Role of the C Terminus of the Photoreceptor ABCA4 Transporter in Protein Folding, Function, and Retinal Degenerative Diseases

ABCA4 is an ATP-binding cassette transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases, including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4, including a conserved VFVNFA motif, resulted in a loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal-stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast, C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C terminus of ABCA4. ABCA4 is an ATP-binding cassette transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases, including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4, including a conserved VFVNFA motif, resulted in a loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal-stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast, C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C terminus of ABCA4. ABCA4, also known as ABCR or the rim protein, is a member of the ABCA subfamily of ATP binding cassette transporters expressed in vertebrate photoreceptor cells (1Allikmets R. Singh N. Sun H. Shroyer N.E. Hutchinson A. Chidambaram A. Gerrard B. Baird L. Stauffer D. Peiffer A. Rattner A. Smallwood P. Li Y.X. Anderson K.L. Lewis R.A. Nathans J. Leppert M. Dean M. Lupski J.R. Nat. Genet.. 1997; 15: 236-246Google Scholar, 2Illing M. Molday L.L. Molday R.S. J. Biol. Chem.. 1997; 272: 10303-10310Google Scholar, 3Molday L.L. Rabin A.R. Molday R.S. Nat. Genet.. 2000; 25: 257-258Google Scholar, 4Azarian S.M. Travis G.H. FEBS Lett.. 1997; 409: 247-252Google Scholar). It is localized along the rims and incisures of rod and cone outer segment discs, where it has been implicated in the binding and transport of the Schiff base adduct of all-trans-retinal and phosphatidylethanolamine (PE) 3The abbreviations used are: PE, phosphatidylethanolamine; N-ret-PE, N-retinylidenephosphatidylethanolamine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; NBD, nucleotide-binding domain; ER, endoplasmic reticulum; WT, wild type. known as N-retinylidene-PE or N-ret-PE across disc membranes as part of visual cycle (3Molday L.L. Rabin A.R. Molday R.S. Nat. Genet.. 2000; 25: 257-258Google Scholar, 5Weng J. Mata N.L. Azarian S.M. Tzekov R.T. Birch D.G. Travis G.H. Cell.. 1999; 98: 13-23Google Scholar, 6Sun H. Molday R.S. Nathans J. J. Biol. Chem.. 1999; 274: 8269-8281Google Scholar, 7Beharry S. Zhong M. Molday R.S. J. Biol. Chem.. 2004; 279: 53972-53979Google Scholar, 8Molday R.S. J. Bioenerg. Biomembr.. 2007; 39: 507-517Google Scholar, 9Papermaster D.S. Schneider B.G. Zorn M.A. Kraehenbuhl J.P. J. Cell Biol.. 1978; 78: 415-425Google Scholar). To date, over 500 different mutations in the ABCA4 gene are known to cause Stargardt macular degeneration, an early onset recessive disease characterized by the loss in central vision, the presence of lipofuscin deposits in retinal pigment epithelial cells, a delay in dark adaptation, and progressive degeneration of photoreceptor and retinal pigment epithelial cells (1Allikmets R. Singh N. Sun H. Shroyer N.E. Hutchinson A. Chidambaram A. Gerrard B. Baird L. Stauffer D. Peiffer A. Rattner A. Smallwood P. Li Y.X. Anderson K.L. Lewis R.A. Nathans J. Leppert M. Dean M. Lupski J.R. Nat. Genet.. 1997; 15: 236-246Google Scholar, 10Allikmets R. Am. J. Hum. Genet.. 2000; 67: 793-799Google Scholar, 11Rozet J.M. Gerber S. Souied E. Ducroq D. Perrault I. Ghazi I. Soubrane G. Coscas G. Dufier J.L. Munnich A. Kaplan J. Mol. Genet. Metab.. 1999; 68: 310-315Google Scholar, 12Rivera A. White K. Stohr H. Steiner K. Hemmrich N. Grimm T. Jurklies B. Lorenz B. Scholl H.P. Apfelstedt-Sylla E. Weber B.H. Am. J. Hum. Genet.. 