Topography and subunit stoichiometry of the coated vesicle proton pump.

Abstract

We have previously shown that the coated vesicle (H+)-ATPase contains nine polypeptides of molecular weight 17,000-100,000 which form a single, macromolecular complex that can be immunoprecipitated using monoclonal antibodies which recognize the native enzyme (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26, 6632-6638). In the present paper, we have calculated from quantitative amino acid analysis that these polypeptides are present in the native complex in a stoichiometry of three copies each of the 73,000- and 58,000-dalton subunits, six copies of the 17,000-dalton subunit, and one copy each of the 100,000-, 40,000-, 38,000-, 34,000-, 33,000-, and 19,000-dalton subunits. To determine the disposition of the (H+)-ATPase subunits with respect to the membrane, we have carried out labeling studies using the membrane impermeant reagents Na125I/lactoperoxidase and 125I-sulfo-succinimidyl-3-(4-hydroxyphenyl)propionate and the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID). Polypeptides exposed to the cytoplasmic surface were identified by labeling with impermeant reagents in intact vesicles from which clathrin had been dissociated followed by immunoprecipitation of the native enzyme. Polypeptides exposed to the luminal surface were identified by increased labeling by these reagents following detergent solubilization under nondenaturing conditions. Labeling by [125I]TID was used to indicate which polypeptides are embedded in the lipid bilayer. Results of these experiments indicate that the principal polypeptides labeled from the cytoplasmic surface are those of molecular weight 73,000 and 58,000, although some cytoplasmic labeling of the 100,000, 40,000, 38,000 and 34,000/33,000 polypeptides was also observed. The polypeptides which show the greatest increase in labeling following detergent solubilization are those of molecular weight 100,000, 19,000, and 17,000, with some increase observed for the 40,000, 38,000, and 34,000/33,000 polypeptides. [125I]TID labeled the 17,000-dalton subunit most heavily, with significant labeling of the 100,000- and 40,000-dalton subunits also observed. In addition, we find that the 73,000-dalton polypeptide can be dissociated from the complex with 0.5 M KI in the absence of detergent, indicating a peripheral association of this subunit with the membrane. We have combined these results to construct a structural model of the coated vesicle (H+)-ATPase.