Sequence analysis of three deficient mutants of cytochrome oxidase subunit I of Saccharomyces cerevisiae and their revertants

Abstract

Three respiratory-deficient mutants of cytochrome oxidase subunit I in the yeast mitochondrion have been sequenced. They are located in, or near, transmembrane segment VI, the catalytic core of the enzyme. Respiratory-competent revertants have been selected and studied. The mutant V244M was found to revert at the same site in valine (wild-type), isoleucine or threonine. The revertants of the mutant G251R were of three types: glycine (wild-type), serine and threonine at position 251. A search for second-site mutations was carried out but none were found. Among 60 revertants tested, the mutant K265M was found to revert only to the wild-type allele.

Molecular and biochemical analysis of Saccharomyces cerevisiae cox1 mutants

“…Activity measurements were also performed in vitro, estimating the efficiency of the electron transfer chain by the ability of purified mitochondrial membranes to oxidize Lemarre et al (1994) b The amino-acid conservation has been considered in terms of the alignment of 31 sequences of Cox1p including bacteria, fungi, plants and animals Table 2 Nature and localization of the COX1 mutations and analysis of COX1 mRNA synthesis. The nucleotides are numbered from 1 to 13 003, starting at position -126 of the short strain (Bonitz et al 1980) and interrupted for exons A5 to A7 by 3060 nucleotides of the long strain (Hensgens et al 1983) an exogenous electron donor, cytochrome c. The initial rate of the cyanide-sensitive cytochrome c oxidation of the wt strain is 650 nmoles of oxidized cytochrome c/ min per mg of protein.…”

Mutations in the mitochondrial split gene COXI are preferentially located in exons: a mapping study of 170 mutants

“…Their origin is still a matter of speculation. The initial goal of this work was to characterize mutants that are unable to grow on a non-fermentable medium as a consequence of point or discrete mutations affecting subunit I of the cytochrome c oxidase (Netter et al 1992;Lemarre et al 1994). A large collection of mutants was available, which had been previously mapped in the C O X I region (Kotylak and Slonimski 1977;Kruszewska and Slonimski 1984).…”

“…The very high level of sequence conservation in these proteins in the whole of the living world, from bacteria to man, illustrates this high degree of specialization. Conversely, many single amino acid changes in these proteins have been shown to lead to respiratory-deficient phenotypes (Netter et al 1992;Meunier et al 1993;Lemarre et al 1994; C. Lemaire, S. Robineau and P. Netter, unpublished work; for a review on cytochrome b, see Degli Esposti et al 1993). The critical need to maintain a fully active respiratory chain is illustrated by the natural overproduction of the mRNA for the corresponding proteins.…”