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V.M. Milenkovic, A. Rivera, G. von Heijne, J.R. Casey, B.H. F. Weber; Insertion and Topology of Normal and Mutant Bestrophin in the Endoplasmic Reticulum Membrane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5132.
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© ARVO (1962-2015); The Authors (2016-present)
PURPOSE: Best vitelliform macular dystrophy (BMD) is an autosomal dominant, early onset macular degeneration associated with striking accumulation of lipofuscin–like material at the level of the retinal pigment epithelium (RPE). The vitelliform macular dystrophy type 2 (VMD2) gene associated with BMD encodes a 585 amino acid transmembrane protein termed bestrophin. Recently, the protein has been shown to be involved in Ca2+–dependent transport of chloride ions across the basolateral membrane of the RPE. The vast majority of known disease–associated alterations are missense mutations which cluster near six predicted transmembrane domains (TMDs). With the present study, we aim to further examine bestrophin topology, and to assess the consequences of BMD mutations on membrane integration. Methods: In vitro translation of constructs encoding wild–type and mutated bestrophin was done with E.coli inner membrane protein leader peptidase (Lep) as an insertion vehicle. The transmembraneous H2 segment of Lep was exchanged by a series of putative TMDs and C–terminally truncated bestrophin. Upon in vitro translation in the presence of canine pancreas microsomes, the glycosylation status of the chimeric constructs was used as a measure of membrane insertion capability. Cysteine–free constructs and those containing single cysteine residues located in putative loops were generated and used for cysteine–scanning mutagenesis. Results: In vitro translation of the various truncated bestrophin molecules in a microsome–containing environment provides evidence that bestrophin spans the ER membrane 4 times with one relatively hydrophobic segment,the putative TMD3 (aa 179–201) located within the cytoplasm. TMD3 is showing signal anchor (SA) activity as an individual segment, but not in the context of the C–terminally truncated constructs. Three (I73N, Y85H, F281del) out of 16 disease–related mutations were found to show measurable effects on membrane insertion. Conclusions: Our data suggest a topological model of bestrophin with 4 TMDs and one large cytoplasmatic loop between TMD2 and 4. In addition, some of the disease–associated mutations interfere with correct membrane insertion likely causing a defective channel subunit assembly.
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