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Prions are transmissible self-perpetuating protein isoforms associated with diseases and heritable traits. Yeast prions and non-transmissible protein aggregates (mnemons) are frequently based on cross-β ordered fibrous aggregates (amyloids). The formation and propagation of yeast prions are controlled by chaperone machinery. Ribosome-associated chaperone Hsp70-Ssb is known (and confirmed here) to modulate formation and propagation of the prion form of the Sup35 protein [PSI+]. Our new data show that both formation and mitotic transmission of the stress-inducible prion form of the Lsb2 protein ([LSB+]) are also significantly increased in the absence of Ssb. Notably, heat stress leads to a massive accumulation of [LSB+] cells in the absence of Ssb, implicating Ssb as a major downregulator of the [LSB+]-dependent memory of stress. Moreover, the aggregated form of Gγ subunit Ste18, [STE+], behaving as a non-heritable mnemon in the wild-type strain, is generated more efficiently and becomes heritable in the absence of Ssb. Lack of Ssb also facilitates mitotic transmission, while lack of the Ssb cochaperone Hsp40-Zuo1 facilitates both spontaneous formation and mitotic transmission of the Ure2 prion, [URE3]. These results demonstrate that Ssb is a general modulator of cytosolic amyloid aggregation, whose effect is not restricted only to [PSI+].
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Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock
Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Saccharomyces cerevisiae . Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [ PSI + ], a prion isoform of the yeast translation termination factor Sup35 . This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [ PSI + ] destabilization. Moreover, deletion of the SIR2 gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [ PSI + ], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104 -GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [ PSI + ], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone.
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- Award ID(s):
- 1817976
- PAR ID:
- 10163420
- Date Published:
- Journal Name:
- Genetics
- Volume:
- 212
- Issue:
- 3
- ISSN:
- 0016-6731
- Page Range / eLocation ID:
- 757 to 771
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI+] is the prion form of the Sup35 protein. While the study of [PSI+] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin’s precise role is unclear. Here, we investigate how actin influences the cell’s ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell.more » « less
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Abstract Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding‐site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client‐protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome‐wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2258Escherichia coli(E. coli) proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent‐exposed than other nonpolar sites, in protein native states. Our findings show that the majority ofE. coliproteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70‐bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70‐chaperone‐binding traits have evolved to favor Hsp70‐assisted protein folding devoid of aggregation.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1534/genetics.119.302237
