Distinct Endosomal Compartments in Early Trafficking of LowDensity Lipoprotein-derived Cholesterol*

Received for publication, January 17, 2003, and in revised form, April 10, 2003Published, JBC Papers in Press, April 28, 2003, DOI 10.1074/jbc.M300542200

Shigeki Sugii‡, Patrick C. Reid‡, Nobutaka Ohgami‡, Hong Du§, and Ta-Yuan Chang‡¶

From the ‡Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755 andthe §Division of Human Genetics, Children’s Hospital Medical Center, Cincinnati, Ohio 45229

We previously studied the early trafficking of low den-sity lipoprotein (LDL)-derived cholesterol in mutant Chi-nese hamster ovary cells defective in Niemann-Pick typeC1 (NPC1) using cyclodextrin (CD) to monitor the arrivalof cholesterol from the cell interior to the plasma mem-brane (PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y.(2000) J. Biol. Chem. 275, 4013–4021). We found that newlyhydrolyzed cholesterol derived from LDL first appears incertain CD-accessible pool(s), which we assumed to be thePM, before accumulating in the late endosome/lysosome,where NPC1 resides. To determine the identity of theearly CD-accessible pool(s), in this study, we performedadditional experiments, including the use of revised CDincubation protocols. We found that prolonged incuba-tion with CD (>30 min) caused cholesterol in internalmembrane compartment(s) to redistribute to the PM,where it became accessible to CD. In contrast, a shortincubation with CD (5–10 min) did not cause such aneffect. We also show that one of the early compartmentscontains acid lipase (AL), the enzyme required for liber-ating cholesterol from cholesteryl ester in LDL. Biochem-ical and microscopic evidence indicates that most of theAL is present in endocytic compartment(s) distinct fromthe late endosome/lysosome. Our results suggest that cho-lesterol is liberated from LDL cholesteryl ester in thehydrolytic compartment containing AL and then moves tothe NPC1-containing late endosome/lysosome beforereaching the PM or the endoplasmic reticulum.

In mammalian cells, low density lipoprotein (LDL)1 binds to itsreceptor at the cell surface and is recruited into clathrin-coated

endocytotic vesicles. After endocytosis, LDL enters the endoso-mal/lysosomal system, where cholesteryl ester, a major lipidfound in LDL, is hydrolyzed by the enzyme acid lipase (AL) (1).Mutations in AL cause cholesteryl ester to eventually accumulatein the lysosome (2, 3). After the hydrolytic action by AL, thetransport of LDL-derived cholesterol from the endosome/lyso-some to the plasma membrane (PM) or to the endoplasmic retic-ulum for re-esterification requires the protein named Niemann-Pick type C1 (NPC1). Mutations in NPC1 cause unesterifiedcholesterol and other lipids to accumulate in the late endosomeand lysosome. Despite significant advances, the events that led toeventual accumulation of cholesterol in the late endosome/lyso-some remain unclear. To delineate the early trafficking events ofLDL-derived cholesterol, we previously performed pulse-chaseexperiments using [3H]cholesteryl linoleate-labeled LDL([3H]CL-LDL) in Chinese hamster ovary (CHO) mutant cellsdefective in the npc1 locus, CT43, along with their parental cells,25RA (4). To monitor the arrival of [3H]cholesterol at the PM, weutilized a cyclodextrin (CD)-based intact cell assay. CD is a wa-ter-soluble molecule that has a high affinity for cholesterol andhas been widely used to monitor the arrival of cholesterol at thePM from the cell interior (5–8). Our results show that [3H]cho-lesterol, newly released from the hydrolysis of [3H]CL-LDL,emerges in the early pool(s) in a manner unaffected by the npc1mutation. Subsequently (within 2 h), in the parental cells,[3H]cholesterol is distributed to the PM and the endoplasmicreticulum. In CT43 cells, [3H]cholesterol accumulates in thecharacteristic aberrant endosome/lysosome. [3H]Cholesterol thatis present in the early pool(s) is extractable by CD, whereas[3H]cholesterol that accumulates in the aberrant endosome/lyso-some is resistant to extraction by CD. Based on this CD sensi-tivity test, the early pool(s) was assumed to be the PM (4). Theseresults led us to hypothesize that, in NPC1 cells, cholesterolliberated from cholesteryl ester in LDL first moves to the PMindependent of NPC1 and then moves back to the cell interiorand accumulates in the aberrant late endosome/lysosome. Usinga similar CD-based assay, other investigators independentlyreached the same conclusion (7).

