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Disease-causing Mutations Associated With Four Bestrophinopathies Exhibit Disparate Effects on the Localization, but Not the Oligomerization, of Bestrophin-1

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Disease-causing Mutations Associated With Four Bestrophinopathies Exhibit Disparate Effects on the Localization, but Not the Oligomerization, of Bestrophin-1

Adiv A Johnson et al. Exp Eye Res.

Erratum in

  • Exp Eye Res. 2014 Oct;127:300

Abstract

BEST1 encodes Bestrophin-1 (Best1), a homo-oligomeric, integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium. Mutations in BEST1 cause five distinct retinal degenerative diseases, including adult vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). The mechanisms underlying these diseases and why mutations cause one disease over another are, for the most part, unknown. To gain insights into these four diseases, we expressed 28 Best1 mutants fused to YFP in polarized MDCK monolayers and, via confocal microscopy and immunofluorescence, live-cell FRET, and reciprocal co-immunoprecipitation experiments, screened these mutants for defects in localization and oligomerization. All 28 mutants exhibited comparable FRET efficiencies to and co-immunoprecipitated with WT Best1, indicating unimpaired oligomerization. RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane of cells, while two AVMD and most ARB mutants were mislocalized. When co-expressed, all mislocalized mutants caused mislocalization of WT Best1 to intracellular compartments. Our current and past results indicate that mislocalization of Best1 is not an absolute feature of any individual bestrophinopathy, occurring in AVMD, BVMD, and ARB. Furthermore, some ARB mutants that do not also cause dominant disease cause mislocalization of Best1, indicating that mislocalization is not a cause of disease, and that absence of Best1 activity from the plasma membrane is tolerated. Lastly, we find that the ARB truncation mutants L174Qfs*57 and R200X can form oligomers with WT Best1, indicating that the first ∼174 amino acids of Best1 are sufficient for oligomerization to occur.

Keywords: MDCK; bestrophin; fluorescence resonance energy transfer; retinal pigment epithelium; retinitis pigmentosa; vitelliform dystrophy.

