The Fate of Cholesterol Exiting Lysosomes*

(Received for publication, January 28, 1997, and in revised form, April 2, 1997)

Yvonne Lange Dagger , Jin Ye and Janet Chin

From the Departments of Pathology and Biochemistry, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgements
REFERENCES


ABSTRACT

Cholesterol released from ingested low density lipoproteins in lysosomes moves both to the plasma membrane and to the endoplasmic reticulum (ER) where it is re-esterified. Whether cholesterol can move directly from lysosomes to ER or first must traverse the plasma membrane has not been established. To examine this question, the endocytic pathway of rat hepatoma cells was loaded at 18 °C with low density lipoproteins (LDL) labeled with [3H]cholesteryl linoleate, and the label then was chased at 37 °C. The hydrolysis of the accumulated ester proceeded linearly for several hours. Almost all of the released [3H]cholesterol moved to the plasma membrane rapidly and without a discernable lag. In contrast, the re-esterification in the ER of the released [3H]cholesterol showed a characteristic lag of 0.5-1 h. These data are inconsistent with direct cholesterol transfer from lysosomes to ER; rather, they suggest movement through the plasma membrane.

Furthermore, we found that progesterone, imipramine and 3-beta -[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) strongly inhibited the re-esterification of lysosomal cholesterol in the ER. However, contrary to previous reports, they did not block transfer of [3H]cholesterol from lysosomes to the cell surface. Therefore, the site of action of these agents was not at the lysosomes. We suggest instead that their known ability to block cholesterol movement from the plasma membrane to the ER accounts for the inhibition of lysosomal cholesterol esterification.

These findings are consistent with the hypothesis that cholesterol released from lysosomes passes through the plasma membrane on its way to the ER rather than proceeding there directly. As a result, ingested cholesterol is subject to the same homeostatic regulation as the bulk of cell cholesterol, which is located in the plasma membrane.


INTRODUCTION

Cell cholesterol levels are tightly regulated by homeostatic mechanisms. For example, when cells ingest cholesterol in the form of low density lipoprotein (LDL),1 sterol biosynthesis is reduced and excess cholesterol is converted to cholesteryl esters for storage (1, 2). While these processes are well understood, the associated pathways of intracellular cholesterol movement are obscure. It appears that cholesterol moves bidirectionally between the plasma membrane and the ER (3). Furthermore, the cholesterol released from ingested LDL in lysosomes moves rapidly to the plasma membrane (4). Since the cholesterol derived from the degradation of LDL is re-esterified by ACAT (1, 2), lysosomal sterol must also be transported to the ER.

Two recent studies addressed the question of the pathway taken by cholesterol from lysosomes to ER. One suggested direct movement of cholesterol from lysosomes to ER by a mechanism that is inhibited by amphiphiles (5). The other concluded that approximately 70% of lysosomal cholesterol passes through the plasma membrane prior to esterification, the remainder moving directly between the lysosomes and the ER (6).

In the present study, we have analyzed the fate of ingested LDL [3H]cholesteryl esters. We found no evidence for a pathway of cholesterol movement from the lysosomes to the ER that does not include the plasma membrane.


EXPERIMENTAL PROCEDURES

Materials

[1a-2a(n)-(3H)]cholesteryl linoleate (48 Ci/mmol), [1,2-3H]cholesterol (40 Ci/mmol), [4-14C]cholesterol (52 mCi/mmol), and [1-14C]oleic acid (58 mC/mmol) were from Amersham Corp. Cholesterol oxidase (EC 1.1.3.6; Brevibacterium sp.) was from Beckman Clinical Diagnostics (Carlsbad, CA). U18666A was from The Upjohn Co. (Kalamazoo, MI).

