The bovine brain uncoating ATPase, a constitutive 70-kDa heat shock protein, uncoats clathrin-coated vesicles in an ATP-dependent reaction. The uncoating ATPase-clathrin complex formed from the uncoating reaction was compared to the complex formed by directly binding free clathrin to uncoating ATPase. The amount of the latter complex shows a simple hyperbolic dependence on either free clathrin or free uncoating ATPase concentration, whichever is in excess, with a binding stoichiometry of one uncoating ATPase per clathrin heavy chain. ATP markedly increases the rates of formation and dissociation of this complex while ADP profoundly inhibits these rates. At low uncoating ATPase concentrations, much more complex is formed by uncoating than by directly binding clathrin to enzyme. However, during column chromatography or dilution for electron microscopy, both types of complex dissociate in ATP but not ADP, and electron microscopy of both types of complex diluted into ADP shows binding only to the vertex of the clathrin triskelion. We conclude that the uncoating ATPase forms only one type of complex with clathrin and has only one site for nucleotide; ADP at this site prevents either formation or dissociation of complex, whereas ATP at this site allows both processes to occur rapidly.
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