Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution

Previous studies have shown that scavenger receptor BI (SR-BI) stimulates the bidirectional flux of free cholesterol (FC) between HDL and SR-BI-expressing cells. A major component of the enhanced FC flux appears to occur independently of HDL binding to SR-BI and may be due to changes in membrane lipid domains resulting from SR-BI expression (1). In the present study, the impact of SR-BI on cellular cholesterol metabolism was determined by examining SR-BI-mediated changes in cellular cholesterol mass, the esterification of HDL-derived FC, and changes in membrane lipid pools. Growth of SR-BI-expressing cells in medium containing HDL led to increased cellular cholesterol mass, most of which accumulated as ester. The esterification of HDL-derived FC was enhanced by SR-BI-expression to a far greater extent than the SR-BI mediated increase in FC uptake, suggesting an SR-BI-mediated effect on cholesterol utilization in the cell. This observation was tested by comparing FC esterification rates in SR-BI positive and negative cells when equivalent amounts of extracellular FC were taken up via cyclodextrins or apolipoprotein AI/phospholipid disks, neither of which contained cholesteryl ester. Under these conditions, SR-BI did not preferentially stimulate cholesterol esterification. These results indicate that the enhanced esterification of HDL-derived FC in SR-BI-expressing cells is due to the expanded pool of cellular FC and not to a specific effect of SR-BI on cholesterol utilization. Two approaches were used to test the effects of SR-BI expression on membrane lipid organization. In the first, the sensitivity of cellular FC to exogenous cholesterol oxidase was tested under conditions in which there is a preferential oxidation of caveolar cholesterol. SR-BI-expression was found to greatly increase the fraction of cellular cholesterol available to the oxidase as compared to either vector-transfected cells or cells expressing the related class B scavenger receptor CD36. These results suggest that SR-BI expression alters the distribution of membrane-free cholesterol to a caveolar fraction or alters the accessibility of this membrane fraction to exogenous cholesterol oxidase. In the second approach, the efflux of cellular FC to high concentrations of cyclodextrins was monitored under conditions where desorption of FC from the plasma membrane is rate limiting for efflux. SR-BI-expressing cells showed a shift in the distribution of FC between two kinetic pools with more FC in the fast pool and less in the slow pool. These data support a model in which SR-BI expression leads to a redistribution of cholesterol to membrane domains that serve to facilitate the flux of FC between cells and lipoproteins.     

Separation of lipid transport functions by mutations in the extracellular domain of scavenger receptor class B,type I

Scavenger receptor class B, type I (SR-BI) shows a variety of effects on cellular cholesterol metabolism, including increased selective uptake of high density lipoprotein (HDL) cholesteryl ester, stimulation of free cholesterol (FC) efflux from cells to HDL and phospholipid vesicles, and changes in the distribution of plasma membrane FC as evidenced by increased susceptibility to exogenous cholesterol oxidase. Previous studies showed that these multiple effects require the extracellular domain of SR-BI, but not the transmembrane and cytoplasmic domains. To test whether 1) the extracellular domain of SR-BI mediates multiple activities by virtue of discrete functional subdomains, or 2) the multiple activities are, in fact, secondary to and driven by changes in cholesterol flux, the extracellular domain of SR-BI was subjected to insertional mutagenesis by strategically placing an epitope tag into nine sites. These experiments identified four classes of mutants with disruptions at different levels of function. Class 4 mutants showed a clear separation of function between HDL binding, HDL cholesteryl ester uptake, and HDL-dependent FC efflux on one hand and FC efflux to small unilamellar vesicles and an increased cholesterol oxidase-sensitive pool of membrane FC on the other. Selective disruption of the latter two functions provides evidence for multiple functional subdomains in the extracellular receptor domain. Furthermore, these findings uncover a difference in the SR-BI-mediated efflux pathways for FC transfer to HDL acceptors versus phospholipid vesicles. The loss of the cholesterol oxidase-sensitive FC pool and FC efflux to small unilamellar vesicle acceptors in Class 4 mutants suggests that these activities may be mechanistically related.     

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.     

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.     

