Faculté de Médecine et des Sciences de la Santé, Département d'Anatomie et de Biologie Cellulaire, Université de Sherbrooke, Sherbrooke, QC, Canada.
Center for Biological Systems Analysis (ZBSA), Faculty of Biology, Albert Ludwigs Universitaet Freiburg, Freiburg, Germany.
EMBO Rep. 2019 Feb;20(2). doi: 10.15252/embr.201847192. Epub 2019 Jan 3.
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 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.
RAB GTPases 是膜运输的核心调节剂。它们受激活鸟嘌呤核苷酸交换因子 (GEFs) 和失活 GTPase 激活蛋白 (GAPs) 的动态调节。一旦被激活，RAB 招募大量效应物来控制真核细胞的运输功能。使用下拉或酵母双杂交方法的多项蛋白质组学研究已经鉴定出许多 RAB 相互作用物。然而，由于这些方法的性质和每种技术的固有局限性，仍然缺乏对 RAB 相互作用物的全面定义。通过比较 GFP:RAB21 的定量亲和纯化与 APEX2 介导的 RAB4a、RAB5a、RAB7a 和 RAB21 的邻近标记，我们发现 APEX2 邻近标记允许全面鉴定 RAB 调节剂和相互作用物。重要的是，通过生化和遗传方法，我们在货物分拣功能方面建立了 RAB21 与 WASH 和逆行体复合物之间的新联系。因此，APEX2 介导的 RAB 邻近蛋白的邻近标记代表了定义 RAB 功能的新的有效工具。
