Intracellular Trafficking of Cholesterol Monitored with a Cyclodextrin*

, The sterol binding agent 2-hydroxypropyl- (cid:98) -cyclodex-trin is shown to be a convenient and useful experimental tool to probe intracellular pathways of cholesterol transport. Biochemical and cytochemical studies reveal that cyclodextrin specifically removes plasma membrane cholesterol. Depletion of plasma membrane sphingomyelin greatly accelerated cyclodextrin-medi-ated cholesterol removal. Cholesterol arriving at the plasma membrane from lysosomes and the endoplasmic reticulum was also removed by cyclodextrin. Cellular cholesterol esterification linked to the mobilization of cholesterol from lysosomes was strongly attenuated by cyclodextrin, suggesting that the major portion of endocytosed cholesterol is delivered from lysosomes to the endoplasmic reticulum via the plasma membrane. Evidence for translocation of lysosomal cholesterol to the endoplasmic reticulum by a plasma membrane-inde-pendent pathway is provided by the finding that cyclo- dextrin loses its ability to suppress esterification when plasma membrane sphingomyelin is depleted. The Golgi apparatus appears to play an active role in directing the relocation of lysosomal cholesterol to the plasma mem- brane since brefeldin A also abrogated

The sterol binding agent 2-hydroxypropyl-␤-cyclodextrin is shown to be a convenient and useful experimental tool to probe intracellular pathways of cholesterol transport. Biochemical and cytochemical studies reveal that cyclodextrin specifically removes plasma membrane cholesterol. Depletion of plasma membrane sphingomyelin greatly accelerated cyclodextrin-mediated cholesterol removal. Cholesterol arriving at the plasma membrane from lysosomes and the endoplasmic reticulum was also removed by cyclodextrin. Cellular cholesterol esterification linked to the mobilization of cholesterol from lysosomes was strongly attenuated by cyclodextrin, suggesting that the major portion of endocytosed cholesterol is delivered from lysosomes to the endoplasmic reticulum via the plasma membrane. Evidence for translocation of lysosomal cholesterol to the endoplasmic reticulum by a plasma membrane-independent pathway is provided by the finding that cyclodextrin loses its ability to suppress esterification when plasma membrane sphingomyelin is depleted. The Golgi apparatus appears to play an active role in directing the relocation of lysosomal cholesterol to the plasma membrane since brefeldin A also abrogated cyclodextrin-mediated suppression of cholesterol esterification. Using cyclodextrin we further show that attenuated esterification of lysosomal cholesterol in Niemann-Pick C cells reflects defective translocation of cholesterol to the plasma membrane that may be linked to abnormal Golgi trafficking.
Cholesterol is an integral membrane component required for normal cellular function (1). In mammalian cells this sterol is derived either from low density lipoprotein (LDL) 1 following receptor-mediated endocytosis and subsequent hydrolysis in lysosomes (2) or via de novo biosynthesis in the endoplasmic reticulum (ER) (3). The mechanism for the ensuing movement of cholesterol from these intracellular sites to their ultimate cellular destinations is an unresolved question of fundamental importance to cell biology and medicine. Defects in these transport pathways can alter cellular cholesterol metabolism resulting in pathological states. Niemann-Pick C (NP-C) disease is characterized by sequestration of LDL-derived cholesterol in lysosomes that is linked to delays in the induction of cholesterol-mediated homeostatic responses (4,5). Development of atherosclerotic lesions involves in part a failure to maintain adequate transfer of cholesterol from intracellular sites of accumulation to the cell surface for removal by extracellular acceptors (6). Understanding pathways of intracellular cholesterol transport is a critical step toward the correction of cellular cholesterol lipidoses.
The intracellular distribution of cholesterol generated by these transport pathways is not uniform, with a major fraction present in the plasma membrane (PM) (7). It has been suggested that this high concentration of cholesterol at the cell surface results in part from its close association with sphingomyelin (SM) (8). Enzymatic digestion of SM by exogenously added sphingomyelinase (SMase) mobilizes PM cholesterol for esterification in the ER (9 -12).
