Department of Biosciences, Centre for Immune Regulation, University of Oslo, 0316 Oslo, Norway.
Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, West China Second University Hospital, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, China.
J Cell Sci. 2018 Sep 3;131(17):jcs216630. doi: 10.1242/jcs.216630.
Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here, we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing of TBC1D5 provided the strongest phenotype and this protein was subsequently validated in various and cell-based assays. TBC1D5 localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b , which is further increased by retromer proteins. Similarly to the constitutively active mutant of Rab7b, inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive vesicles. Together, the results show that TBC1D5 is a GAP for Rab7b in the control of endosomal transport to the trans-Golgi.This article has an associated First Person interview with the first author of the paper.
Rab GTPases 是细胞内运输的关键调节剂，在 GTP 结合的活性状态和 GDP 结合的非活性状态之间循环。这个循环由鸟嘌呤核苷酸交换因子（GEFs）和 GTPase 激活蛋白（GAPs）调节。已经做出了一些努力将正确的 GEFs 和 GAPs 与其特定的 Rab 连接起来。在这里，我们旨在确定 Rab7b 的 GAPs，Rab7b 是一种参与从晚期内体到反式高尔基体运输的小 GTPase。针对含有对 Rab GAPs 至关重要的 TBC 结构域的蛋白质进行了 siRNA 筛选，并与一种表型读数相结合，该读数可可视化 Rab7b 的分布。沉默 TBC1D5 提供了最强的表型，随后在各种细胞和基于细胞的测定中验证了该蛋白。TBC1D5 定位于 Rab7b 阳性囊泡上，与 Rab7b 相互作用，并对 Rab7b 具有 GAP 活性，这种活性通过 retromer 蛋白进一步增加。与 Rab7b 的组成性激活突变体类似，TBC1D5 的失活也减少了 CI-MPR-和 sortilin 阳性囊泡的数量。总之，这些结果表明 TBC1D5 是控制内体向反式高尔基体运输的 Rab7b 的 GAP。本文有一篇与该论文第一作者的相关第一人称访谈。