The original CD-based assay used by us and by others in-volved continuous incubation of cells with CD for 30 min orlonger. Thus, it is possible that prolonged incubation of cellswith CD may cause redistribution of cellular cholesterol, socholesterol originally residing in internal membranes moves tothe PM and becomes extractable by CD. Recently, Haynes et al.(9) showed that, in CHO cells, depending on the incubationtime used (ranging from 30 s to 20 min), CD is capable ofextracting cellular cholesterol from two or three kineticallydistinct pools; rearrangement of cholesterol between thesepools could occur under various treatments. In this work, wefurther investigated the early trafficking events of LDL-de-rived cholesterol. To follow the fate of newly hydrolyzed cho-lesterol more precisely, we redesigned the procedures for the

* This work was supported by National Institutes of Health Grant HL36709 (to T.-Y. C.). The Herbert C. Englert Cell Analysis Laboratorywas established by equipment grants from the Fannie E. Rippel Foun-dation and the National Institutes of Health Shared Instrument Pro-gram, and its operation is supported in part by NCI Core Grant CA23108 from the National Institutes of Health (to the Norris CottonCancer Center). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

¶ To whom correspondence should be addressed: Dept. of Biochemis-try, Dartmouth Medical School, HB 7200, Hanover, NH 03755. Tel.:603-650-1622; Fax: 603-650-1128; E-mail: Ta.Yuan.Chang@dartmouth.edu.

1 The abbreviations used are: LDL, low density lipoprotein; AL, acidlipase; PM, plasma membrane; NPC1, Niemann-Pick type C1; CL,cholesteryl linoleate; [3H]CL-LDL, [3H]cholesteryl linoleate-labeled lowdensity lipoprotein; CHO, Chinese hamster ovary; CD, cyclodextrin;FBS, fetal bovine serum; DNP, dinitrophenyl; DAMP, 3-(2,4-dini-troanilino)-3�-amino-N-methyldipropylamine; V-ATPase, vacuolarATPase; MPR, mannose 6-phosphate receptor; CI-MPR, cation-independent mannose 6-phosphate receptor; HDL, high density lipopro-tein; Hf, human fibroblast; PBS, phosphate-buffered saline; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GFP, green fluores-cent protein; TGN, trans-Golgi network.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 29, Issue of July 18, pp. 27180–27189, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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pulse-chase experiment and the CD treatment. We also per-formed biochemical and immunofluorescence experiments todefine the hydrolytic compartment(s) involved in producingLDL-derived cholesterol. A model, revised from the one previ-ously proposed by this laboratory (4), describing the early itin-erary of LDL-derived cholesterol in the context of the endocyticpathway is presented.

EXPERIMENTAL PROCEDURES

Materials—Fetal bovine serum (FBS), protease inhibitor mixture,Nonidet P-40, 2-hydroxypropyl-�-cyclodextrin, monoclonal antibodyagainst dinitrophenyl (DNP), paraformaldehyde, and human apoA-Iwere purchased from Sigma. The acyl-CoA:cholesterol O-acyltrans-ferase inhibitor F12511 was a gift of Pierre Fabre Research (Castres,Cedex, France). Percoll and [1,2,6,7-3H]CL were from Amersham Bio-sciences. Optiprep (Nycomed) was from Axis-Shield. FuGENE 6 trans-fection reagent was from Roche Applied Science. The ProLong antifadekit, Alexa 488- or Alexa 568-conjugated goat anti-rabbit or anti-mouseIgG, LysoTracker Red (DND-99), 3-(2,4-dinitroanilino)-3�-amino-N-methyldipropylamine (DAMP), and Zenon rabbit IgG labeling kits werefrom Molecular Probes, Inc.. Monoclonal antibodies against EEA1,caveolin-1, and syntaxin-6 were from BD Biosciences. Monoclonal an-tibody against Na�/K�-ATPase was from Upstate Biotechnology, Inc.Monoclonal antibody against hamster LAMP2 (lysosomal-associatedmembrane protein-2) was from the Developmental Studies HybridomaBank maintained by the University of Iowa. Rabbit polyclonal antibod-ies against AL were produced as described (10). Monoclonal antibodyagainst vacuolar ATPase (V-ATPase) was a generous gift from Profes-sor Satoshi Sato (Kyoto University, Kyoto, Japan); this antibody(OSW2) is directed against the 100–116-kDa subunit of the V0 domainof V-ATPase and has been shown to specifically recognize the vacuolartype proton pump (11). Rabbit polyclonal antibodies against the cation-independent mannose 6-phosphate receptor (CI-MPR) and againstRab9 were generous gifts from Professor Suzanne Pfeffer (StanfordUniversity) (12). Delipidated FBS was prepared as described (13). LDL(density of 1.019–1.063 g/ml) was prepared from fresh human plasmaby sequential flotation as previously described (14). High density li-poprotein (HDL; density of 1.063–1.21 g/ml) was prepared by the sameflotation method and purified by heparin affinity chromatography.

Cell Lines and Cell Culture—25RA is a CHO cell line that is resistantto the cytotoxicity of 25-hydroxycholesterol (15) and that contains again-of-function mutation in SCAP (SREBP cleavage-activating pro-tein) (16). The CT43 mutant cell line was isolated as one of the choles-terol trafficking mutants from mutagenized 25RA cells (17). It containsthe same gain-of-function mutation in SCAP. In addition, it contains apremature translational termination mutation near the 3�-end of thenpc1 coding sequence, producing a nonfunctional truncated NPC1 pro-tein (4). CHO cells were maintained in medium A (Ham’s F-12 mediumplus 10% FBS and 10 �g/ml gentamycin) as monolayers at 37 °C with5% CO2. When medium B (Ham’s F-12 medium supplemented with 5%delipidated FBS plus 35 �M oleic acid and 10 �g/ml gentamycin) wasused at lower temperatures (18 °C or lower), Ham’s F-12 medium (ti-trated to pH 7.4 without sodium bicarbonate) was used, and cells wereplaced in a water bath without CO2. A human fibroblast (Hf) cell linederived from an NPC patient (No. 93.22) was the generous gift of Dr.Peter Pentchev (National Institutes of Health). Hf cell lines isolatedfrom patients with Wolman’s disease (GM00863A and GM01606A) andwith mucolipidosis II (I-cell disease) (GM02013D) were from theNIGMS Human Genetic Cell Repository (Camden, NJ). Hepatocyte-likeHepG2 and monocytic THP-1 cells were obtained from American TypeCulture Collection (Manassas, VA). Hf and HepG2 cells were grown inDulbecco’s modified Eagle’s medium supplemented with 10% FBS and100 units/ml penicillin/streptomycin at 37 °C with 10% CO2. THP-1cells were maintained under the same conditions, except that RPMI1640 medium was used instead. Prior to the experiment, THP-1 wastreated with 100 nM phorbol 12-myristate 13-acetate and 100 nM 1�,25-dihydroxyvitamin D3 (both from Sigma) for at least 72 h to inducedifferentiation (18).

LDL-derived Cholesterol Trafficking Assays—[3H]CL-LDL with spe-cific radioactivity of �5 � 104 cpm/�g of protein was prepared aspreviously described (19). Cells were plated in 6- or 12-well dishes aspreviously described (4, 20). Prior to each experiment, the cells werecultured for 2 days in medium B (to deplete stored cholesterol withinthe cell). Cells were chilled on ice, labeled with 30 �g/ml [3H]CL-LDL inmedium B for 5 h at 18 °C, and washed once with 1% bovine serumalbumin-containing cold phosphate-buffered saline (PBS) at 4 °C andthree more times with cold PBS. Cells were then fed cold medium B and

placed in a water bath for the indicated chase times at 37 °C. During thechase period, rapid metabolism of [3H]CL-LDL occurs in a time-depend-ent manner. To obtain reproducible results, we found that it was es-sential to use healthy cells grown at late log phase for plating and tocontrol the temperature and pH of the growth media in a precisemanner. To control the temperature, we incubated tissue culture platesor dishes on platforms covered with water in a constant temperaturewater bath. To control the pH in a precise manner, we used mediadevoid of sodium bicarbonate and titrated to pH 7.4 within 1 weekbefore usage. An acyl-CoA:cholesterol O-acyltransferase inhibitor (2 �M

F12511) was included whenever cells were incubated at 37 °C. F12511was previously shown to inhibit acyl-CoA:cholesterol O-acyltransferaseactivity at the submicromolar level (21). Labeled cellular lipids wereextracted and analyzed by TLC as described (17); the percent hydrolysiswas calculated as [3H]cholesterol counts divided by the sum of [3H]CLand [3H]cholesterol counts. For cholesterol efflux experiments, cellswere incubated with 4% 2-hydroxypropyl-�-cyclodextrin (CD) in me-dium B in the presence of the acyl-CoA:cholesterol O-acyltransferaseinhibitor at 37 °C for the indicated times. The labeled lipids wereextracted and analyzed as described (4, 17). The percent cholesterolefflux was calculated as [3H]cholesterol counts in the medium dividedby the sum of [3H]CL counts in the cell and [3H]cholesterol counts in thecell and in the medium.

