Control of endocytic pathways via ubiquitylation of small GTPases of the Ras superfamily – Ub-GTPases

Endocytosis and endocytic trafficking control the function of many proteins, comprising receptors and transporters, by regulating their spatial/temporal localization between the interior of the cell and the plasma membrane. It is consequently involved in the control of a plethora of cell functions. Furthermore, these pathways are very often hijacked by pathogens to allow their phagocytosis and the colonization of the host cells and the surrounding tissues. The small GTPases of the Ras superfamily including Rab and Rho members are known to be involved in the control of vesicle intracellular trafficking and actin cytoskeleton dynamic, respectively. The teams involved in this proposal have previously defined the importance of Rab4 and Rho/Rac GTPases in respectively endocytic trafficking and bacterial phagocytosis. Team 1 will extend their researches on Rab4 functions studying the member Rab4a and for the first time Rab4b, by using recently developed specific tools. Team2 will focus on the role of Cdc42 in bacteria phagocytosis, having already described the molecular mechanisms involving Rac in this process. Furthermore, phagocytosis probably required endocytic trafficking and, the fate of internalized bacteria will depend on how they hijacked the molecular mechanisms involved in early endosomes sorting to avoid trafficking toward the lysosomal degradative pathway. Our joint aims is now to determine the relationships between Rab4 (a and/or b) and the Rho GTPases in endocytosis and endocytic trafficking in line with a role in bacteria phagocytosis and the subsequent colonization of host cells, by studying infection of bladder epithelial cells by uropathogenic Escherichia coli. This study will be performed in the context of the possible control of the functions of Rab4b/Rho GTPases via ubiquitylation. Indeed, RhoGTPases (including Cdc42) were recently shown to be under the control of ubiquitylation and our unpublished observations indicate that Rab4b, but not Rab4a, is ubiquitylated as well. We will thus biochemically characterize the ubiquitylated forms of these small GTPases. We will determine (i) the lysine residues that are ubiquitylated (ii) the form of the small GTPases that are ubiquitylated (active, inactive, membrane associated or not… (iii) the impact of ubiquitylation of these GTPases on their subcellular localization (iv) the involvement of the ubiquitylation in the degradation of these GTPases. We will characterize the involvement of ubiquitylation in the physical and functional interactions between these small GTPases and their known interacting proteins, and will further search for novel interacting proteins that will specifically interact with the ubiquitylated GTPases. Our research program will allow us to determine the role of ubiquitylation in controlling Rab4b/RhoGTPases activity during endocytosis and endocytic trafficking and their involvement in bacterial phagocytosis and the subsequent bacterial infection processes.