causes the nonsense suppression in strains that bear different N-substituted variants of Sup35p, which is a translation release factor eRF3. As a result of the genomic screen, we identified VTS1, the overexpression of which is a phenotypic copy of [NSI ⁺]. Here, we analyzed the influence of SUP35 and VTS1 on [NSI ⁺]. We demonstrated nonsense suppression in the [NSI ⁺] strains, which appears when SUP35 expression was decreased or against a background of general defects in the fidelity of translation termination. [NSI ⁺] has also been shown to increase VTS1 mRNA amounts. These findings facilitate the insight into the mechanisms of nonsense suppression in the [NSI ⁺] strains and narrow the range of candidates for [NSI ⁺] determinant.     

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.     

Suppression of Nonsense and Frameshift Mutations Obtained by Different Methods for Inactivating the Translation Termination Factor eRF3 in Yeast Saccharomyces cerevisiae

Mutations in genes of omnipotent nonsense suppressors SUP35 and SUP45 in yeast Saccharomyces cerevisiae encoding translation termination factors eRF3 and eRF1, respectively, and prionization of the eRF3 protein may lead to the suppression of some frameshift mutations (CPC mutations). Partial inactivation of the translation termination factor eRF3 was studied in strains with unstable genetically modified prions and also in transgenic yeast S. cerevisiae strains with the substitution of the indigenous SUP35 gene for its homolog from Pichia methanolica or for a recombinant S. cerevisiae SUP35gene. It was shown that this partial inactivation leads not only to nonsense suppression, but also to suppression of the frameshift lys2-90 mutation. Possible reasons for the correlation between nonsense suppression and suppression of the CPC lys2-90 mutation and mechanisms responsible for the suppression of CPC mutations during inactivation of translation termination factors are discussed.