The substrate specificity of the human TRAPPII complex\u2019s Rab-guanine nucleotide exchange factor activity

Meredith L. Jenkins,Calvin K. Yip,Udit Dalwadi,Kaelin D. Fleming,Daniel S. Ziemianowicz,Atefeh Rafiei,Noah J. Harris,John E. Burke,Emily M. Martin,David C. Schriemer

doi:10.1038/s42003-020-01459-2

Abstract

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.

Highlights

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
These disorders are caused by mutations, truncations, and deletions in the genes encoding TRAPPC2, TRAPPC2L, TRAPPC6A, TRAPPC6B, TRAPPC9, TRAPPC11, and TRAPPC12, revealing that mutations of both the TRAPPII and TRAPPIII complexes are involved in disease
X-ray crystallography and electron microscopy show that the yeast TRAPPI core complex is an extended flat assembly composed of 1 copy of each TRAPPC1, TRAPPC2, TRAPPC4, TRAPPC5, and TRAPPC6, and 2 copies of TRAPPC337

Summary

Introduction

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. The ability to transport cargo to specific intracellular membranes is critical to the survival of eukaryotic cells This process of membrane trafficking is mediated by Rab GTPases, which act as molecular switches, cycling between GTP bound active and GDP bound inactive states. Highlighting the critical roles of the TRAPP complexes in myriad cellular processes has been the discovery of a spectrum of human diseases caused by mutations in different TRAPP subunits, collectively known as TRAPPopathies[5,7,28,29,30,31,32,33,34,35] Disorders associated with these mutations include the developmental disorder spondyloepiphyseal dysplasia tarda (SEDT), neurodevelopmental delay, microcephaly, epilepsy, and severe intellectual disability. The specific molecular determinants for why the mammalian TRAPP complexes achieve differential Rab-GEF specificity are not fully resolved

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Conclusion