Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria.
Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH2-cytochrome c reductase activity with a turnover number of 15.7 sec(-1) and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c1 per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (35S)-methionine in the absence or presence of inhibitors of cytoplasmic or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using anti-serum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (35S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.
D003573:Cytochrome b Group; D003574:Cytochrome c Group / Q000096:biosynthesis / Q000378:metabolism; D003580:Cytochromes / Q000378:metabolism; D003575:Cytochromes c1 / Q000378:metabolism; D014450:Electron Transport Complex III; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis*; D011808:Quinone Reductases / Q000096:biosynthesis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013104:Spheroplasts / Q000201:enzymology; D013367:Submitochondrial Particles / Q000201:enzymology; D013385:Succinate Dehydrogenase / Q000378:metabolism
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D003580:Cytochromes; D009097:Multienzyme Complexes; D003575:Cytochromes c1; D013385:Succinate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III