Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

TitleOrganization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.
Publication TypeJournal Article
Year of Publication2015
AuthorsLee, J, Ding, S, Walpole, TB, Holding, AN, Montgomery, MG, Fearnley, IM, Walker, JE
JournalJ Biol Chem
Date Published2015 May 22
KeywordsAmino Acid Sequence, Animals, Catalytic Domain, Cattle, Cell Membrane, Cross-Linking Reagents, Mitochondria, Mitochondrial Proton-Translocating ATPases, Models, Molecular, Molecular Sequence Data, Mutation, Peptide Fragments, Protein Conformation, Protein Structure, Secondary, Protein Subunits, Sequence Homology, Amino Acid, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.

Alternate JournalJ. Biol. Chem.
Citation Key10.1074/jbc.M115.645283
PubMed ID25851905
PubMed Central IDPMC4505582
Grant ListMC_U105663150 / / Medical Research Council / United Kingdom
U105663150 / / Medical Research Council / United Kingdom