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

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 and to 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.

RAB小GTP酶（RAB GTPases）是膜运输过程的核心调控因子。其活性处于激活型鸟苷酸交换因子（guanine exchange factors, GEFs）与失活型GTP酶激活蛋白（GTPase activating proteins, GAPs）的动态调控之下。一旦被激活，RAB蛋白会招募一系列效应分子，以调控真核细胞的膜运输功能。既往多项蛋白质组学研究通过下拉实验（pull-down）或酵母双杂交（yeast two-hybrid）技术，已鉴定出多种RAB互作蛋白。然而，由于此类实验均基于体外体系，且各技术本身存在固有局限性，目前仍未能全面定义RAB的互作蛋白组。本研究通过对比GFP标记的RAB21（GFP:RAB21）的定量亲和纯化结果，与针对RAB4a、RAB5a、RAB7a及RAB21的APEX2介导邻近标记（APEX2-mediated proximity labeling）结果，发现APEX2邻近标记技术可实现RAB调控蛋白与互作蛋白的全面鉴定。尤为重要的是，本研究通过生化与遗传学实验手段，确立了RAB21与WASH复合物及retromer复合物之间的全新关联，且该关联对货物分选具有功能性影响。因此，针对RAB邻近蛋白的APEX2介导邻近标记技术，为解析RAB的生物学功能提供了一种全新且高效的研究工具。