Michael D. Ter-Avanesyan

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.

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.

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.

Structure and Replication of Yeast Prions

By analogy with the yeast model, the fragmentation of prion and amyloid polymers performed by Hsp104p homologs or other factors should influence their propagation and accumulation. The fragmentation might accelerate accumulation of prion and amyloid aggregates, but it might also serve to dissolve these aggregates if the fragmenting activity is high enough. The level of chaperone activity could control the appearance of amyloids and explain their age-dependent onset. The fragmentation of prions would be essential for their transmissibility, but would not be so critical for amyloid polymers, which are not transmissible and can appear by nucleation.The different frequency of fragmentation could explain the different properties of prions in two human diseases. The absence of observable fibrils in some cases of human CJD probably reflects a high level of PrPSc fragmentation. Another human prion disease, Gerstmann-Straussler-Sheinker syndrome, is characterized by filamentous, amyloid-like PrP deposits and a slower disease progression than CJD. These phenotypes could be related: more frequent fragmentation of CJD fibrils could mean higher overall polymerization speed and faster disease progression.In vitro, yeast Hsp104p interacted specifically with Sup35p, PrP and β-amyloid 1–42 peptide (Schirmer and Lindquist 1997xSchirmer, E.C and Lindquist, S. Proc. Natl. Acad. Sci. USA. 1997; 94: 13932–13937Crossref | PubMed | Scopus (75)See all ReferencesSchirmer and Lindquist 1997), and moreover, promoted the prion conversion of PrP (DebBurman et al. 1997xDebBurman, S.K, Raymond, G.J, Caughey, B, and Lindquist, S. Proc. Natl. Acad. Sci. USA. 1997; 94: 13938–13943Crossref | PubMed | Scopus (202)See all ReferencesDebBurman et al. 1997). These results suggest that Hsp104p analogs in mammals could also interact with such proteins and participate in prion and amyloid formation. Hsp104p is likely to occur in animals, since it shows similarity to the Clp/Hsp100 family of bacterial and eukaryotic chaperones and is functionally conserved between yeast and plants. However, in contrast to yeast prions, which propagate inside cells, mammalian prions and most amyloids occur extracellularly. Hsp104p is not secreted in yeast and it is likely to be so in mammals as well. At least one might expect different levels of chaperone fragmenting activity and probably even different factors causing fragmentation in intra- and extracellular compartments.Thus, in terms of the type and levels of fragmenting activity the best analogy with yeast prions may be found in intracellular amyloid diseases, related to the expansion of polyglutamine tracts in some human proteins. These diseases are accompanied by neuronal nuclear inclusions, formed by the respective polyglutamine proteins. The expanded polyglutamine domain of one such protein, huntingtin, was shown to form amyloid fibrils in vitro (Scherzinger et al. 1997xScherzinger, E, Lurz, R, Turmaine, M, Mangiarini, L, Hollenbach, B, Hasenbank, R, Bates, G.P, Davies, S.W, Lehrach, H, and Wanker, E.E. Cell. 1997; 90: 549–558Abstract | Full Text | Full Text PDF | PubMed | Scopus (833)See all ReferencesScherzinger et al. 1997). The abundance of glutamine and the related amino acid asparagine is also a characteristic feature of prion domains of Sup35p and Ure2p of S. cerevisiae. This feature is common for Sup35p from different yeast species and fungi, although not from higher eukaryotes. The importance of these residues was further demonstrated by the finding that the mutations which interfere with the prion properties of Sup35p fall within a short amino-terminal fragment, which is rich in Gln and Asn (DePace et al. 1998xDePace, A.H, Santoso, A, Hillner, P, and Weissman, J.S. Cell. 1998; 93: 1241–1252Abstract | Full Text | Full Text PDF | PubMed | Scopus (276)See all ReferencesDePace et al. 1998). The yeast prions and polyglutamine proteins might be considered as a single structural class, in contrast to other amyloidogenic proteins, which are not abundant in Gln and Asn. These similarities suggest that chaperones like Hsp104p might play an important role in diseases associated with expanded polyglutamine tracts.

Prion properties of the Sup35 protein of yeast Pichia methanolica

The Sup35 protein (Sup35p) of Saccharomyces cerevisiae is a translation termination factor of the eRF3 family. The proteins of this family possess a conservative C‐terminal domain responsible for translation termination and N‐terminal extensions of different structure. The N‐terminal domain of Sup35p defines its ability to undergo a heritable prion‐like conformational switch, which is manifested as the cytoplasmically inherited [PSI+] determinant. Here, we replaced the N‐terminal domain of S.cerevisiae Sup35p with an analogous domain from Pichia methanolica. Overexpression of hybrid Sup35p induced the de novo appearance of cytoplasmically inherited suppressor determinants manifesting key genetic and biochemical traits of [PSI+]. In contrast to the conventional [PSI+], ‘hybrid’ [PSI+] showed lower mitotic stability and preserved their suppressor phenotype upon overexpression of the Hsp104 chaperone protein. The lack of Hsp104 eliminated both types of [PSI+]. No transfer of prion state between the two Sup35p variants was observed, which reveals a ‘species barrier’ for the [PSI+] prions. The data obtained show that prion properties are conserved within at least a part of this protein family.

