Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.

We have studied the movement of low density lipoprotein (LDL)-derived cholesterol in cultured Chinese hamster ovary cells. Our hypothesis is that when LDL cholesterol is effluxed from lysosomes, the bulk of LDL cholesterol is mobilized to the plasma membrane, while another pathway delivers LDL cholesterol from lysosomes to acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum. Three lines of evidence support this model. First, LDL cholesterol transport to ACAT can be blocked without inhibiting the movement of cholesterol from lysosomes to plasma membrane or from plasma membrane to endoplasmic reticulum. Second, LDL cholesterol transport to ACAT is normal in a Chinese hamster ovary mutant with defective plasma membrane-to-ACAT movement. Third, LDL cholesterol is not diluted by the plasma membrane cholesterol pool before reaching ACAT. Our evidence supports a vesicular model of cholesterol transport from lysosomes to the endoplasmic reticulum that is independent of the plasma membrane.

(ER) enzyme acyl-CoA/cholesterol acyltransferase (ACAT) or incorporated into lipoproteins, bile acids, or steroid hormones, depending on the cell type.
The mechanism of cholesterol efflux from lysosomes and movement to other cellular sites is not well defined. Cholesterol transport to the plasma membrane is energy-independent and does not require an intact cytoskeleton (4), yet it is unknown whether transport is vesicle-mediated or via soluble carrier proteins. Furthermore, the route of cholesterol trafficking is an area of intense investigation. Several lines of evidence suggest that LDL cholesterol is rapidly transported to the plasma membrane (5,6). However, two recent studies have provided evidence for a plasma membrane-independent pathway from lysosomes to ER (7,8).
Underwood et al. (7) investigated cholesterol transport using hydrophobic amines, drugs that inhibit specific cholesterol transport pathways. We found that LDL stimulation of cellular cholesterol esterification by ACAT is much more sensitive to hydrophobic amine inhibition than bulk cholesterol movement from lysosomes to plasma membrane and from plasma membrane to ER. We concluded that hydrophobic amines must inhibit either a previously uncharacterized transport pathway from lysosomes to plasma membrane or a signaling event that activates ACAT. Neufeld et al. (8) also presented strong evidence for an alternative pathway for LDL cholesterol movement to ER. Their data suggested that up to 30% of LDL cholesterol may traffic by this pathway.
In this study, we provide further evidence supporting our model that, while the bulk of LDL cholesterol is mobilized to the plasma membrane, a portion is delivered to the ER by a plasma membrane-independent pathway. Hydrophobic amine inhibition of LDL cholesterol transport to ACAT was quantified in Chinese hamster ovary (CHO) cells, and evidence is presented for a pathway that is uniquely sensitive to hydrophobic amine inhibition. Amphotericin B was used to confirm that LDL cholesterol is prevented from reaching the plasma membrane in the presence of hydrophobic amines. In addition, LDL cholesterol transport to ACAT was assessed in a CHO mutant with defective plasma membrane-to-ER cholesterol transport (9) and found to be normal. The route and mechanism of LDL cholesterol movement was tested using pharmacological agents. We found that LDL cholesterol transport to ACAT proceeded while the Golgi complex was severely disrupted but that it was inhibited by agents that interfere with vesicular trafficking. Overall, our evidence strongly supports a vesicular LDL cholesterol transport pathway from lysosomes to ER that is independent of the plasma membrane.  1 The abbreviations used are: LDL, low density lipoprotein; ER, endoplasmic reticulum; ACAT, acyl-CoA/cholesterol acyltransferase; CHO, Chinese hamster ovary; U18666A, 3-␤- [ (11). Lipoprotein-deficient serum was prepared as described, omitting the thrombin incubation (10). The following media were prepared: H-5% NCS (Ham's F-12 medium containing 5% (v/v) newborn calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 20 mM HEPES, pH 7.1); H-5% LPDS and H-1% LPDS (H-5% NCS in which 5% (v/v) newborn calf serum was replaced with 5 or 1% (v/v) lipoproteindeficient calf serum, respectively); H-5% LPDS/mev (H-5% LPDS containing 20 M mevinolin and 0.5 mM mevalonate); and H-BSA (Ham's F-12 medium containing 0.2% BSA). The corresponding Dulbecco's modified Eagle's medium-based media were prepared using fetal calf serum.