High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI

The role of high density lipoprotein (HDL) phospholipid in scavenger receptor BI (SR-BI)-mediated free cholesterol flux was examined by manipulating HDL(3) phosphatidylcholine and sphingomyelin content. Both phosphatidylcholine and sphingomyelin enrichment of HDL enhanced the net efflux of cholesterol from SR-BI-expressing COS-7 cells but by two different mechanisms. Phosphatidylcholine enrichment of HDL increased efflux, whereas sphingomyelin enrichment decreased influx of HDL cholesterol. Although similar trends were observed in control (vector-transfected) COS-7 cells, SR-BI overexpression amplified the effects of phosphatidylcholine and sphingomyelin enrichment of HDL 25- and 2.8-fold, respectively. By using both phosphatidylcholine-enriched and phospholipase A(2)-treated HDL to obtain HDL with a graded phosphatidylcholine content, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r(2) = 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the K(d) or B(max) values for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.     

Caveolin-1 does not affect SR-BI-mediated cholesterol efflux or selective uptake of cholesteryl ester in two cell lines

Free cholesterol (FC) has been reported to efflux from cells through caveolae, which are 50-100 nm plasma membrane pits. The 22 kDa protein caveolin-1 is concentrated in caveolae and is required for their formation. The HDL scavenger receptor BI (SR-BI), which stimulates both FC efflux and selective uptake of HDL-derived cholesteryl ester (CE), has been reported to be concentrated in caveolae, suggesting that this localization facilitates flux of FC and CE across the membrane. However, we found that overexpression of caveolin-1 in Fischer rat thyroid (FRT) cells, which lack caveolin-1 and caveolae, or HEK 293 cells, which normally express very low levels of caveolin-1, did not affect FC efflux to HDL or liposomes. Transient expression of SR-B1 did not affect this result. Similarly, caveolin-1 expression did not affect selective uptake of CE from labeled HDL particles in FRT or HEK 293 cells transfected with SR-BI. We conclude that basal and SR-BI-stimulated FC efflux to HDL and liposomes and SR-BI-mediated selective uptake of HDL CE are not affected by caveolin-1 expression in HEK 293 or FRT cells.     

Highly purified scavenger receptor class B,type I reconstituted into phosphatidylcholine/cholesterol liposomes mediates high affinity high density lipoprotein binding and selective lipid uptake

The murine class B, type I scavenger receptor mSR-BI is a high and low density lipoprotein (HDL and LDL) receptor that mediates selective uptake of cholesteryl esters. Here we describe a reconstituted phospholipid/cholesterol liposome assay of the binding and selective uptake activities of SR-BI derived from detergent-solubilized cells. The assay, employing lysates from epitope-tagged receptor (mSR-BI-t1)-expressing mammalian and insect cells, recapitulated many features of SR-BI activity in intact cells, including high affinity and saturable (125)I-HDL binding, selective lipid uptake from [(3)H]cholesteryl ether-labeled HDL, and poor inhibition of HDL receptor activity by LDL. The novel properties of a mutated receptor (Q402R/Q418R, normal LDL binding but loss of most HDL binding) were reproduced in the assay, as was the ability of the SR-BI homologue CD36 to bind HDL but not mediate efficient lipid uptake. In this assay, essentially homogeneously pure mSR-BI-t1, prepared by single-step immunoaffinity chromatography, mediated high affinity HDL binding and efficient selective lipid uptake from HDL. Thus, SR-BI-mediated HDL binding and selective lipid uptake are intrinsic properties of the receptor that do not require the intervention of other proteins or specific cellular structures or compartments.     

Scavenger receptor BI (SR-BI) clustered on microvillar extensions suggests that this plasma membrane domain is a way station for cholesterol trafficking between cells and high-density lipoprotein

Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at the organismal and cellular levels. In contrast to the well-studied pathway of LDL receptor-mediated endocytosis, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by the HDL receptor, scavenger receptor BI (SR-BI). SR-BI mediates HDL cholesteryl ester uptake in a process in which HDL lipids are selectively transferred to the cell membrane without the uptake and degradation of the HDL particle. We report here the cell surface locale where the trafficking of HDL cholesterol occurs. Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy showed SR-BI in patches or clusters primarily on microvillar extensions of the plasma membrane. The organization of SR-BI in this manner suggests that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. The types of phospholipids in this domain are unknown, but SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids. We speculate that SR-BI is in a more fluid membrane domain that will favor rapid cholesterol flux between the membrane and HDL.     

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.     

