V-ATPases are composed of a peripheral complex containing the ATP-binding sites, the V 1 sector, attached to a membrane complex containing the proton pore, the V o sector. In vivo, free, inactive V 1 and V o sectors exist in dynamic equilibrium with fully assembled, active V 1 V o complexes, and this equilibrium can be perturbed by changes in carbon source. Free V 1 complexes were isolated from the cytosol of wild-type yeast cells and mutant strains lacking V o subunit c (Vma3p) or V 1 subunit H (Vma13p). V 1 complexes from wild-type or vma3⌬ mutant cells were very similar, and contained all previously identified yeast V 1 subunits except subunit C (Vma5p). These V 1 complexes hydrolyzed CaATP but not MgATP, and CaATP hydrolysis rapidly decelerated with time. V 1 complexes from vma13⌬ cells contained all V 1 subunits except C and H, and had markedly different catalytic properties. The initial rate of CaATP hydrolysis was maintained for much longer. The complexes also hydrolyzed MgATP, but showed a rapid deceleration in hydrolysis. These results indicate that the H subunit plays an important role in silencing unproductive ATP hydrolysis by cytosolic V 1 complexes, but suggest that other mechanisms, such as product inhibition, may also play a role in silencing in vivo. V-ATPases1 are highly conserved proton pumps distributed throughout the vacuolar network in all eukaryotic cells. VATPases maintain organelle acidification and affect cytosolic pH and ion balance, and their activity has been linked to a diverse array of cellular processes ranging from zymogen activation to protein sorting to viral membrane fusion events (1, 2). V-ATPases are comprised of two structural domains, the V 1 domain, which consists of a complex of peripheral subunits containing the nucleotide-binding sites attached to the cytoplasmic face of membrane, and the V o domain, which is comprised of several integral membrane and tightly associated peripheral proteins that contain the proton pore (1, 2). The yeast V-ATPase has at least eight V 1 subunits (designated A, B, C, D, E, F, G, and H) and five V o subunits (designated a, c, cЈ, cЉ, and d) (1,3,4 (10,13,14). Starvation appears to stimulate disassembly of V 1 from V o , but this disassembly is fully reversible upon refeeding (13-15). This reversible association between V 1 and V o is believed to regulate V-ATPase function in vivo: disassembly of V-ATPase complexes may conserve ATP when energy reserves are low and reassembly of the enzyme may provide the renewed proton pumping capacity necessary to prevent cytosolic acidification when active metabolism resumes (16,17).A constitutively active free V 1 in the cytosol could quickly become lethal to the cell by hydrolyzing cytosolic reserves of ATP. Graf et al. (14) have isolated cytosolic V 1 complexes from M. sexta and shown that these complexes exhibit Ca 2ϩ -dependent ATP hydrolysis at nonphysiological Ca 2ϩ concentrations but hydrolyze MgATP only in the presence of methanol. The properties of V 1 complexes have also been examined by recons...