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Katharina Baub, Tina Maerker, Erwin v.Wijk, Ferry Kersten, Ronald Roepman, Hannie Kremer, Uwe Wolfrum; The Ush1g Protein Sans Interacts With The Vesicular Traffic Associated Protein Magi2. Invest. Ophthalmol. Vis. Sci. 2011;52(14):49.
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© ARVO (1962-2015); The Authors (2016-present)
The human Usher syndrome (USH) is the most common cause of combined deaf-blindness. The encoded molecules are integrated into protein networks by scaffolds including the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain). Previous data indicated that SANS is involved in photoreceptor transport processes related to cilia. To decipher the cellular role of SANS, we screened with a yeast-2-hybrid (Y2H) for new interacting partners in the retina and validated putative interactions.
Results obtained from Y2H screens of a retinal cDNA library were validated by independent assays: GST-pull downs, co-transfection and co-immunoprecipitations. Correlative immunofluorescence and immunoelectron microscopy were used to study subcellular distribution of confirmed interactors. Functional assays elucidated the role of SANS associated complexes more precisely.
Y2H revealed three PDZ-domain containing proteins as putative interactors. Here we identified Magi2 (membrane associated guanylate kinase inverted-2) as a novel component of the USH interactome and confirmed its direct interaction to SANS. Immunofluorescence showed partial co-localization of both interactors in mouse retina especially at the periciliary-ciliary region in the apical inner segment of photoreceptor cells. In addition, electron microscopy revealed an association of the Magi2-SANS complex with transport vesicles in this region. Further analysis of the complex indicated an association with the endocytosis/exocytosis machinery in retinal cells.
Direct binding of SANS to vesicle-associated Magi2 and subcellular distribution of both interaction partners in photoreceptor cells further support a role of SANS-organized protein complexes in periciliary-ciliary transport processes. Moreover, our results provide additional evidence for a role of SANS-Magi2 assembly in endo-/exocytotic processes associated with cilia function.
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