Functional reassembly of the coated vesicle proton pump.
We have shown previously that treatment of the coated vesicle proton-translocating adenosine triphosphatase (H(+)-ATPase) with chaotropic agents results in the release of a set of peripheral polypeptides which includes the 73-, 58-, 40-, 34-, and 33-kDa subunits (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973), with a coordinate loss of H(+)-ATPase activity. In the present paper we report the functional reassembly of the coated vesicle proton pump following dissociation of the peripheral subunits. Reassembly was demonstrated by restoration of ATP-driven proton transport using both native membranes and reconstituted vesicles and by Western blot analysis using a monoclonal antibody specific for the 73-kDa subunit. Reassembly occurs by attachment of a peripheral subcomplex containing the 73-, 58-, 34-, and 33-kDa subunits together with the 40-kDa polypeptide. The reassembled H(+)-ATPase, like the native proton pump, is inhibited by N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and N,N'-dicyclohexylcarbodiimide. Reassociation shows a biphasic time dependence, with restoration of 50-60% of the starting proton transport activity in the 1st h followed by recovery of a further 20-30% of the activity after 24 h. Reassembly also shows a marked dependence on protein concentration but, unlike solubilization of the intact H(+)-ATPase complex, does not require the presence of glycerol. Despite the ability of nucleotides to promote dissociation of the peripheral complex by chaotropic agents, reassociation is not blocked by the presence of 1 mM ATP. These results thus provide the first evidence for functional reassembly of a vacuolar H(+)-ATPase complex and should be useful in further analysis of the role of individual subunits in the assembly and activity of these ATP-driven proton pumps.
Puopolo
K
K
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Forgac
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology*; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D005990:Glycerol