Saccharomyces cerevisiae Hsp70 Mutations Affect [PSI⁺] Prion Propagation and Cell Growth Differently and Implicate Hsp40 and Tetratricopeptide Repeat Cochaperones in Impairment of [PSI⁺]

Abstract

We previously described an Hsp70 mutant (Ssa1-21p), altered in a conserved residue (L483W), that dominantly impairs yeast [PSI⁺] prion propagation without affecting growth. We generated new SSA1 mutations that impaired [PSI⁺] propagation and second-site mutations in SSA1-21 that restored normal propagation. Effects of mutations on growth did not correlate with [PSI⁺] phenotype, revealing differences in Hsp70 function required for growth and [PSI⁺] propagation and suggesting that Hsp70 interacts differently with [PSI⁺] prion aggregates than with other cellular substrates. Complementary suppression of altered activity between forward and suppressing mutations suggests that mutations that impair [PSI⁺] affect a similar Hsp70 function and that suppressing mutations similarly overcome this effect. All new mutations that impaired [PSI⁺] propagation were located in the ATPase domain. Locations and homology of several suppressing substitutions suggest that they weaken Hsp70's substrate-trapping conformation, implying that impairment of [PSI⁺] by forward mutations is due to altered ability of the ATPase domain to regulate substrate binding. Other suppressing mutations are in residues important for interactions with Hsp40 or TPR-containing cochaperones, suggesting that such interactions are necessary for the impairment of [PSI⁺] propagation caused by mutant Ssa1p.