Three-dimensional structure and subunit topology of the V₁ ATPase from Manduca sexta midgut

The three-dimensional structure of the Manduca sexta midgut V₁ ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V₁ complex has a barrel-like structure 11 nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V₁ complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl₂-induced disulfide formation. Treatment of V₁ with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V₁ decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl₂ was added to V₁ in the presence of CaADP, the cross-linked products A-E-F and B-H were generated. In experiments where CuCl₂ was added after preincubation of CaATP, the cross-linked products E-F and E-G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.