Several methods have been employed to monitor intracellular movement of cholesterol. Arrival of cholesterol at the ER is accompanied by esterification catalyzed by the resident enzyme acetyl-coenzyme A:cholesterol acyltransferase. Transfer of cholesterol to the PM has been studied by subcellular fractionation (13), chemical modification by exogenously added cholesterol oxidase (14), or via removal from the cell surface to extracellular cholesterol acceptors (15,16). This latter approach has been limited by the lack of an acceptor that efficiently removes cholesterol. The effectiveness of cholesterol depletion from the PM by HDL varies with cell type and is neither an extensive nor a rapid process (16,17).
In the present study we demonstrate the utility of 2-hydroxypropyl-␤-cyclodextrin (CD), as an effective extracellular cholesterol acceptor, to monitor the flux of cholesterol through the PM of living cells. Cyclodextrins are cyclic oligomers of glucose that have the capacity to sequester lipophiles in their hydrophobic core (18). The water-soluble cyclodextrins preferentially form inclusion complexes with sterols thereby greatly enhancing their solubility in aqueous solution (19 -21). It has recently been shown that CD is capable of stimulating cholesterol efflux from cultured cells with very high efficiency (22). We now report our findings on the use of CD to follow movement of de novo synthesized cholesterol to and from the PM and the egress of LDL-derived cholesterol from lysosomes. We further used CD to reveal that cell surface SM and the Golgi apparatus regulate intracellular transport of cholesterol.

EXPERIMENTAL PROCEDURES
Materials-Fetal bovine serum was obtained from HyClone Laboratories, Inc., Logan, UT. Lipoprotein-deficient bovine serum (LPDS) and human low density lipoprotein (LDL) were prepared by Advanced Bioscience Laboratories, Rockville, MD. Glass and plastic microscope culture wells (Lab-Tek) were purchased from Thomas Scientific. [ Tissue Culture-Normal and NP-C fibroblasts were derived from volunteers and confirmed patients of the Developmental and Metabolic Neurology Branch under the guidelines approved by clinical and research committees of the National Institutes of Health. Fibroblasts (3-15 passages) were cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 2 mM glutamine, and 100 units of penicillin/streptomycin/ml in humidified 95% air and 5% CO 2 at 37°C. For biochemical analyses, fibroblasts were seeded at a density of 40,000 cells/well ("subconfluent") in plastic 6-well dishes (Costar, Cambridge, MA) in McCoy's medium supplemented with 5% LPDS, 2 mM glutamine, and 100 units of penicillin/streptomycin/ml (McCoy's/5% LPDS medium) in humidified 95% air and 5% CO 2 at 37°C for 4 days. For cytochemical analyses, fibroblasts were seeded at a density of 20,000 cells/well in McCoy's/5% LPDS medium in 9.5-cm glass microscope wells (Nunc, Inc., Naperville, IL) coated with human fibronectin. For cholesterol depletion studies, 2-hydroxy-propyl-␤-cyclodextrin was routinely added at 2 g/100 ml to McCoy's/5% LPDS medium supplemented with 25 mM HEPES, pH 7.0, to adjust pH to 7 (23). A modification of the procedure described by Rome et al. (24) for the fractionation of human fibroblasts was employed. Cells were harvested by scraping with a razor blade and disrupted by N 2 cavitation. A combined postnuclear supernatant fraction (600 ϫ g, 10 min) was obtained from 1 to 2 confluent 75-cm 2 flasks that generally contained 0. . Centrifugation was carried out in a Sorvall SV288 vertical rotor at 28,000 ϫ g for 1 h at 4°C in a Sorvall RC2C Plus centrifuge equipped with a rate controller. Fractions (2.0 ml) were collected from the bottom of the gradients with the aid of a Beckman Fraction Recovery System. A light density membrane fraction as well as a heavy density membrane fraction was found in both normal and NP-C human fibroblast prepa-rations. Alkaline phosphatase assayed by the method of Touster et al. (25) sedimented with the light density protein peak.
