APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting
APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting
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DOI：
10.15252/embr.201847192
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发表时间：
2019-02-01
期刊：
影响因子：
7.7
通讯作者：
Jean, Steve
中科院分区：
文献类型：
--
作者：
Del Olmo, Tomas;Lauzier, Annie;Jean, Steve
RAB GTP酶是膜运输的中心调节剂。它们在激活鸟嘌呤交换因子（GEF）和失活GTP酶激活蛋白（GAP）的动态调节下。一旦被激活，RABs募集大量的效应子来控制真核细胞的运输功能。多个蛋白质组学研究，使用下拉或酵母双杂交方法，已经确定了一些RAB相互作用。然而，由于这些方法的体外性质和每种技术的固有局限性，仍然缺乏RAB相互作用物的全面定义。通过比较GFP：RAB 21的定量亲和纯化与RAB 4a、RAB 5a、RAB 7a和RAB 21的APEX2介导的邻近标记，我们发现APEX2邻近标记允许RAB调节子和相互作用子的全面鉴定。重要的是，通过生物化学和遗传学方法，我们在RAB 21与WASH和retromer复合物之间建立了一种新的联系，并对货物分选产生了功能性影响。因此，APEX2介导的RAB邻近蛋白的邻近标记代表了定义RAB功能的新的且有效的工具。
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.