Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions.
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 defects 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.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Patrick G
PG
Needham
Program in Molecular Pharmacology and Chemistry.
Hardik J
HJ
Patel
Program in Molecular Pharmacology and Chemistry; Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA.
Gabriela
G
Chiosis
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Patrick H
PH
Thibodeau
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. Electronic address: Jbrodsky@pitt.edu.
Jeffrey L
JL
Brodsky
D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000737:chemistry; D015966:Gene Expression Regulation, Fungal; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry; D018869:Heat-Shock Response; D008928:Mitochondria / Q000378:metabolism; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D017510:Protein Folding; D059748:Proteolysis; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000737:chemistry; D013379:Substrate Specificity
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae
ERAD; molecular chaperone; protein degradation; protein folding; protein translocation