Lipid Analyses-Cell monolayers were washed directly in their plastic 35-mm wells twice with chilled phosphate-buffered saline (PBS) and then extracted with 0.5 ml of isopropyl alcohol by gentle rocking for 30 min at room temperature in sealed plates. The isopropyl alcohol-extracted lipids were stored at Ϫ20°C in sealed tubes until analyzed. The lipid-free cell residues remaining in the culture wells were taken up in 0.5 ml of 0.5 N NaOH and protein measured by the method of Lowry et al. (26). Unesterified and esterified cholesterol levels were measured in aliquots of the isopropyl alcohol extract corresponding to approximately 10 g of protein with a fluorescently linked assay employing cholesteryl oxidase and cholesteryl esterase as described previously (27) (29).
Cytochemical Analyses-Cells in glass slide chambers were washed with PBS, fixed in 3% paraformaldehyde for 30 min at room temperature, washed with PBS, and then incubated overnight with 0.05% filipin in PBS at room temperature. Cells were washed with PBS, mounted in p-phenylenediamine glycerol, and viewed with a Leitz fluorescence microscope by using excitation filter BP-350 -410 for filipin.

RESULTS
CD Removes Cellular Cholesterol from the Plasma Membrane-2-Hydroxypropyl-␤-cyclodextrin (CD) is an effective sterol acceptor that removes the unesterified cholesterol mass from viable cells in a saturable, concentration-dependent manner (Table I). Approximately 70% of cellular cholesterol can be removed after 4 h treatment with 2% CD at 37°C. The kinetics of cholesterol removal from both normal and NP-C fibroblasts are similar with a biphasic nature (Fig. 1 cannot be mobilized to an appreciable extent by CD (Table I).
The small amount of cholesterol ester removed does not appear to depend on the concentration of CD. Exposure of normal and NP-C cultures to neutral sphingomyelinase (SMase) for 1 h resulted in the digestion of 80% of [ 3 H]sphingomyelin formed by de novo biosynthesis (data not shown). SMase treatment dramatically enhances both the rate and extent of removal of cellular [ 3 H]sterol by CD (Fig. 2). Treatment with CD following SM digestion resulted in the removal of cellular sterol in a biphasic manner with an initial rapid rate of depletion (70% within 15 min) followed by a subsequent slower rate of depletion (t1 ⁄2 ϭ 5-6 h). Similar results were obtained when cells were treated with CD and SMase concurrently (data not shown). There was no detectable release of lactic acid dehydrogenase to the medium nor altered triglyceride synthesis for up to 2 h of this combined treatment (data not shown).
We employed fluorescence microscopy using filipin, a fluorescent marker specific for unesterified cholesterol (30), to demonstrate that CD primarily targets the PM for removal of cellular cholesterol. In normal cells, we have confirmed (31) that filipin-cholesterol fluorescence is associated with both the PM and intracellular structures (Fig. 3A). Treatment of these cells with CD alone greatly diminishes PM fluorescence without substantially affecting intracellular staining (Fig. 3B). Filipin-cholesterol fluorescence associated with the PM is essentially eliminated after treatment with CD/SMase (Fig. 3C). In NP-C fibroblasts cultured with LDL, we also confirmed (31) that considerable filipin-cholesterol fluorescence is seen at the PM as well as intense fluorescence in perinuclear lysosomes resulting from defective intracellular mobilization of LDL-derived cholesterol in these cells (Fig. 4A). Treatment of these NP-C cells with CD/SMase resulted in complete loss of filipincholesterol fluorescence from the PM with no substantial loss of fluorescence from intracellular sites (Fig. 4B  1-5) and light density (fractions 10 -15) membranes (Fig. 5). A lysosomal marker (␤-hexosaminidase) was found predominantly in the heavy membrane fraction (HM), whereas markers for the PM, Golgi, and ER were found in the light membrane fraction (LM) (data not shown). In normal cells, 60 -70% of the total recovered radioactivity was distributed in the LM while 18% was associated with the HM (Fig. 5A) (Fig. 5B), reflecting the defective intracellular distribution of lysosomal cholesterol (15). Unlike normal fibroblasts, CD/SMase treatment did not substantially alter the LDL-derived [ 3 H]cholesterol distributed in HM of NP-C fibroblasts. This treatment did, however, produce a decrease by 2/3 in the percentage of the total [ 3 H]cholesterol distributed in LM. Although less lysosomal cholesterol appears at the PM of NP-C cells, a comparable proportion of LDLderived cholesterol was removed by CD/SMase from this membrane in both cell types.
