Spontaneous Variants of the [RNQ+] Prion in Yeast Demonstrate the Extensive Conformational Diversity Possible with Prion Proteins

Abstract

Prion strains (or variants) are structurally distinct amyloid conformations arising from a single polypeptide sequence. The existence of prion strains has been well documented in mammalian prion diseases. In many cases, prion strains manifest as variation in disease progression and pathology, and in some cases, these prion strains also show distinct biochemical properties. Yet, the underlying basis of prion propagation and the extent of conformational possibilities available to amyloidogenic proteins remain largely undefined. Prion proteins in yeast that are also capable of maintaining multiple self-propagating structures have provided much insight into prion biology. Here, we explore the vast structural diversity of the yeast prion [RNQ+] in Saccharomyces cerevisiae. We screened for the formation of [RNQ+] in vivo, allowing us to calculate the rate of spontaneous formation as ~2.96x10-6, and successfully isolate several different [RNQ+] variants. Through a comprehensive set of biochemical and biological analyses, we show that these prion variants are indeed novel. No individual property or set of properties, including aggregate stability and size, was sufficient to explain the physical basis and range of prion variants and their resulting cellular phenotypes. Furthermore, all of the [RNQ+] variants that we isolated were able to facilitate the de novo formation of the yeast prion [PSI+], an epigenetic determinant of translation termination. This supports the hypothesis that [RNQ+] acts as a functional amyloid in regulating the formation of [PSI+] to produce phenotypic diversity within a yeast population and promote adaptation. Collectively, this work shows the broad spectrum of available amyloid conformations, and thereby expands the foundation for studying the complex factors that interact to regulate the propagation of distinct aggregate structures.

Extensive Diversity of Prion Strains Is Defined by Differential Chaperone Interactions and Distinct Amyloidogenic Regions

“…Therefore, our data suggest that thermal stability, while a crucial parameter, does not serve as a perfect measure of fiber fragmentation, and cannot explain the existence of many different aggregate structures, nor the large variability in phenotype. Moreover, our work and that of another group show that many additional properties that helped elucidate the physical basis of [ PSI +] variants, similarly do not distinguish the [ RNQ +] variants and their phenotypic effects on [ PSI +] [33], [44], [51], [52]. These data indicate that additional factors influence prion strain propagation and the resulting phenotypes, and highlight the wide variety of polymorphic structures that can exist [7], with [ PSI +] and [ RNQ +] variants possibly exhibiting different types of polymorphism (e.g.…”

Myopathy-causing Mutations in an HSP40 Chaperone Disrupt Processing of Specific Client Conformers

“…Cultures were maintained in log phase and grown in the presence of 5 g/ml doxycycline. Samples were subjected to SDD-AGE and Western blot using an ␣Rnq1 antibody following established protocols for the [RNQϩ] prion (18), which does not reliably show Rnq1 monomer for unknown reasons. SDS-PAGE and Western blot were also performed using an ␣Sis1 antibody (a gift from E. Craig) and quantified by ImageJ.…”

“…However, amyloidogenic proteins, including Rnq1, exist in a variety of different self-propagating structures (18,19,33,34). Prion strains of [RNQϩ] have been classified largely on two properties: their ability to induce the formation of the [PSIϩ] prion (low to very high rates) and the in vivo aggregation pattern observed by expressing Rnq1-GFP (22,35 Fig.…”

“…Genetic and environmental modifiers likely play a role in the generation and selection of different amyloid conformers, a characteristic we recently described with the [RNQϩ] prion (17)(18)(19)(20). Like the regulation of proteostasis in humans, molecular chaperones are intimately involved in the propagation of yeast prions.…”

Prion-Associated Toxicity is Rescued by Elimination of Cotranslational Chaperones

“…Strains were cytoduced with the “medium” variant of [ RNQ +] [47] and transformed with pEMBL-SUP35. Induction of [ PSI +] was performed as previously described [54]. At least three independent experiments were performed and a minimum of 600 colonies were counted.…”