N-terminal domain of yeast Hsp104 chaperone is dispensable for thermotolerance and prion propagation but necessary for curing prions by Hsp104 overexpression

Hsp104 is a hexameric protein chaperone that resolubilizes stress-damaged proteins from aggregates. Hsp104 promotes PSI(+)] prion propagation by breaking prion aggregates, which propagate as amyloid fibers, into more numerous prion "seeds." Inactivating Hsp104 cures cells of PSI(+)] and other amyloid-like yeast prions. Overexpressing Hsp104 also eliminates PSI(+)], presumably by completely resolubilizing prion aggregates. Inexplicably, however, excess Hsp104 does not cure the other prions. Here we identify missense mutations in Hsp104's amino-terminal domain (NTD), which is conserved among Hsp100 proteins but whose function is unknown, that improve PSI(+)] propagation. Hsp104Delta147, engineered to lack the NTD, supported PSI(+)] and functioned normally in thermotolerance and protein disaggregation. Hsp104Delta147 failed to cure PSI(+)] when overexpressed, however, implying that excess Hsp104 does not eliminate PSI(+)] by direct dissolution of prion aggregates. Curing of PSI(+)] by overexpressing catalytically inactive Hsp104 (Hsp104KT), which interferes with endogenous Hsp104, did not require the NTD. We further found that Hsp104 mutants defective in threading peptides through the hexamer pore had reduced ability to support PSI(+)] in proportion to protein resolubilization defects, suggesting that PSI(+)] propagation depends on this threading and that Hsp104 "breaks" prion aggregates by extracting protein monomers from the amyloid fibers.