Joo Yun Hong

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.

Yeast Ty1 Retrotransposition Is Stimulated by a Synergistic Interaction between Mutations in Chromatin Assembly Factor I and Histone Regulatory Proteins

ABSTRACT A screen for host mutations which increase the rate of transposition of Ty1 and Ty2 into a chromosomal target was used to identify factors influencing retroelement transposition. The fortuitous presence of a mutation in the CAC3 gene in the strain in which this screen was undertaken enabled us to discover that double mutaions of cac3 andhir3, but neither of the two single mutations, caused a dramatic increase in the rate of retrotransposition. We further showed that this effect was not due to an increase in the overall level of Ty1 mRNA. Two subtle cac3 phenotypes, slight methyl methanesulfonate (MMS) sensitivity and reduction of telomeric silencing, were significantly enhanced in the cac3 hir3 double mutant. In addition, the growth rate of the double mutant was reduced. HIR3 belongs to a class ofHIR genes that regulate the transcription of histones, while Cac3p, together with Cac1p and Cac2p, forms chromatin assembly factor I. Other combinations of mutations in cac andhir genes (cac3 hir1, cac3 hir2, and cac2 hir3) also increase Ty transposition and MMS sensitivity and reduce the growth rate. A model explaining the synergistic interaction between cac and hirmutations in terms of alterations in chromatin structure is proposed.

Ssa1 overexpression and [PIN+] variants cure [PSI+] by dilution of aggregates

Several cellular chaperones have been shown to affect the propagation of the yeast prions [PSI(+)], [PIN(+)] and [URE3]. Ssa1 and Ssa2 are Hsp70 family chaperones that generally cause pro-[PSI(+)] effects, since dominant-negative mutants of Ssa1 or Ssa2 cure [PSI(+)], and overexpression of Ssa1 enhances de novo [PSI(+)] appearance and prevents curing by excess Hsp104. In contrast, Ssa1 was shown to have anti-[URE3] effects, since overexpression of Ssa1 cures [URE3]. Here we show that excess Ssa1 or Ssa2 can also cure [PSI(+)]. This curing is enhanced in the presence of [PIN(+)]. During curing, Sup35-GFP fluorescent aggregates get bigger and fewer in number, which leads to their being diluted out during cell division, a phenotype that was also observed during the curing of [PSI(+)] by certain variants of [PIN(+)]. The sizes of the detergent-resistant [PSI(+)] prion oligomers increase during [PSI(+)] curing by excess Ssa1. Excess Ssa1 likewise leads to an increase in oligomer sizes of low, medium and very high [PIN(+)] variants. While these phenotypes are also caused by inhibition of Hsp104 or Sis1, the overexpression of Ssa1 did not cause any change in Hsp104 or Sis1 levels.

Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability.

Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI +] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI +] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI +].

Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN+]-independent pathway.

Characterization of novel rad6/ubc2 ubiquitin-conjugating enzyme mutants in yeast.

Abstract Null mutations in the RAD6/UBC2 gene encoding an E2 ubiquitin-conjugating enzyme cause deficiencies in DNA repair, N-end-rule protein degradation, sporulation and telomeric silencing, and alter the preferred integration positions for Ty1 retrotransposons. Here we selected for mutants of RAD6 that cause a release of telomeric silencing. Some alleles retained nearly wild-type ability for sporulation, DNA repair and the degradation of proteins. Alteration in Ty1 integration-site bias accompanied some of these alleles. The possibility that some mutations specifically affect binding of an unknown protein that works with Rad6 in its silencing role, but is not required for DNA repair or N-end-rule activity, is discussed in terms of the Rad6 crystal structure.

A new colour assay for [URE3] prion in a genetic background used to score for the [PSI+] prion

Most scoring assays for yeast prions are dependent on specific genetic markers and constructs that differ for each prion. Here we describe a simple colour assay for the [URE3] prion that works in the 74D‐964 strain frequently used to score the [PSI+] prion. Although this assay can only be used to score for [URE3] in the [psi−] version of the strain, it makes it easier to examine the effects of host mutations or environmental changes on [URE3] or [PSI+] using a colour assay in the identical genetic background. Copyright