Characterization of the functional coupling of bovine brain vacuolar-type H+-translocating ATPase

Vacuolar-type H+-translocating ATPases (V-ATPases or V-pumps) are complex proteins containing multiple subunits and are organized into two functional domains: a peripheral catalytic sector V-1 and a membranous proton channel V-0. The functional coupling of ATP hydrolysis activity to proton transport in V-pumps requires a regulatory component known as subunit H (SFD) as has been shown both in vivo and in vitro ( Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., and Anraku, Y. ( 1993) J. Biol. Chem. 268, 18286 - 18292; Xie, X. S., Crider, B. P., Ma, Y. M., and Stone, D. K. ( 1994) J. Biol. Chem. 269, 25809 - 25815). Ca2+ is thought to uncouple V-pumps because it is found to support ATP hydrolysis but not proton transport, while Mg2+ supports both activities. The direct effect of phospholipids on the coupling of V-ATPases has not been reported, likely due to the fact that phospholipids are constituents of biological membranes. We now report that Ca2+-induced uncoupling of the bovine brain V-ATPase can be reversed by imposition of a favorable membrane potential. Furthermore we report a simple "membrane-free" assay system using the V0 proton channel-specific inhibitor bafilomycin as a probe to detect the coupling of V-ATPase under certain conditions. With this system, we have characterized the functional effect of subunit H, divalent cations, and phospholipids on bovine brain V-ATPase and have found that each of these three factors plays a critical role in the functional coupling of the V-pump.