Subunit interactions and requirements for inhibition of the yeast V1-ATPase

Abstract

Disassembly of the yeast V-ATPase into cytosolic V1 and membrane V0 sectors inactivates MgATPase activity of the V1-ATPase. This inactivation requires the V1 H subunit (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767), but its mechanism is not fully understood. The H subunit has two domains. Interactions of each domain with V1 and V0 subunits were identified by two-hybrid assay. The B subunit of the V1 catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V1 subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V0 subunit Vph1p. V1-ATPase complexes from yeast expressing H-NT are partially inhibited, exhibiting 26% the MgATPase activity of complexes with no H subunit. The H-CT domain does not copurify with V1 when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V1 lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V1 was more stable than binding of either H-NT or H-CT, suggesting that both domains contribute to binding and inhibition. Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but this fragment of Vph1p does not bind to V1 complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V1-ATPase activity and precludes V0 interactions. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.