The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergentsolubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V 1 and V 0 , with overall dimensions of 28 ؋ 14 ؋ 14 nm. The V 1 is a more or less spherical molecule with a central cavity. The V 0 has the shape of a flattened sphere or doughnut with a radius of about 100 Å. The V 1 and V 0 are joined by a 60-Å long and 40-Å wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V 1 , and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30 -50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V 1 -V 0 interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F 1 F 0 -ATP synthase, confirming their common origin.A vacuolar ATPase (or V-ATPase) 1 is found in the membrane of subcellular compartments of eucaryotic cells, where it functions to acidify the interior and at the same time energize the membranes of organelles such as clathrin-coated vesicles, endosomes, lysosomes, chromaffin granules, and Golgi-derived vesicles (1, 2). ATP hydrolysis-driven acidification of these organelles plays an important role in processes like receptormediated endocytosis, neurotransmitter release, protein trafficking, pH maintenance, and storage of metabolites. Early electron microscopic images (3-5) show the V-ATPase complex organized in two parts, a membrane extrinsic V 1 and a membrane-embedded V 0 , named after the related F 1 F 0 -type ATP synthase. As in F 1 F 0 , ATP hydrolysis on the membrane extrinsic domain is coupled to proton translocation across the membrane bilayer, but unlike F 1 F 0 , the vacuolar enzyme cannot utilize the potential energy of a proton gradient to synthesize ATP. The V 1 V 0 -ATPase is composed of at least 12 different subunits. The V 1 contains subunits A-H with molecular weights of 73,000, 58,000, 40,000, 34,000, 33,000, 14,000, 10,000, and 50,000 -57,000, and the V 0 contains subunits a, cЈ, cЉ, and d having molecular weights of 100,000, 17,000, 19,000 and 38,000, respectively. The subunit stoichiometry of the complex is A 3 B 3 CDEF x G y H z ac (1) 6 cЉd, giving a calculated molecular weight of approximately 840,000 (assuming one copy of subunits F, G, and H each), in good agreement...