Esterification of LDL [ 3 H]Cholesterol
On day 0, CHO cells were seeded into six-well plates (25,000 cells/ well) in H-5% NCS. On day 1, cells were washed in Hanks' balanced salt solution (HBSS) and refed H-5% LPDS. On day 3, cells were refed H-5% LPDS. Experiments were conducted on day 3 or 4 as described in the figure legends. Cells were then washed with TBS, and lipids were extracted with hexane/isopropyl alcohol (3:2 (12). Radioactivity was measured with liquid scintillation counting in ReadySafe. After lipid extraction, monolayers were incubated with 0.1 N NaOH, and aliquots were taken for protein determination (13). The 50% inhibitory concentrations (IC 50 ) were calculated by regression analysis.

Cholesterol Oxidase Treatment
Comparison of CHO and Hepatoma Cells-On day 0, CHO cells were seeded into 12-well plates (15, (7). Hepatoma cells were subjected to the identical experiment in parallel using the appropriate Dulbecco's modified Eagle's medium-based media. Statistical comparisons were made using a standard two-tailed, unpaired Student's t test (GraphPad In-Stat, GraphPad Software version 2.02).

Basal Esterification of Plasma Membrane Cholesterol
On day 0, CHO cells were seeded into 12-well plates (10,000 cells/ well) in H-5% NCS. On day 2, cells were washed with HBSS and refed 0.5 ml of H-5% LPDS. Additions of 1 Ci/ml [ 3 H]cholesterol in ethanol were made at staggered times. U18666A and imipramine were added 11.5 h before harvest. On day 4, cells were washed with TBS. Lipids were extracted with hexane/isopropyl alcohol (3:2), and a chromatography standard was added (50 g of cholesterol, 30

FITC-Dextran Uptake and Fluorescence Microscopy
On day 0, CHO cells were seeded into two-well chamber slides (Falcon 4102, 20,000 cells/well) in H-5% NCS. On day 2, cells were washed with HBSS and fed H-5% LPDS. On day 3, cells were incubated as described in the Fig. 6 legend and then washed with PBS and fixed with 3% paraformaldehyde for 30 min. Cells were washed with PBS and mounted. Fluorescence images were obtained using a Zeiss IM35 microscope (ϫ 40 objective) and photographed using Kodak Elite II 400 film.

Incorporation of LDL [ 3 H]Cholesterol and [ 14 C]Oleate into Cholesteryl Esters
On day 0, CHO cells were seeded into six-well plates (25,000 cells/ well) in H-5% NCS. On day 2, cells were washed with HBSS and fed H-5% LPDS. On day 4, cells were incubated as described in the Fig. 8   At these hydrophobic amine concentrations, LDL cholesterol is able to move freely from lysosomes to plasma membrane and from plasma membrane to ER. These inhibition results indicate that LDL cholesterol moves from lysosomes to ACAT via a pathway that is independent of the plasma membrane.
Hydrophobic Amine Inhibition of LDL Cholesterol Transport to the Plasma Membrane-In our previous study (7), the lysosome to plasma membrane cholesterol transport pathway was assessed by pulsing cells with [ 3 H]CL-LDL and determining the amount of LDL [ 3 H]cholesterol that is sensitive to exogenously added cholesterol oxidase (14), i.e. at the plasma membrane (2). However, our results were questioned by Lange et al. (17), who found that LDL [ 3 H]cholesterol moves to a cholesterol-oxidase accessible pool, even in the presence of hydrophobic amines. They conclude that all LDL cholesterol is transported to the plasma membrane before moving to the cell interior, and that the hydrophobic amine inhibition of LDL cholesterol transport to ACAT is due solely to a block in plasma membrane-to-ER movement. This has led us to study the FU5AH rat hepatoma cells used by Lange  Lange et al. (17) found no effect of U18666A and imipramine on accessibility of LDL cholesterol to cholesterol oxidase. We attribute this to their using a hepatoma cell line that is less sensitive to hydrophobic amine inhibition than CHO cells and to their using a cholesterol oxidation method that we find oxidizes intracellular pools of cholesterol. Fig. 3 shows fluorescence microscopy of filipin-stained CHO cells cultured in H-5% NCS. Filipin is a fluorescent polyene antibiotic that binds specifically to cholesterol and is used to detect cellular cholesterol pools (18). Untreated CHO cells exhibit filipin