2000; 67: 800-813Google Scholar, 13Gelisken O. De Laey J.J. Int. Ophthalmol.. 1985; 8: 225-235Google Scholar, 14Fishman G.A. Farber M. Patel B.S. Derlacki D.J. Ophthalmology.. 1987; 94: 809-814Google Scholar, 15Fishman G.A. Farbman J.S. Alexander K.R. Ophthalmology.. 1991; 98: 957-962Google Scholar). Mutations in ABCA4 are also responsible for other related but more severe retinal degenerative diseases, including autosomal recessive cone-rod dystrophy and retinitis pigmentosa (16Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J. Hayden M.R. Nat. Genet.. 1999; 22: 336-345Google Scholar, 17Cremers F.P. van de Pol D.J. van Driel M. den Hollander A.I. van Haren F.J. Knoers N.V. Tijmes N. Bergen A.A. Rohrschneider K. Blankenagel A. Pinckers A.J. Deutman A.F. Hoyng C.B. Hum. Mol. Genet.. 1998; 7: 355-362Google Scholar, 18Martinez-Mir A. Paloma E. Allikmets R. Ayuso C. del Rio T. Dean M. Vilageliu L. Gonzalez-Duarte R. Balcells S. Nat. Genet.. 1998; 18: 11-12Google Scholar, 19Fishman G.A. Stone E.M. Eliason D.A. Taylor C.M. Lindeman M. Derlacki D.J. Arch. Ophthalmol.. 2003; 121: 851-855Google Scholar). Finally, individuals heterozygous for selected disease-linked mutations in ABCA4 have been suggested to be at higher risk in developing age-related macular degeneration (20Allikmets R. Shroyer N.F. Singh N. Seddon J.M. Lewis R.A. Bernstein P.S. Peiffer A. Zabriskie N.A. Li Y. Hutchinson A. Dean M. Lupski J.R. Leppert M. Science.. 1997; 277: 1805-1807Google Scholar). Disease-associated mutations are distributed throughout the ABCA4 gene and comprise missense, splice site, and nonsense mutations as well as small deletion and insertions resulting in a truncated protein. Biochemical studies indicate that disease-linked mutations in ABCA4 cause complete or partial loss in retinal-stimulated ATPase activity (21Sun H. Smallwood P.M. Nathans J. Nat. Genet.. 2000; 26: 242-246Google Scholar). ABCA4 is most similar to ABCA1, an ABC transporter implicated in the efflux of cholesterol and phospholipids from cells (16Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J. Hayden M.R. Nat. Genet.. 1999; 22: 336-345Google Scholar, 22Attie A.D. Trends Biochem. Sci.. 2007; 32: 172-179Google Scholar, 23Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest.. 1999; 104: Scholar). ABCA4 and ABCA1 are over in amino have a similar of a segment by a a and a nucleotide-binding S. Molday L.L. Molday R.S. J. Biol. Chem.. Scholar, E. S. Y. A. 18: Scholar). In transporters an C-terminal of amino acids in from the in the C-terminal of the C terminus is by the that a in ABCA4, which the removal of the C-terminal 30 amino acids of ABCA4, is responsible for cone-rod dystrophy S. S. I. E. R. M. L. Clin. and a to the deletion of the C-terminal amino acids of ABCA1 is with an autosomal recessive characterized by a loss in and of cholesterol in J. B. P. D. G. B. J.M. J. 2000; Scholar). K. J.J. J. Biol. Chem.. 2004; 279: have C-terminal deletion mutants of ABCA1, including the with studies that a conserved VFVNFA motif in the C-terminal of ABCA1 with an protein to the binding of to ABCA1 and efflux of cholesterol from cells. VFVNFA motif is also the C-terminal 30 amino acids of ABCA4, but role in transporter has been In we have characterized a number of C-terminal deletion and substitution mutants of ABCA4, including mutants known to cause cone-rod dystrophy and Stargardt macular degeneration S. S. I. E. R. M. L. Clin. Scholar, A. M. N. A. B. E. R. Dean M. Allikmets R. L. Hum. Genet.. Scholar). Our studies indicate that the VFVNFA motif the 30 C-terminal amino acids a role in the folding of ABCA4 into a functionally active protein. and ATP were from was from and including and were from removal was from and was a of and were and as by and 