0 avis
The dense granule proteins GRA2 and GRA6 cooperate to generate membranous nanotubes
Archive ouverte
Edité par CCSD -
International audience. The parasitophorous vacuole in which Toxoplasma gondii develops is characterized by a membranous nanotubular network (MNN) that connects the parasites together and to the parasitophorous vacuole membrane. The molecular components, the formation and the function of the MNN remain poorly explored. We had previously demonstrated indirectly, by the study of the phenotype of knocked-out parasites, that both the dense granule secreted proteins GRA2 and GRA6, which contain three amphipathic alpha-helices and one long hydrophobic alpha-helix, respectively, are key elements of the MNN formation (Mercier et al., 2002; Travier et al., 2008). The aim of this work was to demonstrate directly the role of both these proteins in the MNN formation, using an in vitro system to study the proteins-membranes interactions. Recombinant GRA2 (rGRA2) and GRA6 (rGRA6) proteins were purified from Escherichia coli. Dynamic light scattering and circular dichroism showed that rGRA2, which folds with an alpha-helical pattern, is purified as two soluble complexes, a potential dimer and complexes of higher molecular weight. rGRA6, which folds partially with an alpha-helical pattern, is purified as monomers. When incubated with Large Unilamellar Vesicles (LUVs) formed with complex lipids, rGRA2 associated preferentially with the membranes of 100 nm diameter-LUVs and induced the formation of short membranous tubules observed by transmission electron microscopy (TEM). rGRA6 also associated with 100 nm diameter-LUVs and tethered vesicles. Upon co-incubation of rGRA2 and rGRA6 with LUVs, the frequency of tubule formation did increase. These results thus validate the cooperation of both GRA2 and GRA6 to deform membranes and to generate membranous tubules.
Consulter en ligne
Suggestions
Du même auteur
