Membrane Topography and Near-neighbor Relationships of the Mitochondrial ATP Synthase Subunits e, f, and g

Abstract

The well characterized subunits of the bovine ATP synthase complex are the ␣, ␤, ␥, ␦, and ⑀ subunits of the catalytic sector, F 1 ; the ATPase inhibitor protein; and subunits a, b, c, and d, OSCP (oligomycin sensitivityconferring protein), F 6 , and A6L, which are present in the membrane sector, F 0 , and the 45-Å-long stalk that connects F 1 to F 0 . It has been shown recently that bovine ATP synthase preparations also contain three small polypeptides, designated e, f, and g, with respective molecular masses of 8.2, 10.2, and 11.3 kDa. To ascertain their involvement as bona fide subunits of the ATP synthase and to investigate their membrane topography and proximity to the above ATP synthase subunits, polyclonal antipeptide antibodies were raised in the rabbit to the COOH-terminal amino acid residues 57-70 of e, 75-86 of f, 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 bovine submitochondrial particles enriched in oligomycin-sensitive ATPase; (ii) the NH 2 termini of f and g are exposed on the matrix side of the mitochondrial inner membrane and can be curtailed by proteolysis; (iii) the COOH termini of all three polypeptides are exposed on the cytosolic 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 bovine ATP synthase complex appears to have 16 unlike subunits, twice as many as its counterpart in Escherichia coli.

Su e of the Yeast F1Fo-ATP Synthase Forms Homodimers

“…Furthermore, formation of Su e-Su e homodimers, between two neighboring F o complexes was hypothesized to play an important role in facilitating dimerization of the F 1 F o -ATP synthase complex (4). In support of this model, it was previously shown that bovine Su e could be chemically cross-linked to a protein of a similar size to Su e, thus providing preliminary support for Su e-Su e dimers (10). Direct demonstration of homodimers of Sue, however, has been lacking to date.…”

The Subunit F of Mitochondrial Yeast ATP Synthase

“…Oligomycin Activity remaining terminal (Belogrudov et al, 1996) which is predicted to form a transmembrane a-helix by computing methods (not shown). Sodium bromide treatment of the F,F,ATP synthase removed most of the F, subunits (Fig.…”

“…Additional small polypeptides (subunits e, f and g) have been identified in the bovine enzyme (Walker et al, 1991;Collinson et al, 1994). Investigation of their membrane topography and their proximity with other known subunits of the complex showed the last three polypeptides as being bonafide subunits of the bovine ATP synthase (Belogrudov et al, 1996).…”

The proximal N-terminal amino acid residues are required for the coupling activity of the bovine heart mitochondrial factor B

“…Cross-links between two copies of e and cross-links of g with both e and f were also reported [37]. Analysis of animal subunit g revealed the presence of a completely conserved acidic residue (Glu82) in the middle of its single transmembrane helix [38].…”

“…Previously, we demonstrated that the N-termini of Fo subunits e and g face the matrix side while their respective C-termini face the cytosolic side of the mitochondrial inner membrane [37]. Cross-links between two copies of e and cross-links of g with both e and f were also reported [37].…”