ACTIVITY AND IN-VITRO REASSEMBLY OF THE COATED VESICLE (H-ATPASE REQUIRES THE 50-KDA SUBUNIT OF THE CLATHRIN ASSEMBLY COMPLEX AP-2())

We have previously shown that the 50-kDa subunit of the clathrin assem bly complex AP-2 (AP50) stoichiometrically binds to and is immunopreci pitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-91 86), We now report that treatment of stripped coated vesicles with cys tine results in a purified V-ATPase complex lacking the AP50 polypepti de, Removal of AP50 can be reversed upon treatment of the vesicles wit h dithiothreitol., Removal of AP50 reduces the ATPase activity of the purified V-ATPase by 90% relative to the enzyme containing AP50. This inhibition is not reversed upon treatment of the AP50-depleted enzyme with dithiothreitol in the absence of AP50. The reconstituted V-ATPase depleted of AP50 is devoid of ATP-dependent proton transport activity . We observe further that the peripheral V-1 subunits are unable to re assemble onto the integral V-0 domain in the absence of AP50. The addi tion of purified AP-2 containing the AP50 polypeptide restores the abi lity of the V-1 subunits to assemble with the V-0 sector to give a V-A TPase complex that is functional in ATP-dependent proton transport, Th ese results indicate that the AP50 polypeptide is necessary for both a ctivity and in vitro reassembly of the V-ATPase complex.