Identification and Characterization of a New Gene (CBP3) Required for the Expression of Yeast Coenzyme QH2-Cytochrome c Reductase

M Wu,A Tzagoloff

doi:10.1016/s0021-9258(18)60438-7

Abstract

Respiratory defective mutants of Saccharomyces cerevisiae assigned to complementation group G28 display a deficiency, in the respiratory chain complex coenzyme QH2-cytochrome c reductase. The mutants define a new nuclear gene, designated CBP3, required for the assembly of the complex. Mutations in CBP3 are expressed in the absence of spectrally and immunologically detectable cytochrome b, a catalytic subunit of coenzyme QH2-cytochrome c reductase. The mutational block responsible for the cytochrome b deficiency has been ascribed to a post-translational step based on the observation that cbp3 mutants have wild type concentrations of cytochrome b mRNA and are capable of synthesizing the apoprotein. Western analysis has revealed that cbp3 mutants have reduced levels of a subset of subunit polypeptides of the coenzyme QH2-cytochrome c reductase complex that include apocytochrome b, the iron-sulfur protein, core 4 (14-kDa subunit), and core 5 (11-kDa subunit). A similar phenotype has previously been reported in strains that fail to assemble the complex as a result of mutations in the noncatalytic core subunits. The CBP3 gene has been cloned by transformation of a mutant from complementation group G28 with a yeast genomic library. The gene is 1005 nucleotides long and codes for a primary translation product of 39 kDa. A transcript of a size commensurate with the length of the CBP3 reading frame is detected in total and poly(A+)-enriched RNA. The amino-terminal region of the CBP3 product is basic and probably corresponds to a cleavable mitochondrial targeting signal. An antibody obtained against a trpE/CBP3 fusion protein detects a protein of 40 kDa in wild type yeast mitochondria. This protein is absent in a mutant construct containing a partially deleted copy of the gene. The CBP3 protein is a membrane constituent, although attempts to demonstrate its physical association with the other subunits of coenzyme QH2-cytochrome c reductase have been unsuccessful.

Highlights

QHz-cytochromec reductase complex that include apo- strains lacking spectrally measurable cytochrome b and cytocytochrome b, the iron-sulfur protein, core4
E364 and other members of complementation group G28 display an absence of cytochrome b, a catalytic subunit of coenzyme QHz-cytochromec reductase (Fig. 1).Other respiratory activities such as cytochrome oxidase and the oligomycin-sensitive ATPase are not affected by the mutations suggesting that thefunction of the wild type gene is confined to theexpression of this singlerespiratory complex
The results of the immunologicalassays indicate that while the levels of core 1, core 2, and cytochrome c1 are not significantly different in cbp3 mutants and in wild type yeast, the concentrations of other subunits such as theiron-sulfur protein, apocytochrome b, core 4, and core 5 are severely reduced in the mutants

Summary

Introduction

QHz-cytochromec reductase complex that include apo- strains lacking spectrally measurable cytochrome b and cytocytochrome b, the iron-sulfur protein, core4 Construction of lacZ Fusions-A 450-bp PstI-EcoRI fragment containing 300 nucleotides of the upstream region and 150 nucleotides of the CBP3 coding sequence was ligated in frame with the hcZgene in the yeast integrative vector YIp357R [15].

Results

Conclusion