The vacuolar-type proton-translocating ATPase of clathrin-coated vesicles is composed of an integral membrane proton channel (V(B)) and a peripheral catalytic sector (V(C)). Native enzyme can catalyze the hydrolysis of both MgATP and CaATP and support proton pumping when reconstituted into liposomes. In contrast, isolated V(C) catalyzes only Ca2+-activated ATP hydrolysis and cannot support proton pumping when reconstituted into liposomes (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We now report that solubilized isolated V(C) can be reassembled with purified V(B) to restore properties of native enzyme, including Mg2+-activated ATP hydrolysis and proton-pumping capability. Investigation of this reassembly revealed that a heterodimer, composed of polypeptides of 50 and 57 kDa, stimulates Ca2+- activated ATPase activity of isolated V(C) 2-fold and Mg2+-activated ATPase activity catalyzed by the reassembled pump 9-fold. Moreover, this heterodimer stimulated proton transport by the reassembled pump >20-fold. When separated from the proton pump, the dimer has no detectable kinase activity. Maximal stimulation occurs at a molar ratio of heterodimer to reassembled pump of 3, implying a structural, nonenzymatic mechanism. These data indicate that the 50-kDa and/or the 57-kDa polypeptide likely plays an essential and potentially regulatory role in the function of the proton-translocating ATPase of clathrin-coated vesicles.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - Oct 14 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology