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. Inhibiting Hsp104 activity by overexpressing the dominant negative Hsp104-2KT caused foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells 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. Starving the guanidine-treated cells briefly 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 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. This Hsp104 activity revealed by guanidine treatment might be important for other Hsp104 functions in yeast.
