membrane transport from endosomes to the plasma A ...

A role for retrosomes in intracellular cholesterol transport from endosomes to the plasma membrane

C. A. Hornick, D. Y. Hui and J. G. DeLamatre Am J Physiol Cell Physiol 273:C1075-C1081, 1997. You might find this additional info useful...

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A role for retrosomes in intracellular cholesterol transport from endosomes to the plasma membrane CONRAD A. Departments New Orleans, University of

HORNICK,ly2 DAVID Y. HU13 AND JOHN G. DELAMATREI of lPhysiology and 2Pathology, Louisiana State Medical Center, Louisiana 70112; and 3Department of Pathology and Laboratory Cincinnati College of Medicine, Cincinnati, Ohio 45267

receptor-mediated branes

endocytosis;

cholesterol

metabolism;

mem-

COMPRISE a series of distinct bilaminar membrane-enclosed organelles that function in the transport and metabolism of macromolecules entering cells through the invagination of clathrin-coated pits on the cell surface. The isolation of individual endosomal compartments has made it possible to assessboth their biochemical compositions and the roles they play in intracellular transport and metabolism. Many different types of receptors and ligands enter cells and pass through this series of tubulovesicular structures 1) to degradation in lysosomes, as is the case for epidermal growth factor (EGF) and low-density lipoprotein (LDL) (3, 5), 2) in traffic between endosomes and the Golgi apparatus as do receptors for lysosomal enzymes (21), 3) crossing cells in transcytosis as in the epithelial transport of immunoglobulin (Ig) A or IgM (2), and 4) in recycling to the cell surface in tubular vesicles (retrosomes), which bud off both the early endosomal compartment for uncoupling receptor and ligand (CURL) as well as from the more centrally located multivesicular bodies (MVB) and perhaps from secondary lysosomes themselves. A recycling itinerary is typified by the intracellular pathways taken by the apolipoprotein B/E receptor (apo B/E receptor), transferrin, high-density lipoprotein (HDL), and plasma membrane glyco- and ENDOSOMES

0363-6143/97

$5.00

Copyright

o 1997

sphingolipids (5, 28, 30, 40). It should be noted that ligands and their respective receptors often segregate after an uncoupling process in CURL. The apo B/E receptor, for example, recycles to the plasmalemma, whereas the very low-density lipoprotein (VLDL), which binds to it, typically goes on to further processing in MVB and lysosomes. We have taken advantage of this fact in the studies presented here to clearly distinguish the retrosome as a recycling compartment. Ligands transported back to the surface by retrosomes may be resecreted intact or in a modified form as a result of intraendosomal processing (7, 14, 30). Recent work (13, 17, 18, 24, 25) has shown that lipids as well as proteins undergo enzymatic processing in the neutral-to-acidic endosomal environment. Because the hydrolysis of triacylglycerol and cholesterol esters generates free fatty acids and amphipathic free cholesterol, which can both readily partition into lipid bilayers, it is probable that intraendosomal lipid processing exerts a considerable effect on the membrane composition as well as the contents of endosomal vesicles. Previous studies have shown that the limiting membranes of the constituent organelles of eukaryotic cells have distinctive lipid compositions (9, 42). Moreover, the free cholesterol and phospholipid content of the individual membranes appears to be closely regulated (for reviews, see Refs. 1, 33). For example, the plasma membrane in various cell types has been shown to be highly enriched in free cholesterol, which can be accounted for in part by its high sphingomyelin and saturated phospholipid content (32). The high concentration of these two molecular species increases the tendency for sterol to partition into the plasma membrane, although the differences in partitioning properties among the internal organellar membranes are not in themselves sufficient to explain the magnitude of the in vivo dissimilarities in their sterol content (42). A mechanism underlying the maintenance of this asymmetric sterol distribution is essentially unknown, although the intracellular transport of cholesterol by sterol-enriched membrane vesicles has been proposed by a number of investigators (1, 33, 42). Fielding and Fielding (16), for example, have suggested that a dramatic increase in the cholesterol content of caveolae in the cell membrane of fibroblasts after exposure to cholesterol-labeled LDL may be mediated by vesicular transport after intracellular catabolism of LDL. In this report, we present data that support a role for the retrosome as a vehicle for the transportation of cholesterol from the endocytic-lysosomal pathway back to the cell surface, thereby reducing intracellular cholesterol the American

