Interactions among prions and prion "strains" in yeast

Abstract

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.

Figure 1

Meiotic inheritance of [PSI⁺] variants. Isogenic haploid yeast, parent 1 (P1; L1842 derivatives) and parent 2 (P2; L1843 derivatives) carrying the indicated [PSI⁺] variants were mated. One representative diploid and two tetrads from each cross are shown.

Figure 2

Characterization of [PIN⁺] variants and their inheritance through cytoduction. Independent derivatives of the original 74-D694 are shown (Originals). These were each cytoduced into a [pin⁻] version of c10B-H49, a kar1–1 yeast strain, and from there back into a [pin⁻] version of 74-D694 (Cytoductants). Both the originals and cytoductants carry the pEMBL-SUP35 plasmid. When the plasmid is maintained at moderate level on −Ura distinct levels of [PSI⁺] induction are observed on transfer to −Ade. When the plasmid is amplified on −Leu, different levels of growth inhibition are observed. The different [PIN⁺] variants are cytoplasmically inherited because cytoductants and donors exhibit the same [PIN⁺] phenotypes. Row 1 is the [pin⁻] control; rows 2, 3, and 5 are the low, medium (med.), and very high (v.h.) spontaneous [PIN⁺] variants obtained in [pin⁻] 74-D694; row four is high [PIN⁺] from the original 74-D694.

Figure 3

Genetic analysis of variants of [PIN⁺]. Independent diploids carrying the pEMBL-SUP35 plasmid reveal the outcome of crosses between haploid parents (P1 and P2) carrying the [pin⁻], low, medium (med.), or very high (v.h.) [PIN⁺] variants. The [PIN⁺] phenotypes are scored as in Fig. 2.

Figure 4

Comparison of levels of soluble and aggregated Rnq1 among [PIN⁺] variants. Lysates were fractionated into soluble (S) and pellet (P) fractions by ultra-centrifugation. Rnq1 was detected by Western blotting with polyclonal Rnq1 antibody (kind gift from S. Lindquist). Stripped blots were then re-probed with monoclonal Sup35 antibody (Control) as a loading control. Note, the soluble Rnq1 in this figure were exposed twice as long as the Rnq1 in the pellet. The gradient depicted was generally reproducible; however, in three of eight independent protein isolations, the level of soluble Rnq1 in the v.h. and low variants appeared similar.

Figure 5

Cartoon depicting a competition for soluble protein between variants of the same prion. Haploids carrying soluble protein (free circles) and aggregated-prion protein as either “weak” (rhomboids) or “strong” (squares) variants are mated. Mitotic growth of the diploid results in loss of the weak variant, presumably because it is starved for convertible soluble protein. When the diploid sporulates, each spore inherits the strong prion variant.

Figure 6

Models illustrating the different seeding preferences proposed for two [PIN⁺] variants. (A) The very high [PIN⁺] variant inefficiently seeds Ure2 (red circles), but efficiently seeds Sup35 (green triangles) converting them into the [PSI⁺] shape. (B) The original YHE711 [PIN⁺] variant inefficiently seeds Sup35, but efficiently seeds Ure2, converting them into the [URE3] shape. Both [PIN⁺] variants propagate their forms by converting Rnq1 (blue rectangles) with the highest efficiency.