Crystallographic and electron microscopy studies of intraflagellar transport

• Applicant: Dr. Esben Lorentzen
• Subject Area: Structural Biology
• Term: from 2009 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 115487418

• Final Report Year: 2015

Final Report Abstract

This Emmy Noether project was funded with the aim of providing mechanistic insights into the process of Intraflagellar Transport (IFT) by which the cilium organelle is formed and maintained. A particular focus was put on the reconstitution and structural elucidation of IFT complexes and the mapping of cargo binding sites. When we started our research there was not a single high-resolution structure available for any IFT protein or domain. In the past 5 years we have reconstituted numerous IFT-complexes of which the larges comprises 9 subunits. Crystal structures have been determined of 3 subcomplexes (IFT27/25, IFT70/52 and IFT46/52) providing the first atomic view of IFT complexes. Additionally, we determined the crystal structure of the N-terminal domain of IFT81 and showed that this domain constitutes a bona fide tubulin cargo-binding site representing the first comprehensive study of how the IFT machinery recognizes ciliary cargoes. Using this approach, we have provided a high-resolution interaction map for the IFT-B core complex and outlined how IFT complexes may function in intracellular transport of ciliary components. Additionally, with biochemically pure samples of IFT complexes in hand, we have successfully initiated single-particle EM work and determined a cryo-EM structure of the IFT-B core complex at 12Å resolution. This structure will serve as a starting point for the structural elucidation of the entire IFT complex and will provide important insights into the molecular mechanisms of the IFT process.

Publications

• (2011) Architecture and function of IFT complex proteins in ciliogenesis, Differentiation, 83(2):S12-22
  Taschner, M., Bhogaraju, S., Lorentzen, E.
  
• (2011) Crystal structure of the intraflagellar transport complex 25/27, EMBO J., 30(10):1907-18
  Bhogaraju, S., Taschner, M., Morawetz, M., Basquin, C. and Lorentzen, E.
  
• (2012) Atomic resolution structure of human α-tubulin acetyltransferase bound to acetyl-CoA, PNAS, 109(48):19649-54
  Taschner, M., Vetter, M., and Lorentzen, E.
  
• (2012) Structural Studies of Ciliary Components, J Mol Biol, 422(2):163-180
  Mizuno, N., Taschner, M., Engel, B. D., and Lorentzen, E.
  
• (2013) Molecular Basis of Tubulin Transport Within the Cilium by IFT74 and IFT81, Science 341(6149):1009-12
  Bhogaraju S, Cajanek L, Fort C, Blisnick T, Weber K, Taschner M, Mizuno N, Lamla S, Bastin P, Nigg EA, Lorentzen, E
  
• (2014) Crystal structure of a Chlamydomonas reinhardtii flagellar RabGAP TBC-domain at 1.8 Å resolution PROTEINS, 10.1002/prot.24597
  Bhogaraju, S., and Lorentzen, E.
  
• (2014) Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly, JCB, 207(2):269-282
  Taschner, M., Kotsis. F., Braeuer, P., Kuehn, W.E., and Lorentzen, E.
  
• (2014) Getting tubulin to the tip of the cilium: One IFT train, many different tubulin cargo-binding sites? Bioessays 36(5):463-467
  Bhogaraju, S., Weber K, Engel, BD., Lechtreck KF., and Lorentzen, E.
  
• (2014) Structural basis for membrane targeting of the BBSome by ARL6, NSMB
  Mourão, A., Nager, A.R., Nachury, M.V., and Lorentzen, E.