CD can also prevent internalization of PM cholesterol to intracellular compartments. Fibroblasts were treated with SMase to induce movement of PM cholesterol to the ER (10). This incubation, performed in the presence of [ 3 H]oleate to follow arrival of cholesterol at the ER by esterification, was carried out in the absence or presence of CD. Treatment of fibroblasts with SMase stimulated cholesterol esterification (Fig. 6). This SMase-stimulated esterification of cholesterol is Arrival of lysosomal cholesterol at the PM can also be indirectly monitored by assessing the effect of CD on the level of cholesterol esterified during its release from lysosomes. LDLderived lysosomal cholesterol has been shown to be transported to the PM (15,(33)(34)(35) where it enriches a precursor pool of cholesterol destined to be esterified in the ER (35)(36)(37)(38). One would predict that CD can retard cellular cholesterol esterification by removing substrate from the PM. This was tested by loading the lysosomes of cultured fibroblasts with LDL-derived cholesterol using progesterone. During a subsequent period of progesterone washout, cells were incubated with [ 3 H]oleate (to assay esterification) in the absence or presence of 2% CD (to remove PM cholesterol). CD strongly inhibited the esterification normally seen during progesterone washout (Fig. 9A) consistent with the relocation of a large portion of lysosomal cholesterol to the PM prior to its esterification in the ER. The metabolic suppression of [ 3 H]oleate incorporation is specific for cholesterol esterification since CD did not alter triglyceride synthesis (data not shown). The level of esterification induced in cholesterol-loaded NP-C cells during progesterone washout was approximately 20-fold less than that observed in normal cells (Fig. 9B). CD did not suppress this attenuated level of esterification (Fig. 9B), consistent with a disrupted movement Plasma Membrane Sphingomyelin Alters the Intracellular Trafficking of Lysosomal Cholesterol-Removal of PM sphingomyelin can affect the intracellular transport path of lysosomal cholesterol. Our experimental protocol was to load the lysosomes of cultured normal fibroblasts with LDL-cholesterol using progesterone. These cells were then briefly treated with SMase to deplete PM SM, followed by washing the progesterone from the cultures to establish cholesterol transport out of lysosomes. During progesterone washout cells were incubated with [ 3 H]oleate in the absence or presence of CD. As established above (Fig. 9), CD alone can effectively suppress esterification of cholesterol in untreated cells (Fig. 10A). Depletion of PM SM alone does not alter the level of esterification of lysosomal cholesterol (cf. Fig. 10, A and B). However, when cells were exposed to SMase, CD no longer blocked cholesterol esterification (Fig. 10B). This loss in the sensitivity of esterification to CD modulation occurs under conditions that render cells particularly sensitive to removal of PM sterol (Fig. 2). These data suggest that depletion of PM SM diverts the trafficking of lysosomal cholesterol away from the PM without diminishing its accessibility to the ER.
