Abstract
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.
Highlights
Bovine mitochondrial F-ATPase has six uncharacterized membrane subunits
The subunit is encoded in the mitochondrial DNA of many, but not all, eukaryotic species [35], and in mammals, the genes for ATP8 and ATPase-6 overlap [12, 36]
Subunit ATP8 in S. cerevisiae has a C-terminal extension that is predicted to be mostly ␣-helical. It is significantly shorter and poorly related to the same region of the mammalian proteins, the sequences of ATP8 proteins are well conserved among the fungi (Fig. 5C)
Summary
Bovine mitochondrial F-ATPase has six uncharacterized membrane subunits. Results: Labeled lysines have been mapped onto its incomplete structure. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ϳ70 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. In this study the identification of covalent cross-links introduced into the subunits of the bovine F-ATPase by reaction of exposed amino groups with isotopically labeled bifunctional reagents has been employed as a means of gaining information about the orientation and location of the supernumerary subunits within the bovine enzyme complex
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