Immunochemical study of subunit VI ( Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase

Abstract

A preparation containing the M r 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol—cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl- N-methylglucamide—sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol—cytochrome c reductase resolved in the sodium dodecyl sulfate—polyacrylamide gel electrophoresis system of Schägger et al. (1987, FEBS Lett. 21, 161–168) and has the same amino acid sequence as that of the M r 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337–343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the M r 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol—cytochrome c reductase. Antibodies against M r 13,400 react only with M r 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol—cytochrome c reductase. However, when delipidated ubiquinol—cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the M r 13,400 protein are buried in the phospholipid environment. Antibodies against M r 13,400 cause an increase of apparent K m for ubiquinol-2 in ubiquinol—cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab′ fragment of anti- M r 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the M r 13,400 protein is extruded toward the matrix side of the membrane.