Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 14, Issue 7, Pages 15121-15140Publisher
MDPI
DOI: 10.3390/ijms140715121
Keywords
BVMD; Best1 protein; cell polarity; MDCK cells
Funding
- Agence Nationale de la Recherche
- Fondation Voir et Entendre
- Centre National de la Recherche Scientifique (CNRS)
- Fondation Bettencourt Schueller
- Universite Pierre et Marie Curie-Paris6
- Foundation Fighting Blindness [CD-CL-0808-0466-CHNO]
- Centre d'Investigation Clinique 503 [C-CMM-0907-0428-INSERM04]
- Fundacion Progreso y Salud
- Instituto de Salud Carlos III [CM06/00183]
- Bulgarian National Science Fund [DDVU 02/10]
- Institut National de la Sante et de la Recherche Medicale
- Universite Pierre et Marie Curie-Paris 6
- Centre National de la Recherche Scientifique
- Department de Paris
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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.
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