Cells

FU5AH rat hepatoma cells and human foreskin fibroblasts derived from primary explants were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Prior to feeding labeled LDL, fibroblasts were incubated for 16-20 h in medium supplemented with 5% lipoprotein-deficient serum to up-regulate LDL receptors (1). Such preincubation did not substantially stimulate LDL uptake in hepatoma cells (data not shown) and was omitted in experiments with these cells. Similar results were obtained with both cells types. However, because they esterify large amounts of cholesterol, hepatoma cells were used in these studies with the exception of the subcellular fractionation of cell homogenates on Percoll gradients where resolution of membranes was much better for fibroblasts.

Preparation of Labeled LDL

Plasma obtained from healthy volunteers was labeled with [3H]cholesteryl linoleate as described (7), and the LDL was isolated by ultracentrifugation (8). The product had a specific activity of ~106 dpm/mg protein. Lipoprotein-deficient serum (LPDS) was prepared from fetal bovine serum as described (9).

Specific Labeling of Cell Surfaces with Sterols

Cell monolayers were rinsed and covered with phosphate-buffered saline plus the radiolabeled cholesterol solubilized in Triton WR1339 (0.025% final). The flasks were incubated for 20 min at 18 °C, the buffer was removed, and the cells were rinsed and layered with medium for further incubation at 37 °C as described in the figure legends.

Pulse-chase Experiments

The medium was removed from replicate flasks of cells, and the cells were rinsed. Medium containing 5% LPDS plus [3H]LDL (50 µg/ml protein) was layered over the cells, and the flasks were incubated for 1.5-2 h at 18 °C (10). The labeled medium was removed, and the cells were rinsed three times with phosphate-buffered saline. Medium containing 10% fetal calf serum or 5% LPDS was then added to the flasks for further incubation at 37 °C.

Subcellular Fractionation

Cell homogenates prepared as described (11) were mixed with 30% Percoll in 5 mM NaPi (pH 7.5), 0.25 M sucrose, 1 mM EDTA and layered on a 0.2-ml cushion of 56% sucrose in 5 mM NaPi (pH 7.5). The tubes were centrifuged for 30 min at 35,000 rpm at 4 °C in a Beckman SW 50.1 rotor. The gradients were fractionated from the bottom of the tubes.

Cholesterol Oxidase Treatment

Cells were dissociated and fixed for 10 min on ice with 1% glutaraldehyde in phosphate-buffered saline. They were washed twice in 5 mM NaPi (pH 7.5), 0.25 M sucrose and resuspended in 0.1 mM EDTA (pH 7.5), 0.25 M sucrose. After warming for 5 min at 37 °C, cholesterol oxidase was added at 10 IU/ml, and the cells were incubated for 10 min at 37 °C prior to extraction and assay.

Lipid Analysis

For measurement of incorporation of [3H]cholesterol into total esters, cholesterol and cholestenone, the extracted lipids were analyzed by thin layer chromatography on silica gel G plates using hexane:ethyl acetate (90:10) as solvent. The plates were developed in iodine, and the spots corresponding to authentic standards were scraped into vials for the determination of radioactivity. The re-esterification in the ER of [3H]cholesterol released from [3H]cholesteryl linoleate in the lysosomes was determined by its appearance in the palmitate plus oleate forms. [3H]cholesteryl esters were measured by HPLC on a C18 reverse phase column at 30 °C using acetonitrile:isopropyl alcohol (1:1) as the mobile phase (12). Re-esterification of [3H]cholesterol was calculated as: ([3H]cholesteryl oleate + [3H]cholesteryl palmitate) divided by ([3H]cholesterol + [3H]cholesteryl oleate + [3H]cholesteryl palmitate). Hydrolysis of [3H]LDL was calculated as: ([3H]cholesterol + [3H]cholesteryl oleate + [3H]cholesteryl palmitate) divided by ([3H]cholesteryl linoleate + [3H]cholesterol + [3H]cholesteryl oleate + [3H]cholesteryl palmitate). Results from time courses using replicate flasks were normalized to total input radioactivity. In some experiments, [3H] values were corrected for losses using [14C]cholesteryl oleate added at the moment of extraction. Cholesterol mass was determined by HPLC (3).