Isolation of the PM—To isolate the PM from the cells, we employedthe 30% Percoll gradient procedure essentially as described (20). Allprocedures were performed at 4 °C. Briefly, after the pulse-chase ex-periment, cells in two 150-mm dishes were collected. The cells werescraped in buffer containing 0.25 M sucrose, 1 mM EDTA, and 20 mM

Tricine (pH 7.8) and broken with 15 strokes using a stainless steeltissue grinder (Dura-Grind, Wheaton). The post-nuclear supernatantwas loaded onto a 30% Percoll gradient. After centrifugation at84,000 � g for 30 min, 25 fractions were collected from the top. The PMfractions usually corresponded to fractions 9 and 10, as evidenced by avisible white membrane band; this band showed high enrichment inNa�/K�-ATPase and caveolin-1 protein (20). In addition, we performedbiotinylation of PM proteins in intact cells at 4 °C for 10 min usingsulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce), which showedthat only fractions 9 and 10 were highly enriched in the biotinylatedproteins (data not shown). The 3H-labeled lipids were extracted usingchloroform/methanol and analyzed by TLC as previously described (20).

11% Percoll Gradient Analyses—All procedures were performed at4 °C. The fractionation method was performed as described previously (4,22). Briefly, after the pulse-chase experiment, cells from one 150-mm dishwere scraped into homogenization buffer (0.25 M sucrose, 1 mM EDTA,and 20 mM Tris (pH 7.4)) and homogenized with 15 strokes using the samestainless steel tissue grinder described above. To minimize breakage ofmembrane vesicles, 250 mM sucrose was included in the buffer. To in-crease recovery, the pellet was resuspended in buffer and homogenized asecond time. The combined post-nuclear supernatant from cells wasloaded onto 11% Percoll and centrifuged at 20,000 � g for 40 min using aBeckman Model Ti-70.1 rotor. 10 fractions were collected from the top.�80% of the PM marker (Na�/K�-ATPase) was concentrated in fractions1 and 2, whereas �80% of the late endosomal/lysosomal markers(LAMP1/LAMP2) were concentrated in fractions 9 and 10 as previouslydescribed (4). The 3H-labeled lipids were extracted using chloroform/methanol and analyzed by TLC as previously described (20).

Optiprep Gradient Analyses—The procedure was based on a previ-ously described method (23) with modifications. Cells grown in one150-mm dish were homogenized at 4 °C as described above. The post-nuclear supernatant (1 ml) was placed onto 9 ml of a linear 5–20%Optiprep gradient prepared in homogenization buffer at 4 °C. Gradi-ents were centrifuged at 27,000 rpm for 20 h at 4 °C using a BeckmanSW 41 rotor. 20 fractions (0.5 ml each) were carefully collected from thetop. Immunoblot analyses were performed using antibodies againstindividual organelle markers as indicated. The 3H-labeled lipids wereextracted using chloroform/methanol and analyzed by TLC as previ-ously described (20).

Immunoblot and Spectrofluorometric Analyses of Percoll Fractions—For immunoblot analysis, each Percoll fraction was ultracentrifugedeither at 100,000 � g for 90 min or at 150,000 � g for 30 min to removethe Percoll particles. Afterward, the samples (located on top of thePercoll particles) were carefully collected using Pasteur pipettes. Pro-teins present in these fractions were concentrated by chloroform/meth-anol precipitation (24). The precipitated proteins were dissolved in lysisbuffer (100 mM Tris (pH 8.0), 0.2 M NaCl, 1% Nonidet P-40, 1 mM EDTA,and 1� protease inhibitor mixture), separated on SDS-polyacrylamidegel, and immunoblotted with polyclonal anti-AL antibodies (1:1000). Toquantitate the LysoTracker signal (a late endosomal/lysosomal mark-

