Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
Commun Biol. 2020 Dec 4;3(1):735. doi: 10.1038/s42003-020-01459-2.
The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.
TRAnsport Protein Particle (TRAPP) 复合物作为 GTP 酶的鸟嘌呤核苷酸交换因子 (GEF)，在真核细胞中是膜运输的主要调节因子。在后生动物中，有两种大型多蛋白 TRAPP 复合物：TRAPPII 和 TRAPPIII，TRAPPII 复合物能够激活 Rab1 和 Rab11。在这里，我们对人 TRAPPII 复合物的 Rab-GEF 特异性进行了详细的生化特征分析，并对 Rab 结合进行了分子洞察。针对 20 种不同 Rab GTP 酶的 TRAPPII 复合物 GEF 测定显示 Rab43 和 Rab19 具有 GEF 活性。电子显微镜和化学交联揭示了哺乳动物 TRAPPII 的结构。氢氘交换 MS 表明 Rab1、Rab11 和 Rab43 具有保守的结合界面。Rab11 的临床突变和 Rab43 的磷酸模拟物显示出降低的 TRAPPII GEF 介导的交换。最后，我们设计了一种 Rab11 突变，它在保持 TRAPPII 介导的 GEF 活性的同时降低了 Rab11-GEF SH3BP5 的活性，为剖析 Rab11 信号提供了工具。总的来说，我们的结果提供了对 TRAPPII 的 GTP 酶特异性的深入了解，以及临床突变如何破坏这种调节。
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