Intragenic suppressors of Hsp70 mutants: Interplay between the ATPase- andpeptide-binding domains

ATP hydrolysis and polypeptide binding, the two keg activities of Hsp70 mol ecular chaperones, are inherent properties of different domains of the prot ein. The coupling of these two activities is critical because the bound nuc leotide determines, in part, the affinity of Hsp70s for protein substrate, In addition, cochaperones of the Hsp40 (DnaJ) class, which stimulate Hsp70 ATPase activity, have been proposed to play an important role in promoting efficient Hsp70 substrate binding, Because little is understood about this functional interaction between domains of Hsp70s, we investigated mutations in the region encoding the ATPase domain that acted as intragenic suppress ors of a lethal mutation (1485N) map ping to the peptide-binding domain of the mitochondrial Hsp70 Ssc1, Analogous amino acid substitution in the ATPa se domain of the Escherichia coil Hsp70 DnaK had a similar intragenic suppr essive effect on the corresponding 1462T temperature-sensitive peptide-bind ing domain mutation, 1462T protein had a normal basal ATPase activity and w as capable of nucleotide-dependent conformation changes. However, the reduc ed affinity of 1462T for substrate peptide (and DnaJ) is likely responsible for the inability of 1462T to function in vivo. The suppressor mutation (D 79A) appears to partly alleviate the defect in DnaJ ATPase stimulation caus ed by 1462T, suggesting that alteration in the interaction with DnaJ may al ter the chaperone cycle to allow productive interaction with polypeptide su bstrates. Preservation of the intragenic suppression phenotypes between euk aryotic mitochondrial and bacterial Hsp70s suggests that the phenomenon stu died here is a fundamental aspect of the function of Hsp70:Hsp40 chaperone machines.