Fo membrane domain of ATP synthase from bovine heart mitochondria: purification, subunit composition, and reconstitution with F1-ATPase

Biochemistry. 1994 Jun 28;33(25):7971-8. doi: 10.1021/bi00191a026.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Amino Acid Sequence
  • Animals
  • Carrier Proteins*
  • Cattle
  • Intracellular Membranes / enzymology
  • Macromolecular Substances
  • Mass Spectrometry
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Mitochondria, Heart / enzymology*
  • Mitochondrial Proton-Translocating ATPases
  • Molecular Sequence Data
  • Proton-Translocating ATPases / chemistry*
  • Submitochondrial Particles / enzymology


  • Carrier Proteins
  • Macromolecular Substances
  • Membrane Proteins
  • Adenosine Triphosphatases
  • Mitochondrial Proton-Translocating ATPases
  • Proton-Translocating ATPases
  • oligomycin sensitivity-conferring protein

Associated data

  • GENBANK/S70447
  • GENBANK/S70448