A Steric Gating Mechanism Dictates the Substrate Specificity of a Rab-GEF

Correct localization of Rab GTPases in cells is critical for proper function in membrane trafficking, yet the mechanisms that target Rabs to specific subcellular compartments remain controversial. Guanine nucleotide exchange factors (GEFs) activate and consequently stabilize Rab substrates on membranes, thus implicating GEFs as the primary determinants of Rab localization. A competing hypothesis is that the Rab C-terminal hypervariable domain (HVD) serves as a subcellular targeting signal. In this study, we present a unifying mechanism in which the HVD controls targeting of certain Rabs by mediating interaction with their GEFs. We demonstrate that the TRAPP complexes, two related GEFs that use the same catalytic site to activate distinct Rabs, distinguish between Ypt1 (Rab1) and Ypt31/32 (Rab11) via their divergent HVDs. Remarkably, we find that HVD length gates Rab access to the TRAPPII complex by constraining the distance between the nucleotide-binding domain and the membrane surface. [Display omitted] •Development of GRab-IT, an assay for investigating GEF-GTPase interactions in vivo•Reconstitution of TRAPP complex activity with physiological substrate specificity•The Rab GTPase hypervariable domain determines GEF substrate specificity•Hypervariable domain length acts as a molecular ruler to control substrate selection Thomas et al. show that certain Rabs are recognized by their guanine nucleotide exchange factors in part through the Rab C-terminal hypervariable domain (HVD). This suggests a model in which the HVD contributes to Rab localization by mediating Rab activation at target organelles.