BFA Alters the Intracellular Trafficking of Lysosomal and Plasma Membrane Cholesterol-The current studies with CD provide evidence that the Golgi plays a key role in the movement of LDL-derived cholesterol from lysosomes to the PM. Lysosomes of normal and NP-C fibroblasts were loaded with LDL-cholesterol using progesterone. Lysosomal cholesterol transport was re-established with progesterone washout, and cells were incubated with [ 3 H]oleate in the presence or absence of BFA and/or CD. The effective suppression of cholesterol esterification by CD during progesterone washout in normal fibroblasts (Fig. 11A) was largely lost when BFA was added (Fig. 11B). This finding does not result from a loss in the ability of CD to remove [ 3 H]sterol from the PM of BFA-treated cells labeled to equilibrium with [ 3 H]acetate (data not shown). The loss of CD-mediated suppression of esterification in the presence of BFA suggests that disruption of the Golgi complex (39) reduces the intracellular trafficking of lysosomal cholesterol to the PM without substantially diminishing its accessibility to the ER. In NP-C cells, CD does not suppress the attenuated cholesterol esterification either in the presence (Fig. 11C) or absence (Fig. 11D)  lates cholesterol esterification 4-fold (Fig. 11D). The BFA-induced increase in cholesterol esterification is consistent with previous observations (40,41,42) and suggests that Golgi cholesterol can be absorbed into the ER along with other Golgi components.
The Golgi also appears to play a role in the movement of PM cholesterol to the ER in normal and NP-C cells. Such movement can be induced by treating cells with SMase and assessed by measuring esterification (10). When cells are treated with either BFA or SMase alone there is a relatively weak stimulation in esterification of PM sterol (Fig. 12). However, when BFA and SMase are both present, there is a dramatic increase in the esterification of PM sterol in normal and mutant cell cultures (Fig. 12). In NP-C cells, stimulation of esterification by cotreatment with BFA and SMase is 50% of that seen in normal cells. It appears that BFA-mediated perturbations of the Golgi complex enhance access of SMase-mobilized PM sterol to the ER for esterification. DISCUSSION We have demonstrated that CD provides a simple and effective method for monitoring the flow of cellular cholesterol pools to and from the plasma membrane of living cells. Using this approach we have followed the movement of cholesterol derived from LDL or de novo biosynthesis. We have found that the bulk of LDL-derived cholesterol destined for esterification in the ER is first delivered to the PM. In addition we provide evidence that transport of cholesterol from lysosomes via this pathway can be dramatically affected by the integrity of the Golgi complex and the SM content of the PM.
Our experimental approach depends on the effective removal of unesterified cholesterol by CD from viable cells. CD depletes up to 80% of cellular cholesterol from intact, viable cells in a concentration (Table I) and time (Fig. 1) -dependent manner. The kinetics of sterol efflux from human fibroblasts to CD are similar to those recently reported for mouse L-cells (22). The efflux process likely involves cholesterol desorption from cells followed by diffusion to an extracellular acceptor (6,43). An excess pool of CD, routinely used in our present studies, serves as a sink for cellular cholesterol. Biochemical and cytochemical analyses strongly suggest that CD specifically targets removal of cholesterol from the cell surface. Depletion of cellular cholesterol with CD corresponds to a marked loss of filipin-cholesterol fluorescence from the PM but not from intracellular pools. Subcellular fractionation of normal and Niemann-Pick C cells reveals that CD preferentially removes LDL-derived [ 3 H]cholesterol primarily from light membranes (PM). The kinetics and extent of cholesterol removal from the PM of normal or NP-C fibroblasts by CD are indistinguishable (Fig. 1). This suggests that the defective intracellular transport of cholesterol in NP-C cells (4,15) is not an intrinsic property of the PM.
