INTERACTION OF NUCLEOTIDE-FREE HSC70 WITH CLATHRIN AND PEPTIDE AND EFFECT OF ATP ANALOGS

The functions of the 70 kDa heat-shock proteins (hsp70s) are regulated by their bound nucleotide. We previously observed major differences i n the effect of bound ATP and ADP on the interaction of hsc70 (constit utive hsp70) with its protein substrates. In the present study, we inv estigated the interaction of protein substrates with nucleotide-free h sc70 and with hsc70 with bound ATP analogues. We found, first, that nu cleotide-free hsc70 appeared to interact differently with different su bstrates. Specifically, nucleotide-free hsc70 behaved much more like h sc70-ATP than hsc70-ADP in that clathrin very rapidly bound to and dis sociated from nucleotide-free hsc70 in contrast to its very slow bindi ng to and dissociation from hsc70-ADP. On the other hand, nucleotide-f ree hsc70 behaved more like hsc70-ADP than hsc70-ATP in that cytochrom e c peptide dissociated very slowly from nucleotide-free hsc70 compare d to its rapid dissociation from hsc70-ATP. Second, binding of the ATP analogues AMP-PNP, dATP, and ATP gamma S to nucleotide-free hsc70 had very Little further effect on the properties of the nucleotide-free h sc70. Therefore, previously observed effects of ATP analogues may have been due to removal of the bound ADP rather than to the presence of a nalogues.