Abstract
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.
Highlights
IntroductionInhibition by intact H in the presence of H-NT was somewhat surprising, but one explanation might be displacement of H-NT from V1(-H)/H-NT complexes by the intact subunit
None H-NT co-isolates with V1 None H binds tightly through repurification Very little detected after repurification More H-CT retained after repurification H subunit partially displaces H-NT ND
Complexes, the % activity in the V1(-H)/H-NT complexes as isolated (26%) was multiplied by the proportion of the activity remaining after addition of expressed H or H-CT
Summary
Inhibition by intact H in the presence of H-NT was somewhat surprising, but one explanation might be displacement of H-NT from V1(-H)/H-NT complexes by the intact subunit We tested this by incubating V1(-H)/H-NT complexes with both intact H and H-CT, as for the inhibition assays, and re-isolating V1 by affinity chromatography via the FLAG-tagged G subunits. Whereas comparable levels of H-NT are present after the re-isolation whether or not H-CT is present, incubation with intact H partially displaced H-NT, so that less Myc-tagged H-NT is present after re-isolation of V1 complexes. This suggests that H-NT binds to V1 less tightly than the intact H subunit and can be displaced
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