Susan W. Liebman

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.

Interactions among prions and prion “strains” in yeast

Prions are “infectious” proteins. When Sup35, a yeast translation termination factor, is aggregated in its [PSI+] prion form its function is compromised. When Rnq1 is aggregated in its [PIN+] prion form, it promotes the de novo appearance of [PSI+]. Heritable variants (strains) of [PSI+] with distinct phenotypes have been isolated and are analogous to mammalian prion strains with different pathologies. Here, we describe heritable variants of the [PIN+] prion that are distinguished by the efficiency with which they enhance the de novo appearance of [PSI+]. Unlike [PSI+] variants, where the strength of translation termination corresponds to the level of soluble Sup35, the phenotypes of these [PIN+] variants do not correspond to levels of soluble Rnq1. However, diploids and meiotic progeny from crosses between either different [PSI+], or different [PIN+] variants, always have the phenotype of the parental variant with the least soluble Sup35 or Rnq1, respectively. Apparently faster growing prion variants cure cells of slower growing or less stable variants of the same prion. We also find that YDJ1 overexpression eliminates some but not other [PIN+] variants and that prions are destabilized by meiosis. Finally, we show that, like its affect on [PSI+] appearance, [PIN+] enhances the de novo appearance of [URE3]. Surprisingly, [PSI+] inhibited [URE3] appearance. These results reinforce earlier reports that heterologous prions interact, but suggest that such interactions can not only positively, but also negatively, influence the de novo generation of prions.

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 UBIQUITIN-CONJUGATING ENZYME RAD6 (UBC2) IS REQUIRED FOR SILENCING IN SACCHAROMYCES CEREVISIAE

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.

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.

Mutations in eukaryotic 18S ribosomal RNA affect translational fidelity and resistance to aminoglycoside antibiotics.

Mutations have been created in the Saccharomyces cerevisiae 18S rRNA gene that correspond to those known to be involved in the control of translational fidelity or antibiotic resistance in prokaryotes. Yeast strains, in which essentially all chromosomal rDNA repeats are deleted and all cellular rRNAs are encoded by plasmid, have been constructed that contain only mutant 18S rRNA. In Escherichia coli, a C‐‐>U substitution at position 912 of the small subunit rRNA causes streptomycin resistance. Eukaryotes normally carry U at the corresponding position and are naturally resistant to streptomycin. We show that a U‐‐>C transition (rdn‐4) at this position of the yeast 18S rRNA gene decreases resistance to streptomycin. The rdn‐4 mutation also increases resistance to paromomycin and G‐418, and inhibits nonsense suppression induced by paromomycin. The same phenotypes, as well as a slow growth phenotype, are also associated with rdn‐2, whose prokaryotic counterpart, 517 G‐‐>A, manifests itself as a suppressor rather than an antisuppressor. Neither rdn‐2‐ nor rdn‐4‐related phenotypes could be detected in the presence of the normal level of wild‐type rDNA repeats. Our data demonstrate that eukaryotic rRNA is involved in the control of translational fidelity, and indicate that rRNA features important for interactions with aminoglycosides have been conserved throughout evolution.

Analysis of Amyloid Aggregates Using Agarose Gel Electrophoresis

Amyloid aggregates are associated with a number of mammalian neurodegenerative diseases. Infectious aggregates of the mammalian prion protein PrP(sc) are hallmarks of transmissible spongiform encephalopathies in humans and cattle (Griffith, 1967; Legname et al., 2004; Prusiner, 1982; Silveira et al., 2004). Likewise, SDS-stable aggregates and low-n oligomers of the Abeta peptide (Selkoe et al., 1982; Walsh et al., 2002) cause toxic effects associated with Alzheimers disease (Selkoe, 2004). The discovery of prions in lower eukaryotes, for example, yeast prions [PSI(+)], [PIN(+)], and [URE3] suggested that prion phenomena may represent a fundamental process that is widespread among living organisms (Chernoff, 2004; Uptain and Lindquist, 2002; Wickner, 1994; Wickner et al., 2004). These protein structures are more stable than other cellular protein complexes, which generally dissolve in SDS at room temperature. In contrast, the prion polymers withstand these conditions, while losing their association with their non-prion partners. These bulky protein particles cannot be analyzed in polyacrylamide gels, because their pores are too small to allow the passage and acceptable resolution of the large complexes. This problem was first circumvented by Kryndushkin et al. (2003), who used Western blots of protein complexes separated on agarose gels to analyze the sizes of SDS-resistant protein complexes associated with the yeast prion [PSI(+)]. Further studies have used this approach to characterize [PSI(+)] (Allen et al., 2005; Bagriantsev and Liebman, 2004; Salnikova et al., 2005), and another yeast prion [PIN(+)] (Bagriantsev and Liebman, 2004). In this chapter, we use this method to assay amyloid aggregates of recombinant proteins Sup35NM and Abeta42 and present protocols for Western blot analysis of high molecular weight (>5 MDa) amyloid aggregates resolved in agarose gels. The technique is suitable for the analysis of any large proteins or SDS-stable high molecular weight complexes.

