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.
Disruption of QCR7, the gene encoding the 14-kDa subunit of ubiquinol-cytochrome-c oxido-reductase of the yeast Saccharomyces cerevisiae, 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(0) 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(0) 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(0) 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.
Hemrika
W
W
E.C. Slater Institute, University of Amsterdam, The Netherlands.
De Jong
M
M
Berden
J A
JA
Grivell
L A
LA
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D003575:Cytochromes c1 / Q000378:metabolism; D014450:Electron Transport Complex III / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003576:Electron Transport Complex IV / Q000378:metabolism; D004926:Escherichia coli; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017384:Sequence Deletion
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D003575:Cytochromes c1; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III