Figures

Figure 1
Figure 1. Whole-cell chloride currents mediated by untagged, CFP-tagged and YFP-tagged Best1 channels
(A) Whole-cell currents recorded from HEK293 cells expressing untagged Best1 (WT) or Best1 tagged with CFP or YFP, as indicated. Dashed lines represent the zero current level. Voltage protocol is shown in the upper panel. (B) Mean whole-cell current density versus voltage relationship. The number of experiments was 5-8 in each case. Tagged and untagged Best1 currents were significantly higher than those observed in untransfected cells, indicating that the C-terminal addition of CFP or YFP does not abolish the associated anion channel activity of Best1.
Figure 2
Figure 2. Localization of Best1 and ADVIRC-, RP-, as well as AVMD-associated mutants in MDCK cells as determined by confocal microscopy
Representative X-Y and X-Z scans of WT or mutant Best1-YFP (green) are shown. WT or mutant Best1 were expressed in confluent, polarized monolayers of MDCK cells via adenovirus-mediated gene transfer and stained for the endogenous apical protein gp135 (red) and nuclei (blue) for positional referencing. All ADVIRC (V86M, V235A, Y236C, and V239M) and RP (L140V, I205T, Y227C, and D228N) mutants localized to the basolateral plasma membrane. For AVMD-associated mutants, T6P and D312N were intracellular while R47H, A146L, and A243V were properly localized. Ap and Bl stand for apical and basolateral, respectively. Scale bars: 20 μm.
Figure 3
Figure 3. Localization of ARB-associated mutants in MDCK cells as determined by confocal microscopy
Representative X-Y and X-Z scans of mutant Best1-YFP (green) are shown. Mutant Best1 was expressed in confluent, polarized monolayers of MDCK cells via adenovirus-mediated gene transfer and stained for the endogenous apical protein gp135 (red) and nuclei (blue) for positional referencing. L41P, P101T, N179del, A195V, L472PfsX10, and H490QfsX24 were localized to the basolateral plasma membrane. In contrast, R141H, R141S, P152A, L174Qfs*57, L191P, R200X, E213G, V317M, and M325T were found in intracellular compartments. Scale bars: 20 μm.
Figure 4
Figure 4. Co-localization X-Y scans between mislocalized AVMD- and ARB-associated mutants and WT Best1 as determined by confocal microscopy
Representative merged X-Y scans of WT and mutant Best1-YFP co-expressed with Best1-c-myc are shown. Confluent, polarized monolayers of MDCK cells were made to express both Best1-c-myc and WT or mutant Best1-YFP (green) via adenovirus-mediated gene transfer. Cells were stained for c-myc (red) and, for positional referencing, the endogenous apical protein gp135 (cyan). Best1-YFP co-localized with Best1-c-myc in the basolateral plasma membrane, while all 11 mislocalized mutants (T6P, R141H, R141S, P152A, L174Qfs*57, L191P, R200X, E213G, D312N, V317M, and M325T Best1) co-localized with Best1-c-myc predominantly in intracellular compartments. Scale bar: 20 μm.
Figure 5
Figure 5. Co-localization X-Z scans between mislocalized AVMD- and ARB-associated mutants and WT Best1 as determined by confocal microscopy
Representative merged X-Z scans of WT and mutant Best1-YFP co-expressed with Best1-c-myc are shown. Confluent, polarized monolayers of MDCK cells were made to express both Best1-c-myc and WT or mutant Best1-YFP (green) via adenovirus-mediated gene transfer. Cells were stained for c-myc (red) and, for positional referencing, the endogenous apical protein gp135 (cyan). Best1-YFP co-localized with Best1-c-myc in the basolateral plasma membrane, while all 11 mislocalized mutants (T6P, R141H, R141S, P152A, L174Qfs*57, L191P, R200X, E213G, D312N, V317M, and M325T Best1) co-localized with Best1-c-myc predominantly in intracellular compartments. Scale bar: 20 μm.
Figure 6
Figure 6. Reciprocal co-immunoprecipitation of Best1-c-myc and WT or mutant Best1-YFP in MDCK cells
(A) MDCK cells were transduced to express both Best1-c-myc (~75 kDa) and WT or mutant Best1-YFP (~95 kDa) using adenovirus-mediated gene transfer. Co-expressing cells were lysed and Best1 immunoprecipitated using an anti-YFP antibody and western blotted using an antibody specific to c-myc. Control lanes were loaded with immunoprecipitates prepared from uninfected MDCK cells. All other lanes were loaded with immunoprecipitated Best1. (B) Excluding the four ARB truncation mutants, MDCK cells were transduced to express both Best1-c-myc (~75 kDa) and WT or mutant Best1-YFP (~95 kDa) using adenovirus-mediated gene transfer. Co-expressing cells were lysed and Best1 immunoprecipitated using an anti-c-myc antibody and western blotted using an antibody specific to YFP. (C) Lanes were loaded with MDCK cells co-expressing WT Best1-c-myc and YFP-tagged ARB truncation mutants (L174Qfs*57, R200X, L472PfsX10, and H490QfsX24 Best1-YFP ). Cells were immunoprecipitated using an antibody specific to c-myc and blotted back using an antibody specific to YFP. (D) MDCK cells were infected with either Best1-c-myc or Best1-YFP via adenovirus-mediated gene transfer. Best1 was immunoprecipitated using anti-cmyc or anti-YFP antibodies and blotted back using an antibody specific to gp135 (135 kDa). Lysate lanes were loaded using lysates from cells infected with either Best1-c-myc or Best1-YFP. (E) MDCK cells were infected with Best1-YFP or with one of the truncation mutants (L174Qfs*57, R200X, L472PfsX10, and H490QfsX24 Best1-YFP). Best1 was immunoprecipitated using an anti-YFP antibody and blotted back using a separate anti-YFP antibody.
Figure 7
Figure 7. Live-cell, confocal FRET acceptor photobleaching of Best1-CFP and WT or mutant Best1-YFP in MDCK cells
(A) Representative X-Y scan of Best1-CFP (blue, donor) and Best1-YFP (yellow, acceptor) co-expressed in polarized MDCK cells using adenovirus-mediated gene transfer. Using a 514 nm laser on a confocal microscope, live-cell acceptor photobleaching was performed, generating the resultant image in (B), which highlights regions in the plasma membrane where donor intensity was increased post-bleaching. (C) For all experiments, n ≥ 19. FRET efficiencies (% E) were determined for Best1-CFP paired with WT or mutant Best1-YFP via adenovirus-mediated gene transfer. As a positive control, MDCK cells were transfected with a CFP-YFP fusion protein. As a negative control, MDCK cells were cotransfected with Best1-CFP and YFP. WT and mutant Best1 had % E's that did not significantly differ from each other, but did significantly differ from the negative and positive controls (p < 0.001). Scale bar: 10 μm. Error bars indicate ± S.D.

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