Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro

Abstract

Prions are infectious protein conformations that are generally ordered protein aggregates. In the absence of prions, newly synthesized molecules of these same proteins usually maintain a conventional soluble conformation. However, prions occasionally arise even without a homologous prion template. The conformational switch that results in the de novo appearance of yeast prions with glutamine/aspargine (Q/N)-rich prion domains (e.g., [PSI+]), is promoted by heterologous prions with a similar domain (e.g., [RNQ+], also known as [PIN+]), or by overexpression of proteins with prion-like Q-, N-, or Q/N-rich domains. This finding led to the hypothesis that aggregates of heterologous proteins provide an imperfect template on which the new prion is seeded. Indeed, we show that newly forming Sup35 and preexisting Rnq1 aggregates always colocalize when [PSI+] appearance is facilitated by the [RNQ+] prion, and that Rnq1 fibers enhance the in vitro formation of fibers by the prion domain of Sup35 (NM). The proteins do not however form mixed, interdigitated aggregates. We also demonstrate that aggregating variants of the polyQ-containing domain of huntingtin promote the de novo conversion of Sup35 into [PSI+]; whereas nonaggregating variants of huntingtin and aggregates of non-polyQ amyloidogenic proteins, transthyretin, alpha-synuclein, and synphilin do not. Furthermore, transthyretin and alpha-synuclein amyloids do not facilitate NM aggregation in vitro, even though in [PSI+] cells NM and transthyretin aggregates also occasionally colocalize. Our data, especially the in vitro reproduction of the highly specific heterologous seeding effect, provide strong support for the hypothesis of cross-seeding in the spontaneous initiation of prion states.

Fig. 1.

High levels of proteins with expanded polyQ tracts, but not non-polyQ proteins, permit the de novo induction of [PSI⁺] in the absence of [RNQ⁺]. [PSI⁺] suppressed a nonsense mutation in the ADE1 gene allowing growth on glucose –Ade medium (scored after 2 weeks at 20°C). All cells contained a [PSI⁺]-inducing construct and either an empty vector or one of the constitutive Ht-expressing multicopy vectors with polyQ stretches of different length (A) or one of the multicopy vectors expressing the indicated proteins (B). [psi^–][rnq^–] carrying an empty vector and transformants of the [psi^–][RNQ⁺] derivative served as negative and positive controls. Note that growth on –Ade in [RNQ⁺] cells in B reflects growth inhibition by TTR-WT, Synph-1, and αSyn overproduction (data not shown) and not reduced [PSI⁺] induction.

Fig. 2.

Colocalization of amyloidogenic aggregates. (A) Non-polyQ amyloidogenic proteins form aggregates in [RNQ⁺] and [rnq^–] cells. (B) Occasional colocalization of TTR-WT (columns 1 and 4) and TTRd (columns 2 and 3) aggregates with [PSI⁺]. Cells shown originate from [psi^–][RNQ⁺] (column 1), [PSI⁺][rnq^–] (column 2), and [PSI⁺][RNQ⁺] (columns 3 and 4). (C) Coaggregation of [RNQ⁺] and newly forming [PSI⁺] aggregates in [psi^–][RNQ⁺] cells during [PSI⁺] induction. For the first two samples, an array of focal planes 1 μm apart (Z-stack) is shown. DIC, differential interference contrast.

Fig. 3.

Effect of amyloidic, aggregated, or soluble proteins on NM conversion kinetics in vitro. The proportion of proteins is given as percent mol/mol. (A) Effect of increasing amounts of sonicated Rnq1 fibers on soluble NM conversion. (B) Control for A. The addition of sonicated Rnq1 fibers converts soluble NM to fibers, because thioflavin T emission did not increase when Rnq1 fibers at the same concentration were incubated without NM. (C) Effect of TTR-WT and αSyn-sonicated fibers on NM conversion. (D) Effect of Ig fibers, insulin fibers, and nonspecifically aggregated and soluble proteins on NM conversion.