The 40-kDa subunit enhances but is not required for activity of the coated vesicle proton pump.
We have previously demonstrated reassembly of a functional vacuolar (H+)-ATPase from clathrin-coated vesicles using the dissociated peripheral domain (V1) and the membrane-bound integral domain (V0) (Puopolo, K., and Forgac, M. (1990) J. Biol. Chem. 265, 14836-14841). We have used this reassembly procedure to test the function of the 40-kDa subunit of the coated vesicle (H+)-ATPase. In the absence of V0, a fraction of the peripheral subunits reassemble into a V1 subcomplex which contains the 73-kDa A subunit, the 58-kDa B subunit, and the 34- and 33-kDa subunits but lacks the 40-kDa subunit. This subcomplex, which sediments with a mass of approximately 500 kDa, can be separated from the remaining monomeric subunits (and the 40-kDa subunit) by density gradient sedimentation. When dissociated with 0.36 M KI, 2.5 mM ATP, and 2.5 mM MgSO4, and added to membranes from which V1 has been dissociated, this V1(-40 kDa) subcomplex is able to reassemble with V0 to give a (H+)-ATPase with a proton pumping activity approximately half that obtained in the presence of the 40-kDa subunit. The undissociated subcomplex is not competent for assembly of a functional (H+)-ATPase. Interestingly, the monomeric fraction obtained from density gradient sedimentation contains the 40-kDa subunit but lacks the 34-kDa subunit. This monomeric fraction is nevertheless also able to assemble with V0 to give a functional proton pump. The V1V0 complexes assembled in the absence of either the 40- or 34-kDa subunits, while active, are not stable to detergent solubilization and immunoprecipitation, suggesting that both of these subunits play a role in stabilization of the (H+)-ATPase complex. Evidence for interaction between the 40- and 33-kDa subunits is also presented.
Puopolo
K
K
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Sczekan
M
M
Magner
R
R
Forgac
M
M
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D002966:Clathrin / Q000302:isolation & purification / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*
D002966:Clathrin; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases