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.     

Membrane cholesterol dynamics: cholesterol domains and kinetic pools

Nonreceptor mediated cholesterol uptake and reverse cholesterol transport in cells occur through cellular membranes. Thus, elucidation of cholesterol dynamics in membranes is essential to understanding cellular cholesterol accumulation and loss. To this end, it has become increasingly evident that cholesterol is not randomly distributed in either model or biologic membranes. Instead, membrane cholesterol appears to be organized into structural and kinetic domains or pools. Cholesterol-rich and poor domains can even be observed histochemically and physically isolated from epithelial cell surface membranes. The physiologic importance of these domains is 2-fold: (i) Select membrane proteins (receptors, transporters, etc.) are localized in either cholesterol-rich or cholesterol-poor domains. Consequently, the structure and properties of the domains rather than of the bulk lipid may selectively affect the function of proteins residing therein. (ii) Kinetic evidence suggests that cholesterol transport through and between membranes may occur through specific domains or pools. Regulation of the size and properties of such domains may be controlling factors of cholesterol transport or accumulation in cells. Recent technologic advances in the use of fluorescent sterols have allowed examination of cholesterol domain structure in model and biologic membranes. These techniques have been applied to examine the role of high-density lipoprotein, cholesterol lowering drugs, and intracellular lipid transfer proteins in membrane sterol domain structure and sterol movement between membranes.     

Membrane proteins and phospholipids as effectors of reverse cholesterol transport

The review highlights the membrane aspect of cholesterol efflux from cell membranes to high density lipoproteins (HDL), an initial stage of reverse cholesterol transport to liver. In addition to traditional viewpoints considering cholesterol transport as the step of sequential lipoprotein transformation, which involves blood plasma apoproteins and proteins transporters, employment of proteomic approaches has shown the active role of cell plasma membranes as cholesterol donors and plasma membrane bound proteins in cholesterol transport. These include ATP-binding ABC-A1 transporter and membrane receptor SR-B1. There is experimental and clinical evidence that impairment of genes encoding these proteins cause impairments of reverse cholesterol transport (e.g. Tangier disease and genetic manipulations with experimental animals.) Although precise mechanism involving these membrane proteins remains unknown it is suggested that ABC-AI with free plasma apoA1 facilitates the efflux of membrane phospholipids and formation of their complex with apoAI. This complex accepts membrane cholesterol, with simultaneous formation of a full HDL particle. In certain cells there is correlation between cholesterol efflux into HDL and expression of SR-BI, which reversibly binds to HDL. This receptor protein may influence molecular organization of membrane phospholipids and cholesterol, facilitating cholesterol efflux. The review also deals with properties of ABC-A1 and SR-B1, putative mechanisms of their effects, the role of these proteins in reverse cholesterol transport and their functional coupling to the phospholipid matrix of biomembranes.     

Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface

Cultured cells have on their cell surface a specific high-affinity binding site (receptor) for high density lipoproteins (HDL) which appears to promote cholesterol efflux. In this study we characterized the cellular mechanisms involved in HDL receptor-mediated transport of cholesterol from cultured human fibroblasts and bovine aortic endothelial cells. HDL3, chemically modified by tetranitromethane (TNM-HDL3), is not recognized by this receptor and was used as a control for efflux not mediated by HDL receptor binding. HDL3 and TNM-HDL3 were found to be equally effective in causing efflux of plasma membrane cholesterol radiolabeled with [3H]cholesterol. However, HDL3 was much more effective than TNM-HDL3 in causing efflux of [3H]cholesterol associated with intracellular membranes. By measuring movement of endogenously synthesized [3H]cholesterol to the plasma membrane, and into the medium, we found that HDL3 induced a rapid movement of [3H]cholesterol from a preplasma membrane compartment to the plasma membrane that preceded [3H]cholesterol efflux. This effect was not observed with TNM-HDL3. Thus, receptor binding of HDL3 appears to facilitate removal of cellular cholesterol from specific intracellular pools by initiation of translocation of intracellular cholesterol to the plasma membrane.     

The molecular structure of apolipoprotein A-II modulates the capacity of HDL to promote cell cholesterol efflux

The influence of apolipoprotein A-II (apoA-II) molecular structure on the capacity of high density lipoproteins (HDL) to promote cellular cholesterol efflux was investigated in cultured mouse peritoneal macrophages (MPM). Conversion by reduction and carboxamidomethylation of the naturally occurring dimeric apoA-II to its monomeric form in both native or reconstituted HDL did not change apolipoprotein secondary structure and lipoprotein size/composition. All particles containing monomeric apoA-II, i.e., native HDL₃ or reconstituted HDL with or without apoA-I, showed a higher ability to promote cholesterol efflux originating from plasma membrane and intracellular stores, compared to particles containing dimeric apoA-II. These findings indicate that apolipoprotein molecular structure is a major determinant of HDL capacity to promote cholesterol efflux from cells.     

