Mitochondrial ATP synthase complex. Membrane topography and stoichiometry of the F0 subunits.
The topography of the subunits of the membrane sector F0 of the ATP synthase complex in the bovine mitochondrial inner membrane was studied with the help of subunit-specific antibodies raised to the F0 subunits b, d, 6, F6, A6L, OSCP (oligomycin-sensitivity-conferring protein), and N,N' -dicyclohexylcarbodiimide (DCCD)-binding proteolipid and to the ATPase inhibitor protein (IF1) as an internal control. Exposure of F0 subunits in inverted and right-side-out inner membranes was investigated by direct antibody binding as well as by susceptibility of these subunits to degradation by various proteases as monitored by gel electrophoresis of the membrane digests and immunoblotting with the subunit-specific antibodies. Results show that subunits b, d, F6, A6L (including its C-terminal end) and OSCP were exposed on the matrix side. Sufficient masses of these subunits to recognize antibodies or undergo proteolysis were not exposed on the cytosolic side. This was also the case for subunit 6 and the DCCD-binding proteolipid on either side of the inner membrane. Quantitative immunoblotting in which bound radio-activity from [125I]protein A was employed to estimate the concentration of an antigen in a sample allowed the determination of the stoichiometry of several F0 subunits and IF1 relative to F1-ATPase. Results showed that per mol of F1 there are in bovine heart mitochondria 1 mol each of d, OSCP, and IF1, and 2 mol each of b and F6. Subunit 6 and the DCCD-binding proteolipid could not be quantitated, because the former transferred poorly to nitrocellulose and the latter's antibody did not bind [125I]protein A.
Hekman
C
C
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Tomich
J M
JM
Hatefi
Y
Y
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000494:pharmacology; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000648:ultrastructure; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000648:ultrastructure; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry; D011402:Pronase / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000648:ultrastructure*; D013820:Thermolysin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D002918:Chymotrypsin; D014357:Trypsin; D011402:Pronase; D013820:Thermolysin; D006180:Proton-Translocating ATPases