Some proteins can change their fold from normal to a specific alternative form, called prion, which is able to catalyze this change (1). In man and animals such process causes prion diseases like Creutzfeldt-Jacob disease, bovine spongiform encephalopathy, and scrapie of sheep. A similar autocatalytic mechanism is shared by human amyloid diseases, which are noninfectious, in contrast to prion diseases (2). In yeast, there are several proteins, which can undergo prion-like structural conversion. The most studied of them are translation termination factor eRF3, also called Sup35, and Ure2 involved in regulation of nitrogen metabolism (3 was not lost (9). Hsp104 was shown to break large aggregates of denatured protein into smaller pieces (10, 11). We proposed that Hsp104 acts similarly on fiber-shaped prion polymers, thus fragmenting them into shorter polymers and increasing their number (12). This is essential for their inheritance and accelerates the prion conversion by multiplying the ends of prion polymers, where the conversion occurs. The overproduction of Hsp104 should cause excessive fragmentation, increased levels of soluble Sup35, and possibly [PSI ϩ ] loss. An alternative model proposed that Hsp104 is primarily required to facilitate the prion conversion in one or another way (13,14).These two models may be distinguished, since they make different predictions for alteration of the size of prion particles upon inhibition of the Hsp104 function. By the former model, the size should increase due to blocked fragmentation, while by the latter it should stay constant or decrease due to block of polymerization. Recent studies provided some support for the "fragmentation" model. Decrease of the Hsp104 expression caused increase in the size of Sup35 prion aggregates, suggesting decreased disaggregation by Hsp104 (15). The activity of Hsp104 is inhibited by growing yeast cells in the presence of 3-5 mM guanidine HCl (GuHCl) 1 (16). Such treatment cures efficiently [PSI ϩ ] (17) and other known yeast prions. Study of the kinetics of [PSI ϩ ] loss in the presence of GuHCl allowed concluding that it blocks replication of prion "seeds" (18,19). Thus, Hsp104 inhibition correlates with the block of fragmentation (replication) of prion particles (seeds). However, in these experiments the relation of the studied prion entities to the Sup35 polymers considered by the above models was not characterized. The prion seeds were defined genetically, but their physical nature was not studied. In the work (15) the size of

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 methods currently used for protein extraction from yeast are either laborious or insuf®ciently reliable. Here I report a method for protein extraction for electrophoretic analysis that is both easy and reliable. In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer. The method was tested for different strains of Saccharomyces cerevisiae and for yeast Hansenula polymorpha DL-1. It yields virtually complete extraction independently of the strain, growth conditions and protein molecular weight and allows working with small amounts of yeast cells grown on agar plates.

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.

SUP35 is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1 alpha 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-1 alpha-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.

The yeast cytoplasmically inherited genetic determinant [PSI+] is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [psi-] cells (Sup35ppsi-) to the prion-like [PSI+]-specific form (Sup35pPSI+) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous [PSI+] propagation. Size fractionation of lysates of [PSI+] cells demonstrated that the converting activity was associated solely with Sup35pPSI+ aggregates, which agrees with the nucleation model for [PSI+] propagation. Sup35pPSI+ was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.

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