Physiological

Society

Cl075


Hornick, Conrad A., David Y. Hui, and John G. DeLamatre. A role for retrosomes in intracellular cholesterol transport from endosomes to the plasma membrane. Am. J. PhysioZ. 273 (CeZZ Physiol. 42): C1075-C1081, 1997.-The recycling component (retrosome) of the en .docytic pathway was evaluated as a potential vehicle for the recycling of lipoprotein-derived cholesterol and the maintenance of a high concentration of free cholesterol in plasma membranes. Receptor-to-ligand ratios were established in three distinct endosomal compartments using a recycling receptor (apolipoprotein B/E) to confirm isolated retrosomes as recycling vesicles. Compositional studies showed that retrosomes have twice the free cholesterol in their limiting membranes as do the endosomal compartments from which they derive. Furthermore, of the three isolated endosomal fractions, retrosomes showed the highest ratio of free to esterified cholesterol derived from injected very low density lipoprotein as well as the highest free-to-esterified cholesterol mass ratio overall, confirming endosomal cholesteryl ester hydrolysis and sorting. Endosomal neutral cholesterol esterase was identified by immunoblot, whereas electron microscopy employing membrane cholesterol-specific filipin revealed a high concentration of cholesterol in appendages that appear to be the formative stage of retrosomal biogenesis.

Medicine,

Cl076

INTRACELLULAR

buildup and maintaining membrane. EXPERIMENTAL

a cholesterol-enriched

CHOLESTEROL

plasma

PROCEDURES

IN RETROSOMES

immunoprecipitation of bile salt-stimulated cholesterol esterase activity from rat liver homogenates. ChoZesteroZ esterase immunobZotting procedure. Proteins were solubilized in 62.5 mM tris(hydroxymethyl)aminomethane (Tris). HCl (pH 6.8), 3% sodium dodecyl sulfate, 5% P-mercaptoethanol, and 10% glycerol, electrophoresed on polyacrylamide gels, and transferred from the gels onto nitrocellulose paper. Electrotransfer was performed at 300 mA for 3 h at 4°C. Protein standards transferred onto the nitrocellulose paper were identified by staining with Ponceau S. The nitrocellulose paper containing the samples was incubated for 1 h at ambient temperature with buffer made of 50 mM TrisHCl (pH 7.5), 2 mM CaC12, 80 mM NaCl containing 5% Carnation nonfat dry milk, 0.2% Nonidet P-40, and 0.01% Antifoam A (Sigma). The paper was then transferred to a solution containing 1:2,000 dilution of the rabbit antiserum and incubated for 2 h. At the end of incubation, the paper was washed four times with the above buffer and then transferred to a second antibody solution containing 1251labeled anti-rabbit IgG at a specific activity of 3 X lo6 countsmi.& (cpm)mll. The nitrocellulose paper was incubated with the second antibody for 2 h at 23°C. After the incubation, the paper was washed as described above and then air dried. Immunoreactive proteins on the nitrocellulose paper were visualized by exposure to Kodak XAR-2 films for 18 h at -70°C. AnalyticaLprocedures. Gas-liquid chromatography was used according to the method of Davis et al. (11) to determine the cholesterol concentration of total endosome fractions and endosomal membranes after extraction of lipids by the chloroform-methanol procedure of Bligh and Dyer (4). P-Stigmasterol was used as an internal standard. Cholesteryl esters were estimated by subtracting free cholesterol from total cholesterol. Statistics. One-way analysis of variance was used, followed by the Bonferroni correction for multiple samples to assess intergroup P values. RESULTS

The results presented here are in agreement with a considerable amount of data in the literature indicating the existence of a recycling pathway for the apo B/E lipoprotein receptor, which reappears on the cell surface after entering cells via the coated pit pathway (20). This divergence of ligand and receptor requires separation and sorting in endosomes, with the receptors recycled to the plasma membrane while ligands move on to eventual degradation in lysosomes. In Table 1, the results of the ligand-receptor dual-labeling studies are reported. VLDL was labeled in its protein moiety with 1251and 1 mg was injected into the femoral vein of 250to iOO-g male Wistar rats. After 15 min, CURL, MVB, and retrosomes were isolated according to published procedures (24). However, after the centrifugation step in which a band of endosomes containing all three of these organelles is collected from the top of a dense sucrose cushion, the cytoplasmic portion of the receptor for VLDL (apo B/E receptor) was labeled with 1311using an antibody specific for this portion of the transmembrane receptor (IgC7). With further separation of the three endosome populations into individual aliquots, each fraction was counted for both 1251and 1311.After these labeling and isolation steps, a comparison of the activities of the two markers in each compartment was used to describe the endosomal distribution of VLDL