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er), we used a highly sensitive fluorometer to measure the fluorescenceintensities present in various Percoll fractions. The method is brieflydescribed as follows. Cells were incubated with 100 nM LysoTrackerRed for 2 h at 37 °C and then fractionated on a Percoll gradient at 4 °C.The Percoll fractions were ultracentrifuged at 150,000 � g for 30 min toremove the Percoll particles. Each fraction was then quantitated for itsfluorescence at Ex577 nm/Em590 nm using a PC1 photon counting spec-trofluorometer from ISS Inc. (Champaign, IL). For detection of thegreen fluorescent protein (GFP) signal in GFP-transfected or NPC1-GFP-transfected cells, a modified method was needed (because Percollparticles exhibited autofluorescent signals that strongly interfered withthe GFP signal). Each Percoll fraction was solubilized with the non-fluorescent detergent Thesit (Roche Applied Science) at 0.2%, and thesolubilized material was ultracentrifuged at 150,000 � g for 6 h. Thefluorescent signal present in the supernatant was quantitated in thefluorometer at Ex488 nm/Em507 nm.

Construction and Transfection of GFP-tagged NPC1—The constructencoding mouse NPC1 protein fused with GFP was created and sub-cloned into the pREX-IRES vector by a procedure described elsewhere(25). CT43 cells were transfected with the npc1-gfp cDNA using Fu-GENE 6 according to the manufacturer’s instructions. Control experi-ments showed that expression of NPC1-GFP, but not GFP alone, com-pletely rescued the cholesterol accumulation defect in CT43 cells,indicating that the NPC1-GFP fusion protein is functional (25). Trans-fected cells were used within 2–3 days of transfection for imaginganalysis and within 4 days for Percoll gradient analysis.

Fluorescence Microscopy—Cells were grown on glass coverslips in6-well plates or in 60-mm dishes and processed for fluorescence studies.For LysoTracker labeling, cells were preincubated with 200 nM Lyso-Tracker Red in the medium at 37 °C for 2 h prior to the experiment. ForDAMP staining, intact cells were incubated with 50 �M DAMP for 30min at 37 °C (26, 27); its signal was detected with monoclonal antibodyagainst DNP, followed by Alexa 568-conjugated secondary antibody.For immunostaining, cells were washed three times with PBS, fixed

with 4% paraformaldehyde for 10 min at room temperature, washedthree times again, and permeabilized either with methanol (chilled at�20 °C) for 1 min or with 1% Triton X-100 in PBS at room temperaturefor 10 min. After three more washes, the cells were blocked with 10%goat serum in PBS for 30 min at room temperature and incubated withpredetermined concentrations of various primary antibodies in theblocking medium for 1 h. When anti-V-ATPase antibody was used asthe primary antibody, the incubation time was for 20 min only. Anti-body dilutions used in immunofluorescence were as follows: LAMP2(1:200), AL (1:500 to 1:1000), EEA1 (1:50), syntaxin-6 (1:50), caveolin-1(1:100), V-ATPase (1:1000), CI-MPR (1:500), and DNP (1:100). Fordouble labeling studies using rabbit anti-AL and rabbit anti-CI-MPRantibodies, Zenon rabbit IgG labeling kits (Alexa 488 and Alexa 568,respectively) were employed according to the manufacturer’s protocol.For other labeling studies, cells were washed with PBS three times,treated with various Alexa-conjugated secondary IgGs, and thenwashed three times. The coverslips were mounted with a drop of Pro-Long antifade medium onto the glass slides before image processing.Samples were viewed and photographed using a Zeiss Axiophot micro-scope with a �63 objective equipped with a CCD camera (DEI-750,Optronics Engineering, Goleta, CA). Fluorescein isothiocyanate andrhodamine filters were used to visualize GFP/Alexa 488 and Lyso-Tracker Red/Alexa 568, respectively. The images were processed usingMetaView Version 4.5 software (Universal Imaging Corp., Downing-town, PA). In selective experiments as indicated, the samples were alsoviewed under a Bio-Rad MRC-1024 krypton/argon laser scanning con-focal microscope. The images were constructed using LaserSharp soft-ware and further processed using Adobe Photoshop Version 5.02.