Lysosomal beta -Galactosidase

The assay used 0.8 mg/ml 4-methylumbelliferyl beta -D-galactopyranoside (Molecular Probes) as substrate. The reaction was carried out for 30 min at 37 °C in 100 mM glycine buffer (pH 3.0) containing 0.2% Triton X-100 and stopped by adding 4 volumes of 200 mM sodium phosphate (pH 12).


RESULTS

Time Course of Hydrolysis and Re-esterification of LDL Cholesteryl Linoleate

A pulse of [3H]cholesteryl linoleate-labeled LDL ([3H]LDL) was allowed to accumulate in pre-lyosomal compartments during a preincubation at 18 °C (10). There was little hydrolysis of the [3H]cholesteryl linoleate during the loading period (Figs. 1A and 2A). Shift to 37 °C evoked rapid hydrolysis. The arrival of the released [3H]cholesterol at the ER was scored by its appearance in cholesteryl oleate and cholesteryl palmitate. The free [3H]cholesterol was re-esterified with lagged kinetics (Fig. 1B).


Fig. 1. Time course of metabolism of [3H]LDL by hepatoma cells. Replicate flasks were incubated for 2 h at 18 °C with medium containing 5% lipoprotein-deficient serum and [3H]LDL. The cells were rinsed and fresh medium containing 10% fetal bovine serum was added to the flasks. At the times indicated, the cells were suspended, treated with cholesterol oxidase, and assayed for the radiolabel in esters, cholestenone, and cholesterol as described under "Experimental Procedures." Panel A shows the hydrolysis of [3H]cholesteryl linoleate calculated as the sum of unesterified and re-esterified label expressed as a percentage of the total label recovered in the flask. Panel B shows the sum of [3H]cholesteryl oleate and [3H]cholesteryl palmitate, representing re-esterified label and given as the total dpm in each flask. Panel C shows [3H]cholestenone (black-diamond ) and free [3H]cholesterol (open circle ) given as the total in each flask. The zero time values were measured in cells that had been incubated with [3H] LDL and not warmed.
[View Larger Version of this Image (13K GIF file)]


Fig. 2. Time course of incorporation into cholesteryl esters of [14C]oleic acid and [3H]cholesterol released from LDL in hepatoma cells. Replicate flasks of cells were labeled with [3H]LDL as described in the legend to Fig. 1. The cells were rinsed, and fresh medium containing 10% fetal bovine serum plus 0.07 µCi/ml [14C]oleic was added to the flasks. At the times indicated, the cells were dissociated and assayed for free [3H]cholesterol and for the incorporation of 3H and 14C into newly synthesized esters as described under "Experimental Procedures." Panel A shows the hydrolysis of LDL [3H]cholesteryl linoleate calculated as described in the legend to Fig. 1A. Panel B shows the incorporation of [3H]cholesterol into [3H]cholesteryl oleate and palmitate expressed as a percentage of total [3H]cholesterol (bullet ), and the incorporation into esters of [14C]oleic acid (open circle ) given as total dpm/flask.
[View Larger Version of this Image (19K GIF file)]

Movement of Lysosomal Cholesterol to the Plasma Membrane

In the experiment shown in Fig. 1, ~87% of the small amount of free [3H]cholesterol released from LDL in the lysosomes was accessible to cholesterol oxidase after the 2-h incubation at 18 °C (Fig. 1C). Approximately the same fraction of total free cholesterol was in the oxidizable pool at each time point during the subsequent 37 °C chase. This finding confirms that lysosomal cholesterol moves rapidly and continuously to the plasma membrane (4). The data also demonstrate that almost all of the free cholesterol released from LDL accumulates in the plasma membrane.

