Characterization of an IFT Protein Complex from Photoreceptors

Abstract

Abstract: : Purpose: Intraflagellar Transport (IFT) is required for the maintenance of organelles such as motile flagella, primary cilia, and sensory cilia. Recently, this concept was extended to photoreceptor outer segments. In IFT, kinesin II and cytoplasmic dynein 2 move a large complex of IFT proteins along axonemal microtubules. To date the hydrodynamic properties and protein interactions within the IFT complex have only been examined in motile flagella. The goal of this study was to extend the analysis of IFT protein interactions and to apply those findings to the photoreceptor outer segment. Methods: Proteins extracted from bovine photoreceptor outer segments or mouse testis were fractionated on sucrose density gradients and analyzed by Western blotting with antibodies raised against the mouse sequences of IFT 88, IFT57, IFT52 and IFT20. Whole retina, kidney, or testis lysates were incubated with antibodies against either IFT88 or IFT57 and precipitated with protein-G Sepharose beads. The precipitate was analyzed by Western blotting for the presence of IFT proteins. To identify direct protein interactions, each IFT protein was expressed in yeast as fusion proteins with either the GAL4-activation domain or GAL4-DNA binding domain. Interactions were measured by the ability of the yeast to grow on selective media. The activity of the reporter enzyme, alpha-galactosidase was measured to quantitate interactions. Results: Velocity sedimentation demonstrates that IFT proteins extracted from photoreceptors co-sediment at ~17S, similar to IFT proteins from testis. Furthermore, the IFT proteins continue to co-fractionate when the salt concentration is increased, but peak higher in the gradient suggesting that they are part of a complex that partially dissociates in high salt. Antibodies directed against IFT57 or IFT88 co-immunoprecipitate all four IFT proteins confirming that they are contained within the same particle. Direct binding between IFT57 and IFT20 was the only interaction observed in yeast two hybrid assays. The interaction domain was mapped to predicted coiled-coil regions within each protein using deletion constructs in the yeast two-hybrid assay. Conclusions: Our data demonstrate that IFT57 and IFT20 directly interact within a complex that also contains IFT88 and IFT52. This complex sediments at ~17S when extracted from photoreceptor outer segments. The hydrodynamic behavior of the photoreceptor IFT complex is indistinguishable from the complex derived from testis suggesting that this complex is highly conserved in diverse axonemal structures.

Keywords: cytoskeleton • photoreceptors • retinal development