staining at the plasma membrane and in a punctate distribution, most likely representing endosomes and lysosomes (Fig. 3A). Each cell has one bright spot that we identified as the Golgi complex by co-localization with Bodipy FL C5-ceramide (Ref. 19 and data not shown). Fig. 3B shows CHO cells after cholesterol oxidase treatment by the method of Slotte (14). Filipin fluorescence was no longer visible at the plasma membrane, but the intracellular staining was still present, as shown previously (20). This treatment oxidized 81% of cholesterol mass and 76% of [ 3 H]cholesterol. Fig. 3C shows CHO cells after cholesterol oxidase treatment by the method of Lange (2). Some cells showed a diffuse fluorescence, but no distinct intracellular fluorescence was visible.  drophobic amines inhibit LDL cholesterol transport to the plasma membrane are in agreement with several other experimental approaches. We have now employed an amphotericin B test to demonstrate hydrophobic amine inhibition of LDL cholesterol transport to the plasma membrane. Amphotericin B is a polyene antibiotic that forms pores in cholesterol-rich membranes (21)(22)(23). Cells capable of transporting LDL cholesterol to the plasma membrane are lysed and killed by amphotericin B treatment, while cells with impaired transport of LDL cholesterol to the cell surface survive (24). In Fig. 4, we show that U18666A and imipramine prevented LDL cholesterol from causing amphotericin B-mediated cell killing.
CHO cells were incubated in various media for 16 h and treated with amphotericin B for 5 h, and then cell survival was evaluated using a colorimetric MTT assay. Cells cultured in H-5% LPDS/mev had a reduced plasma membrane cholesterol content because cholesterol synthesis is inhibited by mevinolin, and the media contain no lipoproteins. These cells survived amphotericin B treatment and exhibited high levels of MTT cleavage. LDL addition restored the plasma membrane cholesterol content and caused cells to be killed by amphotericin B (Fig. 4A). However, when LDL was added along with U18666A ( Fig. 4B) or imipramine (Fig. 4C), cell killing was prevented. To prevent amphotericin B-mediated cell killing, hydrophobic amines had to be present throughout the LDL incubation. When cells were incubated with LDL for 16 h and then either U18666A or imipramine was added during the last hour of the incubation, cells were effectively killed by amphotericin B (data not shown). Our results indicate that, in the presence of hydrophobic amines, LDL cholesterol is not transported to the plasma membrane, i.e. not accessible to cholesterol oxidase or amphotericin B.
Hydrophobic Amine Inhibition of the Plasma Membrane to the ER Cholesterol Transport Pathway-Lange et al. (17) attribute hydrophobic amine inhibition of LDL cholesterol esterification to inhibition of the plasma membrane to the ER pathway. However, we found no effect of imipramine on this pathway. In our previous study (7), the plasma membrane to ER pathway was evaluated by treating [ 3 H]cholesterol-labeled cells with sphingomyelinase (25). Digestion of plasma membrane sphingomyelin causes redistribution of cholesterol within the plasma membrane and movement of 5-10% of plasma membrane cholesterol to the ER (26 5A). U18666A inhibited the basal movement of plasma membrane cholesterol to ACAT with an IC 50 of 0.6 M (Fig. 5B), which is similar to the IC 50 for U18666A inhibition of sphingomyelinase-stimulated esterification, 0.5 Ϯ 0.1 M (7). Imipramine consistently showed a very slight inhibition at 10 and 30 M but no significant difference at 100 M (Fig. 5C). This finding is consistent with imipramine's lack of inhibition of sphingomyelinase stimulation of cholesterol esterification (7,26). It underscores our assertion that imipramine inhibition of lysosome to ER cholesterol movement cannot be due to effects on plasma membrane-to-ER transport.
This result also indicates that [ 3 H]cholesterol added to the medium in ethanol is transferred to the plasma membrane. If a substantial portion of the [ 3 H]cholesterol had adhered to serum components and been delivered to lysosomes, we would have seen imipramine inhibition of [ 3 H]cholesterol esterification.
