bMultiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/ asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions. Misfolding of a wide range of proteins leads to formation of amyloid fibrils, which are ordered, ␤-sheet-rich protein aggregates. Many human diseases are associated with the formation of amyloid fibrils, including Alzheimer's disease, type II diabetes, and the transmissible spongiform encephalopathies (TSEs) (1). However, only a small subset of amyloids are infectious (called prions), including the causative agents of TSEs in mammals (2-4) and, and others in Saccharomyces cerevisiae (5-9).Most of the known yeast prion proteins contain glutamine/ asparagine (Q/N)-rich domains that drive amyloid formation. Q/N-rich domains are found in 1 to 4% of the proteins in most eukaryotic proteomes (10), but very few of these proteins have been shown to undergo amyloid structural conversion. Bioinformatics screens for prions in yeast have had some notable successes (reviewed in reference 11); however, despite advances in predicting which Q/N-rich domains may turn out to be bona fide prions (12, 13), predictions remain imperfect.A well-studied model prion from yeast (S. cerevisiae) is [PSI ϩ ], the prion form of the translational terminator protein Sup35 (5). Like other yeast prion proteins, Sup35 is modular, as it contains a distinct prion-forming domain (PFD), middle domain (M), and C-terminal domain (C) (Fig. 1A) (14-17). The PFD (amino acids 1 to 114) drives the conversion of Sup35 into its amyloid form (15), the charged M domain has no known function other than its ability to stabilize [PSI ϩ ] fibers, and the C domain is an essential component resp...

[PSI+], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI+] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI+] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI+] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI+]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI+] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.