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

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.

Colorectal cancer cells respond differentially to autophagy inhibition in vivo

“…To genetically affect autophagosome/lysosome fusion, we targeted RAB21 44 . RAB21 has been implicated in other cellular functions 56,57 . Hence, its effect on tumor cell growth in CAM, could be caused by its other cellular roles, and we cannot rule out this possibility.…”

Complex roles of Rabs and SNAREs in the secretory pathway and plant development: a never‐ending story

“…As a consequence, it would be an enormous task to describe the combinatorial interactions that must exist in cells on a one-to-one basis. New interactomic approaches that rely on proximity labelling might be used to shed light on this complexity in plants (Del Olmo et al, 2019;Gillingham et al, 2019).…”

The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles

“…Shortly afterwards, an improved APEX2 variant exhibiting enhanced catalytic efficiency was generated [ 3 ], which since then has been applied to map a variety of protein–protein interactions. Amongst others, APEX2 was tagged to a G-protein coupled receptor (GPCR) to track GPCR signalling [ 4 ], to the fibroblast growth factor 1 to identify interaction partners on the cell surface [ 5 ], and to Rab proteins to determine new Rab functions [ 6 ]. Furthermore, APEX2 was used to define the protein environment in other specific cellular compartments like lipid droplets, organelles regulating lipid storage [ 7 ], and autophagosomes.…”