The esterification of bulk cell cholesterol in intact cells is commonly measured by the incorporation of [14C]oleate into cholesteryl esters (13). We compared the kinetics of bulk cholesterol esterification with that of the re-esterification of lysosomal [3H]cholesterol (Fig. 2). Incorporation of [14C]oleate into cholesteryl esters proceeded linearly while the esterification of the [3H]cholesterol from lysosomes once again was lagged. These data suggest that the lag in re-esterification of LDL cholesterol reflected the kinetics of its delivery to ACAT rather than the activity of the enzyme.

Esterification of Plasma Membrane Cholesterol Compared with Cholesterol Released in the Lysosomes

We prelabeled the lysosomal pool with ingested LDL [3H]cholesteryl linoleate and the plasma membrane pool with [14C]cholesterol. At intervals thereafter, 3H and 14C were determined in free and (re-)esterified cholesterol. The re-esterification of ingested LDL [3H]cholesterol was again strongly lagged while the esterification of plasma membrane [14C]cholesterol had only a small lag, perhaps reflecting recovery from the 18 °C incubation (Fig. 3A). With increasing time of chase, the fraction of each label esterified approached the same value (Fig. 3B).


Fig. 3. Esterification of plasma membrane [14C]cholesterol and the [3H]cholesterol released from ingested LDL in hepatoma cells. Replicate flasks were incubated for 1.5 h at 18 °C with medium containing 5% lipoprotein-deficient serum plus [3H]LDL. The cells were rinsed and buffer added containing [14C]cholesterol solubilized in Triton WR-1339. After a further 20 min incubation at 18 °C, the medium was replaced with medium containing 10% serum, and the flasks were incubated at 37 °C. At the times indicated, the cells were dissociated, extracted, and assayed for free [3H] and [14C]cholesterol and for the incorporation of 3H and 14C into newly synthesized esters as described under "Experimental Procedures." Panel A shows the synthesis of [3H]cholesteryl oleate plus palmitate from [3H]cholesterol released in lysosomes (bullet ) and of [14C]esters (open circle ), both expressed as percentages of total labeled cholesterol. Panel B shows the ratio of the percentage esterification of the two labels in four experiments, each with a different symbol. The zero time points (measured at the end of the 18 °C labeling incubation) for the four experiments are superimposed.
[View Larger Version of this Image (17K GIF file)]

Effect of Amphiphiles on the Movement of Lysosomal Cholesterol to the Plasma Membrane

A variety of amphiphiles inhibit cholesterol esterification, apparently by blocking movement of plasma membrane cholesterol to the ER (14-16), perhaps through interaction with a site resembling the multidrug-resistance P-glycoprotein (16). Although there is evidence that progesterone inhibits the transport of nascent cholesterol to caveolae (17), other studies have shown that the amphiphiles have no effect on sterol movement from the ER to the plasma membrane (14, 18). We now have tested their effect on transfer of [3H]cholesterol from lysosomes to the plasma membrane and ER.

None of the amphipaths tested had a significant impact on either the hydrolysis of the labeled LDL esters or on the movement of the liberated [3H]cholesterol to the cholesterol oxidase-sensitive compartment (the plasma membrane) (Fig. 4). In some experiments (e.g. Fig. 6), and at higher concentrations, imipramine and U18666A inhibited the hydrolysis of [3H]LDL, perhaps by reducing lysosomal acidity (19). However, in no case did the agents affect the rate of appearance of [3H]cholesterol in the oxidizable pool.


Fig. 4. Effect of progesterone, U18666A, and imipramine on transfer of lysosomal cholesterol to the plasma membrane. Replicate flasks of hepatoma cells were incubated in medium containing 5% lipoprotein-deficient serum and [3H]LDL for 2 h at 18 °C in the presence of progesterone (16 µM), imipramine (50 µM), U18666A (2.7 µM), or ethanol (<1%) as a solvent control. The medium was removed, and the cells were rinsed and overlaid with medium containing lipoprotein-deficient serum plus the agents at the same concentrations. After a 3-h incubation at 37 °C, the cells were dissociated, treated with cholesterol oxidase, and extracted for assay as described under "Experimental Procedures." Panel A shows percent hydrolysis of [3H]cholesteryl linoleate for (from the left) control (C), progesterone (PG), U18666A (U), and imipramine (IM). Panel B gives the percent oxidation of [3H]cholesterol, i.e. 100 × 3H in cholestenone/total 3H in unesterified cholesterol for (from the left) control, progesterone, U18666A, and imipramine.
[View Larger Version of this Image (34K GIF file)]


