FORMATION OF THE YEAST MITOCHONDRIAL-MEMBRANE .8. ELECTROPHORETIC BEHAVIOR OF CYTOCHROME-B IN A PARTIALLY PURIFIED PREPARATION AND EVIDENCE FOR HIGH MOLECULAR-WEIGHT ASSOCIATED MITOCHONDRIAL TRANSLATION PRODUCTS

A partially purified preparation of cytochrome b lacking cytochromes c, c1 and a-a3 was isolated from yeast submitochondrial particles. The preparation contained 7 nmol of heme b/mg of protein and upon dodecyl sulfate gel electrophoresis separated into four major bands with apparent molecular weights of 31000, 35 000, 48 000, and 50 000. The 31 000-dalton band, identified as cytochrome b by comparison with total mitochondrial translation products, showed normal migration behavior during dodecyl sulfate electrophoresis in different concentrations of acrylamide. Furthermore, this polypeptide migrated at a molecular weight of 31 000 when the preparation was heated in dissociation medium at 20, 37, 70, or 100 °C, when phenylmethanesulfonyl fluoride, the protease inhibitor, was present or absent, and in dodecyl sulfate-urea gels. By contrast, cytochrome b in the intact mitochondrial membrane displayed anomalous migration behavior in gels of different acrylamide concentrations. Two proteins of higher molecular weight are present in the immunoprecipitates of labeled mitochondria treated with the specific antiserum against cytochrome b. These polypeptides are products of mitochondrial protein synthesis as they are labeled in the presence of cycloheximide, not labeled in the presence of chloramphenicol, and absent in petite mutants. These mitochondrial translation products do not copurify with cytochrome b as they are not present in the partially purified cytochrome b preparation obtained from yeast cells labeled in the presence of cycloheximide. These proteins do not appear to be precursors of cytochrome b as the addition of a short or long chase of unlabeled amino acid did not alter the labeling of these high molecular weight proteins relative to cytochrome b. Furthermore, varying the time of pulse label in the presence of cycloheximide from 3 to 30 min also did not indicate any precursor-product relationship between these high molecular weight proteins and cytochrome b. © 1979, American Chemical Society. All rights reserved.