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Alecia Gross, Nicholas Reish; NUDC is a novel component of the rab11a-rhodopsin interaction in the inner segment. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1224.
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The C-terminus of rhodopsin mediates several important intermolecular interactions essential for its trafficking and for the formation of rod outer segments. To identify previously unrecognized interactions we employed a pull-down approach using native rhodopsin purified from mouse retinas of wild type and knock-in mutant rhodopsin strains. Using this approach we previously identified rab11a in mouse retina as a direct binding partner of rhodopsin’s C-terminus. Here, we report NUclear Distribution C (NUDC) is an additional component of the rhodopsin-rab11a trafficking complex in the inner segment of rod photoreceptors.
Rhodopsin protein affinity chromatography was used to isolate proteins binding to the C-terminus. Rhodopsin binding proteins were identified by in-gel trypsin digestion and mass spectroscopy. Protein identities were confirmed by Western blotting. Protein localization was assayed by immunohistochemistry from retinal sections and by production of transgenic Xenopus laevis tadpoles expressing EGFP-rab11a and/or mCherry-NUDC.
NUDC was identified as a protein that bound rhodopsin in association with rab11a. Reciprocal pull-down experiments confirmed the association between rhodopsin, rab11a, and NUDC. Rhodopsin C-terminal mutants Q344Ter and rhodopsin-EGFP bound neither rab11a nor NUDC. Immunohistochemistry revealed NUDC localization in the inner segment of photoreceptors. Examination of transgenic mCherry-NUDC expressing tadpoles revealed a punctate distribution in the inner segment with minimal outer segment localization. In tadpoles expressing EGFP-rab11a and mCherry-NUDC, there is overlap between EGFP and mCherry positive puncta in the inner segment, in a distribution between the Golgi apparatus and the base of the outer segment.
NUDC was shown to interact with rab11a and rhodopsin in preparations from mouse retina. This interaction was abolished in two mouse lines expressing either truncated or extended rhodopsin C-termini. Immunohistochemical and transgenic expression experiments reveal a role for NUDC in rab11a positive vesicular trafficking in the inner segment.
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