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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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The middle domain of Hsp104 can ensure substrates are functional after processing
Molecular chaperones play a central role in protein disaggregation. However, the molecular determinants that regulate this process are poorly understood. Hsp104 is an AAA+ ATPase that disassembles stress granules and amyloids in yeast through collaboration with Hsp70 and Hsp40.In vitrostudies show that Hsp104 processes different types of protein aggregates by partially translocating or threading polypeptides through the central pore of the hexamer. However, it is unclear how Hsp104 processing influences client protein functionin vivo. The middle domain (MD) of Hsp104 regulates ATPase activity and interactions with Hsp70. Here, we tested how MD variants, Hsp104A503Sand Hsp104A503V, process different protein aggregates. We establish that engineered MD variants fail to resolve stress granules but retain prion fragmentation activity required for prion propagation. Using the Sup35 prion protein, ourin vitroandin vivodata indicate that the MD variants can disassemble Sup35 aggregates, but the disaggregated protein has reduced GTPase and translation termination activity. These results suggest that the middle domain can play a role in sensing certain substrates and plays an essential role in ensuring the processed protein is functional.
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- Award ID(s):
- 2127616
- PAR ID:
- 10618859
- Editor(s):
- Chernoff, Yury O
- Publisher / Repository:
- PLOS
- Date Published:
- Journal Name:
- PLOS Genetics
- Volume:
- 20
- Issue:
- 10
- ISSN:
- 1553-7404
- Page Range / eLocation ID:
- e1011424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pgen.1011424