Effect of U18666A on Plasma Membrane Vesiculation Induced by Sphingomyelinase and Energy Poisons-U18666A inhibits basal movement of plasma membrane cholesterol to the ER, as well as accelerated movement induced by plasma membrane sphingomyelin digestion (7,26). One possible mechanism for U18666A action is inhibition of plasma membrane internalization. As stated above, Skiba et al. (27) showed that sphingomyelinase treatment induced plasma membrane internalization, which was viewed as punctate fluorescence throughout the cell by including FITC-dextran in the culture medium. Sphingomyelinase treatment in the presence of energy poisons caused the vesicles to remain in the cell periphery. However, energy poisons did not interfere with sphingomyelinase-induced delivery of plasma membrane cholesterol to ACAT. Since U18666A does inhibit sphingomyelinase-induced delivery of plasma membrane cholesterol to ACAT (7,26), we wanted to determine if U18666A inhibits plasma membrane vesiculation or delivery of vesicles throughout the cell.
CHO cells were preincubated with and without U18666A and energy poisons for 10 min and then incubated with the same medium containing FITC-dextran, with or without sphingomyelinase, for 30 min before fixation. Fluorescence microscopy revealed punctate endocytic vesicles throughout control cells but not cells treated with energy poisons (data not shown), as seen by Skiba et al. (27). Sphingomyelinase treatment produced an increased number of vesicles with brighter fluorescent intensity (Fig. 6A). U18666A did not prevent FITC-dextran uptake (Fig. 6B). In the presence of energy poisons, the sphingomyelinase-induced vesicles remained at the cell periphery (Fig. 6C), and U18666A did not inhibit their formation or distribution (Fig. 6D). Therefore, U18666A does not act by preventing plasma membrane internalization in response to sphingomyelin degradation.
As a control, parallel cultures of the same experiment were labeled with [ 3

H]cholesterol and subjected to sphingomyelinase treatment. Sphingomyelinase stimulated [ 3 H]cholesterol conversion to [ 3 H]cholesteryl esters, and U18666A inhibited the [ 3 H]cholesteryl ester formation (data not shown).
The Lysosome-to-ER Pathway in Cholesterol Transport-defective CHO Cell Mutants-Additional evidence for a lysosome to ER cholesterol transport pathway comes from analysis of two complementation classes of cholesterol transport-defective CHO cells. The Ced-1 class exhibits a classical Niemann-Pick type C biochemical phenotype (24). Ced-1 mutants 2-2 and 4-4 show no LDL stimulation of cholesterol esterification, because efflux of LDL cholesterol from lysosomes is greatly impaired. Ced-2 mutant 3-6 shows LDL stimulation of cholesterol esterification that is reduced compared with control but that is clearly discernible (28). Mutant 3-6 displays normal movement of LDL cholesterol out of lysosomes but completely defective transport of cholesterol from the plasma membrane to ACAT (9). In the following experiment, we tested the hypothesis that the discernible LDL stimulation of ACAT in mutant 3-6 is due to an intact lysosome-to-ER cholesterol transport pathway. Table I  Pharmacological Analysis of Lysosome-to-ER Cholesterol Transport-The mechanism by which LDL cholesterol is transported from lysosomes to ER was investigated. Table II shows the effects of agents that disrupt the cytoskeleton and acidic compartments and thus affect vesicular transport pathways. In these experiments, CHO cells were labeled for 2 h with [ 3 H]CL-LDL in the presence of 100 M imipramine, which allows uptake of [ 3 H]CL-LDL but prevents movement of free [ 3 H]cholesterol out of lysosomes. Loading lysosomes with [ 3 H]CL-LDL was necessary, because many of the agents tested inhibit receptor-mediated endocytosis of LDL (4). Imipramine was washed out using medium containing various test compounds, after which the cells were incubated for 5 h.
Colchicine disruption of microtubules had no effect on LDL cholesterol movement to ACAT. Parallel cultures incubated with an anti-tubulin antibody and examined by indirect immunofluorescence showed observable changes in the microtubule network at 5 M colchicine and dramatically altered appearance at 100 M (data not shown). Nigericin, a potassium ionophore, consistently inhibited LDL cholesterol transport to ACAT. This inhibition is not due to action on the enzyme, since nigericin does not directly inhibit ACAT activity in cell homogenates (29). Cytochalasin D at 10 and 50 M disrupted actin filaments, as assessed by fluorescence microscopy after phalloidin staining (data not shown), and inhibited LDL cholesterol transport to ACAT by 36 -40%. Monensin, a sodium ionophore, inhibited LDL cholesterol transport to the ER very effectively but does not directly inhibit ACAT activity in cell homogenates (29). The above results implicate the actin microfilament network and acidic compartments, such as transport vesicles and the trans-Golgi network, in LDL cholesterol transport to the ER.
