Differential effects of best disease causing missense mutations on bestrophin-1 trafficking

Mutations in bestrophin-1 (Best1) cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited retinal degenerative disease. Best1 is a homo-oligomeric anion channel localized to the basolateral surface of retinal pigment epithelial (RPE) cells. A number of Best1 mutants mislocalize in Madin-Darby canine kidney (MDCK) cells. However, many proteins traffic differently inMDCK and RPE cells, andMDCKcells do not express endogenousBest1. Thus, effects of Best1mutationsonlocalizationinMDCKcellsmaynot translate toRPEcells. To determine whether BVMD causing mutations affect Best1 localization, we compared localization and oligomerization of Best1 with Best1 mutants V9M,W93C, and R218C. InMDCKcells, Best1 and Best1^R218C were basolaterally localized. Best1^W93C and Best1^V9M accumulated in cells. In cultured fetal human retinal pigment epithelium cells (fhRPE) expressing endogenous Best1, Best1^R218C and Best1^W93C were basolateral. Best1^V9M wasintracellular. All three mutants exhibitedsimilar fluorescence resonance energy transfer (FRET) efficiencies to, and co-immunoprecipitated with Best1, indicating unimpaired oligomerization. When human Best1 was expressed in RPE in mouse eyes it was basolaterally localized. However, Best1^V9M accumulated in intracellular compartments inmouseRPE. Co-expression of Best1 and Best1^W93C inMDCKcells resultedin basolateral localization of both Best1 andBest1^W93C, but co-expression of Best1 with Best1^V9M resultedinmislocalization of both proteins.Weconclude that different mutations in Best1 cause differential effects on its localization and that this effect varies with the presence or absence of wild-type (WT) Best1. Furthermore, MDCK cells can substitute for RPE when examining the effects of BVMD causing mutations on Best1 localization if co-expressed with WT Best1.