BackgroundTermination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45.ResultsWe have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 → UAA, E266 → UAA, L283 → UAA, L317 → UGA, E385 → UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37°C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4–20 cells.ConclusionsNonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.

Yeast self-perpetuating protein aggregates (prions) provide a convenient model for studying various components of the cellular protein quality control system. Molecular chaperones and chaperone-sorting factors, such as yeast Cur1 protein, play key role in proteostasis via tight control of partitioning and recycling of misfolded proteins. In this study, we show that, despite the previously described ability of Cur1 to antagonize the yeast prion [URE3], it enhances propagation and phenotypic manifestation of another prion, [PSI ]. We demonstrate that both curing of [URE3] and enhancement of [PSI ] in the presence of excess Cur1 are counteracted by the cochaperone Hsp40-Sis1 in a dosage-dependent manner, and show that the effect of Cur1 on prions parallels effects of the attachment of nuclear localization signal to Sis1, indicating that Cur1 acts on prions via its previously reported ability to relocalize Sis1 from the cytoplasm to nucleus. This shows that the direction in which Cur1 influences a prion depends on how this specific prion responds to relocalization of Sis1.

propagation. In this work, we identified SFP1 as a multicopy inducer of [PSI + ]-dependent lethality. Sfp1 is likely to up-regulate transcription of genes encoding release factors; however, its overproduction increases Sup35, but not Sup45 protein level. Using the synthetic lethality test, we compared the effects of SFP1 and SUP35 over-expression on the viability of [PSI + ] strains.Together with an observation that Sfp1 overproduction leads to an increased accumulation of Sup35 in [PSI + ] aggregates, we suggest that excess Sfp1 causes [PSI + ] toxicity. Even though SUP45 over-expression is known to compensate for the [PSI + ]-dependent lethality, it fails to do so when the lethality is caused by SFP1 over-expression. We discovered that the increased levels of Hsp40 chaperone Sis1 alleviate prion toxicity caused by either SFP1 or SUP35 over-expression and revert back to normal distribution of Sup35 between monomers and aggregate fractions. Finally, we showed that Sfp1 partially colocalizes with Sup35 aggregates, which may contribute to another mechanism of Sfp1-derived [PSI + ] prion toxicity. Introduction [PSI +], one of the best studied yeast prions, is formed by amyloid aggregates of the translation termination factor Sup35 (eRF3). Both translation termination factor genes SUP45 and SUP35 encoding eRF1 and eRF3, respectively, are essential in yeast Saccharomyces cerevisiae (see . The Sup35 protein consists of three domains. The Nproximal region (Sup35N), or prion-forming domain (PrD), is not essential for viability and translation termination (Ter-Avanesyan et al. 1993), but is required for [PSI + ] induction (Chernoff et al. 1992;Derkatch et al. 1996) and propagation (Ter-Avanesyan et al. 1994). The charged M (middle) region (Sup35M) is required for neither viability nor translation termination; however, it may influence [PSI + ] propagation. The C-proximal region (Sup35C) is required (and sufficient) for translation termination and cell viability; it also contains eRF1-interacting domain (see ], which is the prion form of the Rnq1 protein (Derkatch et al. 1997(Derkatch et al. , 2001).Malfunctioning of translation termination apparatus allows readthrough of stop codons leading to the suppression of nonsense mutations (see . It can be caused by a decrease in the level of functional Sup35 or Sup45 via different mechanisms. For example, the collection of sup45 and sup35 suppressor mutants includes viable nonsense mutants, in which the amount of eRF1 or ]-dependent toxicity during PIN4C over-expression (Yang et al. 2013(Yang et al. , 2014 ] cells. SFP1-fs induction in OT56 led to a statistically significant increase in both SUP35 and SUP45 mRNA levels (Fig. 1A). Quantitative RT-PCR showed a 1.51-and 1.50-fold increase in median mRNA levels of SUP35 and SUP45, respectively. We also confirmed an increase in SFP1 expression, because the median SFP1 mRNA level increased 2.96-fold (Fig. 1A).To determine whether SFP1 increases Sup35 and Sup45 production, we estimated the protein levels using Western blotting. How...

Background: The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.

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Background : The termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs), eRF1 and eRF3. In mammals two genes encoding eRF3 structural homologues were identified and named GSPT1 and GSPT2.

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.

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