Effect of Brefeldin A on the Lysosome to ER Pathway-To determine if LDL cholesterol is transported through the Golgi complex, we used brefeldin A, a fungal metabolite that prevents ER to Golgi vesicular transport (30). Retrograde transport still continues in brefeldin A-treated cells; thus, the cis and medial Golgi cisternae fuse with the ER. At 2 g/ml, brefeldin A causes the Golgi to merge with the ER in CHO cells, as assessed by fluorescence microscopy with Bodipy FL C5ceramide/BSA (Ref. 31 and data not shown). Fig. 7 (Fig. 7A), consistent with its known effects on endocytosis and delivery of ligands to lysosomes (30); however, the residual [ 3 H]CL internalized was efficiently hydrolyzed and esterified (Fig. 7, B and C). There was essentially no difference in the percentage of LDL [ 3 H]cholesterol transported to ACAT and esterified in brefeldin Atreated cells for up to 4 h (Fig. 7D), suggesting that an intact Golgi apparatus is not necessary for cholesterol transport from lysosomes to ER. At 6 h, there was a decrease in [ 3 H]cholesterol esterification, but this could be due to secondary effects of the lengthy brefeldin A treatment on the membrane environment surrounding the enzyme. The actual effects of brefeldin A on cholesterol    Fig. 8C shows the 3 H/ 14 C ratio, which was high at early time points and then declined with time. This is consistent with a model in which LDL cholesterol reaches ACAT without first being transported to the plasma membrane and diluted by cellular cholesterol. DISCUSSION At steady state, 65-80% of cellular cholesterol is in the plasma membrane (3,32). Cholesterol in the plasma membrane is not static, however; it constantly moves between the cell interior and surface (33). Plasma membrane cholesterol levels rise when LDL is internalized, as the LDL cholesterol is rapidly transported to the plasma membrane (6,34). As the cholesterol pool expands, ACAT is activated in the ER, and excess cellular cholesterol is stored as cholesteryl esters (35).
How does the signal reach ACAT that LDL cholesterol is expanding the cellular cholesterol content? One possibility is that LDL cholesterol is efficiently transported to the plasma membrane. The rising plasma membrane cholesterol level increases cholesterol cycling into the cell interior, which activates ACAT. Another possibility is that LDL cholesterol is transported along multiple pathways.
Our hypothesis is that, while the bulk of LDL cholesterol is mobilized to the plasma membrane, a portion is delivered to the ER by a plasma membrane-independent route. Three lines of evidence from our studies support this model.
(i) Conditions of hydrophobic amine inhibition of cholesterol transport were defined in which LDL cholesterol freely moves from lysosomes to plasma membrane and plasma membrane cholesterol can freely move to ACAT in the ER. Yet under these conditions, LDL cholesterol does not activate ACAT and does not get esterified. Furthermore, LDL cholesterol is not esterified even if ACAT is independently activated by 25-HC. These results suggest that a cholesterol transport pathway exists that is exquisitely sensitive to hydrophobic amine inhibition.
(ii) Cholesterol transport mutant 3-6 exhibits normal movement of LDL cholesterol from lysosomes to plasma membrane but completely defective transport of plasma membrane cholesterol to ACAT (9,28). Here, we found that LDL cholesterol transport from lysosomes to ACAT is normal in mutant 3-6, which suggests that the fraction of LDL cholesterol that is esterified does not traffic through the plasma membrane on its way to the ER. The portion transported to ACAT in mutant 3-6 is probably responsible for the detectable LDL stimulation of [ 14 C] ratio should be high at early times and then decline. If LDL cholesterol was transported to the plasma membrane before moving to the ER, then we expected a low 3 H/ 14 C ratio that increased with time. However, our results indicated that LDL cholesterol was not diluted by the plasma membrane cholesterol pool before reaching ACAT.
