A Promiscuous Prion: Efficient Induction of [URE3] Prion Formation by Heterologous Prion Domains

The [URE3] and [PSI⁺] prions are the infections amyloid forms of the Saccharomyces cerevisiae proteins Ure2p and Sup35p, respectively. Randomizing the order of the amino acids in the Ure2 and Sup35 prion domains while retaining amino acid composition does not block prion formation, indicating that amino acid composition, not primary sequence, is the predominant feature driving [URE3] and [PSI⁺] formation. Here we show that Ure2p promiscuously interacts with various compositionally similar proteins to influence [URE3] levels. Overexpression of scrambled Ure2p prion domains efficiently increases de novo formation of wild-type [URE3] in vivo. In vitro, amyloid aggregates of the scrambled prion domains efficiently seed wild-type Ure2p amyloid formation, suggesting that the wild-type and scrambled prion domains can directly interact to seed prion formation. To test whether interactions between Ure2p and naturally occurring yeast proteins could similarly affect [URE3] formation, we identified yeast proteins with domains that are compositionally similar to the Ure2p prion domain. Remarkably, all but one of these domains were also able to efficiently increase [URE3] formation. These results suggest that a wide variety of proteins could potentially affect [URE3] formation.AMYLOID fibril formation is associated with numerous human diseases, including Alzheimer''s disease, type II diabetes, and the transmissible spongiform encephalopathies. Yeast prions provide a powerful model system for examining amyloid fibril formation in vivo. [URE3] and [PSI⁺] are the prion forms of the Saccharomyces cerevisiae proteins Ure2p and Sup35p, respectively (Wickner 1994). In both cases, prion formation is thought to result from conversion of the native protein into an inactive amyloid form (Glover et al. 1997; King et al. 1997; Taylor et al. 1999). Both proteins contain an N-terminal glutamine/asparagine (Q/N)-rich prion-forming domain (PFD) and a C-terminal functional domain (Ter-Avanesyan et al. 1993; Ter-Avanesyan et al. 1994; Masison and Wickner 1995; Liebman and Derkatch 1999; Maddelein and Wickner 1999). Sup35p contains an additional highly charged middle domain (M) that is not required either for prion formation or for normal protein function, but stabilizes [PSI⁺] aggregates (Liu et al. 2002).Amyloid fibril formation is thought to occur through a seeded polymerization mechanism. In vitro, amyloid fibril formation from native proteins is generally characterized by a significant lag time, thought to result from the slow rate of formation of amyloid nuclei; addition of a small amount of preformed amyloid aggregates (seeds) eliminates the lag time, resulting in rapid polymerization (Glover et al. 1997; Taylor et al. 1999; Serio et al. 2000).Despite considerable study, the mechanism by which amyloid seeds initially form is unclear. At least some of the amyloid proteins involved in human disease can interact with unrelated amyloidogenic proteins, resulting in cross-seeding and modulation of toxicity. Injecting mice with amyloid-like fibrils formed by a variety of short synthetic peptides promotes amyloid formation by amyloid protein A, a protein whose deposition is found in systemic AA amyloidosis (Johan et al. 1998). In yeast, [PSI⁺] and [PIN⁺], the prion form of the protein Rnq1p (Sondheimer and Lindquist 2000; Derkatch et al. 2001), both promote the aggregation of and increase toxicity of expanded polyglutamine tracts, like those seen in Huntington''s disease (Osherovich and Weissman 2001; Meriin et al. 2002; Derkatch et al. 2004; Gokhale et al. 2005; Duennwald et al. 2006); however, in Drosophila, [PSI⁺] aggregates reduce polyglutamine toxicity (Li et al. 2007). Thus, interactions between heterologous amyloidogenic proteins can influence amyloid formation both positively and negatively in vivo.A variety of interactions have been observed among the yeast prions. Under normal cellular conditions, efficient formation, but not maintenance, of [PSI⁺] requires the presence of [PIN⁺] (Derkatch et al. 2000). Overexpression of various Q/N-rich proteins can effectively substitute for [PIN⁺], allowing [PSI⁺] formation in cells lacking [PIN⁺] (Derkatch et al. 2001; Osherovich and Weissman 2001). In vitro and in vivo evidence suggest that the ability of [PIN⁺] to facilitate [PSI⁺] formation is the result of a direct interaction between Rnq1p aggregates and Sup35p (Derkatch et al. 2004; Bardill and True 2009; Choe et al. 2009). [PIN⁺] also increases the frequency of [URE3] formation, while [PSI⁺] inhibits [URE3] formation (Bradley et al. 2002; Schwimmer and Masison 2002).It is unclear whether the ability of Ure2p, Sup35p, and Rnq1p to cross-react is an intrinsic feature of all similar amyloidogenic proteins, or whether it has specifically evolved to regulate prion formation. There is debate as to whether yeast prion formation is a beneficial phenomenon, allowing for regulation of the activity of the prion protein (True and Lindquist 2000; True et al. 2004), or a deleterious event analogous to human amyloid disease (Nakayashiki et al. 2005). Either way, it is likely that interactions between the yeast prion proteins have specifically evolved, either to minimize the detrimental effects of amyloid formation or to regulate beneficial amyloid formation.For both Ure2p and Sup35p, the amino acid composition of the PFD is the predominant feature that drives prion formation. Scrambled versions of Ure2p and Sup35p (in which the order of the amino acids in the PFD was randomized while maintaining amino acid composition) are able to form prions when expressed in yeast as the sole copy Ure2p or Sup35p (Ross et al. 2004, 2005). To examine whether amino acid composition can similarly drive interactions between heterologous proteins, we tested whether the scrambled PFDs can interact with their wild-type counterparts to stimulate prion formation. When overexpressed, scrambled Ure2 PFDs promoted de novo prion formation by wild-type Ure2p, suggesting that the Ure2p PFD can promiscuously interact with compositionally similar PFDs during prion formation. When we searched the yeast proteome for proteins with regions of high compositional similarity to Ure2p, four of the top five proteins were able to efficiently stimulate [URE3] formation. However, there were limits to this promiscuity; overexpression of wild-type or scrambled Sup35 PFDs did not increase [URE3] levels. We propose that this ability to promiscuously interact may have evolved as a mechanism to regulate Ure2p activity and/or prion formation.