title: Disease-causing mutations in BEST1 gene are associated with altered sorting of bestrophin-1 protein.
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.
authors.affiliation: Biological Faculty, Sofia University "Saint Kliment Ohridski", 8 Dragan Tzankov str, Sofia 1164, Bulgaria. doumanov@biofac.uni-sofia.bg
authors.forename: Jordan A
authors.initials: JA
authors.lastname: Doumanov
authors.forename: Christina
authors.initials: C
authors.lastname: Zeitz
authors.forename: Paloma
authors.initials: P
authors.lastname: Dominguez Gimenez
authors.forename: Isabelle
authors.initials: I
authors.lastname: Audo
authors.forename: Abhay
authors.initials: A
authors.lastname: Krishna
authors.forename: Giovanna
authors.initials: G
authors.lastname: Alfano
authors.forename: Maria Luz Bellido
authors.initials: ML
authors.lastname: Diaz
authors.forename: Veselina
authors.initials: V
authors.lastname: Moskova-Doumanova
authors.forename: Marie-Elise
authors.initials: ME
authors.lastname: Lancelot
authors.forename: José-Alain
authors.initials: JA
authors.lastname: Sahel
authors.forename: Emeline F
authors.initials: EF
authors.lastname: Nandrot
authors.forename: Shomi S
authors.initials: SS
authors.lastname: Bhattacharya
mesh_terms: D000818:Animals; D000075002:Bestrophins; D002460:Cell Line; D018118:Chloride Channels / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D005136:Eye Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D057826:Vitelliform Macular Dystrophy / Q000235:genetics / Q000378:metabolism / Q000473:pathology
chemical_list: C113133:BEST1 protein, human; D000075002:Bestrophins; D018118:Chloride Channels; D005136:Eye Proteins; D007041:Hypoxanthine Phosphoribosyltransferase