Abstract
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.
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Code for the stochastic model of aggregate persistence is available at https://doi.org/10.6071/M33T08
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Acknowledgements
We thank S. Lindquist (MIT) and E. Craig (University of Wisconsin–Madison) for reagents and J. Laney and members of the Serio and Sindi groups for helpful discussions and comments on the manuscript. This research was sponsored in part by the Joint DMS/NIGMS Initiative to Support Research at the Interface of the Biological and Mathematical Sciences (R01GM126548 to S.S.S.), NSF-INSPIRE (1344279 to S.S.S. and J.K.D.) and NIH/NIGMS (F32GM096582 to J.V., R35GM118042 to T.R.S.) and with support from NIH/NIGMS to T.M.B. (T32GM008659).
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J.V. and J.D. conceptualized and designed the work, acquired, analyzed and interpreted data, and drafted and revised the manuscript. T.M.B. and F.P. acquired, analyzed and interpreted data and revised the manuscript. S.S.S. and T.R.S. conceptualized and designed the work, analyzed and interpreted data, and drafted and revised the manuscript.
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Extended data
Extended Data Fig. 1 Protein abundance for strains with heterozygous disruptions of HSP104 and/or RNQ1.
The relative levels of Hsp104 (a), Sup35 (b), and Rnq1 (c) in lysates from diploid yeast strains analyzed in Fig. 1e–g were determined by SDS-PAGE and immunoblotting with specific antisera. Strains carried wildtype (+/+), heterozygous (+/Δ), or homozygous (Δ/Δ) disruptions of HSP104 or RNQ1 and the indicated variant of [PSI+]. Protein levels are expressed relative to the protein levels in wildtype version of the same prion strain (that is [PSI+]Strong or [PSI+]Weak) in arbitrary units (a.u.). Data are mean and s.d. from 3 independent experiments performed with independent cultures. Data for the graphs in panels a-b are available as source data.
Extended Data Fig. 2 Hsp104 abundance, availability and association with Sup35 in strains with varying copy number or propagating [PIN+].
The relative levels of Hsp104 (a) and Sup35 (b) in lysates from diploid yeast strains used to determine the frequency of [PSI+] appearance in response to transient overexpression of the Sup35 prion domain (Fig. 2c) were determined by SDS-PAGE and immunoblotting with specific antisera. Strains carried wildtype (+/+), heterozygous disruptions of HSP104 (+/Δ), or an extra copy of HSP104 (+/++). Protein levels are expressed relative to wildtype in arbitrary units (a.u.). Data are mean and s.d. from 3 independent experiments performed with independent cultures. c, The frequency of spontaneous loss of the indicated [PSI+] variant was determined in yeast strains carrying wildtype (+/+, black), heterozygous disruptions of HSP104 (+/Δ, gray), or an extra copy of HSP104 (+/++). Data shown are means; error bars represent 95% confidence intervals from ten independent cultures. d, Representative immunoblots of SDS-PAGE gels following immunocapture of NM-HA from lysates isolated from the indicated [PSI+] variant using either specific (anti-HA) or non-specific (anti-MYC) beads. Both [PIN+] (+) and [pin−] (-) strains were analyzed for capture of NM-HA and co-capture of Sup35 and Hsp104, using specific anti-sera. A non-specific band cross-reacting with the HA antiserum is indicated (*). Data for the graphs in panels a-c and uncropped images for panel d are available as source data.
Extended Data Fig. 3 Aggregate amplification probabilities and size distributions.
A stochastic persistence model of Sup35 aggregate dynamics in vivo determines the probability of amplification of a single Sup35 aggregate as a function of fragmentation rate with Sup35 expression levels (a) at 25% (red), 50% (orange), 100% (teal), 200% (blue) or 400% (purple) or Sup35 aggregate numbers (b) of one (red), two (orange), four (blue) or eight (purple) for [PSI+]Strong with a seed size of five. c, Aggregate size distributions for [PSI+]Strong with a seed size of five (solid black), [PSI+]Weak with a seed size of five (dashed red), or [PSI+]Weak with a seed size of fifteen (solid red) are shown.
Extended Data Fig. 4 Aggregate size distribution shifts in response to experimental perturbation.
The aggregate size distributions revealed by our deterministic model (shaded) shift in response to inhibition of Sup35 synthesis (a, b) or Hsp104-mediated fragmentation (c, d) after 90 min (solid, unshaded),180 min (dashed, unshaded), 270 min (dotted, unshaded) or at its steady-state (gray, unshaded) for strains propagating [PSI+]Strong with a seed size of 5 (a, c) or [PSI+]Weak with a seed size of fifteen (b, d). e, The distribution of aggregate sizes before (Xi, solid black) and after (Ci(t), dashed black) t minutes of inhibition of Sup35 synthesis is shown. The shift in the size distribution, S(t), is quantified as the area between the curves when Ci(t) > Xi (shaded area). f, The size distribution of Sup35 aggregates from a strain propagating [PSI+]Weak with a seed size of five (shaded) shifts upon inhibition of Sup35 synthesis for 90 min (solid, unshaded), 180 min (dashed, unshaded), 270 min (dotted, unshaded) or at its steady-state (gray, unshaded). g, and h, The shifts in aggregate size distribution over a range of fragmentation and conversion rates that match the steady-state soluble level of Sup35 were monitored at 90 min (solid), 180 min (dashed), 270 min (dotted) and steady-state (gray) for a strain propagating [PSI+]Weak with a seed size of five (g) or fifteen (h). The x-axis shows the fragmentation rate scaled to the strain-specific value in Supplementary Table 2 (vertical black line). While [PSI+]Weak with a seed size of five (g) shifts slowly to its steady-state distribution, [PSI+]Weak with a seed size of fifteen (h) rapidly reaches is steady-state distribution, matching our empirical observations (Fig. 4a).
Extended Data Fig. 5 Fiber transfection fitting.
Fiber transfection experiments from Fig. 1 were fit mathematically to determine the number of fibers required to induce a stable [PSI+] state. Shown are the observations (black) and fits for fibers of the prion domain of Sup35 assembled at 4 °C (Sc4) or at 37 °C (Sc37) in [pin-] (a, c) or [PIN+] (b, d) yeast strains to a model of one (red), two (green), or three (blue) fibers. Data shown are means and s.d. from at least two independent experiments performed with independent cultures.
Extended Data Fig. 6 Schematics of mathematical models.
a, Schematic of nucleated polymerization dynamics used in the mathematical models. Non-prion state Sup35 (circle) is synthesized at rate α and can join either end an amyloid aggregate of Sup35 (polymer of squares) at a conversion rate β. Amyloid aggregates are fragmented at rate γ at the interface between any two monomers. If the resulting two aggregates are both greater in size than the minimum seed (ns), the amyloid state persists (blue); if either aggregate is smaller in size than ns, it will disassemble into non-prion state Sup35 (red). b, The stochastic persistence model calculates the probability of each of three outcomes, relative to cell division, upon the introduction of a single aggregate of minimal size under nucleated polymerization dynamics: aggregate disassembly, retention of a single aggregate, and aggregate persistence (that is the creation of two aggregates greater in size than ns).
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Supplementary Note 1 (mathematical supplement) and Supplementary Tables 1–5.
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Villali, J., Dark, J., Brechtel, T.M. et al. Nucleation seed size determines amyloid clearance and establishes a barrier to prion appearance in yeast. Nat Struct Mol Biol 27, 540–549 (2020). https://doi.org/10.1038/s41594-020-0416-6
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DOI: https://doi.org/10.1038/s41594-020-0416-6