MiP2005: Session 6
Mitochondrial Physiology Network 10.9: 71 (2005) - download pdf
Functional and proteomic impacts of variations of the expression of uncoupling proteins in living cells.
P Douette, R Navet, Francis E Sluse
Laboratory of Bioenergetics, Department of life Sciences, University of Ličge, Belgium. – email@example.com
Uncoupling proteins (UCPs) are consumers of the proton electrochemical gradient built up by the respiratory chain and in this way they compete with the processes of oxidative phosphorylation, including ADP and inorganic phosphate import and ATP synthesis. In some way, their (over)expression in a cell can be considered as a stress at the energetic level. Indeed, in front of a decrease in the oxidative phosphorylation yield (uncoupling due to the presence and the activity of UCPs), cells do not remain neutral and react by promoting their general metabolic capacity. This concept will be illustrated at the functional and proteomic levels, by recombinant UCP1 from BAT expressed in a UCP-free cell i.e. the yeast Saccharomyces cerevisiae. UCP1 activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at ~1 and ~10 µg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radicals production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related namely to energy metabolism. We also quantified the cellular H2O2 release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radicals generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side-consequence of the overall increase in oxidative metabolism. Some pathologies and exogenous stresses that are accompanied by an increase in the expression of tissue-specific UCPs will also be analysed at the level of the mitochondrial proteome.