Increased Expression of Hsp40 Chaperones, Transcriptional Factors, and Ribosomal Protein Rpp0 Can Cure Yeast Prions

The Sup35 (eRF3) translation termination factor of Saccharomyces cerevisiae can undergo a prion-like conformational conversion, thus resulting in the [PSI +] nonsense-suppressor determinant.In vivo this process depends critically on the chaperone Hsp104, whose lack or overexpression can cure [PSI +]. The use of artificial prion [PSI + PS] based on a hybrid Sup35PS with prion domain from the yeast Pichia methanolicaallowed us to uncover three more chaperones, Ssb1, Ssa1, and Ydj1, whose overexpression can cure prion determinants. Here, we used the [PSI + PS] to search a multicopy yeast genomic library for novel factors able to cure prions. It was found that overexpression of the Hsp40 family chaperones Sis1 and Ynl077w, chaperone Sti1, transcriptional factors Sfl1 and Ssn8, and acidic ribosomal protein Rpp0 can interfere with propagation and manifestation of [PSI + PS] in a prion strain-specific manner. Some of these factors also affected the manifestation and propagation of conventional [PSI +]. Excess of Sfl1, Ssn8, and Rpp0 influenced at least one of the tested chaperone-specific promoters,SSA4, HSP104, and model promoters, with either the heat shock or stress response elements. Thus, the induction of chaperone expression by these proteins could explain their prion-curing effects.

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 Ψ+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58→Asp change in the Sup35p N‐terminal domain eliminates Ψ+. 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 Ψ+ 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 Ψ+‐eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Ψ+‐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.

[Psi(+)] prion generation in yeast: characterization of the 'strain' difference.

The yeast cytoplasmically‐inherited nonsense suppressor [PSI+] determinant is presumed to be a manifestation of the aggregated prion‐like state of the Sup35 protein. Overexpression of the Sup35 protein induces generation of [PSI+] determinants with various suppressor efficiency and mitotic stabilities. Here, we demonstrate that the relative frequency of appearance of [PSI+] with different properties depends on the SUP35 allele used to induce their generation. The difference in properties of [PSI+] determinants was preserved after their transmission from one yeast strain to another. This difference correlated with variation in properties of the Sup35 protein. A novel type of prion instability was observed: some [PSI+] with weak suppressor efficiency could convert spontaneously into strong suppressor determinants. Copyright

The Role of the N-Terminal Oligopeptide Repeats of the Yeast Sup35 Prion Protein in Propagation and Transmission of Prion Variants

The cytoplasmic [PSI+] determinant of Saccharomyces cerevisiae is the prion form of the Sup35 protein. Oligopeptide repeats within the Sup35 N-terminal domain (PrD) presumably are required for the stable [PSI+] inheritance that in turn involves fragmentation of Sup35 polymers by the chaperone Hsp104. The nonsense suppressor [PSI+] phenotype can vary in efficiency probably due to different inheritable Sup35 polymer structures. Here we study the ability of Sup35 mutants with various deletions of the oligopeptide repeats to support [PSI+] propagation. We define the minimal region of the Sup35–PrD necessary to support [PSI+] as amino acids 1–64, which include the first two repeats, although a longer fragment, 1–83, is required to maintain weak [PSI+] variants. Replacement of wild-type Sup35 with deletion mutants decreases the strength of the [PSI+] phenotype. However, with one exception, reintroducing the wild-type Sup35 restores the original phenotype. Thus, the specific prion fold defining the [PSI+] variant can be preserved by the mutant Sup35 protein despite the change of phenotype. Coexpression of wild-type and mutant Sup35 containing three, two, one, or no oligopeptide repeats causes variant-specific [PSI+] elimination. These data suggest that [PSI+] variability is primarily defined by differential folding of the Sup35–PrD oligopeptide-repeat region.

C-terminal truncation of the Sup35 protein increases the frequency of de novo generation of a prion-based [PSI+] determinant in Saccharomyces cerevisiae

Abstract The yeast non-Mendelian [PSI+] determinant is presumed to be the manifestation of the aggregated prion-like state of the Sup35 protein. Plasmid-mediated amplification of the SUP35 gene greatly increases the frequency of Sup35p transition to this prion-like state. Here we show that the 3′-deletions of plasmid SUP35, leading to the C-terminal truncation of Sup35p, further increase the frequency of [PSI+] induction despite a marked decrease in Sup35p expression levels. The data suggest that the presence of Sup35p N-terminal proteolytic fragments can cause [PSI+] appearance in wild-type yeast cells.