OrganeZZe isolation. Endosomes (CURL, MVB, and retrosomes) were isolated from the livers of male Wistar rats weighing 250-300 g. AI1 steps during the preparation were carried out at between 0 and 4°C. The method of isolation was exactly the same as that published previously (24). Membranes were separated from whole endosomal fractions by running each fraction, suspended in 4 ml of 1 mM suramin in 0.15 M KC1 (to remove bound lipoproteins), through a French pressure cell two times at 16,000 psi. The fractions were then centrifuged at 12O,OOOg,, in a Beckman SW 60 rotor for 2 h to pellet the membranes, which were then resuspended in 0.15 M KC1 and frozen at -70°C until analyzed. Electron microscopy. Pellets of endosomal fractions were prepared and sectioned as described in earlier work (23). Filipin staining was carried out according to the method of Steer et al. (38), in which a 3009~1 aliquot of sample is brought to 3 ml with ice-cold cacodylate buffer (pH 7.4), containing 5% sucrose and 2.5% glutaraldehyde, and incubated on ice for 45 min. Test samples then received 1 ml of the fixative buffer containing 300 PM filipin and 0.5% dimethyl sulfoxide (DMSO); controls received the same volume of buffer with DMSO but no filipin. Samples were incubated in the dark at room temperature for 2 h and centrifuged at 150,000 g for 45 min at 4°C. The pellets were washed in cacodylate buffer and prepared for sectioning as previously described (23). DuaZ-ZabeZ experiments. VLDL was labeled in its protein moiety with 9 and injected into the femoral vein of 250-g rats 15 min before endosome isolation. After partial purification of endosomes containing the 12”1-labeled VLDL (total endosome fraction), the cytoplasmic portion of the apo B/E receptor was then labeled with 1311 by incubating the total endosomal fraction with l”lI-labeled antibody (IgC7) specific to the apo BE receptor cytoplasmic tail at 4°C for 1 h. The total endosome fraction was then centrifuged through a sucrose step gradient into its three components (CURL, MVB, and retrosomes) and counted to establish the relative amounts of VLDL and its receptor in each endosomal fraction. Fractional distribution of labeled VLDL appeared to be the same as in previous experiments without the incubation with antibody. Lipoproteins. Lipoproteins were isolated by standard centrifugal techniques and iodinated by the method of McFarlane (34). VLDL cholesteryl ester was labeled endogenously in rats in the following manner: 1 mCi of [lol,2cx(N):3H lcholesteryl oleate (Amersham) was dried under nitrogen and resuspended in 20 ~1 of isopropanol. Bovine serum albumin (4%) in phosphate-buffered saline (pH 7.6) was added to a final volume of 280 ~1. This was diluted with saline to a volume of 1 ml, and aliquots of 0.5 ml each were injected directly into the portal veins of two rats under anesthesia. Blood was collected from the abdominal aorta after 40 min, and VLDL was isolated by ultracentrifugation. ChoZesteroZ esterase antibody preparation. Rat pancreatic cholesterol esterase, isolated from freshly frozen pancreases by the sequential chromatography procedure previously described (15), was injected into New Zealand White rabbits for the production of antibodies. Rabbits were injected three times at 2-wk intervals with 250 pg of the purified cholesterol esterase. Rabbits were killed 2 wk after the final injection to collect serum. The specificity of the antiserum was verified by its recognition of a single band on immunoblots and specific

TRANSPORT

INTRACELLULAR

Table 1. Ratio of ligand endosomal fractions

CURL MVB Retrosomes

to receptor

CHOLESTEROL

in three isolated

VLDL, cpm ‘Wmg protein

Apo BIE Receptor, cpm ‘Wmg protein

139,898 81,936 73,500

88,484 38,692 263,561

-c 28,164 i: 32,392 i 23,471

Ligandl Receptor (qp31~)

