Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions

Abstract

The major cytoplasmic Hsp70 chaperones in the yeast Saccharomyces cerevisiae are the Ssa proteins, and much of our understanding of Hsp70 biology has emerged from studying ssa mutant strains. For example, Ssa1 catalyzes multiple cellular functions, including protein transport and degradation, and to this end the ssa1–45 mutant has proved invaluable. However, the biochemical defect(s) associated with the corresponding Ssa1–45 protein (P417L) are unknown. Consequently, we characterized Ssa1 P417L as well as a P417S variant, which corresponds to a mutation in the gene encoding the yeast mitochondrial Hsp70. We discovered that the P417L and P417S proteins exhibit accelerated ATPase activity that was similar to the Hsp40-stimulated rate of ATP hydrolysis of wild type Ssa1. We also found that the mutant proteins were compromised for peptide binding. These data are consistent with defects in peptide-stimulated ATPase activity and with results from limited proteolysis experiments, which indicated that the mutants’ substrate binding domains were highly vulnerable to digestion. Defects in the reactivation of heat-denatured luciferase were also evident. Correspondingly, yeast expressing P417L or P417S as the only copy of Ssa were temperature sensitive and exhibited defects in Ssa1-dependent protein translocation and misfolded protein degradation. Together, our studies suggest that the structure of the substrate binding domain is altered and that coupling between this domain and the nucleotide binding domain is disabled when the conserved P417 residue is mutated. Our data also provide new insights into the nature of the many cellular defects associated with the ssa1–45 allele.

Synthesis and evaluation of esterified Hsp70 agonists in cellular models of protein aggregation and folding

“…The magnitude of this effect mirrored the effect on the bacterial chaperones 26 , and the data are consistent with the ability of MAL1–271 to suppress the formation of α-synuclein aggregates in a neuroglioma cell model. 34 We also note that the turnover rate measured in these experiments (0.08 min-1) is consistent with published values for this chaperone 32 and that none of the pyrimidinone-peptoid hybrid molecules acts on Hsp90, consistent with their specific binding to the co-chaperone binding pocket in Hsp70. 26 …”

The absence of specific yeast heat-shock proteins leads to abnormal aggregation and compromised autophagic clearance of mutant Huntingtin proteins

“…We speculated the efficient Htt103QP IB formation in each single mutant is likely due to the redundant function of Hsp70 protein family. To test this possibility, we used a strain lacking SSA2 , SSA3 and SSA4 genes but expressing temperature-sensitive ssa1-45 [ 35 ]. We found that this mutant showed a significant Htt103QP IB formation defect when incubated at 35°C.…”

Endoplasmic reticulum–associated degradation of the renal potassium channel, ROMK, leads to type II Bartter syndrome

“…To examine whether ROMK degradation is chaperone-dependent, we utilized a yeast strain containing a temperature-sensitive mutation in the gene encoding Ssa1. Specifically, in the ssa1-45 strain, a Pro to Leu mutation at residue 417 renders the resulting protein unstable at higher temperatures (81). When expressed in a strain that lacks the genes encoding the paralogous cytosolic Hsp70 proteins Ssa2, Ssa3, and Ssa4, defects in growth, protein translocation, and ERAD were evident at 37°C (82).…”