Subunit interactions in the clathrin-coated vesicle vacuolar (H+)-ATPase complex

Abstract

The vacuolar (H+)-ATPases (or V-ATPases) are structurally related to the F1F0 ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V1) and an integral (V0) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents. Cross-linked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits. In the intact V1V0 complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V1 domain, cross-linking of subunits H and E was also observed. Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3- (dimethylaminopropyl)carbodiimide, while subunits a and E could be cross- linked by 4-(N-maleimido)benzophenone. It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V0 domain. In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H. Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis. Based on these and previous observations, a new structural model of the V-ATPase from clathrin-coated vesicles is proposed.