Nonsense Mutations in the Yeast SUP35 Gene Affect the [PSI+] Prion Propagation

The essential SUP35 gene encodes yeast translation termination factor eRF3. Previously, we isolated nonsense mutations sup35-n and proposed that the viability of such mutants can be explained by readthrough of the premature stop codon. Such mutations, as well as the prion [PSI+], can appear in natural yeast populations, and their combinations may have different effects on the cells. Here, we analyze the effects of the compatibility of sup35-n mutations with the [PSI+] prion in haploid and diploid cells. We demonstrated that sup35-n mutations are incompatible with the [PSI+] prion, leading to lethality of sup35-n [PSI+] haploid cells. In diploid cells the compatibility of [PSI+] with sup35-n depends on how the corresponding diploid was obtained. Nonsense mutations sup35-21, sup35-74, and sup35-218 are compatible with the [PSI+] prion in diploid strains, but affect [PSI+] properties and lead to the formation of new prion variant. The only mutation that could replace the SUP35 wild-type allele in both haploid and diploid [PSI+] strains, sup35-240, led to the prion loss. Possibly, short Sup351–55 protein, produced from the sup35-240 allele, is included in Sup35 aggregates and destabilize them. Alternatively, single molecules of Sup351–55 can stick to aggregate ends, and thus interrupt the fibril growth. Thus, we can conclude that sup35-240 mutation prevents [PSI+] propagation and can be considered as a new pnm mutation.

Search for genes encoding potentially amyloidogenic proteins involved in regulation of nonsense -suppresion in Sacharom yces cerevisiae

Previously, the deletion of SUP35N has been shown to create the genetic background for identification of the novel genes and epigenetic determinants controlling the nonsense-suppression. Here, using a genomic overexpression screen, we have found several genes encoding potentially amyloidogenic proteins, whose overexpression affects the suppressor phenotype in the strain producing the chimeric protein Aβ-Sup35MC on the background of the deletion of SUP35 gene encoding releasing factor eRF 3. It has been demonstrated the NAB2, NAB3 and VTS1 genes participate in the regulation of nonsense-suppression in S. cerevisiae.

Novel Non-Mendelian Determinant Involved in the Control of Translation Accuracy in Saccharomyces cerevisiae

Two cytoplasmically inherited determinants related by their manifestation to the control of translation accuracy were previously described in yeast. Cells carrying one of them, [PSI+], display a nonsense suppressor phenotype and contain a prion form of the Sup35 protein. Another element, [PIN+], determines the probability of de novo generation of [PSI+] and results from a prion form of several proteins, which can be functionally unrelated to Sup35p. Here we describe a novel nonchromosomal determinant related to the SUP35 gene. This determinant, designated [ISP+], was identified as an antisuppressor of certain sup35 mutations. We observed its loss upon growth on guanidine hydrochloride and subsequent spontaneous reappearance with high frequency. The reversible curability of [ISP+] resembles the behavior of yeast prions. However, in contrast to known prions, [ISP+] does not depend on the chaperone protein Hsp104. Though manifestation of both [ISP+] and [PSI+] is related to the SUP35 gene, the maintenance of [ISP+] does not depend on the prionogenic N-terminal domain of Sup35p and Sup35p is not aggregated in [ISP+] cells, thus ruling out the possibility that [ISP+] is a specific form of [PSI+]. We hypothesize that [ISP+] is a novel prion involved in the control of translation accuracy in yeast.

Genetic and Environmental Factors Affecting the de novo Appearance of the [PSI + ] Prion in Saccharomyces cerevisiae

It has previously been shown that yeast prion [PSI + ] is cured by GuHCl, although reports on reversibility of curing were contradictory. Here we show that GuHCl treatment of both [PSI + ] and [psi – ] yeast strains results in two classes of [psi – ] derivatives: Pin+, in which [PSI + ] can be reinduced by Sup35p overproduction, and Pin–, in which overexpression of the complete SUP35 gene does not lead to the [PSI + ] appearance. However, in both Pin+ and Pin– derivatives [PSI + ] is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, [PSI + ] curing remains reversible in both cases. Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin+ [psi – ] derivatives are observed in Pin– [psi – ] derivatives. Genetic analyses show that the Pin+ phenotype is determined by a non-Mendelian factor, which, unlike the [PSI + ] prion, is independent of the Sup35p N-terminal domain. A Pin– [psi – ] derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while [PSI + ] curing by Hsp104 overproduction resulted exclusively in Pin+ [psi – ] derivatives. We hypothesize that in addition to the [PSI + ] prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin+ phenotype.

