The IFT81-IFT74 Complex Acts As an Unconventional RabL2 GTPase-activating Protein During Intraflagellar Transport

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 2023 Aug 22

PMID 37606072

Authors

Niels Boegholm

Narcis A Petriman

Marta Loureiro-Lopez

Jiaolong Wang

Miren Itxaso Santiago Vela

Beibei Liu

Tomoharu Kanie

Roy Ng

Peter K Jackson

Jens S Andersen

Esben Lorentzen

Affiliations

Soon will be listed here.

Abstract

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.

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References

Liew G, Ye F, Nager A, Murphy J, Lee J, Aguiar M . The intraflagellar transport protein IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3. Dev Cell. 2014; 31(3):265-278. PMC: 4255629. DOI: 10.1016/j.devcel.2014.09.004. View

Bhogaraju S, Taschner M, Morawetz M, Basquin C, Lorentzen E . Crystal structure of the intraflagellar transport complex 25/27. EMBO J. 2011; 30(10):1907-18. PMC: 3098482. DOI: 10.1038/emboj.2011.110. View

Wong A, Shkolny D, Dorman A, Willingham D, Roe B, McDermid H . Two novel human RAB genes with near identical sequence each map to a telomere-associated region: the subtelomeric region of 22q13.3 and the ancestral telomere band 2q13. Genomics. 1999; 59(3):326-34. DOI: 10.1006/geno.1999.5889. View

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816-21. PMC: 6286148. DOI: 10.1126/science.1225829. View

Dutcher S, Li L, Lin H, Meyer L, Giddings Jr T, Kwan A . Whole-Genome Sequencing to Identify Mutants and Polymorphisms in Chlamydomonas reinhardtii. G3 (Bethesda). 2012; 2(1):15-22. PMC: 3276182. DOI: 10.1534/g3.111.000919. View

Zhou Z, Qiu H, Castro-Araya R, Takei R, Nakayama K, Katoh Y . Impaired cooperation between IFT74/BBS22-IFT81 and IFT25-IFT27/BBS19 causes Bardet-Biedl syndrome. Hum Mol Genet. 2021; 31(10):1681-1693. DOI: 10.1093/hmg/ddab354. View

SOROKIN S . Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol. 1962; 15:363-77. PMC: 2106144. DOI: 10.1083/jcb.15.2.363. View

Hibbard J, Vazquez N, Satija R, Wallingford J . Protein turnover dynamics suggest a diffusion-to-capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins. Mol Biol Cell. 2021; 32(12):1171-1180. PMC: 8351562. DOI: 10.1091/mbc.E20-11-0717. View

Pan X, Eathiraj S, Munson M, Lambright D . TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature. 2006; 442(7100):303-6. DOI: 10.1038/nature04847. View

10.

Taschner M, Lorentzen E . Recombinant Reconstitution and Purification of the IFT-B Core Complex from Chlamydomonas reinhardtii. Methods Mol Biol. 2016; 1454:69-82. DOI: 10.1007/978-1-4939-3789-9_5. View

11.

Dutcher S . The awesome power of dikaryons for studying flagella and basal bodies in Chlamydomonas reinhardtii. Cytoskeleton (Hoboken). 2013; 71(2):79-94. PMC: 4055781. DOI: 10.1002/cm.21157. View

12.

Sun W, Xue B, Liu Y, Zhang R, Li R, Xin W . LZTFL1 mediates phototaxis via controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip. Proc Natl Acad Sci U S A. 2021; 118(35). PMC: 8536384. DOI: 10.1073/pnas.2101590118. View

13.

Wittinghofer A, Vetter I . Structure-function relationships of the G domain, a canonical switch motif. Annu Rev Biochem. 2011; 80:943-71. DOI: 10.1146/annurev-biochem-062708-134043. View

14.

Silva D, Huang X, Behal R, Cole D, Qin H . The RABL5 homolog IFT22 regulates the cellular pool size and the amount of IFT particles partitioned to the flagellar compartment in Chlamydomonas reinhardtii. Cytoskeleton (Hoboken). 2011; 69(1):33-48. DOI: 10.1002/cm.20546. View

15.

Pazour G, Dickert B, Witman G . The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly. J Cell Biol. 1999; 144(3):473-81. PMC: 2132917. DOI: 10.1083/jcb.144.3.473. View

16.

Daumke O, Weyand M, Chakrabarti P, Vetter I, Wittinghofer A . The GTPase-activating protein Rap1GAP uses a catalytic asparagine. Nature. 2004; 429(6988):197-201. DOI: 10.1038/nature02505. View

17.

Kozminski K, Johnson K, Forscher P, Rosenbaum J . A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad Sci U S A. 1993; 90(12):5519-23. PMC: 46752. DOI: 10.1073/pnas.90.12.5519. View

18.

Pai E, Krengel U, Petsko G, Goody R, Kabsch W, Wittinghofer A . Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990; 9(8):2351-9. PMC: 552258. DOI: 10.1002/j.1460-2075.1990.tb07409.x. View

19.

van Dam T, Townsend M, Turk M, Schlessinger A, Sali A, Field M . Evolution of modular intraflagellar transport from a coatomer-like progenitor. Proc Natl Acad Sci U S A. 2013; 110(17):6943-8. PMC: 3637775. DOI: 10.1073/pnas.1221011110. View

20.

Pedersen L, Rosenbaum J . Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling. Curr Top Dev Biol. 2009; 85:23-61. DOI: 10.1016/S0070-2153(08)00802-8. View