Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.
Bottom Line: 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.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.
Affiliation: From the The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, United Kingdom and.Show MeSH
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Mentions: Since its discovery as a subunit of the bovine and yeast F-ATPases (12, 33, 34), subunit ATP8 (also known as A6L in mammals and Aap1 in Saccharomyces cerevisiae) has remained a rather mysterious component of the enzyme complex. It is not found in eubacterial and chloroplast enzymes, and therefore, it was classified as a supernumerary subunit, apparently not required for the core ATP synthetic and hydrolytic functions of F-ATPases. 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 (or subunit a) overlap (12, 36). The bovine protein is probably folded into a single transmembrane α-helix from residues 8–29 followed by a hydrophilic extension up to its C terminus at residue 66 (Fig. 5A). This region from residue 30 to 66 is predicted to have an extended conformation, except for residues 57–61, which may form a short β-sheet. The region consisting of residues 51–63 is well conserved in mammals (Fig. 5B). In contrast, subunit ATP8 in S. cerevisiae has a C-terminal extension that is predicted to be mostly α-helical. It is significantly shorter and poorly related to the same region of the mammalian proteins, although the sequences of ATP8 proteins are well conserved among the fungi (Fig. 5C). On the basis of cysteine scanning mutagenesis and reaction with fluorescein-5-maleimide, residues 1–14 of yeast ATP8 have been proposed to be exposed to the intermembrane space followed by a transmembrane α-helix from residues 15–35 (37). A short β-strand is predicted around residue Arg-42, and this residue is conserved throughout fungi (Fig. 5C). Despite these differences, proteolytic digestion studies conducted on bovine mitochondrial membranes (38, 39) and cross-linking studies on the yeast enzyme (40) have shown that the bovine and yeast proteins have a common topography with their N-terminal regions in the intermembrane space and their C-terminal regions on the matrix side of the inner mitochondrial membrane.
Affiliation: From the The Medical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, United Kingdom and.