Cytochrome b is necessary for the assembly of subunit VII in the cytochrome b-c1 complex of yeast mitochondria.
The synthesis and assembly of subunit VII, the Q-binding protein of the cytochrome b-c1 complex, into the inner mitochondrial membrane has been compared in wild-type yeast cells and in a mutant cell line lacking cytochrome b. Both immunoblotting and immunoprecipitation analysis with specific antiserum against subunit VII indicated that this subunit is not detectable in the mutant as compared to the wild-type mitochondria. However, labeling in vivo of the cytochrome b deficient yeast cells in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone clearly demonstrated that subunit VII was synthesized in the mutant cells to the same extent as in the wild-type cells. Incubation of subunit VII, synthesized in vitro in a reticulocyte lysate programmed with yeast RNA, with mitochondria isolated from both wild-type and cytochrome b deficient yeast cells revealed that the subunit VII was transported into the wild-type mitochondria into a compartment where it was resistant to digestion by exogenous proteinase K. By contrast, subunit VII was bound in lowered amounts to the cytochrome b deficient mitochondria where it remained sensitive to digestion by exogenous proteinase K, suggesting that the import of subunit VII may be impaired due to the lack of cytochrome b. Furthermore, subunit VII was synthesized both in vivo and in vitro with the same molecular mass as the mature form of this protein.
Japa
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Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506.
Beattie
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D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D002458:Cell Fractionation; D003573:Cytochrome b Group / Q000502:physiology*; D014450:Electron Transport Complex III / Q000096:biosynthesis* / Q000378:metabolism; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000378:metabolism; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D009154:Mutation; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism
D003573:Cytochrome b Group; D004792:Enzyme Precursors; D046911:Macromolecular Substances; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D014450:Electron Transport Complex III