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. Synopsis The small GTPase Rab2L regulates assembly and maintenance of cilia by acting on the intraflagellar transport (IFT) complex. Here, the IFT‐B1 complex is shown to act as a GTPase‐activating protein (GAP) for RabL2, inducing its dissociation from anterograde IFT trains. RabL2 interacting protein CEP19 has high affinity for RabL2‐GTP but is not a GAP for RabL2. The IFT‐B1 complex is a GAP for RabL2, but not for IFT27 or IFT22, the small GTPase subunits of IFT‐B1. 70 residues of IFT81/IFT74 coiled‐coil structure constitute a minimal complex that binds RabL2 to activate GTP hydrolysis. An IFT74 point‐mutant deficient in RabL2 binding disrupts ciliogenesis and injection of IFT particles into cilia of mammalian cells. Graphical Abstract The IFT‐B1 complex inactivates RabL2, promoting its dissociation from anterograde intraflagellar transport (IFT) trains for correct ciliogenesis.