2000; Scholar). of were as CHAPS, and and CHAPS, and in was an of and a the C terminus of ABCA4 and the to the C-terminal of have been M. Molday L.L. Molday R.S. J. Biol. Chem.. 1997; 272: 10303-10310Google Scholar, D. A. P. Molday R.S. Scholar, R.S. J.M. Molday L. Molday R.S. J. Biol. Chem.. Scholar). purified was from the C-terminal deletion mutants of ABCA4 an were by the in the as a a that and and an and that and and and and and and an and the for C-terminal deletion mutants of ABCA1 were also by in the as the a that and of ABCA1 with a site, and that to and and a and the for the with the C terminus of ABCA4 with the C terminus of ABCA1 the VFVNFA motif and in which the C terminus of ABCA1 was with the C terminus of ABCA4 were by substitution mutants and in which the VFVNFA motif in ABCA4 and ABCA1 were with alanine residues were by of and ABCA4 and ABCA1 in HEK 293T and of cells were in with and of cells at was with of C. H. Mol. Cell Biol.. 1987; 7: Scholar). cells from or were in of from of cells were as S. Molday L.L. Molday R.S. J. Biol. Chem.. but in a the from or was a and to a of and was for 30 at in a at the the and were at and at a of of in of and To of the expressed ABCA4 of membranes was to of at was for and at for in a was with and by for analysis by To determine the of of membranes was with of and by for by of binding all-trans-retinal was as S. Zhong M. Molday R.S. J. Biol. Chem.. 2004; 279: 53972-53979Google Scholar). were at HEK 293T cells from were at for and in of for was with of in the the protein was with of by and with of all-trans-retinal activity of 500 in for 30 was with of to all-trans-retinal and in the presence or of ATP for was more with of to the ABCA4 with were more the all-trans-retinal from the with of for at by all-trans-retinal in the was by were in for In the were to of the was in the and used as a to for in the of the of were from the to determine with of was used to determine the of protein in ATPase of HEK 293T cells were as for binding protein was with of or for at in an was with of to protein. was with of or in at for of and was and were ABCA4 were into the of Sun H. Molday R.S. Nathans J. J. Biol. Chem.. 1999; 274: 8269-8281Google with of PE, other was with of in ABCA4 protein was and the was for 30 was and the was of with in a with a ABCA4 or ABCA1 protein was at by with a and was to ATPase activity ATPase were and as J. Molday R.S. J. Biol. Chem.. 2000; Scholar). ATPase were in a of of protein and of or was by the of of a ATP to a of 30 at of was of the was a and in was to a for and in a to ATP and were of the of to the of ATP in the was was in were to determine ATP which was from the ATP of protein in and vesicles was by the of of ABCA4 with that of of were by and to membranes at for 30 in a a of membranes were with in for 30 and with in for in the membranes were for 30 with with in and in to analysis with a of of protein in was with of in the was to at a of for was with and the were by and by were for of membranes was by and to analysis for protein of cells were and with the the cells were in and for in in at were and for 30 at in the and the cells were with the for at in and with in and with the ABCA4 protein was with by to and was with a by to was a or a C-terminal the ATPase of the to was used to ABCA4 from rod outer segments and ABCA4 from cells for studies M. Molday L.L. Molday R.S. J. Biol. Chem.. 1997; 272: 10303-10310Google Scholar, 6Sun H. Molday R.S. Nathans J. J. Biol. Chem.. 1999; 274: 8269-8281Google Scholar, 7Beharry S. Zhong M. Molday R.S. J. Biol. Chem.. 2004; 279: 53972-53979Google Scholar, H. Smallwood P.M. Nathans J. Nat. Genet.. 