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Cloning, characterization, and localization of an ABC transporter in the rod photoreceptor rims implicated in Stargardt's and age-related macular distrophy

University of British Columbia

Outer segments of vertebrate photoreceptor cells contain an abundantly expressed membrane protein that migrates with an apparent molecular mass of 220-290 kDa by SDS gel electrophoresis. Twenty years ago, immunocytochemical studies localized the 290 kDa frog protein to the rims of the photoreceptor disks and since then it has been generally believed that this rim protein is responsible for the filaments observed that link adjacent disks and/or disks to the plasma membrane. Due to its localization and abundance in the outer segment, this protein is likely a good candidate for involvement in retinal disease. In this study, the primary structure of the bovine 220 kDa protein has been determined by cDNA cloning and direct peptide sequence analysis. A data base homology search showed that it is a new member of the ABC (ATP-binding cassette) transporter superfamily, which includes the cystic fibrosis transmembrane conductance regulator (CFTR), multi-drug resistance proteins P-glycoprotein and MRP, and bacterial transporters. Generally, these proteins utilize the energy of ATP hydrolysis by two nucleotide binding domains (NBDs) for transport of a substrate across the membrane. Clones containing the human 220 kDa orthologue were found to be identical to a recently cloned retina cell-specific ABC transporter (Allikmets et al., Nature Genetics, 15, 236, 1997). Mutations in the gene encoding this protein (ABCR) have been implicated in Stargardt's disease, and more recently, in age-related macular dystrophy. Studies of immunoaffinity purified 220 kDa protein showed that both ATP and GTP bind this protein with similar affinities. In addition, the protein contains at least one carbohydrate chain and, when solubilized with detergent, does not appear to co-purify with other proteins. Immunocytochemical and biochemical studies have revealed that the 220 kDa glycoprotein is distributed along the rim region and incisures of rod outer segment disk membranes in a pattern similar to that observed for the 290 kDa protein in frog photoreceptors. Two cytoplasmic fragments each containing one NBD were expressed in Escherichia coli and purified. Both of the NBDs were capable of hydrolyzing ATP and GTP at rates comparable to NBDs of other ABC transporters. These experiments suggest that the energy of nucleotide hydrolysis supports active transport of a substrate across the disk membrane. This study serves as a basis for beginning to define the role of the ABC transporter in rod photoreceptor structure and function and elucidating the molecular basis for how specific mutations in the ABCR gene, particularly those in the NBD, lead to either Stargardt's disease or AMD.

Thesis/Dissertation

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2009-06-03

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http://hdl.handle.net/2429/8683

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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