+ 2,372 i 3,160 F 27,049*

1.62 t- 0.35 2.23 + 0.91 0.24 t 0.06”

Values are means t SD of 3 individual experimental procedures. CURL, compartment for uncoupling receptor and ligand; MVB, multivesicular bodies. Endosomes were isolated from normal rat livers 15 min after a femoral injection of 1251-labeled very low density lipoproteins (VLDL). Endosomal receptors for VLDL [apolipoprotein (ape) B/E receptor] were labeled during isolation procedure with i3iI-labeled antibody to cytoplasmic portion of apo B/E receptor. Ratio 1251/1311 was used as a measure of relative amounts of ligand and receptor in 3 endosomal compartments. *Differences between retrosomes and other organelles using analysis of variance (ANOVA) and Bonferroni tests are significant at P < 0.05.

PREIMMUNE

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IN RETROSOMES

moving receptors back from endosomal compartments to the cell surface. Figure 1 shows the result of an immunoblot assay of membranes from the three endosomal fractions probed with antibody to pancreatic neutral cholesteryl esterase. As shown in Fig. 1, the anticholesterol esterase bound to a single band in each gel with an apparent molecular mass of 67 kDa. This provides strong supporting evidence for the presence of neutral cholesteryl esterase in endosomes, which had been inferred in earlier studies by the ability of the isolated endosomal fractions to metabolize cholesteryl esters (l&25) and by the ability of specific blockers of neutral cholesteryl hydrolase to inhibit the hydrolysis of internalized cholesteryl esters in nonlysosomal fractions of hepatoma cells (13). Figure 2 illustrates the free cholesterol-to-cholesteryl ester isotope ratios in rat liver endosomal fractions isolated 15 min after a femoral injection of VLDL endogenously labeled in its cholesteryl ester moiety. Compared with the injected VLDL, each of the isolated organelles show a significant increase in the isotopic ratio of free to esterified cholesterol. Furthermore, although the free-to-esterified cholesterol ratio is significantly greater in the CURL and MVB fractions than in the original VLDL label, this ratio in the retrosomes is again significantly greater than it is in the other isolated organelles (P < 0.01). These findings extend previous work in demonstrating the activity of a neutral cholesteryl esterase in endosomes, its ability to use VLDL as a substrate, and the preponderance of

c&Ease

67 kDa

0 i= s

4-

k?” s v, s2 zi LL

lMVB

CURL

Retrosome

MVB CURL

Retrosome

Fig. 1. Cholesterol esterase (cr-CEase) immunoblots of endosomal membranes. Immunoblotting was performed on endosomal membranes solubilized in 62.5 mM Tris.HCl (pH 6.8), 3% sodium dodecyl sulfate, 5% 9-mercaptoethanol, and 10% glycerol, electrophoresed on polyacrylamide gels, and transferred from gels onto nitrocellulose paper. Bile salt-stimulated neutral cholesterol esterase was identified by incubation of nitrocellulose sheets with a solution containing 1:2,000 dilution of rabbit antiserum. After a 2-h incubation and 4 buffer washes, paper was then exposed for 2 h to a 2nd antibody solution containing lz51-labeled anti-rabbit immunoglobulin G at a specific activity of 3 x lo6 cpmml. After being washed again, immunoreactive proteins were visualized by exposure to Kodak XAR-2 film for 18 h at -70°C. CURL, compartment for uncoupling receptor and ligand; MVB, multivesicular bodies.

oVLDL

CURL

MVB

RETRO

Fig. 2. Ratio of free to esterified cholesterol (FC/CE) label found in hepatic endosomes of rats 15 min after injection of 1 mg very low density lipoprotein (VLDL) endogenously labeled in its cholesteryl ester moiety. Lipids in VLDL and endosomes were extracted according to method of Bligh and Dyer (4), applied to silica gel flexible sheets, run in a solvent containing petroleum ether-ethyl acetateglacial acetic acid (85:15:1.5), visualized with iodine vapor, scraped and counted by liquid scintillation counting. Data are normalized to make initial ratio of 1 in VLDL. In all experimental protocols greater than 95% of initial VLDL label was cholesteryl ester. Retro, retrosomes.