2000; 26: 242-246Google Scholar, J. Molday R.S. J. Biol. Chem.. 2000; Scholar). the is to the C terminus acids of ABCA4, the be used to ABCA4 C-terminal deletion mutants for biochemical To we have a to the C terminus of and mutant ABCA4. This by the D. A. P. Molday R.S. has been to be for the of for studies Molday R.S. S. 1987; Scholar, Molday R.S. J. Biol. Chem.. Scholar). it was to determine the of the has the biochemical of ABCA4. This was by ABCA4 and in HEK 293T cells and the from membranes a and expressed at similar and were to the in of the to the with the similar of ABCA4 as with into ABCA4 and displayed similar and ATPase for ABCA4 and were and of ATP of protein, HEK 293T cells with were used in or ATPase activity was the from cells was a or results indicate that the C-terminal the or activity of ABCA4, and the and retinal-stimulated ATPase are to expressed ABCA4. ABCA4 and ABCA1 the of the C terminus the biochemical of ABCA4, we C-terminal deletion C-terminal mutant in which the C-terminal segment of ABCA4 was with the C-terminal segment of ABCA1, and C-terminal mutant in which a conserved VFVNFA motif was with alanine residues related ABCA1 mutants were also for and mutant a C-terminal for and we have in the of the ABCA4 used in of C-terminal the and of of of ABCA4 and the mutants was by membranes from HEK 293T cells in for analysis by and and mutants as from of the of of the mutants was that of ABCA4 with the of which expressed at of ABCA4. of to the ABCA4 mutants from HEK 293T membranes was also that mutants as a but at that a of ABCA4 a was in at a with ABCA4, the and mutants at and of ABCA4. and the mutants at that of ABCA4. of the mutants was to the of protein as by the and mutants with ABCA4 that a of mutants were of ABCA4 of the C-terminal ABCA4 deletion mutants to ATP was by photoaffinity from HEK 293T cells were with with and by for analysis of a in and ABCA4, and were to a similar mutant displayed a higher of for the of protein the mutant labeling, and the a conserved VFVNFA motif of to C-terminal we have that purified ABCA4 from rod outer segments an N-ret-PE in the of ATP S. Zhong M. Molday R.S. J. Biol. Chem.. 2004; 279: 53972-53979Google Scholar). of ATP N-ret-PE to from ABCA4. have used to N-ret-PE binding to the C-terminal mutants mutants N-ret-PE in the of ATP at with ABCA4. N-ret-PE substrate binding was the of part of we also purified ABCA1 was of binding in binding of N-ret-PE to ABCA1 was of ABCA1 and analysis of ATPase and mutant ABCA4 and ABCA1 were purified from of cells a of the protein from the with the ABCA4 and ABCA1 transporters were as an molecular of protein was in and mutant ABCA4 and ABCA1 of protein to be but it most a protein that is to a of expressed ABCA of C-terminal Mutations and ATPase and ATPase activity was for the C-terminal ABCA4 mutants into vesicles mutants retained of ATPase activity and mutants ATPase activity that was by the of all-trans-retinal at similar to ABCA4. mutant and ATPase the mutant the VFVNFA motif displayed activity and by mutant activity and by To the of the VFVNFA motif in retinal-stimulated ATPase we and expressed an ABCA4 mutant in which the amino acids of motif were with alanine residues in the was of retinal-stimulated ATPase activity. of C-terminal and Mutations the ATPase of ABCA1 and the of ABCA1 C-terminal deletion mutations the efflux cholesterol in the presence and of has been K. J.J. J. Biol. Chem.. 2004; 279: their the ATPase activity of the purified transporter was part of we the ATPase activity of C-terminal deletion mutants of ABCA4 with C-terminal ABCA1 deletion mutants in ATPase activity of ABCA4 was with deletion of the C was for ABCA1 deletion higher ATPase activity was for the deletion mutants the C-terminal and amino In and were the mutant ATPase activity