Subunit VIa of yeast cytochrome c oxidase is not necessary for assembly of the enzyme complex but modulates the enzyme activity. Isolation and characterization of the nuclear-coded gene.
COX13, the nuclear gene for cytochrome c oxidase subunit VIa of Saccharomyces cerevisiae, has been isolated in two steps. First, the partial amino acid sequence information of the subunit was used to design two degenerate oligodeoxynucleotide primers to amplify part of the gene in a polymerase chain reaction. Next, the amplified product was used to screen a yeast genomic library in order to obtain the entire gene and its flanking sequences. COX13 is present as a single copy gene per haploid genome. Alignment of the N-terminal sequence of mature, subunit VIa with the amino acid sequence deduced from the DNA sequence indicates that subunit VIa is synthesized as a precursor comprised of a leader sequence of 9 amino acid residues and a mature polypeptide of 120 amino acid residues. The mature polypeptide shares 34% identical amino acid residues with the human subunit isoform VIa-L. Sequence analysis of the 3'-flanking region of COX13 revealed that the gene is located 599 base pairs downstream of CDC55, a gene which has been mapped to the left arm of chromosome VII. Null mutants of COX13, generated by gene replacement, showed a slightly reduced growth rate on nonfermentable carbon sources. Heme spectra and analysis of immunopurified cytochrome c oxidase from a null strain demonstrated that the enzyme is fully assembled without subunit VIa. At low ionic strength, cytochrome c oxidase missing subunit VIa was more active, whereas at high ionic strength, it was less active than the enzyme complex in which subunit VIa was present. In addition, distinct effects of ATP on the activity of the null and wild type enzyme were found. The results suggest that ATP interacts specifically with subunit VIa and thereby modulates the cytochrome c oxidase activity.
Institute of Molecular Biology, University of Oregon, Eugene 97403.
J W
JW
Taanman
R A
RA
Capaldi
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003062:Codon; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification*; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017385:Sequence Homology
D003062:Codon; D004271:DNA, Fungal; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV