The vacuolar (H ϩ )-ATPases (V-ATPases) 1 are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments (such as endosomes, lysosomes, and secretory vesicles) as well as proton transport across the plasma membrane (1-11). V-ATPases present in intracellular compartments function in such processes as receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, coupled transport of small molecules and ions, and the entry of certain viruses and toxins from endocytic compartments into cells (1, 2). Plasma membrane V-ATPases are important for urinary acidification by renal intercalated cells, bone resorption by osteoclasts, K ϩ secretion by insect goblet cells, sperm maturation in the male reproductive tract, and the invasiveness of certain tumor cells (1, 7-11).The V-ATPases are composed of a peripheral V 1 domain responsible for ATP hydrolysis and an integral V 0 domain that carries out proton translocation (1, 2). V 1 is a 600 -650-kDa complex with the subunit composition A 3 B 3 C 1 D 1 E 1 F 1 G 2 H 1-2 (1, 2). The nucleotide binding sites are located at the interface of the A and B subunits (which form an alternating hexamer), with the catalytic sites located mainly on the A subunit and "non-catalytic" sites located mainly on the B subunit (12-14). The V 0 domain is a 250-kDa complex with the subunit composition a 1 d 1 e x c 4 cЈ 1 cЉ 1 (1, 15). The three proteolipid subunits (c, cЈ, and cЉ) each contain a buried glutamic acid residue that is essential for proton transport (16, 17) and together they form a hexameric ring. The V-ATPases thus resemble the F-ATPases (or ATP synthases), which are involved in ATP synthesis (18 -20). High resolution crystal structures have been obtained for both F 1 (21, 22) and F 1 bound to a proteolipid ring (23).The F-ATPases operate by a rotary mechanism (20). ATP hydrolysis in F 1 causes rotation of a central stalk composed of the ␥ and ⑀ subunits (23-26), which in turn causes rotation of the ring of proteolipid c subunits (27)(28)(29). A peripheral stalk (composed of the ␦ subunit and the soluble regions of subunit b, Ref. 30) functions as a stator, holding subunit a fixed relative to the ␣ 3  3 head during ATP-driven rotation of the proteolipid ring. It is rotation of the ring of proteolipid c subunits relative to subunit a that is believed to drive proton transport (19,31,32). Comparison of amino acid sequences of the V-and FATPases reveals clear sequence homology both between the nucleotide binding subunits and between the proteolipid subunits of the two classes (33,34,16). Because there is virtually no sequence similarity for the remaining subunits, it has not been possible to deduce the function or localization of the remaining V-ATPase subunits from their primary sequence.Electron microscopy studies indicate that like the F-ATPases (35), the V-ATPases also contain multiple stalks connecting the peripheral and integral domains (36 -38). To determine the composition of the central and peripheral stalks...