The [<em>PSI</em>⁺] Prion Exists as a Dynamic Cloud of Variants

<div><p>[<em>PSI</em>⁺] 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 <em>Saccharomyces cerevisiae</em> isolates have <em>SUP35</em> alleles that fall into three groups, called reference, Δ19, and E9, with limited transmissibility of [<em>PSI</em>⁺] 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 [<em>PSI</em>⁺] 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 [<em>PSI</em>⁺], all indistinguishably “weak” [<em>PSI</em>⁺], 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 [<em>PSI</em>⁺]. Notably, a “strong [<em>PSI</em>⁺]” can have any of several different transmission efficiency patterns, showing that “strong” versus “weak” is insufficient to indicate prion variant uniformity.</p> </div

Wild [<i>PSI</i>⁺] prion isolates are largely sensitive to polymorph-determined transmission barriers.

<p>Spores of wild <i>S. cerevisiae</i> reported to be [<i>PSI</i>⁺] <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003257#pgen.1003257-Halfmann1" target="_blank">[49]</a> were crossed with strain 4972 and meiotic segregants showing weak, guanidine-curable suppression of <i>ade1-14</i> were used as cytoduction donors.</p

[<i>PSI</i>⁺] variants with distinct transmission properties can have identical “strong” or “weak” phenotypes.

<p>A. [<i>PSI</i>⁺] strains derived from 779-6A by extensive non-selective subcloning have different transmission patterns, but identical “strong” phenotypes. B. [<i>PSI</i>⁺] prions in wild <i>S. cerevisiae</i> isolates were moved into strain 4830 for direct comparison of prion intensity. Each is “weak”, although transmission to Sup35p polymorphs varies as indicated. [A], [B], [C] and [D] refer to the transmission types shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003257#pgen-1003257-t005" target="_blank">Table 5</a>.</p

Transmission of 779-6A's [<i>PSI</i>⁺ref] carried by other Sup35 polymorphs.

<p>The bold indicates cytoductants used as donors in a subsequent cytoduction.</p

Does passage through a Sup35 polymorph change [<i>PSI</i>⁺] transmission properties?

<p>Cytoductions of the form ref→polymorph→ref→polymorph were carried out (where ref is strain 799-6A or the same cured of [<i>PSI</i>⁺]). One cytoductant of each ref→polymorph was cytoduced to ref, and five of those cytoductants were each used as donors to each of the three polymorphs. Summed data is shown; the complete data set is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003257#pgen.1003257.s007" target="_blank">Table S6</a>.</p

Variable transmission of [<i>PSI</i>⁺E9]E9 isolates A, F, and G to polymorphs Sup35ref, Sup35E9, and Sup35Δ19 shows that they are distinct prion variants.

<p>Three prion isolates (A, F, G) in strain 4828 expressing the E9 polymorph of Sup35 were used as cytoduction donors to strain 4830 expressing the different polymorphs. <b>Bold</b> figures show which cytoductants were used as donors in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003257#pgen-1003257-t002" target="_blank">Table 2</a>. The proportions of transmission by variant E9A and E9G to the reference sequence differs from the proportion observed for variant E9F (*) with p<10⁻¹⁰, calculated as described in Methods.</p

The prion cloud model <b>[56]</b>, <b>[57]</b> applied to yeast.

<p>Segregation of different prion variants on mitotic growth is followed by re-emergence of different variants, presumably due to mis-templating.</p

Instability of transmission variants on extensive mitotic growth.

<p>From each of subclones Y1, Y2 and Y5 from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003257#pgen-1003257-t005" target="_blank">Table 5</a> were isolated ten subclones, which were then propagated a further >75 generations and clones were amplified and used as cytoduction donors to the three polymorphs. The results from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003257#pgen-1003257-t005" target="_blank">Table 5</a> for Y1, Y2 and Y5 are reproduced at the top for comparison. The p values are calculated as described in Methods and indicate the probability that the difference between the indicated result with Yx-y as donor and that with the parent strain Yx as donor is due to chance. Transmission types are listed in the text.</p

Subclones of [<i>PSI</i>⁺ref] develop divergent transmission properties without selection.

<p>Twelve subclones of 779-6A were grown for >75 generations and single clones were then amplified and used as cytoduction donors to the three polymorphs. <b>Bold</b> figures are transmissions between polymorphs that are more efficient than when the donor was the parent strain 779-6A (top three lines). The p values shown are the probability that the results observed would be obtained by chance if there were in fact no difference between the indicated cytoduction from the subclone and the corresponding cytoduction from the parent strain. The p values are calculated as described in Methods and indicate the probability that the difference between the indicated result with Yx as donor and that with the parent strain 779-6a as donor is due to chance. Transmission types are listed in the text.</p

Supplemental Material for Edskes et al., 2018

Supplemental Methods_figures_tables.pdf - Provides a detailed description of the culture conditions, induction of <i>Hermes</i> transposition, selection of colonies carrying a transposition, extraction of cellular DNA, PCR amplification and isolation of the junction points between transposon and chromosomal insertion site, next-generation sequencing of these sites, and analysis of the data by visual display and by counting insertions per open reading frame. <br><br> Exon Intron Counts.xlsx - Gives the insertions in every yeast open reading frame, distinguishing exons from introns where appropriate.<br><br>Sorted Hits.xlsx - Gives prominent hits sorted by functional group, “TY gag-pol Counts.xlsx comparing insertions in the Ty retrotransposons at different locations in the genome. <br><br>Count Insertions in ORFs and Introns.txt - The Python program used for counting insertions. <br><br>LUGsIGV-InsertDistributions.pptx - Is a slide show of insert distributions in each of 500 genes for which insertions were recovered more frequently in [ure-o] than in [URE3] cultures