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Amyloid appearance is a rare event that is promoted in the presence of other aggregated proteins. These aggregates were thought to act by templating the formation of an assembly-competent nucleation seed, but we find an unanticipated role for them in enhancing the persistence of amyloid after it arises. Specifically, Saccharomyces cerevisiae Rnq1 amyloid reduces chaperone-mediated disassembly of Sup35 amyloid, promoting its persistence in yeast. Mathematical modeling and corresponding in vivo experiments link amyloid persistence to the conformationally defined size of the Sup35 nucleation seed and suggest that amyloid is actively cleared by disassembly below this threshold to suppress appearance of the [PSI + ] prion in vivo. Remarkably, this framework resolves multiple known inconsistencies in the appearance and curing of yeast prions. Thus, our observations establish the size of the nucleation seed as a previously unappreciated characteristic of prion variants that is key to understanding transitions between prion states.

The self-assembly of alternative conformations of normal proteins into amyloid aggregates has been implicated in both the acquisition of new functions and in the appearance and progression of disease. However, while these amyloidogenic pathways are linked to the emergence of new phenotypes, numerous studies have uncoupled the accumulation of aggregates from their biological consequences, revealing currently underappreciated complexity in the determination of these traits. Here, to explore the molecular basis of protein-only phenotypes, we focused on the Saccharomyces cerevisiae Sup35/[PSI(+)] prion, which confers a translation termination defect and expression level-dependent toxicity in its amyloid form. Our studies reveal that aggregated Sup35 retains its normal function as a translation release factor. However, fluctuations in the composition and size of these complexes specifically alter the level of this aggregate-associated activity and thereby the severity and toxicity of the amyloid state. Thus, amyloid heterogeneity is a crucial contributor to protein-only phenotypes.

Prion variants are self-perpetuating conformers of a single protein that assemble into amyloid fibers and confer unique phenotypic states. Multiple prion variants can arise, particularly in response to changing environments, and interact within an organism. These interactions are often competitive, with one variant establishing phenotypic dominance over the others. This dominance has been linked to the competition for non-prion state protein, which must be converted to the prion state via a nucleated polymerization mechanism. However, the intrinsic rates of conversion, determined by the conformation of the variant, cannot explain prion variant dominance, suggesting a more complex interaction. Using the yeast prion system [PSI+], we have determined the mechanism of dominance of the [PSI+]Strong variant over the [PSI+]Weak variant in vivo. When mixed by mating, phenotypic dominance is established in zygotes, but the two variants persist and co-exist in the lineage descended from this cell. [PSI+]Strong propagons, the heritable unit, are amplified at the expense of [PSI+]Weak propagons, through the efficient conversion of soluble Sup35 protein, as revealed by fluorescence photobleaching experiments employing variantspecific mutants of Sup35. This competition, however, is highly sensitive to the fragmentation of [PSI+]Strong amyloid fibers, with even transient inhibition of the fragmentation catalyst Hsp104 promoting amplification of [PSI+]Weak propagons. Reducing the number of [PSI+]Strong propagons prior to mating, similarly promotes [PSI+]Weak amplification and conversion of soluble Sup35, indicating that template number and conversion efficiency combine to determine dominance. Thus, prion variant dominance is not an absolute hierarchy but rather an outcome arising from the dynamic interplay between unique protein conformations and their interactions with distinct cellular proteostatic niches. [ABSTRACT FROM AUTHOR]

Prions adopt alternative, self-replicating protein conformations and thereby determine novel phenotypes that are often irreversible. Nevertheless, dominant-negative prion mutants can revert phenotypes associated with some conformations. These observations suggest that, while intervention is possible, distinct inhibitors must be developed to overcome the conformational plasticity of prions. To understand the basis of this specificity, we determined the impact of the G58D mutant of the Sup35 prion on three of its conformational variants, which form amyloids in S. cerevisiae. G58D had been previously proposed to have unique effects on these variants, but our studies suggest a common mechanism. All variants, including those reported to be resistant, are inhibited by G58D but at distinct doses. G58D lowers the kinetic stability of the associated amyloid, enhancing its fragmentation by molecular chaperones, promoting Sup35 resolubilization, and leading to amyloid clearance particularly in daughter cells. Reducing the availability or activity of the chaperone Hsp104, even transiently, reverses curing. Thus, the specificity of inhibition is determined by the sensitivity of variants to the mutant dosage rather than mode of action, challenging the view that a unique inhibitor must be developed to combat each variant.

Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid. [ABSTRACT FROM AUTHOR]

According to the prion hypothesis, atypical phenotypes arise when a prion protein adopts an alternative conformation and persist when that form assembles into self-replicating aggregates. Amyloid formation in vitro provides a model for this protein-misfolding pathway, but the mechanism by which this process interacts with the cellular environment to produce transmissible phenotypes is poorly understood. Using the yeast prion Sup35/[PSI+], we found that protein conformation determined the size distribution of aggregates through its interactions with a molecular chaperone. Shifts in this range created variations in aggregate abundance among cells because of a size threshold for transmission, and this heterogeneity, along with aggregate growth and fragmentation, induced age-dependent fluctuations in phenotype. Thus, prion conformations may specify phenotypes as population averages in a dynamic system. [ABSTRACT FROM AUTHOR]