Characterization of nascent HDL particles and microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I

The nascent HDL created by ABCA1-mediated efflux of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apolipoprotein A-I (apoA-I) has not been defined. To address this issue, we characterized the lipid particles released when J774 mouse macrophages and human skin fibroblasts in which ABCA1 is activated are incubated with human apoA-I. In both cases, three types of nascent HDL containing two, three, or four molecules of apoA-I per particle are formed. With J774 cells, the predominant species have hydrodynamic diameters of approximately 9 and 12 nm. These discoidal HDL particles have different FC contents and PL compositions, and the presence of acidic PL causes them to exhibit alpha-electrophoretic mobility. These results are consistent with ABCA1 located in more than one membrane microenvironment being responsible for the production of the heterogeneous HDL. Activation of ABCA1 also leads to the release of apoA-I-free plasma membrane vesicles (microparticles). These larger, spherical particles released from J774 cells have the same PL composition as the 12 nm HDL and contain CD14 and ganglioside, consistent with their origin being plasma membrane raft domains. The various HDL particles and microparticles are created concurrently, and there is no precursor-product relationship between them. Importantly, a large fraction of the cellular FC effluxed from these cells by ABCA1 is located in microparticles. Collectively, these results show that the products of the apoA-I/ABCA1 interaction include discoidal HDL particles containing different numbers of apoA-I molecules. The cellular PLs and cholesterol incorporated into these nascent HDL particles originate from different cell membrane domains.     

Direct detection of ABCA1-dependent HDL formation based on lipidation-induced hydrophobicity change in apoA-I

ABCA1 mediates the efflux of cholesterol and phospholipids into apoA-I to form HDL, which is important in the prevention of atherosclerosis. To develop a novel method for the evaluation of HDL formation, we prepared an apoA-I-POLARIC by labeling the specific residue of an apoA-I variant with a hydrophobicity-sensitive fluorescence probe that detects the environmental change around apoA-I during HDL formation. apoA-I-POLARIC possesses the intact ABCA1-dependent HDL formation activity and shows 4.0-fold higher fluorescence intensity in HDL particles than in the lipid-free state. Incubation of apoA-I-POLARIC with ABCA1-expressing cells, but not ABCA1-non-expressing cells, caused a 1.7-fold increase in fluorescence intensity. Gel filtration analysis demonstrated that the increase in fluorescence intensity of apoA-I-POLARIC represents the amount of apoA-I incorporated into the discoidal HDL particles rather than the amount of secreted cholesterol. THP-1 macrophage-mediated HDL formation and inhibition of HDL formation by cyclosporine A could also be measured using apoA-I-POLARIC. Furthermore, HDL formation-independent lipid release induced by microparticle formation or cell death was not detected by apoA-I-POLARIC. These results demonstrate that HDL formation by ABCA1-expressing cells can be specifically detected by sensing hydrophobicity change in apoA-I, thus providing a novel method for assessing HDL formation and screening of the HDL formation modulator.     

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 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.     

Effects of apolipoprotein A-I on ATP-binding cassette transporter A1-mediated efflux of macrophage phospholipid and cholesterol: formation of nascent high density lipoprotein particles

The mechanism of formation of high density lipoprotein (HDL) particles by the action of ATP-binding cassette transporter A1 (ABCA1) is not defined completely. To address this issue, we monitored efflux to apoA-I of phosphatidylcholine (PC), sphingomyelin (SM), and unesterified (free) cholesterol (FC) from J774 macrophages, in which ABCA1 is up-regulated, and investigated the nature of the particles formed. The various apoA-I/lipid particles appearing in the extracellular medium were separated by gel filtration chromatography. The presence of apoA-I in the extracellular medium led to the simultaneous formation of more than one type of poorly lipidated apoA-I-containing particle: there were 9- and 12-nm diameter particles containing approximately 3:1 and 1:1 phospholipid/FC (mol/mol), respectively, which were present together with 6-nm monomeric apoA-I. Removal of the C-terminal alpha-helix (residues 223-243) of apoA-I reduced phospholipid and FC efflux and prevented formation of the 9- and 12-nm HDL particles; the apoA-I variant formed larger particles that eluted in the void volume. FC loading of the J774 cells also led to the formation of larger apoA-I-containing particles that were highly enriched in FC. Besides creating HDL particles, ABCA1 mediated release of larger (20-450-nm diameter) FC-rich particles that were not involved in HDL formation and that are probably membrane vesicles. These particles contained 1:1 PC/SM in contrast to the HDL particles, which contained 2:1 PC/SM. This is consistent with lipid raft and non-raft plasma membrane domains being involved primarily in ABCA1-mediated vesicle release and nascent HDL formation, respectively.     

