The IFT81-IFT74 complex acts as an unconventional RabL2 GTPase-activating protein during intraflagellar transport.

Cilia are important cellular organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery, which is followed by initiation of IFT. We reconstituted and purified RabL2 with CEP19 or IFT proteins to show that a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis rate of RabL2. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 was mapped to a 70-amino-acid-long coiled-coil region of IFT81/74. We present structural models for RabL2-containing IFT complexes that we validate in vitro and in cellulo and demonstrate that Chlamydomonas IFT81/74 enhances GTP hydrolysis of human RabL2, suggesting an ancient evolutionarily conserved activity. Our results provide an architectural understanding of how RabL2 is incorporated into the IFT complex and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.