Stoichiometry of subunit e in rat liver mitochondrial H+-ATP synthase and membrane topology of its putative Ca2+-dependent regulatory region

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H+-ATP synthase are homologous with the Ca2+-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca2+-dependent regulation of H+-ATP synthase activity. In this study, we determined the content of subunit e in H+-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca2+-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H+-ATP synthase revealed that 1 mol of H+-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F-0 is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H+-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca2+-dependent regulatory region of subunit e is exposed on the surface of the C-side of F-0 and that subunit e is involved in the regulation of mitochondrial H+-ATP synthase activity probably via its putative Ca2+-dependent regulatory region. (C) 2001 Elsevier Science B.V. All rights reserved.