Fig. 6. Effect of progesterone, imipramine, and U18666A on re-esterification of lysosomal cholesterol in hepatoma cells. Replicate flasks of cells were incubated in medium containing 5% lipoprotein-deficient serum and [3H]LDL for 2 h at 37 °C in the presence of progesterone (12 µM), imipramine (50 µM), U18666A (2.7 µM), or ethanol (<1%) as a solvent control. The medium was discarded, and the cells were rinsed and overlaid with medium containing 10% serum plus the agents or ethanol as before. After 4 h of incubation at 37 °C, the cells were processed and assayed for [3H] in cholesteryl linoleate, cholesteryl oleate, cholesteryl palmitate, and cholesterol as described under "Experimental Procedures." Panel A gives percent hydrolysis calculated as described in the legend to Fig. 1 for (from the left) control (C) cells, cells treated with progesterone (PG), imipramine (IM), and U18666A (U). Panel B gives percent re-esterification of free [3H]cholesterol calculated as described in the legend to Fig. 1 for control and cells treated as in panel A.
[View Larger Version of this Image (28K GIF file)]

The effect of progesterone on the transfer to the plasma membrane of [3H]cholesterol released from the lysosomes of human fibroblasts was analyzed by subcellular fractionation on Percoll gradients (Fig. 5). The lysosomes (Fig. 5A, marked by beta -galactosidase) were well resolved from plasma membranes (Fig. 5, B and C, marked by cholesterol mass). Furthermore, the distribution of these markers was the same for treated and untreated cells. The major fraction of the [3H]cholesterol released from lysosomes was found in association with the plasma membranes both in control cells (Fig. 5B) and progesterone-treated cells (Fig. 5C). Similar results also were obtained with imipramine (not shown). These findings are consistent with the results obtained with cholesterol oxidase (Fig. 4), which suggested that progesterone and imipramine had no effect on movement of lysosomal cholesterol to the plasma membrane.


Fig. 5. Effect of progesterone on transfer of cholesterol from lysosomes to plasma membrane in fibroblasts. Replicate flasks of cells were preincubated for 19 h in medium containing 5% lipoprotein-deficient serum. [3H]LDL was then added to the medium together with progesterone (12 µM) or ethanol as a solvent control, and the flasks were incubated for 3 h at 37 °C. The cells were washed, dissociated, homogenized, and fractionated on Percoll gradients as described under "Experimental Procedures." Fraction 1 is the bottom of the gradient. The contents of beta -galactosidase, cholesterol mass, and [3H]cholesterol were measured in each gradient fraction. Panel A shows the distribution of beta -galactosidase in the control (down-triangle) and progesterone-treated (black-down-triangle ) cells. Panel B shows the distribution of cholesterol mass (bullet ) and [3H]cholesterol (open circle ) in the control cells. Panel C shows the distribution of cholesterol mass (black-diamond ) and [3H]cholesterol (diamond ) in the progesterone-treated cells. In this experiment, 68% of the [3H]cholesteryl ester had been hydrolyzed after the 3-h incubation at 37 °C both in the presence and absence of progesterone.
[View Larger Version of this Image (20K GIF file)]

The agreement of the results using subcellular fractionation and cholesterol oxidase validates the use of this probe. It was shown earlier that this enzyme does not have access to intracellular cholesterol pools in fixed cells (20); however, lysosomal cholesterol was not addressed specifically in those experiments. Recent studies of cells induced to accumulate lysosomal cholesterol by treatment with amphiphiles have confirmed that this intracellular pool is not a substrate for cholesterol oxidase.2

