Dissociation of clathrin from coated vesicles by the uncoating ATPase.
The uncoating ATPase has been shown to dissociate clathrin from both clathrin-coated vesicles and synthetic clathrin baskets (Rothman, J. E., and Schmid, S. L. (1986) Cell 46, 5-9). In the present study, we investigated the mechanism of action of the uncoating ATPase using intact coated vesicles isolated from bovine brain. We observed an initial burst of uncoating followed by much slower steady-state uncoating. The initial burst of uncoating was essentially stoichiometric with each molecule of uncoating ATPase apparently binding to one leg of the clathrin triskelion. When the enzyme was preincubated with equimolar ADP, Pi, and ATP, rather than just ATP alone, both the initial burst and the slow steady-state uncoating were markedly inhibited, suggesting that the combination of ADP and Pi is a strong competitive inhibitor of ATP binding. However, kinetic studies suggested that ADP and Pi dissociates from the enzyme relatively rapidly unless clathrin is also bound to the enzyme. These results suggest that, after the uncoating ATPase rapidly removes a stoichiometric amount of clathrin while ATP is hydrolyzed at the active site, slow release of ADP and Pi from the resulting enzyme.clathrin.ADP.Pi complex limits the rate at which further uncoating occurs.
Greene
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LE
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Eisenberg
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D007700:Kinetics; D008962:Models, Theoretical; D008970:Molecular Weight
D002352:Carrier Proteins; D002966:Clathrin; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D000251:Adenosine Triphosphatases