Paule Brivet-Chevillotte

Cloning by functional complementation, and inactivation, of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc1 complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1− mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc1 complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.

Electron-transfer Restoration By Vitamin-k3 in a Complex-iii-deficient Mutant of S-cerevisiae and Sequence of the Corresponding Cytochrome-b Mutation

The yeast box‐mutant W7 exhibits deficiencies in cytochrome b and in nuclear coded complex III subunits, a phenotype observed previously in a patient with mitochondrial myopathy. DNA sequence analysis of mutant W7 revealed a single base transition in the cytochrome b gene; the mutated residue Gly 131 is perfectly conserved in all known cytochromes b and belongs to the Q0 domain. Mutant W7 provides a model system for evaluating the action of therapeutic agents, such as vitamin K3 which restored NADH‐oxidase activity in the mutant as well as in the antimycin‐inhibited wild type. However, with the mutant, a greater quantity of menadione was necessary due to a decrease in other complex activities, and a much lower electron‐flow fraction passed through cytochrome oxidase.

Specificities of the two center N inhibitors of mitochondial bc1 complex, antimycin and funiculosin: strong involvement of cytochrome b-asparagine-208 in funiculosin binding

Funiculosin, a center N inhibitor of the bc 1 complex, induces a blue‐shift in the cytochrome b spectrum. A thermosensitive revertant [Coppee, J.Y. et al., J. Biol. Chem. 269 (1994) 4221–4226] isolated from a cytochrome b respiratory‐deficient mutant, exhibits a red‐shift instead of the blue‐shift retained in the original mutant and shows resistance to this inhibitor. Replacing cytochrome b‐Asparagine‐208 by Lysine in this revertant, keeping the original mutation S206L, leads, when mitochondria are incubated at non‐permissive temperature, to complete loss of bc 1 complex activity and funiculosin‐binding, while the antimycin‐binding is conserved. These data suggest some inhibitor site specificity and close proximity between the funiculosin‐binding site and the catalytic center N domain (QN).

The bc1 Complex in the Mitochondrial Respiratory Chain

In the inner mitochondrial membrane, the bc1 complex (complex III or ubiquinol cytochrome c oxidoreductase) catalyses the electron transfer from ubiquinol to cytochrome c coupled with a vectorial proton translocation from the matrix side to the positive intermembrane space (Fig. 1). The proton gradient thus created is used by he ATP synthase enzyme to drive ATP synthesis. The bc1 complex is an energy transducing component which is essential to the viability of most eukaryotic cells, which require oxidative phosphorylation to produce ATP. Genetic alterations can lead to human diseases in which the expression or functional efficiency of this complex is affected, especially in the tissues with the highest energy requirements, such as the muscles, brain and heart.