Genetic screening in Saccharomyces cerevisiae for large numbers of mitochondrial point mutations which affect structure and function of catalytic subunits of cytochrome-c oxidase.

A new search for mitochondrial respiratory deficient mutants (Mit-) has been undertaken in order to accumulate a large number of point mutations in the coding portions of cytochrome-c-oxidase catalytic subunits and cytochrome b. Therefore, a mitochondrial DNA which retains the exons and lacks all the introns of the cytochrome oxidase subunit I and of the cytochrome-b split genes has been introduced into a strain carrying a nuclear recessive mutation affecting the adenine-nucleotide translocator, the op1 mutation, which is known to prevent the accumulation of large deletion petite mutants and this was used as the parental strain. After a moderate MnCl2 mutagenesis in order to limit multiple mutations, 105 Mit- mutants were isolated from 15,000 mutagenised clones in Saccharomyces cerevisiae. Mutations were mapped to the three catalytic subunits encoding genes (COX1, COX2 and COX3) of the cytochrome-c oxidase (70 mutations) and to the cytochrome-b gene (15 mutations). More than 50% of the mutants tested still exhibited mitochondrial translation products (subunits I, II and III), suggesting that they carry a missense mutation, rather than a nonsense mutation which would normally have led to a truncated protein. Mutations in the COX1 gene were allocated to four different subregions corresponding to exons 4 and 8 or to groups of exons, exons 1, 2, 3 or exons 5, 6, 7. Seven missense monosubstitution mutations and two frameshift mutations were also identified. The amino acid changes of the missense mutations were located in the vicinity of the CuB-heme alpha 3 binuclear centre ligands.