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

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

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Received: 14 May 2013; in revised form: 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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Abstract: 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 which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

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.

AMA Style

Doumanov JA, Zeitz C, Gimenez PD, Audo I, Krishna A, Alfano G, Diaz MLB, Moskova-Doumanova V, Lancelot M-E, Sahel J-A, Nandrot EF, Bhattacharya SS. Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein. International Journal of Molecular Sciences. 2013; 14(7):15121-15140.

Chicago/Turabian Style

Doumanov, Jordan A.; Zeitz, Christina; Gimenez, Paloma D.; Audo, Isabelle; Krishna, Abhay; Alfano, Giovanna; Diaz, Maria L.B.; Moskova-Doumanova, Veselina; Lancelot, Marie-Elise; Sahel, José-Alain; Nandrot, Emeline F.; Bhattacharya, Shomi S. 2013. "Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein." Int. J. Mol. Sci. 14, no. 7: 15121-15140.



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