CHARACTERIZATION OF CBP4, A NEW GENE ESSENTIAL FOR THE EXPRESSION OF UBIQUINOL-CYTOCHROME-C REDUCTASE IN SACCHAROMYCES-CEREVISIAE

Nuclear respiratory deficient mutants of Saccharomyces cerevisiae have been screened for lesions in genes affecting ubiquinol-cytochrome c r eductase activity. In the present study we describe a new gene, CBP4, whose encoded product is absolutely essential for the activity and exp ression of the respiratory enzyme. We have cloned and sequenced CBP4, which encodes a 20-kDa protein having no obvious homology to any known protein. cbp4 mutants are unable to respire due to specific loss of u biquinol-cytochrome c reductase activity. cbp4 mutants demonstrate a p leiotropic reduction in the steady state levels of four subunits of ub iquinol-cytochrome c reductase, namely core 4, core 5, the Rieske prot ein and cytochrome b. Cytochrome b is not spectrally visible in the mu tants, although transcription and translation of the apoprotein is nor mal. Antiserum prepared against a trpE/CBP4 gene fusion react with an 18.4-kDa mitochondrial protein in wild type yeast, but the protein is not found in a mutant containing a deletion of 86% of the CBP4 coding region. CBP4 protein is tightly associated with the mitochondrial memb rane as evidenced by the association of the protein with the membrane fraction following carbonate extraction. The phenotype of cbp4 mutants is similar to that of cbp3 mutants. CBP3 encodes a second protein ess ential for ubiquinol-cytochrome c reductase activity. We found that lo ss of CBP3 protein does not affect the membrane stability or steady st ate concentration of CBP4 protein. A double mutant containing a deleti on in both CBP3 and CBP4 was constructed. Our study shows that the phe notype of the double mutant is identical to the phenotypes of the indi vidual single mutants.