ATP synthase is an essential protein complex located in the mitochondrial inner membrane, which synthesize ATP by coupling to a rotary proton transport across the membrane at the expense of the electrochemical proton gradient created by the electron transport chain. This reaction guarantees the supply of energy to biological processes in a cell. When mitochondria get deenergized, i.e. the protomotive force across the mitochondrial inner membrane collapses, the ATP synthase switches from ATP synthesis to hydrolysis. This hydrolytic activity is then immediately prevented by a natural soluble mitochondrial ATPase inhibitor, IF1. This efficient reversible inhibition system protects cells from wasting energy. In yeast, IF1 is a small protein consisting of 63 amino acids. It binds to one of the three (αβ) catalytic interfaces of ATP synthase and thereby blocks the rotary catalysis. Although the crystal structure of the dead-end IF1 inhibited F1-ATPase complex has been resolved, IF1 initial binding and locking to ATPase still remain unclear events at the molecular level.During my thesis, we have been interested in the dynamic mechanism of ATPase inhibition by IF1. By means of analyses of published structures and protein sequence alignment, we selected numerous residues located in different regions of Saccharomyces cerevisiae ATP synthase α, β subunits, which might potentially paticipate in IF1 binding process. Using site-directed mutagenesis combined with kinetic experiments, we studied the effect of mutations of the selected candidates on the rate and extent of ATPase inhibition by IF1. In this way we identified residues or motifs in ATP synthase α, β subunits involved in IF1 recognition and/or locking steps, which allows complementing structural studies and drawing an outline of IF1 binding.
| Identifer | oai:union.ndltd.org:CCSD/oai:tel.archives-ouvertes.fr:tel-00998383 |
| Date | 12 December 2013 |
| Creators | Wu, Qian |
| Publisher | Université Paris Sud - Paris XI |
| Source Sets | CCSD theses-EN-ligne, France |
| Language | English |
| Detected Language | English |
| Type | PhD thesis |
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