Effect of Amphiphiles on Re-esterification of Cholesterol Released from Lysosomes

The finding that progesterone, imipramine, and U18666A have little effect on the appearance of lysosomal cholesterol in the plasma membrane suggests that these agents do not inhibit movement out of lysosomes. Therefore, we examined whether they affect the re-esterification of lysosomal cholesterol. Cells were loaded with [3H]LDL for 2 h at 37 °C in the presence of various amphipaths and then chased for a further 4 h at 37 °C prior to assay of [3H]cholesteryl linoleate hydrolysis and re-esterification. We found that 38% of the [3H]cholesteryl linoleate was hydrolyzed in the control cells, and that 7.5% of this became re-esterified (Fig. 6). Progesterone, imipramine, and U18666A all caused a moderate decrease in the amount of labeled [3H]cholesteryl linoleate hydrolyzed, but profoundly inhibited the re-esterification of the released [3H]cholesterol. We have shown by measuring esterification in cell homogenates supplemented with saturating amounts of cholesterol delivered in Triton WR-1339 that 12 µM progesterone inhibits ACAT activity by less than 20% (15). Similar experiments showed that U18666A and imipramine have no effect on ACAT activity at 2.7 and 50 µM, respectively (data not shown); these were the concentrations used in Fig. 6. That U18666A does not inhibit ACAT also was concluded from the observation that the amphiphile had no effect on the synthesis of cholesteryl oleate in cells treated with 25-hydroxycholesterol (5).


DISCUSSION

It is well established that progesterone (21) and hydrophobic amines such as imipramine and U18666A (22) cause the dramatic accumulation of cholesterol in the lysosomes of cultured fibroblasts. This finding was explained by the subsequent observation that these compounds appeared to inhibit cholesterol movement from lysosomes to the plasma membrane (21, 23). The latter hypothesis was supported by more recent studies in which cholesterol oxidase was used to distinguish cholesterol in the plasma membrane from that in intracellular pools (5). In contrast, the results shown in Figs. 4 and 5 suggest that the amphiphiles have no effect on cholesterol transfer from lysosomes to plasma membrane. The reason for the discrepancy between our results and the earlier ones is not clear. Since it appears that the amphiphiles do not inhibit cholesterol egress from lysosomes (Figs. 4 and 5), its accumulation in the lysosomes of treated cells (21, 22) must have another explanation. In this regard, we have shown recently that treating cells with these amphiphiles induces the accumulation of excess sterol at the cell surface, which then is transferred to lysosomes in a process not normally visible.2 Thus, the large amount of lysosomal cholesterol found in amphiphile-treated cells is not trapped there but appears to be dynamically derived from the plasma membrane.

The second major finding presented here relates to the fate of [3H]cholesterol released from LDL [3H]cholesteryl linoleate in lysosomes. The lag in esterification of the sterol suggests that it passes through an intermediate compartment prior to esterification in the ER. This compartment could be intracellular; however, we shall argue below that it is most likely to be the plasma membrane.

Fig. 1C shows that most of the [3H]cholesterol released in lysosomes moves immediately to the plasma membrane (also see Ref. 4). About 85% of cell-free [3H]cholesterol was associated with the plasma membrane at all times during the chase. The fraction of cell cholesterol mass in the oxidizable pool was also 85% (not shown, but see Ref. 20). Of the ~15% of unoxidized cell cholesterol mass, a major fraction has been shown to be endocytic (20). Thus, the data suggest that most, and perhaps all, of the [3H]cholesterol emerging from the lysosomes immediately moved to the cell surface and its derivatives. The question then becomes whether there is evidence for a direct pathway from lysosome to ER, however minor in magnitude. As outlined below, our findings suggest not.

