Insertion and Topology of Normal and Mutant Bestrophin-1 in the Endoplasmic Reticulum Membrane

Abstract

The vitelliform macular dystrophy type 2 (VMD2) gene mutated in Best macular dystrophy encodes a 585-amino acid putative transmembrane protein termed bestrophin-1. The vast majority of known disease-associated alterations are of the missense type, which cluster near predicted transmembrane domains (TMDs). To investigate bestrophin-1 membrane topology and to assess consequences of point mutations on membrane integration, we have analyzed the insertion of putative TMDs into the endoplasmic reticulum (ER) membrane. Out of six potential TMDs, our data suggest a topological model of bestrophin-1 with four transmembrane-spanning segments and one large cytoplasmatic loop between putative TMD2 and TMD5. Consequently, a relatively hydrophobic segment containing putative TMD3 (aa 130 -149) and TMD4 (aa 179 -201) is located within the cytoplasm. Furthermore, we show that three out of 18 disease-associated alterations investigated (I73N, Y85H, F281del) reveal measurable effects on membrane insertion suggesting that defective membrane integration of bestrophin-1 may represent a potential disease mechanism for a small subset of Best macular dystrophy-related mutations.Bestrophin-1 is encoded by the vitelliform macular dystrophy type 2 (VMD2) 2 gene as a 585-amino acid putative integral transmembrane protein localized to the basolateral aspect of the retinal pigment epithelium (RPE) (1). Disease-related mutations in the gene are responsible for the autosomal dominant Best macular dystrophy (BMD) (2, 3) but have also been associated with a minor proportion of cases presenting with adult vitelliforme macular dystrophy (4), bulls eye maculopathy (5), and autosomal dominant vitreoretinochoroidopathy (6). Thus far, a total of 100 distinct mutations have been identified including 92 in BMD, four in adult vitelliforme macular dystrophy, one in bulls eye maculopathy, and three in autosomal dominant vitreoretinochoroidopathy patients (for further information see BMD mutation data base). Of these, more than 93% are missense mutations, distributed in a non-random fashion across the highly conserved N-terminal half of the protein.Clinically, BMD is characterized by striking yellowish lesions in the macular area eventually leading to a decline in central vision at later stages in disease pathology. The disease manifests in teenage years although with reduced penetrance and considerable variability in phenotypic expression. Histopathologically, aberrant remnants of heterogeneous material including proteins, lipids, and lipofuscin-containing particles are found in the central retina at the level of the RPE. Typically, BMD patients display a characteristic electrooculogram (EOG) abnormality with a greatly reduced light peak/dark trough ratio reflecting RPE dysfunction (7).Accumulating experimental evidence suggests bestrophin-1, but also its highly conserved family members bestrophin-2, bestrophin-3 and bestrophin-4 (8, 9), to be involved in Ca 2ϩ -dependent transport of chloride ions across cellular membranes. It is still unclear whether bes...

The GABA A receptor α1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation

“…Although many disease-causing mutations result in protein misfolding, the misfolding typically alters function, assembly, or subcellular trafficking; protein topology is not affected (17). Recently, using an in vitro bacterial protein leader peptidase (Lep) translation system, Milenkovic et al (18) suggested that mutations in the macular degeneration-associated bestrophin-1 protein inhibit transmembrane segment insertion. However, we are only aware of one mutation proven to alter topology in a native protein; the prion-protein (PrP) A117V mutation found in GerstmannStraussler-Scheinker disease increases the fraction of the transmembrane form of the PrP (19).…”

The Best Disease-Linked Cl−ChannelhBest1Regulates CaV1 (L-type) Ca2+Channels via src-Homology-Binding Domains

“…3A) (Tsunenari et al, 2003;Milenkovic et al, 2007). To identify the region of hBest1 responsible for inhibition of Ca V 1.3, we cotransfected Ca V 1.3 subunits with cDNAs corresponding to either the C-terminal (CT ) or N-terminal (NT 1-289 ) part of hBest1.…”

Stoichiometry and specific assembly of Best ion channels

“…Two differing topology models have been proposed for Best channels (15,23). For simplicity, we numbered the domains according to the recent Milenkovic model (Fig.…”

“…hBest1 contains six hydrophobic segments (S1-S6), with both N and C termini residing inside the cell. Two topology models have been proposed for hBest1 (15,23). In the first model, S1, S2, S4, and S6 traverse the membrane, whereas S3 is intracellular and S5 forms a reentrant loop from outside (15).…”

“…In the first model, S1, S2, S4, and S6 traverse the membrane, whereas S3 is intracellular and S5 forms a reentrant loop from outside (15). A more recent model has S1, S2, S5, and S6 traversing the membrane and S3 and S4, although hydrophobic, remaining on the intracellular side (23).…”