ATP-binding cassette A1-mediated lipidation of apolipoprotein A-I occurs at the plasma membrane and not in the endocytic compartments

ATP-binding cassette transporter (ABC) A1 is required for the lipidation of apolipoprotein A-I to generate high density lipoprotein (HDL). This process is proposed to occur through a retro-endocytosis pathway in which apoA-I internalizes with ABCA1 and generates HDL from the endosomal compartments before resecretion. The aim of this study was to determine the route of apoA-I endocytosis and whether endocytosis contributes to HDL biogenesis. Using confocal microscopy, we found that internalized apoA-I only transiently colocalized with transferrin, a retro-endocytosis marker. Instead, apoA-I perfectly colocalized with a bulk phase uptake marker (fluorescein isothiocyanate-dextran) and, at later time points, with LysoTracker in several cell models including macrophages, fibroblasts, and baby hamster kidney cells. ABCA1 colocalized poorly with internalized apoA-I. To determine the contribution of internalized apoA-I to HDL biogenesis, we specifically removed apoA-I from the cell surface and analyzed the fate of internalized apoA-I. We found that 23% of cell-associated apoA-I was internalized at steady state. Of internalized apoA-I, only 20% was converted to HDL, and the rest was degraded, consistent with a lysosomal destination. We also found that apoA-I was released approximately five times faster from the plasma membrane than from the intracellular compartments. From these kinetic parameters, we estimated that approximately 5.6% of apoA-I that interacts with cells is degraded and that internalized apoA-I contributes to approximately 1.4% of total HDL production. We also found that blocking endocytosis with sucrose or cytochalasin D did not decrease cholesterol efflux or HDL biogenesis. We therefore conclude that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs.     

ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol

Alpha-tocopherol (alpha-TOH) is associated with plasma lipoproteins and accumulates in cell membranes throughout the body, suggesting that lipoproteins play a role in transporting alpha-TOH between tissues. Here we show that secretion of alpha-TOH from cultured cells is mediated in part by ABCA1, an ATP-binding cassette protein that transports cellular cholesterol and phospholipids to lipid-poor high density lipoprotein (HDL) apolipoproteins such as apoA-I. Treatment of human fibroblasts and murine RAW264 macrophages with cholesterol and/or 8-bromo-cyclic AMP, which induces ABCA1 expression, enhanced apoA-I-mediated alpha-TOH efflux. ApoA-I lacked the ability to remove alpha-TOH from Tangier disease fibroblasts that have a nonfunctional ABCA1. BHK cells that lack an active ABCA1 pathway markedly increased secretion of alpha-TOH to apoA-I when forced to express ABCA1. ABCA1 also mediated a fraction of the alpha-TOH efflux promoted by lipid-containing HDL particles, indicating that HDL promotes alpha-TOH efflux by both ABCA1-dependent and -independent processes. Exposing apoA-I to ABCA1-expressing cells did not enhance its ability to remove alpha-TOH from cells lacking ABCA1, consistent with this transporter participating directly in the translocation of alpha-TOH to apolipoproteins. These studies provide evidence that ABCA1 mediates secretion of cellular alpha-TOH into the HDL metabolic pathway, a process that may facilitate vitamin transport between tissues and influence lipid oxidation.     

Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol

ATP-binding cassette transporter A1 (ABCA1) mediates transport of cellular cholesterol and phospholipids to high density lipoprotein (HDL) apolipoproteins, such as apoA-I. ABCA1 mutations can cause a severe HDL deficiency and atherosclerosis. Here we show that the protein-tyrosine kinase (TK) Janus kinase 2 (JAK2) modulates the apolipoprotein interactions with ABCA1 required for removing cellular lipids. The protein kinase A (PKA) inhibitor H89, the TK inhibitor genistein, and the JAK2 inhibitor AG490 suppressed apoA-I-mediated cholesterol and phospholipid efflux from ABCA1-expressing cells without altering the membrane ABCA1 content. Whereas PKA inhibition had no effect on apoA-I binding to cells or to ABCA1, TK and JAK2 inhibition greatly reduced these activities. Conversely, PKA but not JAK2 inhibition significantly reduced the intrinsic cholesterol translocase activity of ABCA1. Mutant cells lacking JAK2 had a severely impaired apoA-I-mediated cholesterol and phospholipid efflux and apoA-I binding despite normal ABCA1 protein levels and near normal cholesterol translocase activity. Thus, although PKA modulates ABCA1 lipid transport activity, JAK2 appears to selectively modulate apolipoprotein interactions with ABCA1. TK-mediated phosphorylation of ABCA1 was undetectable, implicating the involvement of another JAK2-targeted protein. Acute incubation of ABCA1-expressing cells with apoA-I had no effect on ABCA1 phosphorylation but stimulated JAK2 autophosphorylation. These results suggest that the interaction of apolipoproteins with ABCA1-expressing cells activates JAK2, which in turn activates a process that enhances apolipoprotein interactions with ABCA1 and lipid removal from cells.     

ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes

High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.     

Stimulation of lipolysis enhances the rate of cholesterol efflux to HDL in adipocytes

Adipose tissue constitutes a major location for cholesterol storage and, as such, it may play a role in the regulation of circulating cholesterol levels. A possible metabolic link between the lipolytic activity of adipocytes and their ability to release cholesterol to reconstituted human high density lipoprotein, HDL, was investigated in 3T3-L1 adipocytes. In the presence of HDL, composed of human apoA-I and phosphatidylcholine, adipocytes release cholesterol in a lipoprotein-dose and time dependent fashion. β-adrenergic activation of the lipolysis promotes a 22% increase in the extent of cholesterol efflux to reconstituted discoidal HDL particles. Activation of lipolysis promotes a rapid decrease in the cholesterol content of the plasma membrane and a concomitant increase in lipid droplet cholesterol. This change is independent of the presence of HDL. Activation of the lipolysis does not affect the levels of ABCA1 and SR-BI. Therefore, the enhancement of cholesterol efflux is not due to the level of plasma membrane cholesterol, or to the levels of the cholesterol transporters ABCA1 and scavenger receptor SR-BI. Brefeldin A did not affect the rate of cholesterol efflux under basal lipolytic conditions, but it abolished the lipolysis-dependent enhancement of cholesterol efflux to HDL. This study suggests that activation of lipolysis is accompanied by an increase in BFA-sensitive vesicular transport that in turn enhances cholesterol efflux to HDL. The study supports a metabolic link between the lipolytic activity of adipocytes and the rate of cellular cholesterol efflux to HDL.     

The role of the ABCA1 transporter and cholesterol efflux in familial hypoalphalipoproteinemia

Defects in the gene encoding for the ATP binding cassette (ABC) transporter A1 (ABCA1) were shown to be one of the genetic causes for familial hypoalphalipoproteinemia (FHA). We investigated the role of ABCA1-mediated cholesterol efflux in Dutch subjects suffering from FHA. Eighty-eight subjects (mean HDL cholesterol levels 0.63 +/- 0.21 mmol/l) were enrolled. Fibroblasts were cultured and loaded with [3H]cholesterol. ABCA1 and non-ABCA1-mediated efflux was studied by using apolipoprotein A-I (apoA-I), HDL, and methyl-beta-cyclodextrin as acceptors. Efflux to apoA-I was decreased in four patients (4/88, 4.5%), and in all cases, a mutation in the ABCA1 gene was found. In the remaining 84 subjects, no correlation between efflux and apoA-I or HDL cholesterol was found. Efflux to both HDL and cyclodextrin, in contrast, did correlate with HDL cholesterol plasma levels (r = 0.34, P = 0.01; and r = 0.27, P = 0.008, respectively). The prevalence of defects in ABCA1-dependent cholesterol efflux in Dutch FHA patients is low. The significant correlation between plasma HDL cholesterol levels and methyl-beta-cyclodextrin-mediated efflux in the FHA patients with normal ABCA1 function suggests that non-ABCA1-mediated efflux might also be important for plasma HDL cholesterol levels in these individuals.     

