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Characterization of the membrane tubulation mechanism induced by the Toxoplasma GRA2 protein
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International audience. Within the host cell, the parasitophorous vacuole in which Toxoplasma gondii develops, is characterized by a membranous nanotubular network (MNN). The molecular components, the formation and the function of the MNN remain poorly explored.We had previously demonstrated indirectly that the secreted dense granule GRA2 protein, which contains three amphipathic alpha helices, is a key element of the MNN formation (Mercier et al., 2002; Travier et al., 2008). The aim of this work was to demonstrate directly the role of GRA2 in the MNN formation, using an in vitro system to study the GRA2-membrane interactions. Native and recombinant GRA2 proteins were puri ed from Toxoplasma and from Escherichia coli, respectively. Dynamic light scattering and circular dichroism showed that recombinant GRA2, which folds with an alpha-helical pattern, is puri ed as both a potential dimer and complexes of higher molecular weight. When incubated with Small Unilamellar Vesicles (SUVs) formed with complex lipids, both native and recombinant GRA2 were shown to associate with the SUV membranes and to induce the formation of long membranous tubules observed by transmission electron microscopy (TEM). In the literature, it has been reported that most proteins capable of inducing membrane curvature, interact with phosphatidylinositol (4,5) bi-phosphate (PI(4,5)P2). Fat blots and binding assays of GRA2 to SUVs of de ned lipid composition and containing PI(4,5)P2 showed that GRA2 recognizes phosphoinositides and associates preferentially with PI(4,5)P2-containing SUVs. TEM showed that GRA2 deforms membranes only when PI(4,5)P2 is present. Together, these results allowed us to propose a model of membrane deformation induced by GRA2, the principal effector of the MNN formation.
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