HDLs induce raft domain vanishing in heterogeneous giant vesicles

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.     

ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cholesterol from cells to high density lipoprotein (HDL) but not to lipid-depleted apolipoprotein A-I. Here we show that human ABCG1 overexpressed in baby hamster kidney cells in the absence of lipoproteins traffics to the plasma membrane and redistributes membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. Cholesterol removed by HDL was largely derived from these domains in ABCG1 transfectants but not in cells lacking ABCG1. Overexpression of ABCG1 also increased cholesterol esterification, which was decreased by the addition of HDL, suggesting that a proportion of the cell-surface cholesterol not removed by HDL is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. A 638-amino acid ABCG1, which lacked the 40 N-terminal amino acids of the predicted full-length protein, was fully functional and of a similar size to ABCG1 expressed by cholesterol-loaded human monocyte-derived macrophages. Mutating an essential glycine residue in the Walker A motif abolished ABCG1-dependent cholesterol efflux and esterification and prevented localization of ABCG1 to the cell surface, indicating that the ATP binding domain in ABCG1 is essential for both lipid transport activity and protein trafficking. These studies show that ABCG1 redistributes cholesterol to cell-surface domains where it becomes accessible for removal by HDL, consistent with a direct role of ABCG1 in cellular cholesterol transport.     

Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking

The hypothesis of lipid domains in cellular plasma membranes is well established. However, direct visualization of the domains has been difficult. Here we report direct visualization of plasma membrane cholesterol microdomains modulated by agents that affect cholesterol trafficking to and from the plasma membrane. The cholesterol microdomains were visualized with a monoclonal antibody that specifically detects ordered cholesterol arrays. These unique cholesterol microdomains were induced on macrophages and fibroblasts when they were enriched with cholesterol in the presence of an ACAT inhibitor, to block esterification of excess cellular cholesterol. Induction of the plasma membrane cholesterol microdomains could be blocked by agents that inhibit trafficking of cholesterol to the plasma membrane and by cholesterol acceptors that remove cholesterol from the plasma membrane. In addition, plasma membrane cholesterol microdomains did not develop in mutant Niemann-Pick type C fibroblasts, consistent with the defect in cholesterol trafficking reported for these cells.The induction of plasma membrane cholesterol microdomains on inhibition of ACAT helps explain how ACAT inhibition promotes cholesterol efflux from cells in the presence of cholesterol acceptors such as HDL. The anti-cholesterol monoclonal antibody also detected extracellular cholesterol-containing particles that accumulated most prominently during cholesterol enrichment of less differentiated human monocyte-macrophages. For the first time, cholesterol microdomains have been visualized that function in cholesterol trafficking to and from the plasma membrane.     

Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages

PURPOSE OF REVIEW: The initial steps of reverse cholesterol transport involve export of cholesterol from peripheral cells to plasma lipoproteins for subsequent delivery to the liver. The review discusses recent developments in our understanding of how these steps occur, with particular emphasis on the macrophage, the major site of cellular cholesterol accumulation in atherosclerosis. RECENT FINDINGS: ATP binding cassette transporter (ABC) A1 exports cholesterol and phospholipid to lipid-free apolipoproteins, while ATP binding cassette transporter G1 and scavenger receptor BI export cholesterol to phospholipid-containing acceptors. ABCA1-dependent cholesterol export involves an initial interaction of apolipoprotein AI with lipid raft membrane domains, although ABCA1 and most exported cholesterol are not raft associated. ABCG1 exports cholesterol to HDL and other phospholipid-containing acceptors. These include particles generated during lipidation of apoAI by ABCA1, suggesting that the two transporters cooperate in cholesterol export. Scavenger receptor BI is atheroprotective, mediating clearance of HDL cholesterol by the liver. The relative contributions of scavenger receptor BI and ABCG to cholesterol export to HDL from macrophages is unclear and may depend on cellular cholesterol status and the cholesterol gradient between cell and acceptor. SUMMARY: The presence of distinct pathways for cholesterol efflux to lipid-free apolipoprotein AI and phospholipid-containing HDL species clarifies our understanding of reverse cholesterol transport, and provides new opportunities for its therapeutic manipulation.     

