Prions affect the appearance of other prions: the story of [PIN(+)].

Prions are self-propagating protein conformations. Recent research brought insight into prion propagation, but how they first appear is unknown. We previously established that the yeast non-Mendelian trait [PIN(+)] is required for the de novo appearance of the [PSI(+)] prion. Here, we show that the presence of prions formed by Rnq1 or Ure2 is sufficient to make cells [PIN(+)]. Thus, [PIN(+)] can be caused by more than one prion. Furthermore, an unbiased functional screen for [PIN(+)] prions uncovered the known prion gene, URE2, the proposed prion gene, NEW1, and nine novel candidate prion genes all carrying prion domains. Importantly, the de novo appearance of Rnq1::GFP prion aggregates also requires the presence of other prions, suggesting the existence of a general mechanism by which the appearance of prions is enhanced by heterologous prion aggregates.

Mutagenic specificity of the base analog 6-N-hydroxylaminopurine in the LYS2 gene of yeast Saccharomyces cerevisiae.

We used the LYS2 gene mutational system to study mutation specificity of the base analog 6-N-hydroxylaminopurine (HAP) in yeast. We characterized phenotypes of mutations using codon-specific nonsense suppressors and the test employing inactivation of the release factor Sup35 due to overexpression and formation of prion-like derivative [PSI]. We have shown that HAP induces predominantly nonsense mutations. While the tests using codon-specific nonsense-suppressors allowed to identify only about 50% of nonsense-mutations, all the nonsense-mutations were identified in the test with defective Sup35. We determined and analyzed the spectrum of HAP-induced nucleotide changes in two regions of the gene. HAP induces predominantly GC-->AT transitions in a hotspots of a central position of trinucleotide GGA or AGG. Directionality of these transitions is consistent with the idea that initial dHAPMP incorporation in the leading strand is more genetically dangerous than in lagging DNA strand. We revealed a specific context inhibitory for HAP mutagenesis, a "T" in -1 position to mutation site.

The relationship between visible intracellular aggregates that appear after overexpression of Sup35 and the yeast prion-like elements [PSI(+)] and [PIN(+)].

Overproduced fusions of Sup35 or its prion domain with green fluorescent protein (GFP) have previously been shown to form frequent dots in [PSI(+)] cells. Rare foci seen in [psi(-)] cells were hypothesized to indicate the de novo induction of [PSI(+)] caused by the overproduced prion domain. Here, we describe novel ring-type aggregates that also appear in [psi(-)] cultures upon Sup35 overproduction and show directly that dot and ring aggregates only appear in cells that have become [PSI(+)]. The formation of either type of aggregate requires [PIN(+)], an element needed for the induction of [PSI(+)]. Although aggregates are visible predominantly in stationary-phase cultures, [PSI(+)] induction starts in exponential phase, suggesting that much smaller aggregates can also propagate [PSI(+)]. Such small aggregates are probably present in [PSI(+)] cells and, upon Sup35-GFP overproduction, facilitate the frequent formation of dot aggregates, but only the occasional appearance of ring aggregates. In contrast, rings are very frequent when [PSI(+)] cultures, including those lacking [PIN(+)], are grown in the presence of GuHCl or excess Hsp104 while overexpressing Sup35-GFP. Thus, intermediates formed during [PSI(+)] curing seem to facilitate ring formation. Surprisingly, GuHCl and excess Hsp104, which are known to promote loss of [PSI(+)], did not prevent the de novo induction of [PSI(+)] by excess Sup35 in [psi(-)][PIN(+)] strains.

Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable.

Four mutant strains from Saccharomyces cerevisiae were used to study ribosome structure and function. They included a strain carrying deletions of the two genes encoding ribosomal protein L24, a strain carrying a mutation spb2 in the gene for ribosomal protein L39, a strain carrying a deletion of the gene for L39, and a mutant lacking both L24 and L39. The mutant lacking only L24 showed just 25% of the normal polyphenylalanine-synthesizing activity followed by a decrease in P-site binding, suggesting the possibility that protein L24 is involved in the kinetics of translation. Each of the two L39 mutants displayed a 4-fold increase of their error frequencies over the wild type. This was accompanied by a substantial increase in A-site binding, typical of error-prone mutants. The absence of L39 also increased sensitivity to paromomycin, decreased the ribosomal subunit ratio, and caused a cold-sensitive phenotype. Mutant cells lacking both ribosomal proteins remained viable. Their ribosomes showed reduced initial rates caused by the absence of L24 but a normal extent of polyphenylalanine synthesis and a substantial in vivo reduction in the amount of 80S ribosomes compared to wild type. Moreover, this mutant displayed decreased translational accuracy, hypersensitivity to the antibiotic paromomycin, and a cold-sensitive phenotype, all caused mainly by the deletion of L39. Protein L39 is the first protein of the 60S ribosomal subunit implicated in translational accuracy.

Dependence and independence of [PSI(+)] and [PIN(+)]: a two-prion system in yeast?

The [PSI(+)] prion can be induced by overproduction of the complete Sup35 protein, but only in strains carrying the non-Mendelian [PIN(+)] determinant. Here we demonstrate that just as [psi (-)] strains can exist as [PIN(+)] and [pin(-)] variants, [PSI(+)] can also exist in the presence or absence of [PIN(+)]. [PSI(+)] and [PIN(+)] tend to be cured together, but can be lost separately. [PSI(+)]-related phenotypes are not affected by [PIN(+)]. Thus, [PIN(+)] is required for the de novo formation of [PSI(+)], not for [PSI(+)] propagation. Although [PSI(+)] induction is shown to require [PIN(+)] even when the only overexpressed region of Sup35p is the prion domain, two altered prion domain fragments circumventing the [PIN(+)] requirement are characterized. Finally, in strains cured of [PIN(+)], prolonged incubation facilitates the reappearance of [PIN(+)]. Newly appearing [PIN(+)] elements are often unstable but become stable in some mitotic progeny. Such reversibility of curing, together with our previous demonstration that the inheritance of [PIN(+)] is non-Mendelian, supports the hypothesis that [PIN(+)] is a prion. Models for [PIN(+)] action, which explain these findings, are discussed.

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.

The PNM2 mutation in the prion protein domain of SUP35 has distinct effects on different variants of the [PSI+] prion in yeast.

We have previously described different variants of the yeast prion [PSI+] that can be obtained and maintained in the same genetic background. These [PSI+] variants, which differ in the efficiency of nonsense suppression, mitotic stability and the efficiency of curing by GuHCl, may correspond to different [PSI+] prion conformations of Sup35p or to different types of prion aggregates. Here we investigate the effects of overexpressing a mutant allele of SUP35 and find different effects on weak and strong [PSI+] variants: the suppressor phenotype of weak [PSI+] factors is increased, whereas the suppressor effect of strong [PSI+] factors is reduced. The SUP35 mutation used was originally described as a "Psi no more" mutation (PNM2) because it caused loss of [PSI+]. However, none of the [PSI+] variants in the strains used in our study were cured by PNM2. Indeed, when overexpressed, PNM2 induced the de novo appearance of both weak and strong [PSI+] variants with approximately the same efficiency as the overexpressed wild-type SUP35 allele. Our data suggest that the change in the region of oligopeptide repeats in the Sup35p N-terminus due to the PNM2 mutation modifies, but does not impair, the function of the prion domain of Sup35p.

Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the [PSI+] prion.

[PSI+], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI+]. However, another non-Mendelian determinant, [PIN+], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI+] by Sup35p overproduction in [PIN+] strains, but has no effect on the propagation of [PSI+] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI+] strains. We argue that excess Sup45p inhibits [PSI+] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.

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.