ATPASE ACTIVITY ASSOCIATED WITH THE UNCOATING OF CLATHRIN BASKETS BY HSP70

In the presence of ATP, bovine brain hsp70 has been shown to remove cl athrin from bovine brain clathrin-coated vesicles in a rapid stoichiom etric initial burst followed by slow steady-state uncoating. In additi on, it has been found recently that a 100-kDa cofactor is required for hsp70 to uncoat clathrin baskets prepared with the assembly protein A P-2. In this study the ATPase activity associated with uncoating was i nvestigated, with baskets formed from clathrin and assembly proteins. Mixed assembly proteins or assembly protein AP-2 could not be used in ATPase studies because they activated the hsp70 ATPase activity even i n the absence of clathrin. However, this was not the case with assembl y protein AP(180). A stoichiometric initial burst of ATP hydrolysis wa s found to accompany the initial burst of uncoating of AP(180)-clathri n baskets by hsp70, with 1 mol of hydrolyzed ATP/mol of released clath rin heavy chain. Furthermore, the presence of a 100-kDa cofactor was n eeded for both processes. These results suggest that an initial burst of uncoating occurs with all clathrin baskets, that an initial burst o f ATP hydrolysis accompanies this initial burst of uncoating, and that a 100-kDa cofactor is required for both.