Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase Export

@article{citeulike:4496586, 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. 10.1074/jbc.M900475200}, author = {Diab, Heba and Ohira, Masashi and Liu, Mali and Cobb, Ester and Kane, Patricia M.}, citeulike-article-id = {4496586}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M900475200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/284/20/13316?rss=1}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/19299516}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=19299516}, doi = {10.1074/jbc.M900475200}, journal = {J. Biol. Chem.}, keywords = {atpase, diab, disassembly, inhibition, proton, vacuolar}, month = {May}, number = {20}, pages = {13316--13325}, posted-at = {2009-05-08 23:26:32}, priority = {2}, title = {Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase}, url = {http://dx.doi.org/10.1074/jbc.M900475200}, volume = {284}, year = {2009} }

TY - JOUR ID - citeulike:4496586 L3 - citeulike-article-id:4496586 N2 - 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. 10.1074/jbc.M900475200 IS - 20 JF - J. Biol. Chem. EP - 13325 TI - Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase VL - 284 SP - 13316 KW - atpase KW - diab KW - disassembly KW - inhibition KW - proton KW - vacuolar AU - Diab, Heba AU - Ohira, Masashi AU - Liu, Mali AU - Cobb, Ester AU - Kane, Patricia PY - 2009/05/15/ UR - http://dx.doi.org/10.1074/jbc.M900475200 ER -