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Abstract
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 V1 and a membranous proton channel V0. 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.
Highlights
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 V1 and a membranous proton channel V0
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
It remains to be determined how subunit H regulates the functional coupling of V-ATPases, and it is unclear what role certain non-protein factors may play in this process, such as divalent cations and phospholipids
Summary
V-ATPase (V-pump), the vacuolar-type Hϩ-translocating ATPase; V1, the peripheral, catalytic sector of Vpumps; V0, the membranous proton channel domain of V-pumps; FATPase, F1F0-ATP synthase; 1799, bis(hexafluoroacetonyl)acetone; PS, phosphatidylserine; MES, 2-(N-morpholino)ethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; 5-CF, 5-carboxyfluorescein; PL, phospholipid. Subunit H is a key regulatory element of V-ATPase that is not required for the assembly of V1V0 but is absolutely essential for the function of V-type proton pumps as has been shown both in vivo [32] and in vitro [33, 34] It remains to be determined how subunit H regulates the functional coupling of V-ATPases, and it is unclear what role certain non-protein factors may play in this process, such as divalent cations and phospholipids. We report a simple assay system using the V0 proton channel-specific inhibitor bafilomycin A1 as an indicator of functional coupling of ATP hydrolysis to proton translocation With this assay system, a direct role of phospholipids in the functional coupling of V-ATPase is demonstrated, and the effects of subunit H and divalent cations are further characterized
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