RESULTS

Early Trafficking of LDL-derived Cholesterol Probed with aLong Versus Short Incubation with CD—We grew CT43 and25RA cells in cholesterol-free medium for 2 days and pulse-

FIG. 1. Early trafficking of LDL-de-rived cholesterol after a long versusshort incubation with CD. A–D, 25RAand CT43 cells were pulse-labeled with[3H]CL-LDL for 5 h at 18 °C; chased at37 °C for 0 min (A), 30 min (B), 60 min (C),or 120 min (D); and incubated with 4%2-hydroxypropyl-�-CD for 5–120 min at37 °C. For the 0-min time point in A, CDwas added at 18 °C for 10 min. Values arethe averages of triplicate dishes; resultsare representative of two independent ex-periments. E and F, 25RA and CT43 cellswere pulse-labeled as described aboveand chased at 37 °C and then incubatedwith 4% CD for 10 min. The chase time asindicated includes the 10-min CD incuba-tion time. Values are the averages of du-plicate dishes; results are representativeof three independent experiments. Errorbars indicate S.E.

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labeled them with [3H]CL-LDL for 5 h at 18 °C. At this tem-perature, LDL was internalized, but accumulated in pre-lyso-somal compartments without significant hydrolysis of CL.When the temperature was increased to 37 °C, CL in LDL wasrapidly hydrolyzed to free cholesterol and transported to des-ignated locations (4). In numerous experiments, we found thatthe half-time of hydrolysis averaged 25 � 5 min in both celltypes; a typical result is shown in Fig. 1F. During the warm-upperiod (i.e. immediately after the labeling), if cells were contin-uously incubated with CD for various time periods as indicated(0–120 min), 25RA and CT43 cells showed the same degree ofcholesterol efflux toward CD; the efflux significantly increasedfrom 30 min on (Fig. 1A). When cells were chased at 37 °C for30 min before adding CD for up to 120 min, a slight defect incholesterol efflux (starting at 15 min after adding CD) occurredin CT43 cells (Fig. 1B). In a separate experiment, a slight effluxdefect in CT43 cells was also found in cells chased for 15 minbefore CD treatment (data not shown). In contrast, a severeefflux defect in CT43 cells occurred when cells were chased at37 °C for 60 min (Fig. 1C) or for 120 min (Fig. 1D) before addingCD. In a separate experiment, a severe efflux defect in CT43

cells was also shown in cells chased for 45 min before CDtreatment (data not shown).

We next compared cholesterol effluxes in 25RA and CT43cells using a procedure that involves a short incubation withCD (28). In a control experiment, we labeled the PM of 25RAand CT43 cells with [3H]cholesterol at 4 °C using the [3H]cho-lesterol/liposome method (4) and then treated the labeled cellswith CD for 10 min at 37 °C. We found that 80–90% of thecellular label could be removed (data not shown). Based on thisfinding, we redesigned the pulse-chase protocol as follows.Cells were labeled with [3H]CL-LDL at 18 °C, chased at 37 °Cfor various time periods as indicated, and incubated with CDfor 10 min; the percent cholesterol efflux was then monitored.The results show that, after the 40-min chase time, cholesterolefflux increased steadily with time in 25RA cells (Fig. 1E). Incontrast, such an increase hardly occurred in CT43 cells. In aseparate experiment, results similar to those shown in Fig. 1Ewere obtained when the CD incubation was reduced to 5 min(data not shown).

LDL-derived Cholesterol Efflux in 25RA and CT43 Cells inResponse to HDL or ApoA-I—Because CD may induce an alter-ation in cellular cholesterol distribution, we examined the cho-lesterol efflux in 25RA and CT43 cells in response to twophysiologically relevant acceptors, HDL and apoA-I. Both ac-ceptors sequester cholesterol slowly from the PM of cells (29).When [3H]CL-LDL-labeled cells were continuously incubatedwith HDL, [3H]cholesterol was slowly but steadily removed in25RA cells; in contrast, this removal did not occur in CT43 cells(Fig. 2A). Similarly, when [3H]CL-LDL-labeled cells were incu-bated with apoA-I, significant cholesterol efflux occurred in25RA cells, but not in CT43 cells (Fig. 2B).

LDL-derived [3H]Cholesterol Present in Isolated PM Frac-tions of 25RA and CT43 Cells—We next used the procedurefirst described by Graham (30) to isolate the PM from cellslabeled with [3H]CL-LDL. The PM isolated by this methodcontained minimal contamination from internal membranes.Using this procedure, we monitored the [3H]cholesterol contentin the PM after various chase times. The results show that the[3H]cholesterol content in the PM increased significantly withtime, reaching a maximum at �75 min in 25RA cells, whereassuch an increase was hardly observed in the CT43 cells (Fig. 2C).

