The mechanistic basis for Rab-GEF substrate specificity and regulation at the Golgi

Abstract

A hallmark of eukaryotic cells is the presence of membrane bound organelles. Proteins and lipids are exchanged between organelles via highly regulated membrane fission and fusion events. Virtually every transport event is mediated by Rab GTPases, which recruit effectors that facilitate vesicle budding, transport, tethering, and fusion. GTPases function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. GTPases are activated by specific guanine nucleotide exchange factors (GEFs); therefore GEFs are master regulators of membrane traffic. In yeast, the conserved Rabs Ypt1 (Rab1) and Ypt31/32 (Rab11) control entry to and exit from the Golgi complex and are thus known as the Golgi “gatekeepers”. Despite the importance of these Rabs in Golgi transport, the identity of the GEFs controlling their activity remained unclear. To address this question, I established an in vitro GEF activity assay to measure Rab activation under physiological conditions. In combination with in vivo experiments, this assay allowed me to discern that TRAPP family multisubunit complexes are Ypt1 and Ypt31/32 GEFs. The TRAPPIII complex is a specific Ypt1 GEF, whereas the related TRAPPII complex activates Ypt31/32. I found that TRAPPIII and TRAPPII distinguish between their distinct substrates using the Rab hypervariable domain (HVD), a flexible linker that connects the Rab to membranes. Remarkably, the longer Ypt31/32 HVD linker allows access to the TRAPPII catalytic site, which is positioned relatively far from the membrane surface. Therefore I propose that a steric gating mechanism controls TRAPP complex substrate specificity. I additionally determined several of the upstream cues coordinating TRAPPII-mediated Ypt31/32 activation: the distinct GTPase Arf1 cooperates with the anionic lipid content of late Golgi membranes to recruit TRAPPII. Given that Ypt31/32 stimulates Arf1 activation, this suggests that TRAPPII participates in a bidirectional feedback loop to coordinate activation of two distinct GTPase pathways. Finally, my recent experiments indicate that TRAPPII acts as a scaffold to recruit multiple Rab-GAPs, factors that mediate Rab inactivation to terminate trafficking events. Together, this suggests that the TRAPPII complex remodels the GTPase composition of late Golgi compartments, promoting Ypt31/32 and Arf1 activation while recruiting the machinery to inactivate other Rabs.