Functional analysis of a conserved amino-terminal region of HSP70 by site-directed mutagenesis.
Hsp70 proteins have been highly conserved throughout evolution. As a first step in a structure-function analysis of hsp70, we constructed and analysed the consequences of mutations in a portion of the SSA1 gene, a member of the Saccharomyces cerevisiae HSP70 multigene family, that encodes a nearly invariant region near the amino terminus. Analysis of strains expressing SSA1 proteins with alterations at positions 8, 11 and 15 showed that these conserved residues within this region are critical for normal functioning of the protein. SSA1 protein containing either of two changes at position 15 was able to slightly complement the inviability of an ssa1ssa2ssa4 strain, but was inactive in other complementation assays. The other mutant proteins tested were unable to complement any tested phenotype. Effective interallelic complementation of several phenotypes was observed when a mutant protein substituted at position 8 was expressed in the same cell with either of two proteins carrying substitutions at position 15, suggesting that hsp70 acts as a multimer. Evidence from previous studies suggests that hsp70 proteins engage in ATP-driven cycles of binding and release from peptides. The ability of the mutant proteins to bind ATP and a peptide was tested. The Ssa1p carrying a substitution at position 8, which inhibits growth of cells carrying wild-type SSA proteins, showed a defect in release from a peptide relative to wild type. Two mutations, one each at position 8 and 15, resulted in accumulation of phosphorylated isoforms which may be a normal, transient hsp70 intermediate.
Nicolet
C M
CM
Department of Physiological Chemistry, University of Wisconsin-Madison 53706.
Craig
E A
EA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013696:Temperature
D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D009842:Oligopeptides; D000255:Adenosine Triphosphate