SUBUNIT-VIA OF YEAST CYTOCHROME-C-OXIDASE IS NOT NECESSARY FOR ASSEMBLY OF THE ENZYME COMPLEX BUT MODULATES THE ENZYME-ACTIVITY - ISOLATIONAND CHARACTERIZATION OF THE NUCLEAR-CODED GENE

COX13, the nuclear gene for cytochrome c oxidase subunit VIa of Saccha romyces 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 i n 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 ha ploid genome. Alignment of the N-terminal sequence of mature subunit V Ia with the amino acid sequence deduced from the DNA sequence indicate s that subunit VIa is synthesized as a precursor comprised of a leader sequence of 9 amino acid residues and a mature polypeptide of 120 ami no acid residues. The mature polypeptide shares 34% identical amino ac id 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 l eft arm of chromosome VII. Null mutants of COX13, generated by gene re placement, showed a slightly reduced growth rate on nonfermentable car bon sources. Heme spectra and analysis of immunopurified cytochrome c oxidase from a null strain demonstrated that the enzyme is fully assem bled without subunit VIa. At low ionic strength, cytochrome c oxidase missing subunit VIa was more active, whereas at high ionic strength, i t was less active than the enzyme complex in which subunit VIa was pre sent. In addition, distinct effects of ATP on the activity of the null and wild type enzyme were found. The results suggest that ATP interac ts specifically with subunit VIa and thereby modulates the cytochrome c oxidase activity.