THE DISSOCIATION OF ATP FROM HSP70 OF SACCHAROMYCES-CEREVISIAE IS STIMULATED BY BOTH YDJ1P AND PEPTIDE-SUBSTRATES

hsp70 proteins of both eukaryotes and prokaryotes possess both ATPase and peptide binding activities, These two activities are crucial for t he chaperone activity of hsp70 proteins, The activity of DnaK, the pri mary hsp70 of Escherichia coli, is modulated by the GrpE and DnaJ prot eins, In the yeast Saccharomyces cerevisiae, the predominant cytosolic hsp70, Ssa1p, interacts with a DnaJ homologue, Ydj1p, In order to bet ter understand the function of the Ssa1p/Ydj1p chaperone, the effects of polypeptide substrates and Ydj1p on Ssa1p ATPase activity were asse ssed using a combination of steady-state kinetic analysis and single t urnover substrate hydrolysis experiments, Polypeptide substrates and Y dj1p both serve to stimulate ATPase activity of Ssa1p, The two types o f effector are biochemically distinct, each conferring a characteristi c K+ dependence on Ssa1p ATPase activity, However, in single turnover ATP hydrolysis experiments, both polypeptide substrates and Ydj1p dest abilized the ATP . Ssa1p complex through a combination of accelerated hydrolysis of bound ATP and accelerated release of ATP from Ssa1p, The acceleration of ATP release by Ydj1p is a previously unidentified fun ction of a DnaJ homologue, In the case of Ydj1p-stimulated Ssa1p, stea dy-state ATPase activity is increased less than g-fold at physiologica l K+ concentrations, despite a 15-fold increase in the hydrolysis of b ound ATP, The primary effect of Ydj1p appears to be to disfavor an ATP form of Ssa1p, On the other hand, peptide stimulation of Ssa1p ATPase activity was enhanced at physiological K+ concentrations, supporting the idea that cycles of ATP hydrolysis play an important role in the i nteraction of hsp70 with polypeptide substrates, The enhanced ATP diss ociation caused by both polypeptide substrates and Ydj1p may play a ro le in the regulation of Ssa1p chaperone activity by altering the relat ive abundance of ATP- and ADP-bound forms.