Dominant prion mutants induce curing through pathways that promote chaperone-mediated disaggregation

Protein misfolding underlies many neurodegenerative diseases, including the transmissible spongiform encephalopathies (prion diseases). Although cells typically recognize and process misfolded proteins, prion proteins evade protective measures by forming stable, self-replicating aggregates. However, coexpression of dominant-negative prion mutants can overcome aggregate accumulation and disease progression through currently unknown pathways. Here we determine the mechanisms by which two mutants of the Saccharomyces cerevisiae Sup35 protein cure the [PSI⁺] prion. We show that both mutants incorporate into wild-type aggregates and alter their physical properties in different ways, diminishing either their assembly rate or their thermodynamic stability. Whereas wild-type aggregates are recalcitrant to cellular intervention, mixed aggregates are disassembled by the molecular chaperone Hsp104. Thus, rather than simply blocking misfolding, dominant-negative prion mutants target multiple events in aggregate biogenesis to enhance their susceptibility to endogenous quality-control pathways.

Figures at a glance

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1. Figure 1: PNM mutants are distinguished by their effective inhibitory ratios.

   

   (a) Wild-type (HSP104/HSP104) or heterozygous-disruption (HSP104/Δ) diploid strains expressing wild-type (WT) and PNM mutants (Q24R, G58D) from P₃₅ at the indicated ratios were spotted on rich (1/4 YPD) or adenine-deficient (−ADE) media to analyze the [PSI⁺] phenotype. Wild-type [PSI⁺] and [psi⁻] diploids (74-D694) were included as controls. (b) To determine the frequency of prion loss, wild-type meiotic progeny (n ≥ 19 for each strain) were isolated from the diploids described in a, and the percentage of [psi⁻] colonies was determined.

2. Figure 2: PNM mutants incorporate into wild-type aggregates and alter multiple events in prion propagation.

   

   (a) HA-tagged Sup35 (WT or mutants) expressed from P₃₅ in haploid [PSI⁺] (+) or [psi⁻] (−) yeast strains, which also expressed untagged Sup35, was immunoprecipitated with anti-HA serum (Ab) and analyzed by SDS-PAGE and anti-Sup35 immunoblotting. (b) [psi⁻] haploids expressing Sup35 (WT or mutants) from P₃₅ and a fluorescent reporter of translation termination efficiency (GST-UGA-DsRed-NLS) were mated to wild-type [PSI⁺] or [psi⁻] (74-D694) cells, and the percentage of fluorescent zygotes was scored. Error bars represent s.d. from three independent experiments, each analyzing at least 15 zygotes per cross (*P = 0.039 in comparison with WT). (c) The fluorescence intensities of zygotes isolated from the indicated crosses as described in b were determined. Horizontal lines on boxes indicate 25th, 50th and 75th percentiles; error bars indicate 10th and 90th percentiles; dots represent outliers (n ≥ 30; *P = 0.0009). (d) Lysates from wild-type haploid strains expressing an additional copy of Sup35 (WT or mutants) from P_tetO2 were incubated in SDS at the indicated temperatures before SDS-PAGE and quantitative immunoblotting for Sup35 (percentage of Sup35 at the indicated temperatures relative to 100 °C). Error bars represent s.d. (n ≥ 6, *P = 0.0003, **P = 0.0001, ***P = 0.008 in comparison with WT at the same temperature).

3. Figure 3: PNM mutants alter the accumulation of propagons but not their transmission.

   

   (a) Lysates from haploid wild-type yeast strains expressing Sup35 (WT or mutants) from P_tetO2 were analyzed by SDD-AGE and immunoblotting for Sup35. Wild-type [PSI⁺]^Strong, [PSI⁺]^Weak and [psi⁻] yeast strains are shown as controls. (b) The number of propagons present in individual cells was determined for the indicated strains, as described in a. Horizontal lines on boxes indicate 25th, 50th and 75th percentiles; error bars indicate 10th and 90th percentiles; dots represent outliers (n ≥ 39; *P ≤ 0.0001 in comparison with WT). (c) The proportion of Sup35 transmitted to daughter cells (circles) or to mother cells (squares) was determined by fluorescence loss in photobleaching (FLIP) of a [PSI⁺] strain expressing Sup35-GFP alone ([PSI⁺]) or with a second copy of Sup35 (WT-GFP + WT) or G58D (WT-GFP + G58D) from P_tetO2. Error bars represent s.e.m. from three independent experiments, each analyzing at least 10 cells.

4. Figure 4: PNM expression promotes Hsp104-mediated disassembly of aggregates.