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ABCA4 structure-function relationships : role in Stargardt disease and related retinal degenerative diseases

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dc.contributor.author Zhong, Ming
dc.date.accessioned 2009-04-15T20:20:41Z
dc.date.available 2009-04-15T20:20:41Z
dc.date.copyright 2009 en
dc.date.issued 2009-04-15T20:20:41Z
dc.identifier.uri http://hdl.handle.net/2429/7111
dc.description.abstract ABCA4, also known as ABCR or the rim protein, is a member of the family of ATP binding cassette (ABC) proteins expressed in rod and cone photoreceptors. Mutations in ABCA4 have been linked to Stargardt macular degeneration and related retinal degenerative diseases and implicated in the transport of retinoid compounds across the outer segment disk membrane. This dissertation investigation describes various aspects of the structure and function relationships for ABCA4 and examines the mechanisms by which mutations in ABCA4 lead to various retinal degenerative diseases. A pull-down was employed to identify the retinoid substrate that interacts with ABCA4. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, ~1 mol of N-retinylidene-phosphatidylethanolamine was bound per mol of ABCA4 with an apparent Kd of 5.4 μM. These results provided the first direct biochemical evidence for the identity of the retinoid substrate for ABCA4. To determine the role of that C-terminus of ABCA4 plays in structure and function, a series of deletion and chimera mutants of ABCA4 was expressed, purified by immunoaffinity chromatography, and their biochemical properties analyzed. Removal of the C-terminal 30 amino acids including a conserved VFVNFA motif or substitution of the VFVNFA motif with alanines resulted in the complete loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal stimulated ATPase activity and caused retention of ABCA4 in the endoplasmic reticulum. In contrast mutants lacking the C-terminal 8, 16 or 24 amino acids but retaining the VFVNFA motif were active. These studies indicated that the VFVNFA motif in ABCA4 is required for proper folding of ABCA4 into a functionally active protein. These results provide a molecular rationale for the disease phenotype displayed by individuals with mutations in the C-terminus of ABCA4. Co-IP studies coupled to mass spectrometry were performed to identify novel protein interactors of ABCA4. Rhodopsin and arrestin (including a splice variant of arrestin, p⁴⁴) were identified and confirmed by western blotting. All-trans-retinal was found as a regulator of this interaction. This study for the first time has identified the retinoid substrate for ABCA4, demonstrated a role for the C-terminus and has found protein partners of ABCA4. en
dc.format.extent 2594261 bytes
dc.format.mimetype application/pdf
dc.language.iso eng en
dc.publisher University of British Columbia
dc.relation.ispartof Electronic Theses and Dissertations (ETDs) 2008+ en
dc.title ABCA4 structure-function relationships : role in Stargardt disease and related retinal degenerative diseases en
dc.type Text
dc.degree.name Doctor of Philosophy - PhD en
dc.degree.discipline Biochemistry and Molecular Biology en
dc.degree.grantor University of British Columbia
dc.date.graduation 2009-05 en
dc.type.text Thesis/Dissertation en
dc.description.affiliation Medicine, Faculty of
dc.degree.campus UBCV en
dc.description.scholarlevel Graduate en

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