Three-dimensional structure of the vacuolar ATPase: Localization of subunit h by difference imaging and chemical cross-linking

The structure of the proton-pumping vacuolar ATPase (V-ATPase) from bovine brain clathrin coated vesicles was analyzed by electron microscopy and single molecule image analysis. A three-dimensional structural model of the complex was calculated by the angular reconstitution method at a resolution of 27 A°. Overall, the appearance of the V₀ and V₁ domains in the three-dimensional model of the intact bovine V-ATPase resembles the models of the isolated bovine V₀ and yeast V₁ domains determined previously (Wilkens, S., and Forgac, M. (2001) J. Biol. Chem. 276, 44064-44068; Zhang, Z., Charsky, C., Kane, P.M., and Wilkens, S. (2003) J. Biol. Chem. 278, 47299-47306). To determine the binding position of subunit H in the V-ATPase, electron microscopy and cysteine-mediated photochemical cross-linking were used. Difference maps calculated from projection images of intact bovine V-ATPase and a V-ATPase preparation in which the two H subunit isoforms were removed by treatment with cystine revealed less protein density at the bottom of the V ₁ in the subunit H-depleted enzyme, suggesting that subunit H isoforms bind at the interface of the V₁ and V₀ domains. A comparison of three-dimensional models calculated for intact and subunit H-depleted enzyme indicated that at least one of the subunit H isoforms, although poorly resolved in the three-dimensional electron density, binds near the putative N-terminal domain of the a subunit of the V₀. For photochemical cross-linking, unique cysteine residues were introduced into the yeast V-ATPase B subunit at sites that were localized based on molecular modeling using the crystal structure of the mitochondrial F₁ domain. Cross-linking was performed using the photoactivatable sulfhydryl reagent 4-(N-maleimido)benzophenone. Cross-linking to subunit H was observed from two sites on subunit B (E494 and T501) predicted to be located on the outer surface of the subunit closest to the membrane. Results from both electron microscopy and cross-linking analysis thus place subunit H near the interface of the V ₁ and V₀ domains and suggest a close structural similarity between the V-ATPases of yeast and mammals.