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Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly.

ABSTRACT: Intraflagellar transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non-conserved G4/G5 motifs. We show that IFT22 specifically associates with G-nucleotides and present crystal structures of IFT22 in complex with GDP, GTP, and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP-binding mode of IFT22 bypassing the classical G4 motif. The GTPase switch regions of IFT22 become ordered upon complex formation with IFT74/81 and mediate most of the IFT22-74/81 interactions. Structure-based mutagenesis reveals that association of IFT22 with the IFT complex is essential for flagellum construction in Trypanosoma brucei although IFT22 GTP-loading is not strictly required.

SUBMITTER: Wachter S

PROVIDER: S-EPMC6484408 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly.

Wachter Stefanie S Jung Jamin J Shafiq Shahaan S Basquin Jerome J Fort Cécile C Bastin Philippe P Lorentzen Esben E

The EMBO journal 20190402 9

Intraflagellar transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non-conserved G4/G5 motifs. We show that IFT22 specifically associates with G-nucleotides and present crystal structures of IFT22 in complex with GDP, GTP, and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP-binding mode of IFT22 bypas ...[more]

PMID: 30940671

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Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly

Project description:Intraflagellar transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non-conserved G4/G5 motifs. We show that IFT22 specifically associates with G-nucleotides and present crystal structures of IFT22 in complex with GDP, GTP and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP-binding mode of IFT22 bypassing the classical G4 motif. The GTPase switch regions of IFT22 become ordered upon complex formation with IFT74/81 and mediate most of the IFT22-74/81 interactions. Structure-based mutagenesis reveals that association of IFT22 with the IFT complex is essential for flagellum construction in Trypanosoma brucei although IFT22 GTP-loading is not strictly required.

| S-SCDT-EMBOJ-2018-101251 | biostudies-other

The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance.

Project description:Approximately 10% of the photoreceptor outer segment (OS) is turned over each day, requiring large amounts of lipid and protein to be moved from the inner segment to the OS. Defects in intraphotoreceptor transport can lead to retinal degeneration and blindness. The transport mechanisms are unknown, but because the OS is a modified cilium, intraflagellar transport (IFT) is a candidate mechanism. IFT involves movement of large protein complexes along ciliary microtubules and is required for assembly and maintenance of cilia. We show that IFT particle proteins are localized to photoreceptor connecting cilia. We further find that mice with a mutation in the IFT particle protein gene, Tg737/IFT88, have abnormal OS development and retinal degeneration. Thus, IFT is important for assembly and maintenance of the vertebrate OS.

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Intraflagellar transport and functional analysis of genes required for flagellum formation in trypanosomes.

Project description:Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3-4 and 7-8, suggesting the existence of specific IFT tracks. Rapid (>3 microm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.

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RABL2 interacts with the intraflagellar transport-B complex and CEP19 and participates in ciliary assembly.

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Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum.

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

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Structural insight into the intraflagellar transport complex IFT-A and its assembly in the anterograde IFT train.

Project description:Intraflagellar transport (IFT) trains, the polymers composed of two multi-subunit complexes, IFT-A and IFT-B, carry out bidirectional intracellular transport in cilia, vital for cilia biogenesis and signaling. IFT-A plays crucial roles in the ciliary import of membrane proteins and the retrograde cargo trafficking. However, the molecular architecture of IFT-A and the assembly mechanism of the IFT-A into the IFT trains in vivo remains elusive. Here, we report the cryo-electron microscopic structures of the IFT-A complex from protozoa Tetrahymena thermophila. We find that IFT-A complexes present two distinct, elongated and folded states. Remarkably, comparison with the in situ cryo-electron tomography structure of the anterograde IFT train unveils a series of adjustments of the flexible arms in apo IFT-A when incorporated into the anterograde train. Our results provide an atomic-resolution model for the IFT-A complex and valuable insights into the assembly mechanism of anterograde IFT trains.

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The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly.

Project description:Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.

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Crystal structure of the intraflagellar transport complex 25/27.

Project description:The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub-complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure-guided mutagenesis. IFT27 displays the fold of Rab-like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase-activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP-binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high-resolution structural understanding of the IFT complex.

| S-EPMC3098482 | biostudies-literature

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