and its receptor. The data show more than a ninefold greater ratio of ligand to receptor in the MVR as opposed to the retrosomal compartment (I’ < 0.01) as well as a nearly sevenfold difference in the same ratio between the CURL and retrosomes (P < 0.05). These findings demonstrate an enrichment of labeled ligand compared with labeled receptor in both the CURL and MVB compartments. Table 1 also establishes that the concentration of receptor relative to ligand is threefold greater in retrosomes than in CURL and nearly sevenfold greater in retrosomes than in MVB. These findings support a role for retrosomes as transport vesicles

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INTRACELLULAR

CHOLESTEROL

IN RETROSOMES

Table 2. Lipid composition of whole fractions and isolated membranes Cholesterol, pglmg protein

Cholesteryl Ester, pg/mg protein

% of Total as Free Cholesterol

Phospholipid, mg/mg protein

Total fractions CURL MVB Retrosomes

127 + 39 110 k 36 182 f- 50

CURL MVB Retrosomes

82*36 74550 170 k 53:‘:

28?6 82 -+ 10 752

8255 66+18 96 + 3:f:

1.84 + 0.6 1.46 + 0.8 1.05 + 0.09

92+5 78 + 15 95 t 1

1.39 t 0.39 1.34 + 0.46 1.012 0.29

Membranes 13 + 13 20-+9 9 + 0.9

Values are means 5 SD from 4 individual experiments. Liver endosomes isolated 15 min after femoral injection of 1 mg VLDL protein were sampled for total endosomal composition studies, diluted to 4 ml with 1 mM suramin to release bound lipoproteins, and then run twice through a French pressure cell at 16,000 psi. Membranes were sedimented by centrifugation at 120,000 g,, for 2 h. + Differences between retrosomes and other organelles using ANOVA and Bonferroni tests are significant at P < 0.05.

and decrease lipolytic activity in the endosomal compartment (18). In both these studies as well as in earlier papers (14, 17, 18, 24, 25), our data support an endosomal sorting process that occurs as membranous appendages bud off the CURL, MVB, and possibly lysosomal compartments. In the work presented here, we proposed that these buds should be rich in free cholesterol, since they most likely represent the biogenesis of retrosomes. To test this hypothesis, the CURL fraction was isolated and then exposed to filipin before electron microscopy. Filipin is a polyene antibiotic used as a specific cytochemical marker for cholesterol containing membranes (35). A number of studies have shown that a direct relationship exists between the extent of membrane modification by filipin and the cholesterol content of these membranes (35, 38). Figure 4 shows isolated CURL both with and without filipin treatment. In the filipin-treated endosome, it is clear that the limiting membrane is extensively corrugated and especially so where it surrounds the budding appendage, indicating a high concentration of cholesterol in this membrane microdomain. DISCUSSION

VLDL Fig. 3. Ratio somes of rats were extracted and gas-lipid to the method graph (Norwalk,

CURL

MVB

RETRO

of free to esterified cholesterol found in hepatic endo15 min after a femoral injection of 1 mg VLDL. Lipids from fractions by the method of Bligh and Dyer (4), chromatography was performed on samples according of Davis et al. (11) using a Perkin-Elmer gas chromatoCT), with P-stigmasterol as a reference standard.

Cholesterol is introduced into cells through de novo synthesis in the endoplasmic reticulum, by transfer of free cholesterol from lipoproteins, and by release during the catabolism of the core cholesterol esters of endocytosed lipoproteins (5, 12). All of these pathways, which can lead to cellular cholesterol buildup initially, result in an increased free cholesterol concentration in the plasma membrane (31). The plasma membrane can apparently buffer the cellular content of free cholesterol up to a critical threshold level, which, when exceeded, results in the intracellular synthesis and storage of cholesteryl esters (43). However, the mechanism of delivery and the means by which this high


exogenous lipoprotein-derived free cholesterol in retrosomes, which appears to be the result of sterol sorting to this membrane-rich organelle in addition to local cholesteryl ester hydrolysis. Because the cholesterol-to-cholesteryl ester ratios determined using labeled VLDL as a tracer describe only a portion of the lipid moieties present in the isolated endosomal fractions, the mass ratios of free cholesterol to cholesteryl ester were also investigated using gas-liquid chromatography. The results of these studies are given in Fig. 3. It can be seen in Fig. 3 that the free-to-esterified cholesterol ratio for whole endosomal fractions is significantly increased in the CURL compartment compared with VLDL, unchanged in the MVB fraction, and greatly increased in retrosomes. These findings along with the tracer data shown in Fig. 2 and the dual-label studies reported in Table 1 strongly suggest the existence of an endosomal mechanism that sorts free cholesterol into the retrosomal compartment. In Table 2, the free cholesterol, cholesterol ester, phospholipid, and protein values for total endosomes and isolated endosomal membranes are contrasted. One-way analysis of variance and subsequent Bonferroni tests demonstrate that both the percentage of free cholesterol in the total retrosomal fraction and the free cholesterol content of the retrosomal membranes are significantly greater than those of the MVB and CURL fractions. The phospholipid-to-protein ratios reported here are essentially the same as those we reported earlier (3) with the exception of the MVB membrane, which in the earlier paper was found to be 0.8 compared with 1.34 in the current work. This discrepancy may be the result of the estrogen treatment, which was used in earlier endosome isolation procedures, since estrogen has been found to both modify cellular membranes (10)

TRANSPORT

INTRACELLULAR

CHOLESTEROL

plasma membrane sterol content is maintained are not clearly understood. As a consequence of its energy requirement, sensitivity to lowered temperature, and cessation during mitosis, a number of researchers have proposed a vesicular mode as the most probable means of intracellular delivery of sterol to the cell membrane (1, 9, 33). This argument presupposes a cholesterol-enriched, mobile intracellular vesicle that fuses with the plasma membrane. We believe that our data as well as that of others in the field support the retrosome as the most likely candidate to serve this function. To reduce the effect of variation in labeling efficiency, hepatic uptake, and yield of organelles, the data in Table 1, which support the concentration of recycling molecules in the retrosome, are expressed per milligram of protein. However, when the raw data are analyzed, of the total lz51-VLDL in endosomes (48,587 f24,127 cpm), 56% was in CURL, 14% in MVB, and 30%

IN RETROSOMES

Cl079

in retrosomes, indicating that a substantial portion of VLDL is potentially recycled. In preliminary experiments using 1251-VLDL in liver perfusions, we found that the labeled lipoprotein was resecreted primarily in the LDL and HDL ranges (Hornick, unpublished data). This is consistent with the data presented here as well as previous work in which we have shown that lipoproteins can have their lipid moieties catabolized in the endosomal compartment (13, 14, 17, 18, 24, 25) and furthermore that this activity is influenced by both diet and sex hormones (17, 18, 25). The average total receptor counts were 58,735 + 22,694 cpm, of which 22% was found in CURL, 3% in MYB, and 75% in retrosomes, clearly underscoring the role of the retrosome as a recycling organelle. Given that the recovery of endosomal fractions cannot be absolute and that neutral cholesterol esterase has been identified in each of them, one might conclude that the high distribution of free cholesterol in the retrosomal fraction could be the result of on-site cholesterol ester metabolism rather than metabolism and sorting. However, this would leave the problem of explaining how an early endosomal CURL with 82% of its total cholesterol nonesterified could give rise to a late endosomal MVB with only 66% of its cholesterol in the free fraction (Table 2). Earlier studies have also shown that neutral cholesterol esterase has approximately the same specific activity in each of the three endosomal organelles and that the time available for lipid hydrolysis in the retrosome cannot be long relative to other endosomes, since receptors leaving the cell surface typically return to it in under 10 min (6,21,29>. We therefore believe that the early endosomal fraction (CURL) evolves into a late endosomal vesicle (MYB) with substantially lower free cholesterol content by budding off a microdomain of its surface to form a retrosome with a high concentration of free cholesterol (96%; see Table 2). Jackie et al. (26) labeled HDL in both its protein and cholesterol moieties with nonhydrolyzable labels before injecting it into rats. After isolation of CURL, MVB, and retrosomes (receptor recycling compartment), their data show that cholesterol label relative to protein label as a percentage of the total in the liver was approximately twofold higher in retrosomes than in CURL or MYB (26). Because the genesis of retrosomes is believed to occur as a result of budding from the other endosomal organelles, these findings also suggest that endosomal cholesterol ester sorting into retrosomes may be occurring. We believe that the ratio of cholesterol label to protein label would have been higher still in isolated retrosomes if these authors had used a hydrolyzable cholesterol ester label and had not used estradioltreated animals for these studies. We have found that estradiol treatment results in a reduced endosomal cholesterol esterase activity and an increased likelihood of endocytosed lipoproteins being catabolized in lysosomes rather than recycling to the cell surface (18). Evidence for endosomal neutral cholesterol esterase activity has been found in rat hepatoma cells (FU 5AH) (13, 14) as well as in endosomes isolated from rat liver


