[en] The energy-dissipating alternative oxydase (AOX) from Hansenula anomala was expressed in Saccharomyces cerevisiae. We have found that the recombinant product is properly addressed to the mitochondria where it was functional. A comparative analysis by two-dimensional differential in-gel electrophoresis (2D-DIGE) of mitochondrial protein patterns found in wild-type and recombinant AOX strains was performed. This analysis has shown that AOX expression affects energy-related enzymes in a very specific manner: 60 proteins exhibiting a significant difference in their abundance were identified and were implicated in major metabolic pathways such as Krebs cycle and amino-acid biosynthesis. At the level of the respiratory chain, we found a ~1.4-fold increase in the proton pumping complex III (ubiquinol-cytochrome c oxidoreductase), which competes with AOX for the reduced Q, as well as a ~1.7-fold increase in the succinate dehydrogenase that delivers electrons produced by the reduction of succinate to the Q pool. In addition, the NADH-cytochrome c reductase (MCR1) that transfers electrons from the externally-produced NADH directly to cytochrome c was ~1.4-fold greater in AOX+ mitochondria. This increase in MCR1 could force electrons to pass through the cytochrome c oxidase, the other proton pumps of the yeast respiratory chain. Up-regulation of the complex III as well as of MCR1 would influence the electron partitioning at the level of the Q pool in favour of the cytochrome pathway in order to diminish the impact of recombinant AOX on oxidative phosphorylation and energy conservation. Surprisingly, this up-regulation of the respiratory-chain was associated with a down-regulation of the ATP synthase complex. This decrease in ATP synthase content would allow the establishment of a novel steady state between the rate of ΔμH+ building and the rate of ΔμH+ consumption, favourable to the ATP synthase to perform ATP synthesis
