Specifically, the assembly of the cytochrome b-c1 complex in the mitochondrial membrane will be investigated in mutant strains of yeast lacking cytochrome b, the only mitochondrial translation product found in this complex. Recently, this lab reported that the mutants lacking cytochrome b contain significantly diminished (50-75%) amounts of the two core proteins as well as the iron-sulfur protein and essentially none of subunit VII (the Q binding protein). By contrast, cytochrome c1 was both spectrally and immunologically detectable in all the mutants studied in amounts equal to that in the wild type. Subsequent studies of the synthesis and import of these proteins into mitochondria from one of the mutants suggested that cytochrome b is necessary for the proper processing and assembly of core protein I, the iron- sulfur protein and the Q-binding protein, but not for cytochrome c1, into complex III of the inner mitochondrial membrane. The role of cytochrome b in the processing and assembly of the iron-sulfur protein (ISP) will be investigated using an in vitro transcription/translation system once the gene for the ISP has been cloned in a suitable plasmid. The import of the ISP into both wild-type and cytochrome b-deficient mitochondria will be compared plus the intramitochondrial sites for cleavage of the precursor form of the ISP. Subsequently, the presequence of the ISP will be modified to establish which amino acids are necessary for binding and import of the ISP. The results obtained will establish a role for cytochrome b in the overall assembly of the cytochrome b-c1 complex. %%% The long term goal of this project is elucidate the factors controlling membrane formation in yeast. This organism is a convenient experimental system because of its ease in manipulation in the laboratory and its well studied genetics. The membrane being studied is the inner mitochondrial membrane. The mitochondrion is the energy factory for the cell. The biosynthesis of the inner membrane affords us an opportunity to understand biological function based on the structural information being generated and to understand how two genetic systems, one in the mitochondrion and the other in nucleus, interact to direct the synthesis of large biopolymers.
