TBC1D5 controls the GTPase cycle of Rab7b

Abstract

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 TBC1D5 provided the strongest phenotype. TBC1D5 was subsequently validated in various in vitro and cell based assays. It localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro, which is further increased by retromer proteins. Inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive vesicles similarly to the effects of the constitutively active mutant of Rab7b. This indicates TBC1D5 as a GAP for Rab7b in control of endosomal transport to the trans-Golgi.