The [PSI] Prion Exists as a Dynamic Cloud of Variants

University of Nevada, Reno, United States of America
PLoS Genetics (Impact Factor: 7.53). 01/2013; 9(1):e1003257. DOI: 10.1371/journal.pgen.1003257
Source: PubMed


[PSI(+)] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion "strains" or "variants" are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, Δ19, and E9, with limited transmissibility of [PSI(+)] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI(+)] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI(+)], all indistinguishably "weak" [PSI(+)], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI(+)]. Notably, a "strong [PSI(+)]" can have any of several different transmission efficiency patterns, showing that "strong" versus "weak" is insufficient to indicate prion variant uniformity.

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Available from: David Bateman, Oct 04, 2015
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    • ", 2008a , 2014 ) . The resulting synthetic [ REP - PSI + ] prions are functional in yeast , generating both a strong [ PSI + ] – like phenotype and various weak phenotypes ( Figures 2 and 3 ) , which are compat - ible with a cloud of prions strains ( Bateman and Wickner , 2013 ) . These chimeric prions assemble themselves in vitro as discrete size particles ( Figure 5 ) , which probably would suffice to act as competent propagons in vivo . "
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    ABSTRACT: The yeast translation termination factor Sup35p, by aggregating as the [PSI (+)] prion, enables ribosomes to read-through stop codons, thus expanding the diversity of the Saccharomyces cerevisiae proteome. Yeast prions are functional amyloids that replicate by templating their conformation on native protein molecules, then assembling as large aggregates and fibers. Prions propagate epigenetically from mother to daughter cells by fragmentation of such assemblies. In the N-terminal prion-forming domain, Sup35p has glutamine/asparagine-rich oligopeptide repeats (OPRs), which enable propagation through chaperone-elicited shearing. We have engineered chimeras by replacing the polar OPRs in Sup35p by up to five repeats of a hydrophobic amyloidogenic sequence from the synthetic bacterial prionoid RepA-WH1. The resulting hybrid, [REP-PSI (+)], (i) was functional in a stop codon read-through assay in S. cerevisiae; (ii) generates weak phenotypic variants upon both its expression or transformation into [psi (-)] cells; (iii) these variants correlated with high molecular weight aggregates resistant to SDS during electrophoresis; and (iv) according to fluorescence microscopy, the fusion of the prion domains from the engineered chimeras to the reporter protein mCherry generated perivacuolar aggregate foci in yeast cells. All these are signatures of bona fide yeast prions. As assessed through biophysical approaches, the chimeras assembled as oligomers rather than as the fibers characteristic of [PSI (+)]. These results suggest that it is the balance between polar and hydrophobic residues in OPRs what determines prion conformational dynamics. In addition, our findings illustrate the feasibility of enabling new propagation traits in yeast prions by engineering OPRs with heterologous amyloidogenic sequence repeats.
    Frontiers in Microbiology 04/2015; 6:311. DOI:10.3389/fmicb.2015.00311 · 3.99 Impact Factor
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    • "Evidence of a single yeast strain harboring multiple prion conformations has also been observed with [PSI+]. Recently, clones isolated from a single [PSI+] colony exhibited a range of transmission profiles across an intra-species barrier, leading authors to propose that an ensemble, or cloud, of [PSI+] variants had been propagating in the parent cell [71]. Similarly, others have described an “unspecified” [PSI+] phenotype that was characterized by white colonies that sectored to pink, which gave rise to progeny carrying weak [PSI+], strong [PSI+], or unspecified [PSI+] [67]. "
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    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.
    PLoS ONE 10/2013; 8(10):e79582. DOI:10.1371/journal.pone.0079582 · 3.23 Impact Factor
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    ABSTRACT: The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet, identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis and denaturation by reagents such as urea or sodium dodecyl sulphate (SDS). Here, we show that these properties can be exploited to identify naturally-occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry, without requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains, and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins, as well as strongly associated proteins, can be identified directly from cells of diverse origin.
    Journal of Biological Chemistry 08/2013; 288(38). DOI:10.1074/jbc.M113.485359 · 4.57 Impact Factor
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