Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoprotein s occurs by an active mechanism distinct from passive diffusion and is cont rolled by the ATP-binding cassette transporter ABCA1, Here we examined whet her ABCA1-mediated lipid efflux involves the selective removal of lipids as sociated with membrane rafts, plasma membrane domains enriched in cholester ol and sphingomyelin, ABCA1 was not associated with cholesterol and sphingo lipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid eff lux to apoA-I was accounted for by decreases in cellular lipids not associa ted with cholesterol/sphingomyelin-rich membranes. Treating cells with fili pin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosp hatidylcholine efflux, In contrast, efflux of cholesterol to high density l ipoproteins (HDL) or plasma was partially accounted for by depletion of cho lesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Ape-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 depl ete 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 r afts do not provide lipid for efflux promoted by apolipoproteins through th e ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated w ith these domains.