Lysine residues of ABCA1 are required for the interaction with apoA-I

ATP-binding cassette protein A1 (ABCA1) plays a pivotal role in cholesterol homeostasis by generating high-density lipoprotein (HDL). Apolipoprotein A-I (apoA-I), a lipid acceptor for ABCA1, reportedly interacts with ABCA1. However, it has also been proposed that apoA-I interacts with ABCA1-generated special domains on the plasma membrane, but apart from ABCA1, and solubilizes membrane lipids. To determine the importance of the apoA-I-ABCA1 interaction in HDL formation, the electrostatic interaction between apoA-I and ABCA1, which mediates the interaction between apoB100 in low-density lipoprotein particles (LDL) and LDL receptor, was analyzed. The apoA-I binding to ABCA1 and the cross-linking between them were inhibited by the highly charged molecules heparin and poly-L-lysine. Treating cells with membrane impermeable reagents that specifically react with primary amino groups abolished the interaction between apoA-I and ABCA1. However, these reagents did not affect the characteristic tight ATP binding to ABCA1. These results suggest that lysine residues in the extracellular domains of ABCA1 contribute to the interaction with apoA-I. The electrostatic interaction between ABCA1 and apoA-I is predicted to be the first step in HDL formation. This article is part of a Special Issue entitled Advances in high density lipoprotein formation and metabolism: a tribute to John F. Oram (1945-2010).     

Enhanced apoA-I-dependent cholesterol efflux by ABCA1 from sphingomyelin-deficient Chinese hamster ovary cells

ATP binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. It is proposed that ABCA1 reorganizes the plasma membrane and generates more loosely packed domains that facilitate apoA-I-dependent cholesterol efflux. In this study, we examined the effects of the cellular sphingomyelin level on HDL formation by ABCA1 by using a Chinese hamster ovary-K1 mutant cell line, LY-A, which has a missense mutation in the ceramide transfer protein CERT. When LY-A cells were cultured in Nutridoma-BO medium and sphingomyelin content was reduced, apoA-I-dependent cholesterol efflux by ABCA1 from LY-A cells increased 1.65-fold compared with that from LY-A/CERT cells stably transfected with human CERT cDNA. Exogenously added sphingomyelin significantly reduced the apoA-I-dependent efflux of cholesterol from LY-A cells, confirming that the decrease in sphingomyelin content in the plasma membrane stimulates cholesterol efflux by ABCA1. The amount of cholesterol available to cold methyl-beta-cyclodextrin (MbetaCD) extraction from LY-A cells was increased by 40% by the expression of ABCA1 and was 1.6-fold higher than that from LY-A/CERT cells. This step in ABCA1 function, making cholesterol available to cold MbetaCD, was independent of apoA-I. These results suggest that the function of ABCA1 could be divided into two steps: (i) a flopping step to move phosphatidylcholine and cholesterol from the inner to outer leaflet of the plasma membrane, where cholesterol becomes available to cold MbetaCD extraction, and (ii) a loading step to load phosphatidylcholine and cholesterol onto apoA-I to generate HDL.     

Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation

ABCA1 plays a major role in HDL metabolism. Cholesterol secretion by ABCA1 is dependent on the presence of extracellular acceptors, such as lipid-free apolipoprotein A-I (apoA-I). However, the importance of the direct interaction between apoA-I and ABCA1 in HDL formation remains unclear. In contrast, ABCB4 mediates the secretion of phospholipids and cholesterol in the presence of sodium taurocholate (NaTC) but not in the presence of apoA-I. In this study, we analyzed apoA-I binding and NaTC-dependent lipid efflux by ABCA1. ABCA1 mediated the efflux of cholesterol and phospholipids in the presence of NaTC as well as in the presence of apoA-I in an ATP-dependent manner. The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. However, the W590S mutation did not impair apoA-I binding and, conversely, retarded the dissociation of apoA-I from ABCA1. These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading, facilitates apoA-I dissociation from ABCA1. NaTC is a good tool for analyzing ABCA1-mediated lipid efflux and allows dissection of the steps of HDL formation by ABCA1.     

The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo

ATP-binding cassette transporter A-1 (ABCA1)-mediated lipid efflux to lipid-poor apolipoprotein A-I (apoA-I) results in the gradual lipidation of apoA-I. This leads to the formation of discoidal high-density lipoproteins (HDL), which are subsequently converted to spherical HDL by the action of lecithin:cholesterol acyltransferase (LCAT). We have investigated the effect of point mutations and deletions in the carboxy-terminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I-deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Delta(232-243)] deletion mutant, or the apoA-I[E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] mutants formed mainly prebeta-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Delta(232-243)], and apoA-I[E191A/H193A/K195A] formed spherical alpha-HDL particles. The findings establish that (a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of alpha-HDL but allow the synthesis of prebeta-HDL particles in vivo, (b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of prebeta-HDL-type particles in an ABCA1-independent process, and (c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.     

ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions

ATP binding cassette transporter A1 (ABCA1) mediates the transport of phospholipids and cholesterol from cells to lipid-poor HDL apolipoproteins. Cholesterol loading of cells induces ABCA1, implicating cholesterol as its major physiologic substrate. It is believed, however, that ABCA1 is primarily a phospholipid transporter and that cholesterol efflux occurs by diffusion to ABCA1-generated phospholipid-rich apolipoproteins. Here we show that overexpression of ABCA1 in baby hamster kidney cells in the absence of apolipoproteins redistributed membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. The cholesterol removed by apolipoprotein A-I (apoA-I), but not by HDL phospholipids, was derived exclusively from these domains. ABCA1 overexpression also increased cholesterol esterification, which was prevented by addition of apoA-I, suggesting that some of the cell-surface cholesterol not removed by apolipoproteins is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. ABCA1 expression was essential for cholesterol efflux even when apolipoproteins had already acquired phospholipids during prior exposure to ABCA1-expressing cells.These studies show that ABCA1 redistributes cholesterol to cell-surface domains, where it becomes accessible for removal by apolipoproteins, consistent with a direct role of ABCA1 in cholesterol transport.     

Cellular cholesterol substrate pools for adenosine-triphosphate cassette transporter A1-dependent high-density lipoprotein formation

PURPOSE OF REVIEW: The removal of cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I), facilitated by the membrane transporter ATP-binding cassette transporter A1 (ABCA1), is the rate-limiting step in the formation of high density lipoprotein particles. This review summarizes recent literature concerning the relative contributions of different cellular pools of cholesterol used by ABCA1 in the initial lipidation of apoA-I for high density lipoprotein particle formation. RECENT FINDINGS: Cell culture studies have shown that apart from lipidating apoA-I directly, ABCA1 can also mediate cholesterol delivery indirectly to apoA-I in the plasma membrane. Moreover, it is now clear that the late endosome/lysosome pool of cholesterol is a critical part of the total cholesterol substrate pool for ABCA1. Internalization of ABCA1 appears to be a requirement for maximum ABCA1-mediated cholesterol mobilization for high density lipoprotein formation. SUMMARY: Current evidence suggests that ABCA1-mediated cholesterol efflux to apoA-I involves mobilization of cholesterol from plasma membrane, endoplasmic reticulum, trans-Golgi network, late endocytic and lysosomal compartments, and cholesteryl ester droplets. Apart from lipidating apoA-I directly, ABCA1 has also been found to efflux cholesterol indirectly to apoA-I in plasma membranes.