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Etudes fonctionnelles et structurales de l'ATPase-Ca2+ du réticulum sarcoplasmique de lapin et de la protéine recombinante exprimée chez Saccharomyces cerevisiae

Abstract : Sarcoendoplasmic reticulum Ca2+-ATPase isoform 1a (SERCA1a), prepared from rabbit fast-twitch muscle, is a membrane protein which catalyses active transport of two calcium ions from the cytosol into the reticulum lumen. Since 2000, several high resolution structures for this protein have been obtained, and these structures have contributed a lot to the current understanding of the catalytic and transport cycle (Toyoshima, Nakasako et al. 2000). However, until 2007, all crystallized forms of the Ca2+-deprived ATPase had been obtained in the presence of inhibitors, bound to the protein transmembrane domain. First, we used fluorescence spectroscopy and proteolytic cleavage to demonstrate that inhibitors bound to membrane domain, which prevent the ATPase from exploring some of the « normal » conformations of its catalytic cycle, had probably induced biases in some of the structures published before 2007 (Montigny, Picard et al. 2007). Crystallization of new forms by our colleagues abroad, in the absence or presence of inhibitors, confirmed our findings. These inhibitors had up to now been used to stabilize the protein during the crystallization process and prevent it from detergent-induced irreversible denaturation (Lund, Orlowski et al. 1989). By using glycerol alone, instead, to stabilize the detergent-solubilized protein, we were able to pinpoint specific and unexpected effects, on the protein, of some of the detergents commonly used in this field (Montigny, Arnou et al. 2008). Again using glycerol, we were able to characterize various features of three particular mutants of the ATPase, previously expressed heterologously in the yeast S. cerevisiae and purified in detergent, one of these mutants being able to bind only one Ca2+ (Montigny, Arnou et al. 2008), and the two other mutants being blocked at particular steps in the ATPase catalytic cycle (Marchand, Winther et al. 2008). Independently, we elucidated the reason for the previously-described extremely low fluorescence of FITC-labelled Ca2+-ATPase under certain conditions: we found that the tethered fluorescein, unexpectedly, had become phosphorylated during catalysis. The experimental conditions required for such phosphorylation imply that the ATPase cytosolic domains have to reorganize before Ca2+ is released on the luminal side (McIntosh, Montigny et al. 2008). In all the above experiments, to estimate the free concentrations of divalent cations like calcium and magnesium, we had to take in account the possibility that anionic species present could chelate these cations. Using pH-meter techniques and metallochromic probes, we checked that the classical Mops buffer does NOT bind magnesium (Montigny and Champeil 2007), contrarily to what has been previously reported. In collaboration with colleagues from our lab, we also used similar techniques to characterize complex formation between cadmium (Cd2+) and reduced glutathion (GSH-) (Leverrier, Montigny et al. 2007), a prerequisite for cadmium detoxification by cells. Abbreviations : ATPase, Adenosine TriPhosphatase ; SERCA1a, Sarco-endoplasmic Reticulum Ca2+-ATPAse, isoform 1a ; FITC, fluorescein isothiocyanate ; Mops, 4-morpholinopropanesulfonic acid.
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Contributor : Cédric Montigny Connect in order to contact the contributor
Submitted on : Thursday, November 12, 2009 - 4:49:55 PM
Last modification on : Tuesday, December 8, 2020 - 3:42:32 AM
Long-term archiving on: : Wednesday, March 29, 2017 - 5:34:17 PM

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  • HAL Id : tel-00430785, version 2

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C. Montigny. Etudes fonctionnelles et structurales de l'ATPase-Ca2+ du réticulum sarcoplasmique de lapin et de la protéine recombinante exprimée chez Saccharomyces cerevisiae. Biochimie [q-bio.BM]. Université Pierre et Marie Curie - Paris VI, 2009. Français. ⟨tel-00430785v2⟩

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