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All the news of the Institute of life sciences Frédéric Joliot
Laboratory | Stress response | Mass spectrometry | Proteomics
Team LeadersFrancis Harauxfrancis.firstname.lastname@example.org Claire Lemaireclaire.email@example.com
We study the role of phosphorylation of protein residues in the respiratory complexes regulation. This program requires a pluridisciplinary approach (biochemistry, biophysics, molecular biology, proteomics and mass spectrometry). Using mass spectrometry, in collaboration with the PAPPSO, we have performed an overall quantitative proteomic and phosphoproteomic study of mitochondria extracted from yeast grown on different carbon substrates. We identified large numbers of phosphorylation sites, one third of which were new. A significant fraction is located in the respiratory complexes (including ATP synthase) and in their assembly factors and regulatory effectors. Interestingly, the phosphorylation pattern partially depends on growth conditions (respiration, fermentation...), which does suggest a possible regulatory role.. Introducing phosphomimetic residues by mutagenesis of identified phosphorylation sites and analyzing the consequences on the respiratory chain should allow understanding the regulation levels governed by phosphorylation.
The respiratory complexes of
S. cerevisiae. From left to right: external and internal NADH dehydrogenases (no structure available), complexes II, III, IV and V, inhibitory peptide IF1. Yellow rings indicate positions of residues that are more phosphorylated when cells have been grown in respiratory medium.Renvoisé M., Bonhomme L., Davanture M., Valot B., Zivy M., Lemaire C. (2014)
J. Proteomics106, 140-150
Mitochondrial ATP synthase is the smallest known rotary molecular motor of the living world. In this enzyme anchored to the inner mitochondrial membrane, a membranous H+-driven turbine rotates an asymmetrical axis, which in turn sequentially distorts the three catalytic sites located at the interface of α and β subunits, inducing the condensation of ADP and inorganic phosphate into ATP. When the proton motive force collapses, a soluble regulatory peptide called IF1 binds to ATP synthase and inhibits ATP hydrolysis. IF1 is ejected when the membrane becomes polarized again. This unidirectional regulation prevents energy waste. Using enzymology combined with site-directed mutagenesis and published structural data, we have clarified the role of the multiple interactions between IF1 and the enzyme in the successive steps (binding and locking) of the inhibition process. According to our results, the recognition step only involves catching of IF1 mid-part by the β (and not α) subunit of a catalytic αβ interface. Three processes then contribute to the stabilization of the inhibited complex: closure of the αβ interface, wrapping of the IF1 Nter extremity around the central shaft of the enzyme, and less expectedly, strengthening interaction between one α subunit (stator) and the γ subunit (rotor), consecutive to IF1-induced distortion of α subunit. We are interested in understanding this regulation in mechanical terms, and more particularly why IF1 binds to ATP synthase when rotation occurs in the direction of ATP hydrolysis and is ejected when rotation is forced in the opposite direction.
We are interested in the understanding of some mitochondrial diseases in molecular terms. We have been involved in kinetic studies of yeast ATP synthase with a mutation mimicking that responsible for the NARP syndrome (collaboration with Di Rago's team of UMR 5095, Bordeaux). We are currently investigating the consequences of some human ATP synthase mutations in the supramolecular organization and the functionality of the respiratory chain (Collaboration with Anne Lombès, Institut Cochin, Paris).
Qian Wu (2013) Regulation of mitochondrial ATPase by its inhibitor protein IF1 in Saccharomyces cerevisiae (Paris-Sud University)
Margaux Renvoisé (2014) Contribution to the study of the regulation of respiratory complexes by phosphorylation in Saccharomyces cerevisiae (Paris-Sud University)
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