It can be argued from the kinetic data in Figs. 1, 2, 3 that the cholesterol emerging from the lysosomes mixed with cholesterol from the plasma membrane prior to re-esterification in the ER. If, upon release from the lysosomes, cholesterol had moved directly to the ER, the time course of its esterification would have been hyperbolic, as was the time course of its arrival at the plasma membrane (Fig. 1C). However, this was not observed (Fig. 1B). We interpret the lag in the re-esterification of the ingested [3H]cholesterol to reflect the profound isotope dilution that this probe experiences upon delivery to the plasma membrane. The accelerating time course would then reflect the progressive rise in the specific labeling of the plasma membrane cholesterol pool. Furthermore, the rate of esterification of lysosomal cholesterol still was increasing sharply after approximately 1 h of chase, even though the rate of hydrolysis of [3H]cholesteryl esters had begun to level off (Fig. 1, A and B). In contrast, the kinetics of movement of [3H]cholesterol to the plasma membrane paralleled the hydrolysis of the [3H]cholesteryl linoleate (Fig. 1, A and C), consistent with a direct relationship between the two processes.

If lysosomal cholesterol had moved directly to the ER, its representation in the nascent ester pool would initially have been at least as great as that of plasma membrane cholesterol. That this was not the case is shown in Fig. 3, B. It is also significant that while the esterification of plasma membrane [14C]cholesterol initially was proportionately greater than that of lysosomal [3H]cholesterol, the contributions from the two pools equalized after 2-3 h of chase (Fig. 3, B). This result suggests that both labels had entered the same substrate pool, namely the plasma membrane.

The inhibition of the esterification of lysosomal cholesterol by progesterone, U18666A, and imipramine (Fig. 6) is unlikely to be due to a block in the export of cholesterol from lysosomes since transfer to the plasma membrane is unaffected by these agents (Fig. 4). Nor can it be attributed to inhibition of ACAT activity that is essentiallly unaffected by the concentrations used in these experiments. Rather, we suggest that the block in esterification is due to the inhibition of movement of plasma cholesterol to the ER by the amphiphiles (14). That is, the most parsimonious hypothesis is that these compounds act at a single site. In this case, there is no need to postulate a direct route of cholesterol movement from lysosomes to ER.

The analysis presented above assumes that if cholesterol movement from lysosomes to ER occurs, it is as rapid as is transfer to the plasma membrane. The amphiphiles could, in principle, inhibit transfer through an intracellular membrane in the pathway from lysosomes to ER. Nonetheless, the simplest hypothesis that explains all our data is that only one major intermediate compartment is involved, the plasma membrane.

While the bulk of cellular free cholesterol resides in the plasma membrane, the proteins that regulate cholesterol homeostasis are in the ER. We have proposed that the abundance of cholesterol in the plasma membrane (and hence in the cell) is communicated to the ER by a stream of cholesterol that moves bi-directionally between these two compartments (14, 24). It makes sense in terms of this model that lysosomal cholesterol does not move directly to the ER but first enters the plasma membrane. In this way, ingested cholesterol is integrated into the bulk pool that determines the extent of cholesterol transfer to the ER for esterification and other regulatory reactions (24).


FOOTNOTES

*   This work was supported by National Institutes of Health Grant HL 28448.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Pathology, Rush-Presbyterian-St. Luke's Medical Center, 1653 W. Congress Pkwy., Chicago, Il 60612. Tel.: 312-942-5256; Fax: 312-942-4228.
1   The abbreviations used are: LDL, low density lipoprotein; ACAT, acyl-CoA:cholesterol acyltransferase; ER, endoplasmic reticulum; LPDS, lipoprotein-deficient serum; [3H]LDL, [3H]cholesteryl linoleate-labeled LDL; U18666A, 3-beta -[2-(diethylamino)ethoxy]androst-5-en-17-one; HPLC, high performance liquid chromatography.
2   Y. Lange, and T. L. Steck, manuscript submitted for publication.

Acknowledgements

We thank Dr. T. L. Steck for helpful discussions and critical reading of the manuscript, for which we are also grateful to Kristen Page.


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