We conducted the first comprehensive study of prion-like Q/N-rich domains in yeast, using three different criteria to investigate their ability (1) to form amyloid in vivo under physiological conditions; (2) to self-assemble in vitro in the absence of other factors; and (3) to replicate indefinitely in cells as self-perpetuating epigenetic elements. We identified 24 protein domains that satisfied the stringent third criterion for prion behavior. This group includes the known prions Ure2p, Sup35p, Rnq1p, and Swi1p, the previously identified prion candidate New1p, and a functionally diverse set of 19 new candidates.
All but one prion candidate (New1p, see Supplemental Discussion
) were strictly dependent on the protein remodeling factor Hsp104p. Interestingly, most proteins that satisfied the stringent third criterion also passed criteria one and two (Figure S3
), underscoring the importance of amyloid’s distinctive self-templating properties for prion phenomena. In addition, the in vitro
aggregation results compare extremely well with the aggregation of these cPrDs in vivo
, a remarkable finding given the absence in vitro
of the prion regulators [RNQ
+] and Hsp104p. Q/N-rich sequences, despite having overtly similar amino acid compositions, have biochemical differences that give rise to a range of amyloid propensities. These differences affect amyloidogenesis intrinsically, rather than simply by controlling interactions with intracellular prion-promoting factors. Thus, prion-forming proteins are predisposed to form amyloid even in the absence of factors that govern prionogenesis in vivo
. The implication is, then, that factors like [RNQ
+] and Hsp104p may have evolved, in part, to regulate the frequency of spontaneous prion appearance and to promote the stable propagation of prions once they appear. The dependence of prions on these factors as well as their interaction with other chaperones and stress proteins links them intricately with cellular stress pathways (Chernoff, 2007
; Shorter and Lindquist, 2008
; Tuite et al., 2008
) and makes them likely to respond to environmental changes that create even minor perturbances in protein homeostasis (Tyedmers et al., 2008
Interestingly, the number of cPrDs forming fluorescent foci was higher than the number of cPrDs forming SDS-resistant aggregates in vivo. This finding, in combination with our detection of SDS-sensitive aggregates in vitro, indicates that some cPrDs are able to generate non-amyloid aggregates, with a low structural stability that prevents detection by SDD-AGE. A sequence analysis of our candidates suggests one explanation for these different behaviors. Highly amyloidogenic proteins were generally more N-rich, whereas non-amyloidogenic proteins were more Q-rich. A direct analysis of the distinct contributions of each of these residues to prion formation is underway and will be reported elsewhere.
The refined set of candidate yeast prions is strongly enriched for proteins involved in gene expression such as transcription factors (p = 5.3 × 10−5
) and RNA-binding proteins (p = 5.1 × 10−4
), a finding that supports the idea that PrDs function as epigenetic switches influencing important cellular pathways. The discoveries of [SWI
+] (Du et al., 2008
) and [MOT3
+] lend further support to the idea that prion-based phenomena are biologically significant. The causal agents of these prions are each global transcription factors; consequently their prion states are likely to have far-reaching phenotypic effects. Our in depth analysis of one of these prions, [MOT3
+], reveals a very appreciable spontaneous induction frequency (~10−4
, and data not shown) that is further dramatically enhanced by overexpression or the introduction of preformed amyloid seeds. We speculate that the prion domain, and expression level, of Mot3p are partial products of selection for prion bistability. The prion properties of these transcription factors may generate an optimized phenotypic heterogeneity that buffers yeast populations against diverse environmental insults.
The role of prions has expanded considerably since their public inception as agents of disease. Prions in yeast and filamentous fungi drive heritable switches that increase phenotypic diversity. Conversely, higher organisms may have harnessed prion-like conformational templating to initiate stable switches involved in, for instance, neuronal synapse activation (Si et al., 2003
). The regulation and frequency of prion switching may have been honed by selective pressures unique to each protein. Self-perpetuating prion and prion-like processes that serve an adaptive role may generally undergo increased switching under stressful conditions that perturb protein homeostasis, as has been established for the [PSI
+] prion (Tyedmers et al., 2008
). We suspect that many of our confirmed prion domains, in their endogenous contexts, will have switching rates that similarly respond to homeostatic cues. The heterogeneity theoretically possible with over twenty different prion switches amplifies the phenotype space of a single proteome, creating an advantageous scenario for any isogenic population under duress. Further, since prions are uniquely self-perpetuating yet metastable, any beneficial phenotype they produce can be easily maintained or lost in subsequent generations depending on selective pressures. This differs fundamentally both from genetic mutations, which are relatively permanent, and from non-heritable phenotypic changes, such as those arising from transcriptional noise.
Self-templating aggregates like yeast prions constitute a paradigm-shifting mechanism for the replication of biological information. Prion-based biological information arises spontaneously yet specifically within select proteins, is metastable, and is intricately linked to stress-response pathways. These features position prions as ideal bet-hedging devices (King and Masel, 2007
) capable of responding to environmental stimuli. Our studies provide the tools for future genome-wide investigations of protein-based self-perpetuating changes in diverse organisms. We predict that these studies will profoundly impact our future understanding of phenotypic variation and will help to unravel the complex relationship between genotype and phenotype.