Fo membrane domain of ATP synthase from bovine heart mitochondria: purification, subunit composition, and reconstitution with F1-ATPase.
The Fo membrane domain of the F1Fo-ATP synthase complex has been purified from bovine heart mitochondria. The purification procedure involves the removal of peripheral membrane proteins, including F1-ATPase, from submitochondrial particles with guanidine hydrochloride, followed by extraction of Fo and other membrane proteins from the stripped membranes in the presence of the detergent n-dodecyl beta-D-maltoside. Fo was then purified by ion-exchange and dye ligand chromatography in the presence of the same detergent. Approximately 15 mg of pure Fo was recovered from 1.8 g of mitochondrial membrane protein. The purified Fo is a complex of nine different polypeptides. They are subunits a, b, c, d, e, F6, and A6L characterized before in F1Fo-ATPase preparations, and two new hitherto undetected subunits, named f and g. The sequences of subunits f and g have been determined. They are not related significantly to any known protein, but subunit f appears to contain a membrane-spanning alpha-helix. Proteins f and g are also present in approximately stoichiometric amounts in a highly purified preparation of intact F1Fo-ATPase, and so it is concluded that they are authentic subunits of the bovine enzyme with unknown functions. Dibutyltin 3-hydroxyflavone, an inhibitor of F1Fo-ATPase, also binds to the purified Fo in detergent and competes for binding with venturicidin. In the presence of F1 and OSCP, the purified Fo was reassembled into the intact F1Fo-ATPase complex. Therefore, this procedure provides a relatively abundant source of pure and functional Fo that is suitable for structural analysis.
MRC Laboratory of Molecular Biology, Cambridge, U.K.
I R
IR
Collinson
M J
MJ
Runswick
S K
SK
Buchanan
I M
IM
Fearnley
J M
JM
Skehel
M J
MJ
van Raaij
D E
DE
Griffiths
J E
JE
Walker
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D002417:Cattle; D007425:Intracellular Membranes / Q000201:enzymology; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D013367:Submitochondrial Particles / Q000201:enzymology
D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C020439:oligomycin sensitivity-conferring protein