SR-BI is required for microvillar channel formation and the localization of HDL particles to the surface of adrenocortical cells in vivo

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl ester into liver and steroidogenic tissues. In steroidogenic cells, juxtaposed microvilli, or microvilli snuggled against the plasma membrane create microvillar channels that fill with HDL. Microvillar membranes contain SR-BI and are believed to be the site of HDL cholesteryl ester uptake. A recent study showed that SR-BI expression in insect cells elicits membrane structures that contain SR-BI, bind HDL, and closely resemble the ultrastructure of microvillar channels. In the present study we compared the ultrastructure of adrenal gland microvillar membranes in Srb1+/+ and Srb1-/- mice to test whether SR-BI is required for the formation of microvillar channels. The results show that SR-BI is absolutely required for microvillar channel formation and that the microvillar membranes of Srb1-/- mice are 17% thinner than in Srb1+/+ mice.We conclude that SR-BI has a major influence on plasma membrane ultrastructure and organization in vivo.     

Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.

Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport.     

Glycine 420 near the C-terminal transmembrane domain of SR-BI is critical for proper delivery and metabolism of high density lipoprotein cholesteryl ester

Scavenger receptor BI, SR-BI, is a physiologically relevant receptor for high density lipoprotein (HDL) that mediates the uptake of cholesteryl esters and delivers them to a metabolically active membrane pool where they are subsequently hydrolyzed. A previously characterized SR-BI mutant, A-VI, with an epitope tag inserted into the extracellular domain near the C-terminal transmembrane segment, revealed a separation-of-function between SR-BI-mediated HDL cholesteryl ester uptake and cholesterol efflux to HDL, on one hand, and cholesterol release to small unilamellar phospholipid vesicle acceptors and an increased cholesterol oxidase-sensitive pool of membrane free cholesterol on the other. To further elucidate amino acid residues responsible for this separation-of-function phenotype, we engineered alanine substitutions and point mutations in and around the site of epitope tag insertion, and tested these for various cholesterol transport functions. We found that changing amino acid 420 from glycine to histidine had a profound effect on SR-BI function. Despite the ability to mediate selective HDL cholesteryl ester uptake, the G420H receptor had a greatly reduced ability to: 1) enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol, 2) mediate cholesterol efflux to HDL, even at low concentrations of HDL acceptor where binding-dependent cholesterol efflux predominates, and 3) accumulate cholesterol mass within the cell. Most importantly, the G420H mutant was unable to deliver the HDL cholesteryl ester to a metabolically active membrane compartment for efficient hydrolysis. These observations have important implications regarding SR-BI function as related to its structure near the C-terminal transmembrane domain.     

Hepatic scavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol and its transport into bile

The clearance of free cholesterol from plasma lipoproteins by tissues is of major quantitative importance, but it is not known whether this is passive or receptor-mediated. Based on our finding that scavenger receptor BI (SR-BI) promotes free cholesterol (FC) exchange between high density lipoprotein (HDL) and cells, we tested whether SR-BI would effect FC movement in vivo using [(14)C]FC- and [(3)H]cholesteryl ester (CE)-labeled HDL in mice with increased (SR-BI transgenic (Tg)) or decreased (SR-BI attenuated (att)) hepatic SR-BI expression. The initial clearance of HDL FC was increased in SR-BI Tg mice by 72% and decreased in SR-BI att mice by 53%, but was unchanged in apoA-I knockout mice compared with wild-type mice. Transfer of FC to non-HDL and esterification of FC were minor and could not explain differences. The hepatic uptake of FC was increased in SR-BI Tg mice by 34% and decreased in SR-BI att mice by 22%. CE clearance and uptake gave similar results, but with much slower rates. The uptake of HDL FC and CE by SR-BI Tg primary hepatocytes was increased by 2.2- and 2.6-fold (1-h incubation), respectively, compared with control hepatocytes. In SR-BI Tg mice, the initial biliary secretion of [(14)C]FC was markedly increased, whereas increased [(3)H]FC appeared after a slight delay. Thus, in the mouse, a major portion of the clearance of HDL FC from plasma is mediated by SR-BI.     

Scavenger receptor class B, type I-mediated uptake of various lipids into cells. Influence of the nature of the donor particle interaction with the receptor