Selective cholesterol dynamics between lipoproteins and caveolae/lipid rafts

Although low-density lipoprotein (LDL) receptor-mediated cholesterol uptake through clathrin-coated pits is now well understood, the molecular details and organizing principles for selective cholesterol uptake/efflux (reverse cholesterol transport, RCT) from peripheral cells remain to be resolved. It is not yet completely clear whether RCT between serum lipoproteins and the plasma membrane occurs primarily through lipid rafts/caveolae or from non-raft domains. To begin to address these issues, lipid raft/caveolae-, caveolae-, and non-raft-enriched fractions were resolved from purified plasma membranes isolated from L-cell fibroblasts and MDCK cells by detergent-free affinity chromatography and compared with detergent-resistant membranes isolated from the same cells. Fluorescent sterol exchange assays between lipoproteins (VLDL, LDL, HDL, apoA1) and these enriched domains provided new insights into supporting the role of lipid rafts/caveolae and caveolae in plasma membrane/lipoprotein cholesterol dynamics: (i) lipids known to be translocated through caveolae were detected (cholesteryl ester, triacylglycerol) and/or enriched (cholesterol, phospholipid) in lipid raft/caveolae fractions; (ii) lipoprotein-mediated sterol uptake/efflux from lipid rafts/caveolae and caveolae was rapid and lipoprotein specific, whereas that from non-rafts was very slow and independent of lipoprotein class; and (iii) the rate and lipoprotein specificity of sterol efflux from lipid rafts/caveolae or caveolae to lipoprotein acceptors in vitro was slower and differed in specificity from that in intact cells-consistent with intracellular factors contributing significantly to cholesterol dynamics between the plasma membrane and lipoproteins.     

Uptake of endogenous cholesterol by a synthetic lipoprotein

The addition of cholesterol-poor phospholipid liposomes to canine plasma in vivo and in vitro substantially alters the distribution of phospholipids, apoproteins, and, especially, cholesterol. In vivo, intravenously injected phospholipid liposomes remain discrete particles, which are readily distinguished from the normally occurring lipoproteins by their buoyant density and electrophoretic mobility. They acquire unesterified cholesterol from endogenous sources, thereby producing an acute rise in the concentration of this sterol in plasma. The liposomes also accumulate endogenous proteins, one of which is identified as apolipoprotein A-I. In vitro, phospholipid liposomes incubated with plasma acquire unesterified cholesterol and apolipoprotein A-I at the expense of high-density lipoproteins (HDL), the major carrier of cholesterol in normal canine plasma. In exchange, the HDL particles are enriched in phospholipids and become larger. At sufficiently high concentrations, the liposomes nearly completely deplete HDL of its unesterified cholesterol. Thus, there are generated two types of particles, both rich in apolipoprotein A-I and phospholipid, but one (modified HDL) containing mainly esterified cholesterol in its core and the other (modified liposomes) containing mainly unesterified cholesterol at its surface. It is concluded that phospholipid liposomes produce important changes in the distribution of lipids and protein in canine plasma, particularly at the expense of HDL. These changes appear to favor the mobilization of tissue cholesterol into the plasma, and may have application to atherosclerosis.     

Cellular cholesterol efflux.

Efflux of free cholesterol (FC) continues even when cellular FC mass is unchanged. This reflects a recirculation of preformed FC between cells and extracellular fluids which has multiple functions in cell biology including receptor recycling and signaling as well as cellular FC homeostasis. Total FC efflux is heterogeneous. Simple diffusion to mature high density lipoprotein (HDL), mainly via albumin as intermediate, initiates FC net transport driven by plasma lecithin:cholesterol acyltransferase activity. A second major efflux component reflects protein-facilitated transport from cell surface domains (caveolae, rafts) driven by FC binding to lipid-poor, pre-beta-migrating HDL (pre-beta-HDL). Facilitated efflux from caveolae, unlike simple diffusion, is highly regulated. Neither ABC1 (the protein defective in Tangier disease) nor other ATP-dependent transporters now appear likely to contribute directly to FC efflux. Their role is limited to the initial formation of a particle precursor to circulating pre-beta-HDL, which recycles without further lipid input from ATP-dependent transporter proteins. Lipid-free apolipoprotein A-I, previously considered a surrogate for pre-beta-HDL, has a reactivity much lower than that of native lipoprotein FC acceptors.     