Percoll Gradient Analyses of Various Membrane FractionsContaining LDL-derived [3H]Cholesterol—To detect the pres-ence of [3H]cholesterol in various membrane fractions, we per-formed Percoll gradient centrifugation using cell homogenatesprepared from labeled cells. The Percoll fractions consisted of10 fractions of increasing density, with light fractions (fractions1–4) enriched in the PM and early endosome and with heavyfractions (fractions 9 and 10) enriched in the late endosome andlysosome (4, 22). For labeling, we pulsed cells with [3H]CL-LDLfor 5 h at 18 °C. As shown in Fig. 3A, when the chase timewas 0 min, [3H]CL was predominantly present in the lighterfractions (fractions 1–4). At the 30-min chase time, a signif-icant decrease in [3H]CL occurred in both cell types; concom-itantly, a significant increase in the [3H]cholesterol countsoccurred in both cell types (Fig. 3B). The cholesterol countswere distributed in the lighter fractions (fractions 1–4), me-dium density fractions (fractions 5–8), and heavy fractions(fractions 9 and 10). Importantly, the [3H]cholesterol distri-bution patterns in 25RA and CT43 cells were similar at the30-min chase time point. In contrast, when the cells werechased for a longer time period (for 1 h) in the absence of CD,a significant difference in the [3H]cholesterol distributionwas seen between 25RA and CT43 cells (Fig. 3C, solid bars):in 25RA cells, [3H]cholesterol was distributed in the lighterfractions (fractions 1–4), medium density fractions (fractions

FIG. 2. A and B, cholesterol efflux in 25RA and CT43 cells mediatedby HDL or apoA-I. 25RA and CT43 cells were pulse-labeled with[3H]CL-LDL for 5 h at 18 °C, washed, and incubated at 37 °C with 1.0mg/ml HDL for various times as indicated (A) or with 10 �g/ml apoA-Ifor 6 h (B). C, appearance of [3H]cholesterol in the PM of 25RA andCT43 cells. Cells were pulse-labeled with [3H]CL-LDL for 5 h at 18 °Cand chased at 37 °C for the indicated times. The PM was isolated asdescribed under “Experimental Procedures.” Cholesterol enrichment inthe PM is expressed as relative percentages, calculated as the [3H]cho-lesterol counts in the PM fractions divided by the sum of the [3H]CL and[3H]cholesterol counts in the post-nuclear supernatants. Results arerepresentative of two independent experiments. BSA, bovine serumalbumin.

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FIG. 3. Early trafficking of LDL-derived transport analyzed by Percoll gradient centrifugation. A and B, 25RA and CT43 cells werepulse-labeled with [3H]CL-LDL for 5 h at 18 °C and chased for either 0 min (no chase) or 30 min. The cells were subjected to Percoll gradientanalysis. [3H]CL (A) and [3H]cholesterol (B) in each Percoll fraction were analyzed according to the procedures described under “ExperimentalProcedures.” Within each cell type, to normalize variation in total 3H counts recovered from different samples, the values reported were normalizedso that the sum of counts in cellular cholesterol and CL was the same for different samples. C, after the pulse, 25RA and CT43 cells were chasedat 37 °C in the presence or absence of CD for 1 h. [3H]Cholesterol in each Percoll fraction and in the medium was counted. For each cell type, thevalues reported were normalized so that the sum of the counts in cellular cholesterol and cholesterol in the medium was the same for differentsamples. D, after the pulse, 25RA and CT43 cells were chased for 1 h and then chased for an additional 1 h with or without CD. The counts wereanalyzed as described for C. Results are representative of two independent experiments.

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5–8), and heavy fractions (fractions 9 and 10), whereas inCT43 cells, [3H]cholesterol accumulated mainly in the heavyfractions (fractions 9 and 10). If CD was included during the1-h chase period, [3H]cholesterol present in the heavy frac-tions (fractions 9 and 10) and light fractions (fractions 1–4)significantly decreased, resulting in a blurring of the differ-ence in the [3H]cholesterol distribution between 25RA andCT43 cells (Fig. 3C, hatched bars). In a separate experiment,the chase time was increased to 2 h. We found that, in theabsence of CD, [3H]cholesterol continued to accumulate in theheavy fractions (fractions 9 and 10) in CT43 cells (Fig. 3D,solid bars); if CD was included during the last hour of the 2-hchase time, the cholesterol that accumulated in the heavyfractions of CT43 cells was resistant to extraction by CD (Fig.3D, compare hatched and solid bars).

