Subunit interactions and requirements for inhibition of the yeast V1-ATPase.
Disassembly of the yeast V-ATPase into cytosolic V(1) and membrane V(0) sectors inactivates MgATPase activity of the V(1)-ATPase. This inactivation requires the V(1) 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 V(1) and V(0) subunits were identified by two-hybrid assay. The B subunit of the V(1) catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V(1) subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V(0) subunit Vph1p. V(1)-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 V(1) when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V(1) lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V(1) 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 V(1) complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V(1)-ATPase activity and precludes V(0) interactions.
Diab
Heba
H
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.
Ohira
Masashi
M
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.
Liu
Mali
M
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.
Cobb
Ester
E
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.
Kane
Patricia M
PM
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210. Electronic address: kanepm@upstate.edu.
D004789:Enzyme Activation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases