Identification of Rab GTPase-Activating Proteins Required for Tubular Endosome Formation

In certain kinds of cells, clathrin-independently endocytosed cargo proteins are recycled back to the plasma membrane via specialized tubular-shaped endosomes, so-called tubular endosomes. Several regulators, including Rab small GTPases, have previously been reported to control tubular endosome structures, and one of the regulators, Rab22A, controls cargo sorting and tubule elongation. Since Rab activity is generally controlled by a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP), these upstream regulators would also be involved in tubular endosome formation. However, although we have previously reported that Vps9d1 is a Rab22A-GEF that controls tubular endosome formation, there have been no reports of Rab-GAPs that are required for tubular endosome formation. Here, we demonstrated by comprehensive screening of TBC/Rab-GAPs that four Rab-GAPs, TBC1D10B, TBC1D18, TBC1D22B and EVI5, are involved in tubular endosome formation in HeLa cells in a GAP-activity-dependent manner. Knockdown or overexpression of each of these Rab-GAPs resulted in the same phenotype, that is, reduced tubular endosome structures. Since one of these four Rab-GAPs, TBC1D10B, was able to reduce the amount of active Rab22A and the size of Rab22A-positive early endosomes, it is the most probable candidate for a Rab22A-GAP. Our findings suggest that a proper GTPase cycle is important for the control of tubular endosome formation.