Hsp70/Hsp90 co‐chaperones are required for efficient Hsp104‐mediated elimination of the yeast [PSI⁺] prion but not for prion propagation

Abstract

The continued propagation of the yeast [PSI(+)] prion requires the molecular chaperone Hsp104 yet in cells engineered to overexpress Hsp104; prion propagation is impaired leading to the rapid appearance of prion-free [psi(-)] cells. The underlying mechanism of prion loss in such cells is unknown but is assumed to be due to the complete dissolution of the prion aggregates by the ATP-dependent disaggregase activity of this chaperone. To further explore the mechanism, we have sought to identify cellular factors required for prion loss in such cells. Sti1p and Cpr7p are co-chaperones that modulate the activity of Hsp70/Ssa and Hsp90 chaperones and bind to the C-terminus of Hsp104. Neither Sti1p nor Cpr7p is necessary for prion propagation but we show that deletion of the STI1 and CPR7 genes leads to a significant reduction in the generation of [psi(-)] cells by Hsp104 overexpression. Deletion of the STI1 and CPR7 genes does not modify the elimination of [PSI(+)] by guanidine hydrochloride, which inhibits the ATPase activity of Hsp104 but does block elimination of [PSI(+)] by overexpression of either an ATPase-defective mutant of Hsp104 (hsp104(K218T/K620T)) or a 'trap' mutant Hsp104 (hsp104(E285Q/E687Q)) that can bind its substrate but can not release it. These results provide support for the hypothesis that [PSI(+)] elimination by Hsp104 overexpression is not simply a consequence of complete dissolution of the prion aggregates but rather is through a mechanism distinct from the remodelling activity of Hsp104.

Modulation and elimination of yeast prions by protein chaperones and co-chaperones.

“…The Hsp90 chaperone system, which serves as a post-translational regulator of many cellular processes, is also important for the curing mechanism. 30,31 Increasing Hsp90 abundance does not affect curing by overexpressed Hsp104. 32 However, although neither deleting Hsp70/ Hsp90 coordinating co-chaperone Sti1p nor chemical inhibition of Hsp90 affect [PSI + ] propagation, either treatment strongly inhibits the curing.…”

Functions of Yeast Hsp40 Chaperone Sis1p Dispensable for Prion Propagation but Important for Prion Curing and Protection From Prion Toxicity

“…These and other data are consistent with a distinction between processes of prion propagation and curing and a difference in mechanism of curing by Hsp104 inhibition and overexpression. We and others recently showed that extragenic suppressors of the Ssa1-21p inhibition of [PSI 1 ] also suppress the ability of overexpressed Hsp104 to cure cells of [PSI 1 ] (Moosavi et al 2010;Reidy and Masison 2010), which implies Ssa1-21p and elevated Hsp104 antagonize [PSI 1 ] by a similar mechanism.…”

Structural basis for intersubunit signaling in a protein disaggregating machine

“…Notably, replacing the M-domain of bacterial ClpB with that of its yeast homolog Hsp104 switched the species specificity of the bichaperone system so that ClpB now cooperated with the eukaryotic Hsp70/40 system and vice versa (7,27). The role of the M-domain in mediating DnaKJ/GrpE interaction is consistent with the M-domain being on the outside of the ClpB hexamer (5, 8), but incompatible with the previously proposed structure of yeast Hsp104 (28,29), which placed the M-domains on the interior or intercalated between subunits.Although we recently showed that ClpB and Hsp104 share a similar 3D structure (30), functional differences exist (19,(31)(32)(33). Moreover, it remains unclear how ClpB exerts the ATP power stroke to thread substrates through the ClpB hexamer ring, and how ClpB unfolds substrates that are typically much larger than the ClpB hexamer itself.…”

“…Although we recently showed that ClpB and Hsp104 share a similar 3D structure (30), functional differences exist (19,(31)(32)(33). Moreover, it remains unclear how ClpB exerts the ATP power stroke to thread substrates through the ClpB hexamer ring, and how ClpB unfolds substrates that are typically much larger than the ClpB hexamer itself.…”