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APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting.

EMBO reports (2019-01-06)
Tomas Del Olmo, Annie Lauzier, Caroline Normandin, Raphaëlle Larcher, Mia Lecours, Dominique Jean, Louis Lessard, Florian Steinberg, François-Michel Boisvert, Steve Jean
ABSTRACT

RAB GTPases are central modulators of membrane trafficking. They are under the dynamic regulation of activating guanine exchange factors (GEFs) and inactivating GTPase-activating proteins (GAPs). Once activated, RABs recruit a large spectrum of effectors to control trafficking functions of eukaryotic cells. Multiple proteomic studies, using pull-down or yeast two-hybrid approaches, have identified a number of RAB interactors. However, due to the in vitro nature of these approaches and inherent limitations of each technique, a comprehensive definition of RAB interactors is still lacking. By comparing quantitative affinity purifications of GFP:RAB21 with APEX2-mediated proximity labeling of RAB4a, RAB5a, RAB7a, and RAB21, we find that APEX2 proximity labeling allows for the comprehensive identification of RAB regulators and interactors. Importantly, through biochemical and genetic approaches, we establish a novel link between RAB21 and the WASH and retromer complexes, with functional consequences on cargo sorting. Hence, APEX2-mediated proximity labeling of RAB neighboring proteins represents a new and efficient tool to define RAB functions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Arginine, reagent grade, ≥98%
Roche
Anti-GFP, from mouse IgG1κ (clones 7.1 and 13.1)
Sigma-Aldrich
Monoclonal Anti-α-Tubulin antibody produced in mouse, clone DM1A, ascites fluid
Sigma-Aldrich
Lys-Lys-Lys-Lys, ≥95% (TLC)