Fig. 4. Thin-section electron microscopy of control (A) and filipinstained CURL (B), isolated from rat liver 15 min after injection of VLDL. Corrugations in membrane, denoting filipin-sterol complexes, can be clearly seen in B and are especially evident in budding appendage. Bar = 100 nM.

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CHOLESTEROL

IN RETROSOMES

We acknowledge the expert technical assistance of Carol Thouron and thank Dr. Bernard Bihain for the generous gift of antibody to the cytoplasmic portion of the apo B/E receptor. This research was supported in part by National Institutes of Health Grants HL-25596 and DK-40917. During a portion of the period in which this research was carried out, C. A. Hornick was an Established Investigator of the American Heart Association (Grant 880212). Address for reprint requests: C. A. Hornick, Dept. of Physiology, Louisiana State, Medical Center, 1542 Tulane Ave., New Orleans, LA 70112. Received

26 March

1997;

accepted

in final

form

27 May

1997.

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(18, 25). In earlier work by Harrison (ZZ), it was suggested that a neutral cholesterol ester hydrolase in liver might be identical to the pancreatic enzyme and that, in some cases, it appeared to be stimulated by bile salts and in others inhibited by them. The immunoblot in Fig. 1 positively identifies the bile salt-stimulated pancreatic enzyme as present in isolated endosomes. However, in other studies, we found that neutral cholesterol esterase activity in endosomes was first enhanced and then inhibited over a Z-day period as the concentration of bile salts rose in endosomes isolated from cholestatic rats (25) in agreement with the earlier findings of Harrison (22). Because the studies of Camulli et al. (6) demonstrated that purified neutral cholesterol esterase from rat liver is identical to the pancreatic enzyme and shows only positive stimulation in response to increasing concentrations of bile salt, it is probable that at least two enzymes capable of attacking the cholesteryl ester bond are present in endosomes. A number of studies have demonstrated a preferential association between cholesterol and sphingomyelin in the membranes of cells (27, 37, 42, 43). Slotte et al. (37), for example, have shown that if sphingomyelin in the plasma membrane is metabolized using sphingomyelinase, cholesterol will leave the membrane to become esterified via acyl-CoA:cholesterol 0-acyltransferase in the interior of the cell, whereas the metabolism of membrane phosphatidylcholine does not affect the asymmetric distribution of cholesterol (39). Conversely, Chen et al. (8) have demonstrated that the amount of membrane sphingomyelin in cultured intestinal cells regulates the amount of unesterified cholesterol that is absorbed. Furthermore, previous studies by ourselves (3) and others (41) h ave found endosomal membranes enriched in sphingomyelin relative to plasma membrane. Kovall and Pagan0 (29) determined that plasma membrane spingomyelin follows a pathway of internalization and recycling, whereas Kallen and colleagues ( 1,27) have recently presented evidence supporting an endosomal site for spingomyelin synthesis and recycling to the plasma membrane. This work suggests that the most likely site for plasma membrane spingomyelin production is the internal leaflet of endosomal membranes. If, as these authors propose, spingomyelin is actively incorporated into these membranes, it is likely that the free cholesterol from lipoprotein catabolism generated by the activity of endosomal neutral cholesterol esterase will readily follow it, producing cholesterol and sphingomyelin-enriched microdomains (36, 42). It would be consistent with the data presented in this report if these microdomains were the sites for CURL and MVB budding and the biogenesis of retrosomes as suggested by Fig. 4. Other work has indicated a Golgi site for spingomyelin production (19). Spingomyelin-cholesterol rich budding from the Golgi could indicate a mechanism in common used by both newly synthesized and lipoprotein-derived cholesterol in targeting deliverv to the plasma membrane pool.

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