The large fraction of cellular cholesterol associated with the PM has often been attributed to its interaction with SM at this site (38). This hypothesis is based in part on observations that cells treated with SMase display a burst of esterification at the ER of cholesterol derived from the PM (9 -11). These studies suggested that a large portion of PM cholesterol relocated to intracellular sites that appeared to be inaccessible to extracellular lipid acceptors such as HDL 3 (40,44). In the present study we find, however, that removal of cellular cholesterol by CD is, in fact, significantly enhanced after enzymatic depletion of PM SM (Fig. 2). Our data are consistent with the recent report that the majority of cellular cholesterol remains at the PM after SM depletion (45). The observed level of enhancement of CD-mediated cholesterol removal after SM depletion suggests that a considerable portion of PM cholesterol (ϳ50%) may be associated with SM. We also show that CD, in contrast to HDL (40,44), can divert PM cholesterol from intracellular transport pathways, such as movement to the ER for esterification (Fig.  6). This difference most likely reflects the relative effectiveness of CD as a cholesterol acceptor (22).
Because of its effective removal of surface cholesterol, CD provides a facile means for monitoring sterol flux through the PM. CD removes a significant fraction of de novo cellular sterol (Fig. 7). It has been estimated that the half-time for delivery of cholesterol from its site of synthesis in the ER to the cell surface is 10 -30 min in several cell types (46 -48). The halftimes reported for fibroblasts appear to depend on the methods employed, ranging from 10 min (13) to 1-2 h (49). The reason for these discrepancies remains unclear. Our data (Fig. 7B) show a marked increase in the rate of removal of de novo synthesized sterol to CD after a few hours of incubation with the [ 3 H]mevalonate precursor, consistent with the extended time of transfer in fibroblasts (49). Further studies will enable us to provide a more precise measurement for the rate of transfer.
Transport of LDL-derived cholesterol from lysosomes to the PM can also be followed with CD. We have previously shown that progesterone treatment of normal cells produces an accumulation of LDL-derived cholesterol in lysosomes, as seen with the NP-C mutation (32). After progesterone removal, this cholesterol pool leaves lysosomes. The reversibility of the progesterone-induced block provides a direct method to study egress of cholesterol from lysosomes. We find that removal of LDLderived cholesterol by CD during progesterone washout occurs with half-times of 4 h in normal cells and 8 h in NP-C cells (Fig.  8). This difference appears to reflect the lesion in lysosomal cholesterol trafficking to the PM reported in the mutant cells (15), since the rates of removal of resident PM sterol itself by CD are identical in the two cell types. Our measurements of removal of lysosomal cholesterol from cells by CD during pro-gesterone washout are considerably longer than those previously reported for the movement of pulse-labeled lysosomal cholesterol to the PM with values of 1-2 min (33), 40 -50 min (34), and 1-2 h (15). Differences among these data may be attributable to variability in the cell type and the methods employed. Our measurements may also reflect extensive loading of lysosomes with cholesterol that occurs when cultures are exposed to progesterone during LDL uptake (32). The slower rates of clearance that we report may therefore reflect saturation of the transport pathways that mediate movement of lysosomal cholesterol to the PM as well as the rate at which CD can remove cholesterol from the PM. Nonetheless, it is important to note that we detect a slower rate in the movement of LDL-derived cholesterol to the PM in NP-C cells compared with normal cells.
Movement of cholesterol from lysosomes during progesterone washout can also be monitored through its subsequent esterification (32). This pathway appears to involve an initial transfer of lysosomal cholesterol to the PM since PM cholesterol is considered to provide the principal source of substrate for acetyl-coenzyme A:cholesterol acyltransferase located in the ER (35)(36)(37)(38). It has, however, been speculated that a portion of LDL-derived cholesterol destined for esterification may be directly transferred from lysosomes to the ER (36). We find that CD in normal cells can block esterification by as much as 70% ( Fig. 9) and conclude that this fraction of endocytosed cholesterol, mobilized from lysosomes, is translocated to the ER through a PM-mediated pathway. The remaining sterol fraction presumably moves to the ER by a PM-independent pathway. In NP-C fibroblasts, the attenuated cholesterol esterification associated with the defective lysosomal cholesterol transport pathway is insensitive to suppression by CD (Fig. 9) and thus appears to bypass the PM.
