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. Depleting or inactivating Hsp104 arrests prion replication, which causes yeast prions to be lost as cells divide because the number of cells eventually becomes greater than the number of infectious prion particles. In order to assess more definitively how depleting Hsp104 leads to prion loss, we monitored the fate of prion particles (foci) in live cells using a Sup35-GFP fusion protein after inactivating or depleting Hsp104 by various methods. Overexpressing dominant negative Hsp104 (Hsp104-KT) inhibits Hsp104 activity by incorporating into Hsp104 hexamers, deleting Hsp104 by excision of its gene depletes Hsp104 activity somewhat more gradually, and millmolar concentrations of guanidine in growth media rapidly and reversibly inactivates Hsp104 ATPas activity. Overexpressing the dominant negative Hsp104-2KT caused foci to increase in size, then decrease in number, and finally disappear when the cells were cured. This pattern was similar to that observed in cells were cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in size of foci but then the foci disappeared before the cells were cured. Briefly starving the guanidine-treated cells makes foci in these uncured cells reappear. Regardless of the curing method the number of cells with foci correlated exactly with the number of PSI+ cells. Therefore, the foci are the prion seeds required for maintenance of PSI+ and inactivation of Hsp104 cures PSI+ by preventing severing of the prion seeds. During curing with guanidine, the loss of visibility of fluorescent foci caused by a reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. Presumably, this trimming, which is only revealed by treating cells with guanidine, occurs at the ends of prion fibers. This Hsp104 activity might be important for non-prion Hsp104 functions in yeast.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$331,928
Indirect Cost
City
State
Country
Zip Code
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
Park, Yang-Nim; Morales, David; Rubinson, Emily H et al. (2012) Differences in the curing of [PSI+] prion by various methods of Hsp104 inactivation. PLoS One 7:e37692
Kirkland, P Aaron; Reidy, Michael; Masison, Daniel C (2011) Functions of yeast Hsp40 chaperone Sis1p dispensable for prion propagation but important for prion curing and protection from prion toxicity. Genetics 188:565-77
Miot, Marika; Reidy, Michael; Doyle, Shannon M et al. (2011) Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation. Proc Natl Acad Sci U S A 108:6915-20
Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan et al. (2011) Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae. Yeast 28:673-81
Reidy, Michael; Masison, Daniel C (2010) Sti1 regulation of Hsp70 and Hsp90 is critical for curing of Saccharomyces cerevisiae [PSI+] prions by Hsp104. Mol Cell Biol 30:3542-52
Greene, Lois E; Park, Yang-Nim; Masison, Daniel C et al. (2009) Application of GFP-labeling to study prions in yeast. Protein Pept Lett 16:635-41