Structural basis of TRAPPIII‐mediated Rab1 activation

• Published in: The EMBO journal Vol. 40; no. 12; pp. e107607 - n/a
• Main Authors: Joiner, Aaron MN, Phillips, Ben P, Yugandhar, Kumar, Sanford, Ethan J, Smolka, Marcus B, Yu, Haiyuan, Miller, Elizabeth A, Fromme, J Christopher
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 15.06.2021; John Wiley and Sons Inc
• Subjects: Autophagy; Binding sites; Cryoelectron Microscopy; Fungi; GTPase; Guanine; Guanine nucleotide exchange factor; Guanine Nucleotide Exchange Factors - chemistry; Guanosine Diphosphate - chemistry; Guanosine Triphosphate - chemistry; membrane trafficking; Membranes; Nucleotides; Phagocytosis; Protein Conformation; rab GTP-Binding Proteins - chemistry; rab GTP-Binding Proteins - ultrastructure; Rab1; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - ultrastructure; Substrates; Vesicular Transport Proteins - chemistry; Vesicular Transport Proteins - ultrastructure; Yeast; Rab1; autophagy; guanine nucleotide exchange factor; GTPase; membrane trafficking
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.2020107607

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α‐helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes. SYNOPSIS The small GTPase Rab1 is a master regulator of both the early secretory and autophagic pathways, and is activated by the conserved multi‐subunit TRAPPIII complex. The cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to Rab1/Ypt1 provides a model for Rab1 activation on membranes. The cryo‐EM structure of the TRAPPIII‐Rab1/Ypt1 complex reveals the binding site for the Rab1/Ypt1 hypervariable domain. Stable membrane binding by the TRAPPIII complex is required for the activation of Rab1/Ypt1. A conserved amphipathic helix within the TRAPPIII regulatory subunit Trs85 is required for TRAPPIII membrane binding and activation of Rab1/Ypt1. Functional experiments suggest that Trs85‐mediated membrane anchoring of TRAPPIII is critical for both secretion and autophagy. The cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1 provides a model for Rab1 GTPase activation on membranes in both secretion and autophagy.