Prion-impairing mutations in Hsp70 chaperone Ssa1: effects on ATPase and chaperone activities

We previously described many Hsp70 Ssa1p mutants that impair PSI⁺] prion propagation in yeast without affecting cell growth. To determine how the mutations alter Hsp70 we analyzed biochemically the substrate-binding domain (SBD) mutant L483W and the nucleotide-binding domain (NBD) mutants A17V and R34K. Ssa1^L483W ATPase activity was elevated 10-fold and was least stimulated by substrates or Hsp40 co-chaperones. Ssa1^A17V and Ssa1^R34K ATPase activities were nearly wild type but both showed increased stimulation by substrates. Peptide binding and reactivation of denatured luciferase were enhanced in Ssa1^A17V and Ssa1^R34K but compromised in Ssa1^L483W. The nucleotide exchange factor Fes1 influenced ATPase of wild type Ssa1 and each mutant differently. Partial protease digestion uncovered similar and distinct conformational changes of the substrate-binding domain among the three mutants. Our data suggest that prion-impairing mutations of Ssa1 can increase or decrease substrate interactions, alter the Hsp70 reaction cycle at different points and impair normal NBD-SBD cooperation.