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Int. J. Mol. Sci. 2013, 14(7), 15121-15140; doi:10.3390/ijms140715121

Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein

1,* , 2,3,4
7, 5
1 Biological Faculty, Sofia University "Saint Kliment Ohridski", 8 Dragan Tzankov str, Sofia 1164, Bulgaria 2 Institut National de la Santé et de la Recherche Médicale (INSERM), UMR_S 968, Paris F-75012, France 3 Centre National de la Recherche Scientifique (CNRS), UMR_7210, Paris F-75012, France 4 Centre de Recherche Institut de la Vision, Université Pierre et Marie Curie-Paris 6, 17 rue Moreau, Paris F-75012, France 5 Andalusian Center of Molecular Biology and Regenerative Medicine, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Avda. Americo Vespucio s/n, Parque Cientifico y Tecnologico, Isla de la Cartuja 41092, Sevilla, Spain 6 Centre de Référence Maladies Rares/Centre d'Investigation Clinique (CMR/CIC), 503 INSERM, CHNO des Quinze-Vingts, Paris F-75012, France 7 Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK 8 Fondation Ophtalmologique Adolphe de Rothschild, Paris F-75019, France
* Author to whom correspondence should be addressed.
Received: 14 May 2013 / Revised: 2 July 2013 / Accepted: 4 July 2013 / Published: 22 July 2013
(This article belongs to the collection Proteins and Protein-Ligand Interactions)
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Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery.
Keywords: BVMD; Best1 protein; cell polarity; MDCK cells BVMD; Best1 protein; cell polarity; MDCK cells
This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Doumanov, J.A.; Zeitz, C.; Gimenez, P.D.; Audo, I.; Krishna, A.; Alfano, G.; Diaz, M.L.B.; Moskova-Doumanova, V.; Lancelot, M.-E.; Sahel, J.-A.; Nandrot, E.F.; Bhattacharya, S.S. Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein. Int. J. Mol. Sci. 2013, 14, 15121-15140.

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