to ABCA4, the mutant activity to results that segments of ABCA4 of the VFVNFA motif the ATPase activity of ABCA4 and ABCA1, segments of ABCA1 of motif the ATPase activity of to the function of the mutant has been to be in cholesterol efflux K. J.J. J. Biol. Chem.. 2004; 279: Scholar). of ABCA4 studies in have that ABCA4 expressed in cells to intracellular vesicles of with the intracellular localization of ABCA4 in photoreceptor cells M. Molday L.L. Molday R.S. J. Biol. Chem.. 1997; 272: 10303-10310Google Scholar, J. S. Molday L.L. Molday R.S. J. Biol. Chem.. 2003; Scholar). In the we have the localization of the and mutant ABCA4 expressed in HEK 293T cells. ABCA4 and deletion mutants and a similar with the of the protein localized to intracellular and In contrast, of the and mutants in the VFVNFA motif and most of the mutant a of the the localization of the of cells part of we also the of the and mutant ABCA1 expressed in HEK cells. localized to the of cells, as N. S. K. Y. E. T. A. of the and most of the mutant displayed a of in the mutant localized to the and studies that the of and mutants are in a that the protein to the and as intracellular vesicles in HEK 293T cells. In contrast, of the and mutants are that are retained in the by the of cells. ABCA1 to the most of the is retained in the In we have purified and characterized a series of C-terminal deletion and substitution mutants of ABCA4 in order to the role of the C terminus of ABCA4 in protein structure and function and to into the molecular for retinal degenerative with mutations in segment of the ABCA4 Our studies indicate that a conserved VFVNFA motif is required for the of a protein. mutant the VFVNFA motif to the substrate N-ret-PE, was with and and retinal-stimulated ATPase activity. the mutant in which the VFVNFA motif was with alanine residues was by In contrast, the and mutants that have the VFVNFA motif are functionally Our results that the VFVNFA motif is required for the of a functionally active ABCA4 protein is in with the studies of K. J.J. J. Biol. Chem.. 2004; 279: that conserved motif is for the function of ABCA1 in binding and cholesterol efflux from cells K. J.J. J. Biol. Chem.. 2004; 279: Scholar). the VFVNFA motif for In the of ABCA1, the VFVNFA motif has been suggested to with an protein to binding to an of ABCA1 and cholesterol efflux from cells K. J.J. J. Biol. Chem.. 2004; 279: Scholar). Our studies ABCA4 indicate that the VFVNFA motif a role in proper folding of the protein into a functionally active that to ABCA4 but the or mutants were in purified protein as the mutant the VFVNFA motif in to and other ABCA4 deletion that most of the expressed protein was the mutant protein the VFVNFA motif that was was functionally for ATPase activity. the of and from that of ABCA4 and the other functionally active deletion mutants as by of cells. In ABCA4, and localized to intracellular in HEK 293T cells, a for expressed and photoreceptor that with ABCA4 along the rim of rod and cone J. S. Molday L.L. Molday R.S. J. Biol. Chem.. 2003; Scholar, Molday R.S. Sci.. Scholar, A.F. Molday R.S. Scholar). In contrast, the and mutants localized in a of of in the studies indicate that the VFVNFA motif of ABCA4 is required for the proper folding of ABCA4 into Our studies that the VFVNFA motif contributes to the proper folding of and mutants the VFVNFA motif and to be of cholesterol efflux activity K. J.J. J. Biol. Chem.. 2004; 279: were retained in the ER, that the loss of function of mutants is to protein and the loss in as suggested K. J.J. J. Biol. Chem.. 2004; 279: Scholar). in the role of VFVNFA in ABCA4 and ABCA1, are in analysis of the ATPase activity of the deletion ABCA1 C-terminal deletion and