Hsp104 is a protein chaperone that helps cells recover from stress by resolubilizing proteins from aggregates. This disaggregation activity requires assistance of Hsp40 and Hsp70 and is necessary for replication of amyloid-based yeast prions. Elevating expression of Hsp104 under conditions where it is not normally induced causes PSI prions to be lost from dividing cells. A universally held view of the underlying mechanism is that Hsp104 protein disaggregation activity dissolves prion aggregates until all these templates that can continue to propagate the prion state are eliminated. However, most prions are unaffected by elevating Hsp104, suggesting that PSI is hypersensitive to this disaggregation or that something else is happening. We showed earlier that Hsp104 can be altered in ways that abolish its ability to cure cells of PSI+, but do not affect its ability to act in prion replication, to confer thermotolerance or to resolubilize proteins. These data are inconsistent with curing being caused by complete dissolution of prion aggregates and suggest that the curing involves some unknown function of Hsp104. We showed earlier that Sti1p, a part of the Hsp90 chaperone machinery, which acts in a folding maturation pathway for many """"""""client"""""""" proteins, was important for the curing indirectly by its ability to regulate Hsp70 and Hsp90. Our recent findings that recessive mutations in the Hsp40 Sis1p abolish the curing show that Sis1p is a specific Hsp40 required for curing of PSI+ by Hsp104. ClpB is an Hsp100 family prokaryotic homolog of Hsp104. Protein disaggregating activity of ClpB and Hsp104 depends on interaction and cooperation with species-specific Hsp70 and Hsp40 homologs. By assessing in vitro and in vivo functions of Hsp104/ClpB hybrid proteins, we identified a subregion of these proteins that directs these specific interactions. Continuing this work will help us define at a molecualr level how the components of this machinery cooperate to resolubilize proteins from and restore their activity. Our data also imply that the only restriction these Hsp100 proteins have in functioning across species is the ability to interact properly with the Hsp70/Hsp40 components of the machinery, and that Hsp100 proteins do not function alone in vivo. In order to assess more definitively how depleting specific proteins influences yeast prion propagation, we developed a gene excision system that allows deleting any gene from yeast by a simple switch of carbon source in the growth medium. HSP104, GFP and URA3 genes all were deleted efficiently from different strain backgrounds using this system. Therefore, this system is useful as a simple and general method for studying biological functions of proteins in yeast by conditional gene deletion, and will help us study how depleting Hsp104 and other chaperones influences propagation of various yeast prions.

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Park, Yang-Nim; Zhao, Xiaohong; Yim, Yang-In et al. (2014) Hsp104 overexpression cures Saccharomyces cerevisiae [PSI+] by causing dissolution of the prion seeds. Eukaryot Cell 13:635-47
Reidy, Michael; Masison, Daniel C (2014) Yeast prions help identify and define chaperone interaction networks. Curr Pharm Biotechnol 15:1008-18
Reidy, Michael; Sharma, Ruchika; Masison, Daniel C (2013) Schizosaccharomyces pombe disaggregation machinery chaperones support Saccharomyces cerevisiae growth and prion propagation. Eukaryot Cell 12:739-45
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