Véronique Gaudreault, Benoît Marchand, Raphaëlle Larcher, Sonya Nassari, Francis Bourassa, Mathilde Moreau, Dominique Jean, François‐Michel Boisvert, Marie A. Brunet, Steve Jean
RAB GTPases, the most abundant small GTPase family, regulate membrane trafficking, including cargo sorting, vesicle budding, transport, docking, and fusion. Guanine exchange factors and GTPase-activating proteins control their activity. Activated RABs recruit effectors to mediate trafficking, but identifying RAB-associated proteins is challenging due to the transient nature of their interactions. Recent proximity labeling techniques enable covalent tagging of nearby proteins, allowing for comprehensive mapping of RAB GTPases neighboring proteins. Here we report APEX2 proximity labeling of 23 human RABs and their neighboring proteomes. Bioinformatic analyses revealed proximal proteins for a broad range of RABs. Notably, we identified a novel interaction between RAB25 and DENND6A, with RAB25 overexpression promoting DENND6A recruitment to recycling endosomes. We also uncovered functional links between RAB14 and the EARP complex, as well as SHIP164 and its ortholog UHRF1BP1. Our dataset offers a valuable resource for exploring new functional connections between RAB GTPases and their associated proteins. Proximity labeling proteomics of RAB GTPases identifies an array of neighboring RAB proteins. Functional validation studies reveal links between RAB25 and DENND6A, or between RAB14 and either the EARP complex or the lipid transport protein UHRF1BP1.