title: RhoGDI3 and RhoG: Vesicular trafficking and interactions with the Sec3 Exocyst subunit.
abstract: RhoGDIs are negative regulators of small GTP-binding proteins of the Rho family, which have essential cellular functions in most aspects of actin-based morphology and motility processes. They extract Rho proteins from membranes, keep them in inactive rhoGDI/Rho complexes and eventually deliver them again to specific membranes in response to cellular signals. RhoGDI3, the most divergent member of the rhoGDI family, is well suited to document the underlying molecular mechanisms, since the active and inactive forms of its cellular target, RhoG, have well-separated subcellular localizations. In this study, we investigate trafficking structures and molecular interactions involved in rhoGDI3-mediated shuttling of RhoG between the Golgi and the plasma membrane.Bimolecular fluorescence complementation and acceptor-photobleaching FRET experiments suggest that rhoGDI3 and RhoG form complexes on Golgi and vesicular structures in mammalian cells. 4D-videomicroscopy confirms this localization, and show that RhoG/rhoGDI3-labelled structures are less dynamic than RhoG and rhoGDI3-labeled vesicles, consistent with the inhibitory function of rhoGDI3. Next, we identify the Exocyst subunit Sec3 as a candidate rhoGDI3 partner in cells. RhoGDI3 relocates a subcomplex of the Exocyst (Sec3 and Sec8) from the cytoplasm to the Golgi, while Sec6 is unaffected. Remarkably, Sec3 increases the level of GTP-bound endogenous RhoG, the RhoG-dependent induction of membrane ruffles, and the formation of intercellular tunneling nanotube-like protrusions.Altogether, our study identifies a novel link between vesicular traffic and the regulation of Rho proteins by rhoGDIs. It also suggests that components of the Exocyst machinery may be involved in RhoG functions, possibly regulated by rhoGDI3.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales; Centre de Recherche de Gif-sur-Yvette; CNRS; Gif-sur-Yvette, France.
authors.forename: Annie
authors.initials: A
authors.lastname: Morin
authors.forename: Fabrice P
authors.initials: FP
authors.lastname: Cordelières
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cherfils
authors.forename: Birgitta
authors.initials: B
authors.lastname: Olofsson