Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

Täschner, Michael; Kotsis, Fruzsina; Braeuer, P.; Kuehn, E. Wolfgang; Lorentzen, Esben

doi:10.1083/jcb.201408002

Cited by 115 publications

(263 citation statements)

References 52 publications

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“…These two IFT‐B1 (core) and IFT‐B2 (“peripheral”) subcomplexes can be purified independently using recombinant proteins (Fig 1 and Taschner et al , 2014) and when incubated result in the reconstitution of a nearly complete 15‐subunit IFT‐B complex lacking only IFT56 (Fig 7A). We uncovered that the IFT57/38 subcomplex is central to IFT‐B complex assembly.…”

Section: Discussionmentioning

confidence: 99%

“…A recent study suggested an interaction of mouse TTC26 with IFT46 based on yeast‐2‐hybrid analysis and pull‐downs (Swiderski et al , 2014), making it the tenth member of the IFT‐B1 complex. Although we currently have a good biochemical understanding of how the 15‐subunit IFT‐B complex assembles and have crystal structures of domains and heterodimeric subcomplexes (Figs 3 and 6; Taschner et al , 2014; Bhogaraju et al , 2011), information on how IFT proteins organize the IFT complex in 3‐dimensions is still missing. The reconstitution of the 15‐subunit IFT‐B complex (Fig 7) will pave the way for structural studies of this assembly.…”

Section: Discussionmentioning

confidence: 99%

“…However, no clear molecular function has so far been assigned to the IFT88 protein. We previously mapped the interaction of IFT88 to a short stretch in the central region of IFT52, but IFT88 was not required for the stability of the IFT‐B1 complex (Richey & Qin, 2012; Taschner et al , 2014). The results presented here do, however, indicate a central role of IFT88 for the stability of the IFT‐B complex by directly linking IFT‐B1 and IFT‐B2.…”

Section: Discussionmentioning

confidence: 99%

“…The resulting plasmids were used to produce recombinant baculoviruses as described previously (Taschner et al , 2014). For protein expression cultures, 3 l of HighFive cells (Invitrogen) was infected with a pre‐determined amount of recombinant virus, harvested after 72 h by centrifugation (1,000 g , 15 min), and resuspended in 5× pellet volume of fractionation buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 250 mM sucrose) freshly supplemented with a protease inhibitor tablet (complete tablet, EDTA‐free; Roche) and 5 mM β‐mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

“…Nine of the IFT‐B subunits (IFT88/81/74/70/52/46/27/25/22) were shown to assemble into a stable IFT‐B core (Lucker et al , 2005; Taschner et al , 2014). IFT56 was recently characterized as an IFT‐B complex protein involved in the transport of axonemal motility factors (Ishikawa et al , 2014), and yeast‐2‐hybrid and pull‐down analyses suggest that it may associate with the IFT‐B core complex (Swiderski et al , 2014).…”

Section: Introductionmentioning

confidence: 99%

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Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

et al. 2016

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Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

et al. 2016

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Chlamydomonas axonemal dynein assembly locus ODA8 encodes a conserved flagellar protein needed for cytoplasmic maturation of outer dynein arm complexes

2015

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The Chlamydomonas reinhardtii oda8 mutation blocks assembly of flagellar outer dynein arms, and interacts genetically with oda5 and oda10, which encode axonemal proteins thought to aid dynein binding onto axonemal docking sites. We positionally cloned ODA8 and identified the gene product as the algal homolog of vertebrate LRRC56. Its flagellar localization depends on ODA5 and ODA10, consistent with genetic interaction studies, but phylogenomics suggests that LRRC56 homologs play a role in intraflagellar transport (IFT)-dependent assembly of outer row dynein arms, not axonemal docking. ODA8 distribution between cytoplasm and flagella is similar to that of IFT proteins and about half of flagellar ODA8 is in the soluble matrix fraction. Dynein extracted in vitro from wild type axonemes will rebind efficiently to oda8 mutant axonemes, without rebinding of ODA8, further supporting a role in dynein assembly or transport, not axonemal binding. Assays comparing preassembled outer dynein arm complexes from the cytoplasm of wild type and mutant strains show that dynein in oda8 mutant cytoplasm has not properly preassembled and cannot bind normally onto oda axonemes. We conclude that ODA8 plays an important role in formation and transport of mature dynein complexes during flagellar assembly.

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RABL4/IFT27 in a nucleotide‐independent manner promotes phospholipase D ciliary retrieval via facilitating BBSome reassembly at the ciliary tip

Liu

Zhang

Fan

2023

Journal Cellular Physiology

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Certain ciliary transmembrane and membrane‐associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome‐dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab‐like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide‐independent manner. IFT25/27 and the IFT subcomplexes IFT‐A and ‐B are irrelevant for maintaining the stability of one another. GTP‐loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT‐B subcomplex core IFT‐B1 entity in cytoplasm, while GDP‐bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT‐B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT‐B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome‐dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.

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Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

Abstract: Crystallographic analysis of the B core complex of the intraflagellar transport machinery provides insight into the molecular basis of ciliogenesis defects caused by several specific IFT mutations.

Cited by 115 publications

References 52 publications

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

Chlamydomonas axonemal dynein assembly locus ODA8 encodes a conserved flagellar protein needed for cytoplasmic maturation of outer dynein arm complexes

RABL4/IFT27 in a nucleotide‐independent manner promotes phospholipase D ciliary retrieval via facilitating BBSome reassembly at the ciliary tip

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