Involvement of caveolin-1 in cholesterol enrichment of high density lipoprotein during its assembly by apolipoprotein and THP-1 cells

High density lipoprotein (HDL) is assembled by interaction of apolipoprotein A-I with human monocytic leukemia cell line THP-1 by removing cellular cholesterol and phospholipid. Although the HDL formed with undifferentiated THP-1 cells contained only phosphatidylcholine and almost no cholesterol, the cells differentiated with phorbol 12-myristate 13-acetate (PMA) generated HDL enriched in cholesterol. The extent of cholesterol enrichment related to the cellular cholesterol level in the differentiated cells, but only weakly in the undifferentiated cells. In contrast, the differentiation had no influence on the diffusion-mediated cellular cholesterol efflux. The undifferentiated cells expressed the messages of ATP-binding cassette transporter 1 and caveolin-1, at low levels, and the PMA-induced differentiation resulted in substantial expression of both messages. Caveolin-1 protein expression was also highly induced by the PMA treatment of THP-1 cells. When the cells were treated with the antisense DNA of caveolin-1 and differentiated, both caveolin-1 synthesis and cholesterol incorporation into the HDL were reduced in parallel to generate the cholesterol-poor HDL.We concluded that caveolin-1 is involved in enrichment with cholesterol of the HDL generated by the apolipoprotein-cell interaction. This function is independent of the assembly of HDL particles with cellular phospholipid and of nonspecific, diffusion-mediated efflux of cellular cholesterol.     

ABCA1-mediated cholesterol efflux generates microparticles in addition to HDL through processes governed by membrane rigidity

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.     

Cell cholesterol efflux to reconstituted high-density lipoproteins containing the apolipoprotein A-IMilano dimer

The apolipoprotein A-IMilano (apoA-IM) is a molecular variant of apoA-I characterized by the Arg(173)-->Cys substitution, resulting in the formation of homodimers A-IM/A-IM. The introduction of the interchain disulfide bridge in the A-IM dimer limits the apolipoprotein conformational flexibility and restricts HDL particle size heterogeneity, thus possibly affecting HDL function in lipid metabolism and atherosclerosis protection. To investigate whether the structural changes in A-IM/A-IM affect apoA-I capacity for cell cholesterol uptake, we tested the ability of four reconstituted HDL (rHDL), that contained either apoA-I or A-IM/A-IM, to remove cholesterol from Fu5AH hepatoma cells and cholesterol-loaded murine primary macrophages (MPM). As the HDL particle size is known to affect the rHDL capacity for cell cholesterol uptake, the reconstitution conditions were carefully selected to produce two sets of rHDL particles of small and large size (7.8 and 12.5 nm in diameter). The small A-IM/A-IM rHDL were more efficient than the corresponding apoA-I particles as acceptors of membrane cholesterol from Fu5AH cells and MPM, and as inhibitors of cholesterol esterification in MPM. The large rHDL and the lipid-free apolipoproteins displayed instead similar capacities for cell cholesterol efflux. These results suggest that cell cholesterol efflux to rHDL particles of different size occurs through different mechanisms. Large HDL accommodate and retain the cholesterol molecules that have desorbed from the cell membrane into the extracellular fluid, in a process that is less sensitive to protein conformation. Small HDL accelerate the desorption of cholesterol from the cell membrane, in a process that is influenced by the conformation of the proteins on the surface of the acceptor particle. The enhanced efficiency of small A-IM/A-IM rHDL seems related to the peculiar structure of the protein on the rHDL surface, with a hydrophobic C-terminal domain extending out of the rHDL particle, available for anchoring the acceptor to the plasma membrane.     

Sphingomyelin-dependence of cholesterol efflux mediated by ABCG1

ABCG1, one of the half-type ATP binding cassette (ABC) proteins, mediates the efflux of cholesterol to HDL and functions in the reverse cholesterol transport from peripheral cells to the liver. We have shown that ABCG1 mediates the efflux of not only cholesterol but also sphingomyelin (SM) and phosphatidylcholine. Because SM preferentially associates with cholesterol, we examined whether it plays an important role in the ABCG1-mediated efflux of cholesterol. The efflux of cholesterol and SM mediated by ABCG1 was reduced in a mutant CHO-K1 cell line, LY-A, in which the cellular SM level is reduced because of a mutation of the ceramide transfer protein CERT. In contrast, CHO-K1 cells overexpressing CERT showed an increased efflux of cholesterol and SM mediated by ABCG1. The sensitivity of cells to methyl-beta-cyclodextrin suggested that cholesterol in nonraft domains was increased due to the disruption of raft domains in LY-A cells. These results suggest that the ABCG1-mediated efflux of cholesterol and SM is dependent on the cellular SM level and distribution of cholesterol in the plasma membrane.     

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.     

Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.     

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.