Abstract
Intraflagellar transport (IFT) is an evolutionarily conserved mechanism thought to be required for the assembly and maintenance of all eukaryotic cilia and flagella. Although IFT proteins are present in cells with sensory cilia, the organization of IFT protein complexes in those cells has not been analyzed. To determine whether the IFT complex is conserved in the sensory cilia of photo-receptors, we investigated protein interactions among four mammalian IFT proteins: IFT88/Polaris, IFT57/Hippi, IFT52/NGD5, and IFT20. We demonstrate that IFT proteins extracted from bovine photoreceptor outer segments, a modified sensory cilium, co-fractionate at approximately 17 S, similar to IFT proteins extracted from mouse testis. Using antibodies to IFT88 and IFT57, we demonstrate that all four IFT proteins co-immunoprecipitate from lysates of mouse testis, kidney, and retina. We also extended our analysis to interactions outside of the IFT complex and demonstrate an ATP-regulated co-immunoprecipitation of heterotrimeric kinesin II with the IFT complex. The internal architecture of the IFT complex was investigated using the yeast two-hybrid system. IFT20 exhibited a strong interaction with IFT57/Hippi and the kinesin II subunit, KIF3B. Our data indicate that all four mammalian IFT proteins are part of a highly conserved complex in multiple ciliated cell types. Furthermore, IFT20 appears to bridge kinesin II with the IFT complex.
Highlights
Intraflagellar transport (IFT)1 involves bidirectional motility of a large protein complex along axonemal microtubules of cilia and flagella
An IFT Particle in the Sensory Cilia of Photoreceptors—We have extended the velocity sedimentation analysis of IFT complexes to photoreceptors in order to determine whether an ϳ17 S particle is present in cells with sensory cilia
We observed that only a portion of IFT20 co-fractionates with the remaining three IFT proteins; a substantial pool of unassembled IFT20 remained near the top of the gradient
Summary
Intraflagellar transport (IFT) involves bidirectional motility of a large protein complex along axonemal microtubules of cilia and flagella. Mutations in IFT particle proteins in Chlamydomonas, C. elegans, and mice prevent ciliary assembly [3, 4, 9, 11,12,13,14,15,16,17,18], suggesting that this mechanism is important for the maintenance of all axonemal structures. The characterization of IFT particles as ϳ17 S protein complexes is based exclusively on studies of the motile flagella of Chlamydomonas and mouse testis, while the IFT complexes of sensory and primary cilia have not yet been characterized. The IFT particle in sensory cilia could be smaller and more simplified as a reflection of the simplified organization of an axoneme lacking large protein complexes involved in regulated motility such as radial spokes and dynein arms. The principal goal of this study was to determine whether IFT88, -57, -52, and -20 are conserved in the IFT complex in
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