Molecular basis of Rab43 inactivation by RN-Tre in endocytic trafficking unveils a general Rab-GAP recognition mechanism

TBC domain-containing Rab GTPase-activating proteins (TBCs) play key roles in regulating intracellular trafficking, and mutations in these proteins can disrupt Rab inactivation and contribute to human disease. However, the molecular principles governing the substrate specificity of TBCs remain poorly understood. Here, we delineate the molecular mechanism by which RN-Tre (also known as USP6NL), an RQ-dual finger TBC protein, selectively recognizes and inactivates Rab43. The crystal structure of the RN-Tre-Rab43 complex reveals a bipartite recognition mechanism: the N-terminal subdomain catalytically remodels Rab43 Switch regions, while the C-terminal subdomain engages Switch II and reorients the hydrophobic triad to confer specificity. Structural and mutational analyses identify Leu146 and several C-terminal residues as key determinants of RN-Tre specificity, which lead us to identify Rab19 as an additional substrate. Functional assays demonstrate that disease-associated RN-Tre mutations impair GAP activity, resulting in aberrant Golgi architecture and endocytic trafficking. Collectively, this study establishes a general structural paradigm for substrate discrimination by TBCs and highlights their pivotal roles in membrane trafficking and disease.