The existence of a lysosome-to-ER pathway of cholesterol transport was first proposed by Tabas (36). Evidence for such a pathway was previously presented by Underwood et al. (7) and Neufeld et al. (8). Neufeld et al. used 2-hydroxypropyl-␤-cyclodextrin (CD) to evaluate cholesterol transport pathways in cultured cells. CD is a cyclic oligomer of glucose that serves as a rapid, efficient extracellular acceptor of plasma membrane cholesterol (37). Cells were preloaded with LDL in the presence of progesterone and then incubated with CD in the absence of LDL and progesterone. [  cholesterol in the ER. They found that CD blunted approximately 70% of the LDL stimulation of ACAT, which suggested that about 70% of the LDL cholesterol was transported to ACAT via the plasma membrane and that the residual LDL cholesterol was transported by a plasma membrane-independent pathway.
Evidence seemingly against a lysosome-to-ER pathway was presented by Lange et al. (17) In a third experiment, Lange et al. (17) found no effect of hydrophobic amines on LDL [ 3 H]cholesterol accessibility to cholesterol oxidase. They attribute hydrophobic amine inhibition of LDL cholesterol esterification to a block in the plasma membrane-to-ER pathway. We and others (26) have found no significant effect of imipramine on the plasma membraneto-ER pathway. Furthermore, we have used multiple experimental approaches to show that hydrophobic amines affect LDL cholesterol transport from lysosomes to the plasma membrane. In the presence of hydrophobic amines, LDL cholesterol is not accessible to cholesterol oxidase or amphotericin B. It is also not available for desorption to extracellular acceptors (38). By filipin staining and subcellular fractionation, it is sequestered within lysosomes (38,39). We attribute the results of Lange et al. (17) results to cholesterol oxidase reaching an intracellular pool of cholesterol.
Our current study does not resolve whether the Golgi apparatus is involved in cholesterol movement. We added brefeldin A to the cultures 1 h prior to the addition of [ 3 H]CL-LDL. By the time LDL [ 3 H]cholesterol was leaving the lysosomes, the Golgi complex was disrupted (30). Yet esterification of LDL [ 3 H]cholesterol proceeded normally for several hours. Therefore, an intact Golgi complex is not necessary for cholesterol transport from lysosomes to ER. However, we cannot conclude that the pathway is Golgi-independent. The lack of an observed brefeldin A effect on LDL cholesterol esterification could be due to a transport "vehicle," which normally docks at the Golgi, being capable of finding its docking site at the merged Golgi/ ER. This is consistent with the results of Neufeld et al. (8), who found little brefeldin A effect on LDL stimulation of [ 3 H]oleate incorporation into cholesteryl [ 3 H]oleate. However, this experiment was complicated by the effect of Golgi cholesterol arriving in the ER and stimulating ACAT. The most compelling evidence for Golgi involvement in cholesterol transport comes from filipin electron microscopy, showing cholesterol enrichment of Golgi cisternae following LDL uptake (40).
LDL cholesterol movement from lysosomes to ER is likely to be a vesicle-mediated event. First, transport was inhibited by monensin and nigericin, ionophores that neutralize acidic compartments such as endosomes and the trans-Golgi network. Second, actin filaments, but not microtubules, appear to play a role in LDL cholesterol delivery to ACAT. Actin filaments were disrupted by cytochalasin D at 10 and 50 M. Lower concentrations of cytochalasin D were ineffective in disrupting filaments, as assessed by fluorescence microscopy of phalloidin-stained cells. This finding is in contrast to that of Tabas et al. (41), who observed filament dissolution in CHO cells with 3 M cytochalasin D, with no effect on ␤-very low density lipoprotein stimulation of cholesteryl [ 3 H]oleate formation.
What is the mechanism by which hydrophobic amines inhibit cholesterol transport? Our work to date has eliminated mechanisms of U18666A action but has not provided positive evidence for a possible mechanism. U18666A does not act by preventing cholesterol desorption from membranes or by inhibiting P-glycoprotein activity (7). U18666A inhibited the basal movement of plasma membrane cholesterol to ACAT but did not block endocytosis of FITC-dextran. U18666A also inhibited the accelerated delivery of plasma membrane cholesterol to ACAT that occurs with plasma membrane sphingomyelin digestion (7,26); however, U18666A did not prevent the sphingomyelinase-induced plasma membrane vesiculation.
Evidence for a cholesterol transport pathway from lysosomes to the ER has implications for macrophages and atherosclerosis. If this pathway is necessary to activate ACAT, then drugs could be designed to selectively knock out this pathway, slowing down esterification and foam cell production in macrophages. Future work will focus on in vitro characterization of this pathway to determine proteins involved.