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蛋白颗粒（TRAPP）复合物充当Rab GTPases的鸟嘌呤核苷酸交换因子（GEF），Rab GTPases是真核细胞中膜运输的主要调节剂。在后生动物中，有两个大型的多蛋白TRAPP复合物：TRAPPII和TRAPPIII，而TRAPPII复合物能够激活Rab1和Rab11。在这里，我们介绍人类TRAPPII复合物的Rab-GEF特异性的详细生化特征，以及对Rab结合的分子洞察。针对20种不同Rab GTP酶的TRAPPII复合物的GEF分析显示GEF对Rab43和Rab19具有活性。电子显微镜和化学交联揭示了哺乳动物TRAPPII的结构。氢氘交换质谱显示Rab1，Rab11和Rab43共享保守的结合界面。Rab11的临床突变和Rab43的磷酸化类似物，显示出TRAPPII GEF介导的交换减少。最后，我们设计了一个Rab11突变，该突变保持TRAPPII介导的GEF活性，同时降低Rab11-GEF SH3BP5的活性，从而提供了一种解剖Rab11信号传导的工具。总体而言，我们的结果提供了对TRAPPII GTPase特异性的了解，以及临床突变如何破坏这种调节。