TBC1D5 controls the GTPase cycle of Rab7b

Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here, we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing of TBC1D5 provided the strongest phenotype and this protein was subsequently validated in various in vitro and cell-based assays. TBC1D5 localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro, which is further increased by retromer proteins. Similarly to the constitutively active mutant of Rab7b, inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive vesicles. Together, the results show that TBC1D5 is a GAP for Rab7b in the control of endosomal transport to the trans-Golgi.