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

Eur J Biochem. 1994 Mar 1;220(2):569-76. doi: 10.1111/j.1432-1033.1994.tb18657.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • Cytochrome b Group / metabolism
  • Cytochrome c Group / metabolism
  • Cytochromes c1 / metabolism
  • Electron Transport Complex III / biosynthesis*
  • Electron Transport Complex III / genetics
  • Electron Transport Complex III / isolation & purification
  • Electron Transport Complex IV / metabolism
  • Escherichia coli
  • Genes, Fungal*
  • Genetic Complementation Test
  • Kinetics
  • Macromolecular Substances
  • Mitochondria / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / growth & development
  • Sequence Deletion

Substances

  • Cytochrome b Group
  • Cytochrome c Group
  • Macromolecular Substances
  • Recombinant Proteins
  • Cytochromes c1
  • Electron Transport Complex IV
  • Electron Transport Complex III