Genesis and Variability of [PSI] Prion Factors in Saccharomyces cerevisiae

We have previously shown that multicopy plasmids containing the complete SUP35 gene are able to induce the appearance of the non-Mendelian factor [PSI]. This result was later interpreted by others as a crucial piece of evidence for a model postulating that [PSI] is a self-modified, prion-like conformational derivative of the Sup35 protein. Here we support this interpretation by proving that it is the overproduction of Sup35 protein, and not the excess of SUP35 DNA or mRNA that causes the appearance of [PSI]. We also show that the “prion-inducing domain” of Sup35p is in the N-terminal region, which, like the “prion-inducing domain” of another yeast prion, Ure2p, was previously shown to be distinct from the functional domain of the protein. This suggests that such a chimeric organization may be a common pattern of some prion elements. Finally, we find that [PSI] factors of different efficiencies and different mitotic stabilities are induced in the same yeast strain by overproduction of the identical Sup35 protein. We suggest that the different [PSI]-containing derivatives are analogous to the mysterious mammalian prion strains and result from different conformational variants of Sup35p.

The dominant PNM2- mutation which eliminates the psi factor of Saccharomyces cerevisiae is the result of a missense mutation in the SUP35 gene.

The PNM2- mutation of Saccharomyces cerevisiae eliminates the extrachromosomal element psi. PNM2 is closely linked to the omnipotent suppressor gene SUP35 (also previously identified as SUP2, SUF12, SAL3 and GST1). We cloned PNM2- and showed that PNM2 and SUP35 are the same gene. We sequenced the PNM2- mutant allele and found a single G-->A transition within the N-terminal domain of the protein. We tested the effects of various constructs of SUP35 and PNM2- on psi inheritance and on allosuppressor and antisuppressor functions of the gene. We found that the C-terminal domain of SUP35 protein (SUP35p) could be independently expressed; expression produced dominant antisuppression. Disruption of the N-terminal domain of PNM2- destroyed the ability to eliminate psi. These results imply that the domains of SUP35p act in an antagonistic manner: the N-terminal domain decreases chain-termination fidelity, while the C-terminal domain imposes fidelity. Two transcripts were observed for SUP35, a major band at 2.4 kb and a minor band at 1.3 kb; the minor band corresponds to 3' sequences only. We propose a model for the function of SUP35, in which comparative levels of N- and C-terminal domains of SUP35p at the ribosome modulate translation fidelity.

The SUP35 omnipotent suppressor gene is involved in the maintenance of the non-Mendelian determinant [psi+] in the yeast Saccharomyces cerevisiae.

The SUP35 gene of yeast Saccharomyces cerevisiae encodes a 76.5-kD ribosome-associated protein (Sup35p), the C-terminal part of which exhibits a high degree of similarity to EF-1 alpha elongation factor, while its N-terminal region is unique. Mutations in or overexpression of the SUP35 gene can generate an omnipotent suppressor effect. In the present study the SUP35 wild-type gene was replaced with deletion alleles generated in vitro that encode Sup35p lacking all or a part of the unique N-terminal region. These 5'-deletion alleles lead, in a haploid strain, simultaneously to an antisuppressor effect and to loss of the non-Mendelian determinant [psi+]. The antisuppressor effect is dominant while the elimination of the [psi+] determinant is a recessive trait. A set of the plasmid-borne deletion alleles of the SUP35 gene was tested for the ability to maintain [psi+]. It was shown that the first 114 amino acids of Sup35p are sufficient to maintain the [psi+] determinant. We propose that the Sup35p serves as a trans-acting factor required for the maintenance of [psi+].