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.

Specificity of prion assembly in vivo ; [PSI^+] and [PIN^+] form separate structures in yeast

The yeast prions [PSI+] and [PIN+] are self-propagating amyloid aggregates of the Gln/Asn-rich proteins Sup35p and Rnq1p, respectively. Like the mammalian PrP prion “strains,” [PSI+] and [PIN+] exist in different conformations called variants. Here, [PSI+] and [PIN+] variants were used to model in vivo interactions between co-existing heterologous amyloid aggregates. Two levels of structural organization, like those previously described for [PSI+], were demonstrated for [PIN+]. In cells with both [PSI+] and [PIN+] the two prions formed separate structures at both levels. Also, the destabilization of [PSI+] by certain [PIN+] variants was shown not to involve alterations in the [PSI+] prion size. Finally, when two variants of the same prion that have aggregates with distinct biochemical characteristics were combined in a single cell, only one aggregate type was propagated. These studies demonstrate the intracellular organization of yeast prions and provide insight into the principles of in vivo amyloid assembly.

The yeast [PSI+] prion: making sense of nonsense.

The [PSI] element was first described by Brian Cox in 1965 (1) in the course of his studies of a Mendelian nonsense suppressor. The efficiency with which this suppressor could misread UAA stop codons as sense was dependent upon the presence of a non-Mendelian factor, which Cox named [PSI]. Nearly 30 years of intriguing investigations followed, but the molecular nature of [PSI] remained unclear (see Refs. 2 and 3 for excellent reviews of this period). Recent reviews (e.g. Refs. 4–9) incorporate these data with current results in the context of the startling hypothesis (Ref. 10 and see below) that [PSI] is a prion. Discovery of [PSI] Although the original suppressor mutation acted on by [PSI] was in a tRNA gene and was specific for UAA codons, [PSI] was later shown to enhance the efficiency of suppression caused by other nonsense and frameshift tRNA suppressors or by aminoglycoside antibiotics (allosuppression). Eventually [PSI] was shown to be an “omnipotent” suppressor because it could cause readthrough of certain UAA, UAG, and UGA codons in the absence of other suppressors or drugs (11). Similar allosuppressor and omnipotent suppressor phenotypes were also associated with different sup35 and sup45 mutant alleles. However, whereas the sup35 and sup45 mutations were recessive and showed the 2:2 segregation pattern expected of Mendelian genes, the [PSI] factor was dominant and segregated 4:0, i.e. to all meiotic progeny. Consistent with this non-Mendelian segregation, the [PSI] factor appeared to be located in the cytoplasm because it was transmitted by cytoduction when the cytoplasm of a [PSI] donor haploid was transferred to a [psi] recipient haploid without altering the recipient’s nucleus (2). Thus it was possible that a cytoplasmic nucleic acid encoded [PSI], although it was shown not to depend upon mitochondrial DNA, 2m DNA, killer viruses, or 20 S RNA (2, 12, 13). The findings that “mutations” of [PSI] to [psi] were induced by conventional mutagens with single-hit kinetics and that UV mutagenesis was dependent upon DNA repair genes and was reduced by photoreactivation suggested that [PSI] was encoded by singlecopy DNA (14). However, growth in the presence of the mild protein denaturant guanidine hydrochloride or various stress-inducing agents, none of which cause the mutation of nuclear genes, caused the efficient loss of [PSI]. The kinetics of loss suggested that [PSI] was encoded by a multicopy element. Another important [PSI] paradox was the observation that although [PSI] was dominant in vivo, it was recessive in vitro, because mixtures of [PSI] and [psi] lysates used in a cell-free translation system did not exhibit the high level of readthrough of nonsense codons characteristic of [PSI] lysates (15).