Cytochrome b is necessary for the effective processing of core protein I and the iron-sulfur protein of complex III in the mitochondria.

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in a mutant of yeast (W-267, Box 6-2) that lacks a spectrally detectable cytochrome b and synthesizes a shortened form of apocytochrome b. We recently reported that several cytochrome b-deficient mutants contained significantly diminished amounts of core proteins I and II as well as the iron-sulfur protein, but contained equal amounts of cytochrome c1 compared to the wild type (K. Sen and D. S. Beattie, Arch. Biochem. Biophys. 242, 393-401, 1985). In the present study, the time course of processing of precursors of both core protein I and the iron-sulfur protein which had accumulated in cells treated with the uncoupler carbonyl m-chlorophenyl hydrazone (CCCP) was noted to be significantly lower in the mutant compared to the wild type. The amounts of the mature forms of these proteins in mitochondria pulse labeled under different conditions was also considerably decreased at all times studied. The synthesis of both proteins appeared to be unaffected in the mutant, as the precursor forms of both proteins accumulated to the same extent when processing in vivo was blocked by CCCP. Furthermore, translation of RNA in a reticulocyte lysate in vitro indicated that the messenger RNAs for both proteins were present in the mutant and translated with equal efficiency. The import into isolated mitochondria of the precursor forms of the iron-sulfur protein synthesized in the cell-free system was also decreased in the mutant mitochondria. In addition, the precursor form was bound to the exterior of the mitochondrial membrane where it was sensitive to digestion with proteases. By contrast, the synthesis and processing of cytochrome c1 appeared to be unaffected in these mutants. These results suggest that cytochrome b is necessary for the proper processing and assembly of both core protein I and the iron-sulfur protein, but not for cytochrome c1, into complex III of the inner mitochondrial membrane.