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Mécanismes de régulation de l'ATP synthase mitochondriale de S.cerevisiae par son peptide endogène IF1 et étude de l'oligomérisation d'IF1 de S.cerevisiae.

Abstract : The F-type ATPase or ATP synthase, anchored to the inner mitochondrial membrane, is a macromolecular complex using the proton motive force (pmf ) generated by the oxydation of small molecules, such as NADH2 and FADH2, in the different respiratory complexes to form ATP. The pmf is converted into mechanical work by the clockwise rotation of the ATP synthase viewed from the membrane. The rotation successively distorts the three catalytic interfaces of the enzyme to allow the synthesis of ATP. Anoxia or hypoxia are cases in which the rotation of ATP synthase proceeds in the direction of ATP hydrolysis. A small peptide named IF1, 63 aminoacids-long in yeast and 84 aminoacids-long in bovine, specifically inhibits the mitochondrial ATP synthase in the direction of ATP hydrolysis. This inhibition is reversible since the peptide is released when the inner mitochondrial membrane is re-energized. In this work, we were interested in characterizing the inhibition mechanism of the mitochondrial ATP synthase of S.cerevisiae by its endogenous peptide IF1. To elaborate and strengthen our statements, we mainly used the structures of IF1 ant of the inhibited IF1-F1-ATPase complex of B.taurus. The data obtained by Elena Cabezón on bovine and yeast IF1 showed that different forms of the peptide coexist and that their pre-eminence depends on the pH. The bovine IF1 mainly adopts a dimeric form at pH below 6.5 and tetrameric one at pH above 6.5. Its inhibitory properties also vary with the pH. The dimeric form is inhibitory and the tetrameric one is not. In yeast, it is known that a monomeric form is predominant at pH above 6.5 and a dimeric form predominant at pH below 6.5. The monomeric form is inhibitory but nothing has been reported about the inhibitory properties of the dimeric form. By using ths structural data of the bovine IF1, we tried to determine the dimerization region of the yeast IF1. For this aim, we decided to combine Site-Directed Spin Labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy. Thus, we attached labels on the C-ter or the mid-region and we could propose that the dimer of yeast IF1 preferentially forms by the mid-region. This work is currently in the preliminary stage and other experiments would be necessary to confirm the precize region of dimerization. In a second part, we tried to precise the inhibitory mechanism by detailing the different steps of recognition, locking and stabilization of the inhibited complex. This was achieved by combining the mutagenesis of yeast IF1 and F1-ATPase with kinetics of inhibition. First, we evaluated the role of some residues located in the C-terminal part of β subunit in the specific molecular recognition of IF1 by the mitochondrial ATPase. These residues belong to the region of the α/ β interface that closes up on IF1 peptide. Then, we showed that the N-terminal part of IF1 plays a minor role in the molecular recognition but its winding around the γ subunit constitutes an important lock in the inhibited complex. Finally, the closing of the catalytic interface on IF1 creates a contact region between the α and the γ subunit which is the last key that definitively locks the peptide in the cage "F1-ATPase". This last locking point is the only one that does not involve any IF1 residue.
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Submitted on : Monday, June 25, 2012 - 2:40:31 PM
Last modification on : Thursday, January 11, 2018 - 6:22:11 AM
Long-term archiving on: : Wednesday, September 26, 2012 - 2:50:09 AM

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  • HAL Id : tel-00711657, version 1

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Tiona Andrianaivomananjaona. Mécanismes de régulation de l'ATP synthase mitochondriale de S.cerevisiae par son peptide endogène IF1 et étude de l'oligomérisation d'IF1 de S.cerevisiae.. Biologie structurale [q-bio.BM]. Université Paris Sud - Paris XI, 2011. Français. ⟨tel-00711657⟩

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