The C‐terminus of the 14‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisiae is involved in the assembly of a functional enzyme

Abstract

Disruption of QCR7, the gene encoding the 14‐kDa subunit of ubiquinol‐cytochrome‐c oxidoreductase of the yeast Saccharomyces cerevisie,, results in an inactive enzyme which lacks holo‐cytochrome b and has severely reduced levels of apo‐cytochrome b, the Rieske Fe‐S protein and the 11‐kDa subunit [Schoppink, P. J., Berden, J. A. & Grivell, L. A. (1989) Eur. J. Biochem. 181, 475–483]. An episomal system was developed to study the effect on complex III of transformation of in vitro mutagenised QCR7 genes to a QCR7° mutant. Transformation of a gene (TNT1) in which the 12 C‐terminal residues are replaced by 3 amino acids encoded by an oligonucleotide containing a stop codon in all three reading frames (STOP‐oligonucleotide), only leads to partial complementation of the respiratory capacity of the yeast strain. The amounts of apo‐cytochrome b, the Rieske Fe‐S protein and the 11‐kDa subunit are reduced and enzymic activity, together with the amount of holo‐cytochrome b, is lowered to about 40% of that of the wild type, indicating a normal turnover number of the mutant enzyme. Transformation of the QCR7° mutant with another gene (TNT2) encoding the first 96 residues of the 14‐kDa subunit fused to 9 amino acids encoded by the STOP‐oligonucleotide, leads to a phenotype almost indistinguishable from that of the QCR7° mutant. The role of the charged C‐terminus of the 14‐kDa (and the 11‐kDa) subunit in the assembly of a functional complex III is discussed. Copyright © 1994, Wiley Blackwell. All rights reserved