Rab5 regulates macropinosome closure through recruitment of the inositol 5-phosphatases OCRL/Inpp5b and the hydrolysis of PtdIns(4,5)P2

A bstract Rab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and SNARE-dependent endomembrane fusion was necessary for completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P 2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P 2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that removal of PtdIns(4,5)P 2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1 prevented macropinosome closure, without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P 2 needed to complete macropinosome scission from the plasmalemma.