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 assembly complex AP-2 (AP50) stoichiometrically binds to and is immunoprecipitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186). We now report that treatment of stripped coated vesicles with cystine results in a purified V-ATPase complex lacking the AP50 polypeptide. Removal of AP50 can be reversed upon treatment of the vesicles with 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 V1 subunits are unable to reassemble onto the integral V0 domain in the absence of AP50. The addition of purified AP-2 containing the AP50 polypeptide restores the ability of the V1 subunits to assemble with the V0 sector to give a V-ATPase complex that is functional in ATP-dependent proton transport. These results indicate that the AP50 polypeptide is necessary for both activity and in vitro reassembly of the V-ATPase complex.
Liu
Q
Q
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Feng
Y
Y
Forgac
M
M
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001921:Brain; D002417:Cattle; D018699:Coated Vesicles / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014617:Vacuoles / Q000201:enzymology*
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; C438885:adaptor protein complex 2, mu 1 subunit; D006180:Proton-Translocating ATPases