Abstract
Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.
Highlights
Cellular cholesterol efflux occurs by at least two distinct mechanisms
Distribution of ABCA1 between Triton X-100 (TX-100)-soluble and -insoluble Membranes—ABCA1 plays a critical role in apolipoproteinmediated cholesterol efflux [15,16,17,18,19,20], perhaps by transporting cellular lipids across the plasma membrane to apolipoproteins and/or shuttling lipids between other cellular compartments and the plasma membrane
high density lipoproteins (HDL) phospholipids promote efflux of plasma membrane cholesterol by a passive diffusion process that is facilitated by HDL binding to SR-BI [9, 10] and suggested to involve caveolae [60]
Summary
Cell Culture—All cell culture incubations were performed at 37 °C in a humidified 5% CO2 incubator. Cholesterol and Phospholipid Efflux—Efflux of [3H]cholesterol from cells was measured by the appearance of label in medium after incubation with acceptors as described in detail previously [5,6,7]. Supernatants and pellets were extracted with CHCl3:CH3OH (3:2; v/v), and lipids were subjected to TLC as above to separate free and esterified cholesterol or phospholipids as indicated in individual experiments. For selective labeling of plasma membrane proteins, fibroblasts were incubated for 30 min at 0 °C with phosphate-buffered saline containing 1 mg/ml sulfo-N-hydroxysuccinimide-biotin to biotinylate cell-surface proteins [48]. The volume of SDS buffer added per lane represented the fraction of protein partitioning between TX-100soluble and -insoluble membranes.
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