displayed a higher ATPase activity ABCA1, the ABCA4 and a in ATPase activity with ABCA4 the VFVNFA motif is for activity of ABCA4 protein segments of motif to a role in the of ABCA4. higher but and retinal-stimulated ATPase that amino acids of the VFVNFA motif or with the to ATP binding and in J. S. Molday L.L. Molday R.S. J. Biol. Chem.. 2003; Scholar). of is in the and analysis of the mutant in which the C-terminal amino acids of ABCA4 are with the C-terminal segment of This mutant retained to N-ret-PE but a in and retinal-stimulated ATPase activity. mutant with and a with a of the protein retained in the results that the ABCA1 C-terminal segment with efficient folding of ABCA4 and has a binding and an was for the mutant in which the C-terminal segment of ABCA1 was with that of ABCA4. In the ATPase activity of mutant was the activity of ABCA1 and in an K. J.J. J. Biol. Chem.. 2004; 279: a higher cholesterol efflux activity in the presence or of was for mutant to the C terminus of ABCA4 to residues that the activity of ABCA1, with the the C terminus of ABCA1 has residues that the folding of ABCA4 and activity. of C-terminal ABCA4 from a conserved of the VFVNFA motif, which to ATPase activity of the mutant ABCA1 also a conserved of the VFVNFA studies the of conserved in the of ABCA Mutations in the ABCA4 gene have been to cause a number of retinal degenerative of has been in which the of the disease phenotype is to the of activity displayed by the mutant ABCA4 A. van Driel M.A. van de Pol D.J. van Haren F.J. Tijmes N. Bergen A.A. Rohrschneider K. Blankenagel A. Pinckers A.J. N. Deutman A.F. Hoyng C.B. F.P. Am. J. Hum. Genet.. 1999; Scholar, N.F. Lewis R.A. Allikmets R. Singh N. Dean M. Leppert M. Lupski J.R. 1999; 39: Scholar). pigmentosa, the most severe disease phenotype with complete loss in vision, mutations in cause the complete loss in as for the of mutations truncated the other of the age-related macular degeneration, the phenotype in individuals are heterozygous for selected disease-linked mutations in ABCA4. individuals retain a of ABCA4 activity from the wild-type rod dystrophy and Stargardt macular degeneration are in with the resulting from mutations that cause a but loss in ABCA4 function and the resulting from mutations that in partial loss in activity. Our studies C-terminal deletion mutants are with and provide into the different displayed by with deletion mutations in the C terminus of ABCA4. with cone-rod dystrophy was to be heterozygous for a which the loss in the C-terminal amino acids of ABCA4, including the VFVNFA motif and a S. S. I. E. R. M. L. Clin. Scholar). severe phenotype of is with the complete loss in activity for the to retain of activity at with to photoaffinity (21Sun H. Smallwood P.M. Nathans J. Nat. Genet.. 2000; 26: 242-246Google Scholar). with a of Stargardt macular degeneration was to be heterozygous for a in and a in the other A. M. N. A. B. E. R. Dean M. Allikmets R. L. Hum. Genet.. Scholar). mutant protein is with the mutant in that the protein is truncated the VFVNFA in the mutant activity. of the in ABCA4 has been based analysis of other disease-linked in the of ABCA4 (21Sun H. Smallwood P.M. Nathans J. Nat. Genet.. 2000; 26: 242-246Google it is that mutant also at activity. mutations are to provide activity to the phenotype of Stargardt macular degeneration. In we have that the VFVNFA motif is required for proper folding of ABCA4 into a functionally active protein. of motif to the activity of ABCA4, or with the of ABCA4. analysis of the C-terminal deletion mutants a molecular based rationale for the phenotype displayed by with mutations in of ABCA4. for the of ABCA4 and ABCA1

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