Structure and mechanism of TRAPPIII-mediated Rab1 activation

Abstract

The GTPase Rab1 is a master regulator of both the early secretory pathway and autophagy. Rab1 activation is controlled by its GEF (guanine nucleotide exchange factor), the multi-subunit TRAPPIII complex. The Trs85 regulatory subunit is critical for robust activation of Rab1 but its mechanistic role within the complex has remained unclear. Here we report the cryo-EM structure of the intact yeast TRAPPIII complex bound to its substrate Rab1/Ypt1. The orientation of the Rab1/Ypt1 hypervariable domain when bound to the complex leads to a model for how TRAPPIII associates with and activates Rab1/Ypt1 at the membrane surface. We identify a conserved amphipathic α-helix motif within Trs85 and demonstrate that this helix is required for stable membrane binding and Rab1/Ypt1 activation by TRAPPIII. Taken together, our results provide a comprehensive analysis of the structure and function of the yeast TRAPPIII complex and reveal that the key function of Trs85 is to serve as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex.

Biochemical insight into novel Rab-GEF activity of the mammalian TRAPPIII complex

“…The decreased exchange observed in TRAPPC8 upon membrane binding (5-21, 115-126, 376-421, 440-457, 471-487, 492-498) could be contact sites between TRAPPC8 and membrane or interactions between TRAPPC8 and different subunits that are enhanced in the presence of membranes. The protection in TRAPPC8 (376-421) in the presence of liposomes is consistent with a conserved amphipathic helix present in the yeast TRAPPC8 homolog trs85 (368-409) that was found to be important for membrane binding [23]. This region is highly conserved (Fig.…”

“…We found multiple peptides with significant protections spanning TRAPPC8 including the region 376-421. This region corresponds to an amphipathic helix (368-409) discovered in yeast Trs85, where mutants of this region decreased membrane recruitment and Rab activation [23]. This highlights a conserved role throughout evolution of the TRAPPC8 subunit in mediating membrane association and activation of Rab1.…”

The fungal RABOME: RAB GTPases acting in the endocytic and exocytic pathways of Aspergillus nidulans (with excursions to other filamentous fungi)

“…In the budding yeast, TRAPPIII, the GEF acting on RAB1 (Joiner et al, 2020;Thomas et al, 2018), consists of core TRAPP plus Tca17 and Trs85. In A. nidulans and metazoans, TRAPPIII additionally includes TRAPPC11, TRAPPC12, and TRAPPC13 (Galindo et al, 2020;Pinar et al, 2019) (Figure 4a).…”

“…In all likelihood TRAPPI does not exist at such in vivo (Pinar et al., 2019; Thomas et al., 2018). In the budding yeast, TRAPPIII, the GEF acting on RAB1 (Joiner et al, 2020; Thomas et al., 2018), consists of core TRAPP plus Tca17 and Trs85. In A .…”

TRAPP structures reveal the big picture

“…Mutational analysis confirmed the importance of this interaction for Trs85 assembly into TRAPPIII and for Ypt1 activation. Joiner et al (2021) propose that Trs85 helps to anchor TRAPPIII to membranes, based on two lines of evidence. First, Trs85 is needed for stable association of purified TRAPPIII with liposomes.…”