Abstract
Abstract: :
Purpose: Intraflagellar Transport (IFT), which is required for the development and maintenance of cilia and flagella, has been proposed as one molecular mechanism for protein transport in photoreceptors. IFT88 is one component of a multi–subunit IFT complex, which is localized in the photoreceptor inner segment and connecting cilium. A hypomorphic mutation in IFT88 results in photoreceptor degeneration. To elucidate the function of IFT88, we searched for IFT88 binding proteins in photoreceptors. Methods: A yeast two hybrid bovine retinal cDNA library was screened using the N–terminal TPR domain of IFT88 as bait. The interaction with IFT88 was confirmed by pull down assays. GST bovine MRJ was incubated with mouse retina, bovine retina and mouse testis lysates together with glutathione–agarose beads, and proteins eluted from the washed beads were assayed by Western blotting. Immunocytochemistry was conducted on bovine retina and cultured epithelial cells to examine the subcellular localization of proteins. Results: Multiple colonies were found to contain the insert encoding bovine MRJ, a DnaJ/Hsp40 family protein. The direct interaction between IFT88 and MRJ was confirmed by the ability of GST–bMRJ to bind purified His–IFT88. In addition, GST–bMRJ pulled down the endogenous IFT88 from mouse retinal lysate, the photoreceptor guanylate cyclase E from the bovine retinal lysate, and IFT88 together with IFT57 and KIF3A, a subunit of kinesin II motor, from the mouse testis lysate. Moreover, both MRJ and IFT88 were localized in the primary cilia of kidney epithelial cells and were concentrated in the inner segments and base of the connecting cilium of bovine photoreceptors. Conclusion: Our data indicate a direct binding of MRJ to IFT88 in the context of an IFT particle bound to the kinesin II motor. In photoreceptors our data are consistent with a complex containing IFT88 and photoreceptor guanylate cyclase. These data suggest that MRJ may be involved in IFT function in photoreceptors and that guanylate cyclase may be a specific molecular cargo for IFT.
Keywords: photoreceptors • cytoskeleton • chaperones