title: ATP synthase from bovine heart mitochondria. In vitro assembly of a stalk complex in the presence of F1-ATPase and in its absence.
abstract: Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli. They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d. Likewise, fragments b', bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment d' (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression. These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase. Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase. When fragments b', bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1.OSCP.b', F1.OSCP.bI or F1.OSCP.bC were formed, but b', bI and bC appeared to be present in sub-stoichiometric amounts. When F6 was added also, then the stoichiometric quaternary complexes F1.OSCP.b'.F6 and F1.OSCP.bI.F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6. Finally, three pentameric complexes F1.OSCP.b'.F6.d, F1.OSCP.b'.F6.d' and F1.OSCP.b.F6.d were isolated. In a further series of reconstitution experiments, the binary complexes b'.OSCP and b'.d, the ternary complex b'.d'.F6, and the quaternary complex OSCP.b'.F6.d were obtained. The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase. It was shown by S-carboxymethylation of cysteine residues with iodo-[2-14C]acetic acid that bovine F1F0-ATPase and the reconstituted F1.stalk complex, F1.OSCP.b'.d.F6, each contained one copy per complex of subunits b (or b'), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1:1:1. The ratio of b to d in purified F0 was 1:1. Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold. These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.
authors.forename: I R
authors.initials: IR
authors.lastname: Collinson
authors.forename: M J
authors.initials: MJ
authors.lastname: van Raaij
authors.forename: M J
authors.initials: MJ
authors.lastname: Runswick
authors.forename: I M
authors.initials: IM
authors.lastname: Fearnley
authors.forename: J M
authors.initials: JM
authors.lastname: Skehel
authors.forename: G L
authors.initials: GL
authors.lastname: Orriss
authors.forename: B
authors.initials: B
authors.lastname: Miroux
authors.forename: J E
authors.initials: JE
authors.lastname: Walker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D006180:Proton-Translocating ATPases