MEMBRANE TOPOGRAPHY AND NEAR-NEIGHBOR RELATIONSHIPS OF THE MITOCHONDRIAL ATP SYNTHASE SUBUNIT-E, SUBUNIT-F, AND SUBUNIT-G

The well characterized subunits of the bovine ATP synthase complex are the alpha, Rho, gamma, delta, and epsilon subunits of the catalytic s ector, F-1; the ATPase inhibitor protein; and subunits a, b, c, and d, OSCP (oligomycin Sensitivity-conferring protein), F-6, and A6L, which are present in the membrane sector, F-0, and the 45-Angstrom-long sta lk that connects F-1 to F-0. It has been shown recently that bovine AT P synthase preparations also contain three small polypeptides, designa ted e, f, and g, with respective molecular masses of 8.2, 10.2, and 11 .3 kDa. To ascertain their involvement as bonafide subunits of the ATP synthase and to investigate their membrane topography and proximity t o the above ATP synthase subunits, polyclonal antipeptide antibodies w ere raised in the rabbit to the COOH-terminal amino acid residues 57-7 0 of e, 75-86 off, and 91-102 of g. It was shown that (i) e, f, and g could be immunoprecipitated with anti-OSCP IgG from a fraction of bovi ne submitochondrial particles enriched in oligomycin-sensitive ATPase; (ii) the NH2 termini of f and g are exposed on the matrix side of the mitochondrial inner membrane and can be curtailed by proteolysis; (ii i) the COOH termini of all three polypeptides are exposed on the cytos olic side of the inner membrane; and (iv) f cross links to A6L and to g, and e crosslinks to g and appears to form an e-e dimer. Thus, the b ovine ATP synthase complex appears to have 16 unlike subunits, twice a s many as its counterpart in Escherichia coli.