Cryo‐EM structure of metazoan TRAPPIII, the multi‐subunit complex that activates the GTPase Rab1

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo . Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo‐EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII‐specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease‐causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur‐TRAPP. SYNOPSIS The metazoan TRAPPIII complex activating the small GTPase Rab1 consists of a catalytic core and four large accessory subunits. This work presents the cryo‐EM structure of the entire Drosophila TRAPPIII complex, and reveals how the catalytic core and the large subunits assemble to form the full complex and confer specificity towards the substrate. The TRAPPIII core binds to the N‐terminal parts of the large subunits TRAPPC8 and TRAPPC11. The C‐terminal halves of TRAPPC8 and TRAPPC11 extend to a vertex where large subunits TRAPPC12 and TRAPPC13 are located. TRAPPC8 arches over the Rab1 binding site within the core and binds Rab1, thus conferring specificity towards the substrate. The overall architecture of the TRAPPII complex is similar to that of TRAPPIII, with its specific subunits, TRAPPC9 and TRAPPC10, resembling TRAPPC8 and TRAPPC11. Graphical Abstract A cryo‐EM structure of the entire Drosophila TRAPPIII complex reveals how the catalytic core and the large accessory subunits assemble to form the full complex and confer specificity towards the substrate.