Scavenger receptor (SR)-BI is the first molecularly defined receptor for high density lipoprotein (HDL) and it can mediate the selective uptake of cholesteryl ester into cells. To elucidate the molecular mechanisms by which SR-BI facilitates lipid uptake, we examined the connection between lipid donor particle binding and lipid uptake using kidney COS-7 cells transiently transfected with SR-BI. We systematically compared the uptake of [(3)H]cholesteryl oleoyl ether (CE) and [(14)C]sphingomyelin (SM) from apolipoprotein (apo) A-I-containing reconstituted HDL (rHDL) particles and apo-free lipid donor particles. Although both types of lipid donor could bind to SR-BI, only apo-containing lipid donors exhibited preferential delivery of CE over SM (i.e. nonstoichiometric lipid uptake). In contrast, apo-free lipid donor particles (phospholipid unilamellar vesicles, lipid emulsion particles) gave rise to stoichiometric lipid uptake due to interaction with SR-BI. This apparent whole particle uptake was not due to endocytosis, but rather fusion of the lipid components of the lipid donor with the cell plasma membrane; this process is perhaps mediated by a fusogenic motif in the extracellular domain of SR-BI. The interaction of apoA-I with SR-BI not only prevents fusion of the lipid donor with the plasma membrane but also allows the optimal selective lipid uptake. A comparison of rHDL particles containing apoA-I and apoE-3 showed that while both particles bound equally well to SR-BI, the apoA-I particle gave approximately 2-fold greater CE selective uptake. Catabolism of all major HDL lipids can occur via SR-BI with the relative selective uptake rate constants for CE, free cholesterol, triglycerides (triolein), and phosphatidylcholine being 1, 1.6, 0.7, and 0.2, respectively. It follows that a putative nonpolar channel created by SR-BI between the bound HDL particle and the cell plasma membrane is better able to accommodate the uptake of neutral lipids (e.g. cholesterol) relative to polar phospholipids.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

HDL modification by secretory phospholipase A(2) promotes scavenger receptor class B type I interaction and accelerates HDL catabolism

During inflammatory states plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) are reduced. Secretory group IIa phospholipase A(2) (sPLA(2)) is a cytokine-induced acute-phase enzyme associated with HDL. Transgenic mice overexpressing sPLA(2) have reduced HDL levels. Studies were performed to define the mechanism for the HDL reduction in these mice. HDL isolated from sPLA(2) transgenic mice have a significantly lower phospholipid content and greater triglyceride content. In autologous clearance studies, (125)I-labeled HDL from sPLA(2) transgenic mice was catabolized significantly faster than HDL from control mice (4.24 +/- 1.16 vs. 2.84 +/- 0.1 pools per day, P < 0.008). In both sPLA(2) transgenic and control mice, the cholesteryl ester component of HDL was more rapidly catabolized than the protein component, indicating a selective uptake mechanism. In vitro studies using CHO cells transfected with scavenger receptor class B type I (SR-BI) showed that sPLA(2)-modified HDL was nearly twice as efficient as a substrate for cholesteryl ester transfer. These data were confirmed in in vivo selective uptake experiments using adenoviral vector overexpression of SR-BI. In these studies, increased hepatic selective uptake was associated with increased (125)I-labeled apolipoprotein uptake in the kidney.We conclude that during inflammation sPLA(2) hydrolysis of HDL phospholipids alters the lipid composition of the particle, allowing for more efficient SR-BI-mediated selective cholesteryl ester uptake. This enhanced SR-BI activity generates HDL remnants that are preferentially catabolized in the kidney.     

Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1

Mutations of the ABC1 transporter have been identified as the defect in Tangier disease, characterized by low HDL and cholesterol ester accumulation in macrophages. A full-length mouse ABC1 cDNA was used to investigate the mechanisms of lipid efflux to apoA-I or HDL in transfected 293 cells. ABC1 expression markedly increased cellular cholesterol and phospholipid efflux to apoA-I but had only minor effects on lipid efflux to HDL. The increased lipid efflux appears to involve a direct interaction between apoA-I and ABC1, because ABC1 expression substantially increased apoA-I binding at the cell surface, and chemical cross-linking and immunoprecipitation analysis showed that apoA-I binds directly to ABC1. In contrast to scavenger receptor BI (SR-BI), another cell surface molecule capable of facilitating cholesterol efflux, ABC1 preferentially bound lipid-free apoA-I but not HDL. Immunofluorescence confocal microscopy showed that ABC1 is primarily localized on the cell surface. In the absence of apoA-I, cells overexpressing ABC1 displayed a distinctive morphology, characterized by plasma membrane protrusions and resembling echinocytes that form when there are excess lipids in the outer membrane hemileaflet. The studies provide evidence for a direct interaction between ABC1 and apoA-I, but not HDL, indicating that free apoA-I is the metabolic substrate for ABC1. Plasma membrane ABC1 may act as a phospholipid/cholesterol flippase, providing lipid to bound apoA-I, or to the outer membrane hemileaflet.