ABC transporters are a superfamily of proteins which actively transport a variety of compounds across cell membranes. Mammalian and most eukaryotic ABC transporters function as exporters, flipping or extruding substrates from the cytoplasmic to the extracellular or lumen side of cell membranes. Prokaryotic ABC transporters function either as exporters or importers. Here, we show that ABCA4, an ABC transporter found in retinal photoreceptor cells and associated with Stargardt macular degeneration, is a novel importer, actively flipping N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic leaflet of disc membranes, thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptors. ABCA4 also actively transports phosphatidylethanolamine (PE) in the same direction. Mutations known to cause Stargardt disease decrease the retinoid and PE transport activity of ABCA4. These studies provide the first direct evidence for a mammalian ABC transporter functioning as an importer and reveal insight into mechanisms underlying substrate transport and Stargardt disease.

Background: Lipid transport by the A-subfamily of ATP-binding cassette (ABC) transporters implicated in human disorders is poorly understood. Results: ABCA1 and ABCA7 export phosphatidylcholine and phosphatidylserine across membranes, whereas ABCA4 imports phosphatidylethanolamine. Conclusion: ABCA transporters actively flip phospholipids across membranes, a function severely reduced for disease-causing mutants. Significance: ABCA-dependent phospholipid transport plays a crucial role in cellular lipid homeostasis.

ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone-rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of abca4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidenephosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders.

Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle tranfficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis among others. P4-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membranes proteins that actively transport phospholipids across cellular membranes. P4-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P4-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport.

The visual cycle is a series of enzyme-catalyzed reactions which converts all-trans-retinal to 11-cis-retinal for the regeneration of visual pigments in rod and cone photoreceptor cells. Although essential for vision, 11-cis-retinal like all-trans-retinal is highly toxic due to its highly reactive aldehyde group and has to be detoxified by either reduction to retinol or sequestration within retinal-binding proteins. Previous studies have focused on the role of the ATP-binding cassette transporter ABCA4 associated with Stargardt macular degeneration and retinol dehydrogenases (RDH) in the clearance of all-trans-retinal from photoreceptors following photoexcitation. How rod and cone cells prevent the accumulation of 11-cis-retinal in photoreceptor disk membranes in excess of what is required for visual pigment regeneration is not known. Here we show that ABCA4 can transport N-11-cis-retinylidene-phosphatidylethanolamine (PE), the Schiff-base conjugate of 11-cis-retinal and PE, from the lumen to the cytoplasmic leaflet of disk membranes. This transport function together with chemical isomerization to its all-trans isomer and reduction to all-trans-retinol by RDH can prevent the accumulation of excess 11-cis-retinal and its Schiff-base conjugate and the formation of toxic bisretinoid compounds as found in ABCA4-deficient mice and individuals with Stargardt macular degeneration. This segment of the visual cycle in which excess 11-cis-retinal is converted to all-transretinol provides a rationale for the unusually high content of PE and its long-chain unsaturated docosahexaenoyl group in photoreceptor membranes and adds insight into the molecular mechanisms responsible for Stargardt macular degeneration.T he visual cycle plays a crucial role in the removal of all-transretinal from photoreceptor cells following photoexcitation and its conversion to 11-cis-retinal for the regeneration of visual pigments in rod and cone photoreceptor cells (1). Deficient clearance of all-trans-retinal and its Schiff-base conjugate N-retinylidenephosphatidylethanolamine (PE) from rod and cone photoreceptor outer segments results in condensation reactions which produce a mixture of bisretinoid products including the pyridinium bisretinoid compound A2PE and its hydrolytic product A2E (2, 3). These bisretinoid compounds accumulate as lipofuscin deposits in retinal pigment epithelial (RPE) cells upon phagocytosis of outer segments and have been implicated in the pathology of a number of retinal degenerative diseases. This is particularly evident in autosomal recessive Stargardt macular degeneration in which mutations in the ATP-binding cassette (ABC) transporter ABCA4 which impair the N-retinylidene-PE transport activity of ABCA4 cause a buildup of lipofuscin, atrophy of the central retina, and severe progressive loss in vision (4-8).In initial studies, Abca4 knockout mice were reported to show a light-dependent accumulation of all-trans-retinal, PE, and N-retinylidene-PE in photoreceptors and lipofuscin and A2E in RPE (9). This led to a model...