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

Niels Boegholm, Narcis A. Petriman, Marta Loureiro-López, Jiaolong Wang, Miren Itxaso Santiago Vela, Beibei Liu, Tomoharu Kanie, Roy Ng, Peter K. Jackson, Jens S. Andersen, Esben Lorentzen*


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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.

TidsskriftEMBO Journal
Udgave nummer18
Antal sider25
StatusUdgivet - 18. sep. 2023

Bibliografisk note

Funding Information:
We would like to thank Maximilian Stoetzel for contributing to preliminary experiments on RabL2 complexes and Anni Christensen and Kathrine Kjærgaard Frederiksen for technical assistance with protein purification. We acknowledge Michael Taschner for initial work on subcloning RabL2 constructs and for carefully reading this manuscript. We also thank Jesper Lykkegaard Karlsen for assistance with running AlphaFold and the EMCC computing facility at Aarhus University for computing time. This work was funded by grants from the Novo Nordisk Foundation (grant number NNF15OC00114164), the Independent Research Fund Denmark (grant no: 1026‐00016B), and the Carlsberg Foundation (grant no: CF22‐0971) to EL. NAP was supported by a postdoc fellowship from the European Commission (H2020, Grant Agreement number 888322). MLL and JSA were supported by the Independent Research Fund Denmark (grant no: 8021‐00425B) to JSA. We also thank Dr. David Sherry and Ms. Megan Stiles for their technical advice on the 3D‐SIM and Nikon Ti2 live cell imaging microscope experiments. The Nikon N‐SIM‐E/STORM super‐resolution microscope is supported by a Large Equipment Grant from the Oklahoma Center for Adult Stem Cell Research (OCASCR) and the OUHSC Department of Cell Biology. The Nikon Ti2 live‐cell imaging microscope is supported by an Equipment Grant from the Presbyterian Health Foundation (PHF, grant no: GRF00006006) and the OUHSC Department of Cell Biology. The cell authentication service performed by MTCRO‐COBRE cell line authentication core of the University of Oklahoma Health Science Center was supported partly by P20GM103639 of the National Institute of Health (NIH) and the National Cancer Institute Grant P30CA225520 of NIH.

Publisher Copyright:
© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.


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