The current studies show that cell surface SM affects the trafficking of lysosomal cholesterol to the PM. In SMasetreated cells, CD no longer suppresses the esterification of lysosomal cholesterol during progesterone washout (Fig. 10). Thus, removal of SM from the PM appears to alter, without apparently attenuating, intracellular trafficking of lysosomal cholesterol. Since esterification of LDL-derived cholesterol in SM-depleted cells is no longer affected by CD, we conclude that it has been re-routed to the ER by a PM-independent pathway. It is conceivable that SM depletion renders the PM incapable of accepting cholesterol delivered from lysosomes. Further investigation is required to evaluate this unexpected finding.
Several studies have provided a link between the Golgi complex and the processing of cellular cholesterol. It has been postulated that the distribution of cholesterol within the complex, increasing in an anterograde cis to trans direction (50), plays a role in the sorting of membrane proteins (51) and lipids (52). We have previously shown that the processing of LDLderived cholesterol in normal cells is accompanied by an enrichment of the cholesterol content of the Golgi complex in an anterograde fashion (31,53,54). By contrast LDL processing in NP-C fibroblasts leads to a premature (31) and abnormal (53) enrichment of cholesterol in Golgi cisternae. These studies were the first to implicate the Golgi with the intracellular trafficking of LDL-derived cholesterol.
We have now obtained further evidence for a role of the Golgi complex in the transport of LDL-derived cholesterol. Treat-ment of normal cells with BFA renders esterification of lysosomal cholesterol insensitive to exogenous CD (Fig. 11). This finding implies that cholesterol is normally transferred from lysosomes to the PM via the Golgi and that BFA disruption of this organelle results in re-routing of cholesterol to the ER via a PM-independent pathway. Our present studies also provide further insights to suggest that defective transport of lysosomal cholesterol in NP-C cells may be linked to disruption of its passage through the Golgi. As shown above, in these mutant cells, lysosomal cholesterol appears to be translocated to the ER via a PM-independent pathway, which is both attenuated and insensitive to exogenous CD. A curtailment in cholesterol transfer from lysosomes to the PM in NP-C cells due to a defect in movement through the Golgi complex is suggested by the finding that BFA curtails this transfer in normal cells. The observed 4-fold stimulation of cholesterol esterification in NP-C cells during disruption of the Golgi with BFA is consistent with the notion that abnormal cholesterol accumulation in the Golgi complex leads to an increase in the supply of substrate for acetyl-coenzyme A:cholesterol acyltransferase when Golgi components fuse with the ER.
The Golgi complex also appears to play a role in shuttling cholesterol between the PM and other organelles. Transfer of cholesterol between the Golgi and PM is thought to be involved in the formation of cholesterol-rich PM caveolae (55). The caveolar-specific cholesterol binding protein, caveolin (56), cycles constitutively between the PM and intracellular sites including the Golgi complex (57). Our present studies support a retrograde movement of cholesterol from the PM to the ER that may be modulated by the Golgi complex. Mobilization of cholesterol from the PM following SMase treatment results in an enrichment of the cholesterol content in the Golgi complex (data not shown), as monitored by the stabilized fluorescence of C 6 -NBD-ceramide (54). The movement of cholesterol between the PM and the Golgi may in turn be linked to its appearance in the ER. This is supported by our observation that BFAinduced disruption of the Golgi complex greatly enhances esterification of PM cholesterol mobilized by SM depletion (Fig.  12). This marked enhancement of cholesterol esterification by BFA suggests a direct role for the Golgi in regulating trafficking of PM-derived cholesterol to the ER. Mobilization of PM cholesterol to the ER remains deficient in NP-C cells, even with BFA treatment, suggesting an inherent defect in the processing of cholesterol by the Golgi in these mutant cells. Golgi processing of cholesterol derived from LDL and the PM may play an essential role in maintaining the appropriate distribution of cholesterol within the cell.