Identification of the AL Compartment(s) in Percoll Frac-tions—The results shown in Fig. 3 suggest that [3H]choles-terol newly liberated from [3H]CL-LDL may be present inmultiple membrane fractions (Percoll fractions 1–8) before itis sequestered in the late endosome/lysosome (Percoll frac-tions 9 and 10). Because more than one membrane compart-ment may be present in any of the Percoll fractions, theidentities of these early fractions could not be positivelydetermined at present. On the other hand, hydrolysis ofcholesteryl ester in LDL requires the action of the enzymeAL. Therefore, the compartment(s) that contains AL is in-volved during the early trafficking of [3H]cholesterol liber-ated from [3H]CL-LDL. In the literature, the localization ofAL has been assumed to be in lysosomes (1); however, directevidence is lacking. We thus focused our effort to identify thecompartment(s) containing AL. We used the specific antibod-ies against AL (10) to perform immunoblot analysis on vari-ous Percoll fractions. These antibodies identified a single41-kDa protein band. The results show that, in both 25RAand CT43 cells, all of the AL-positive signals were distributedin the buoyant fractions (fractions 1–3); no detectable signalcould be found in either the heavy or medium density frac-tions. Representative results are shown in Fig. 4A. A controlexperiment showed that LysoTracker, a marker for the lateendosome and lysosome, was predominantly found in theheavy fractions (fractions 9 and 10) (Fig. 4B). In another ex-periment, the NPC1-GFP fusion protein expressed in CT43cells was also predominantly found in the heavy fractions (Fig.4C); a control experiment showed that GFP alone expressed inCT43 cells was predominantly localized in the buoyant frac-tions (Fig. 4C).

Monitoring the Early Fate of [3H]Cholesterol Using OptiprepGradients—The results shown in Figs. 3 and 4 demonstratethat, on a 11% Percoll gradient, the AL-containing membraneswere located in light fractions (fractions 1–3). However, be-cause the PM fractions were also enriched in these fractions,one could not determine whether [3H]cholesterol newly liber-ated from [3H]CL-LDL was present in the AL compartment(s)or in the PM. To clarify this issue, we used another subcellularfractionation method that separates the PM from the endoso-mal compartments using the Optiprep gradient procedure firstdeveloped by Sheff et al. (23). As shown in Fig. 5A, immunoblotanalyses demonstrated that both AL and the early endosomalmarker EEA1 were predominantly located in early fractions(fractions 3–5), whereas the PM marker Na�/K�-ATPase wasbroadly enriched in heavier fractions (fractions 11–19). Thelate endosomal marker Rab9 was located mainly in fractions 7and 8; the trans-Golgi network (TGN) marker syntaxin-6 waslocated mainly in fractions 9 and 10. Next, we pulse-labeled25RA and CT43 cells with [3H]CL-LDL as described above,chased the cells at 37 °C for various times as indicated (0 min

to 2 h), and analyzed [3H]cholesterol present in cell homoge-nates after Optiprep gradient fractionation. At zero time, asexpected, little [3H]cholesterol was present in various fractionsin both cell types (Fig. 5B). A control experiment showed that,at zero time, the unhydrolyzed [3H]CL was located as a broadpeak in fractions 1–7 in both cell types (data not shown). Whencells were chased for 30 min, the [3H]cholesterol fractionsemerged and were seen as a broad peak (fractions 4–11) thatcentered at fraction 7 in both cell homogenates (Fig. 5C). Whencells were chased for 2 h, [3H]cholesterol fractions continued toaccumulate as a broad peak (fractions 5–12) that centered atfraction 9 in CT43 cells. In contrast, in 25RA cells, a significant

FIG. 4. Distribution of AL and NPC1 on Percoll gradients. A,immunoblot analysis of AL. Each Percoll fraction from 25RA cellsgrown in medium B at 37 °C was analyzed by SDS-PAGE and immu-noblotted for AL as described under “Experimental Procedures.” Asingle band at �41 kDa was detected in the light fractions. The resultsshown are representative of two independent experiments. The sameresults were obtained when CT43 cells were used for analysis. B,distribution of LysoTracker. C, distribution of GFP or NPC1-GFP inPercoll fractions from CT43 cells transfected with GFP alone or withNPC1-GFP. In B and C, the fluorescent signal in each Percoll fractionwas quantitated using the fluorometer as described under “Experimen-tal Procedures.”

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portion of [3H]cholesterol was redistributed to various regionsacross the entire gradient, including the heavier fractionswhere the PM was located (Fig. 5D). These results, along with

the results shown in Figs. 2–4, show that [3H]cholesterol newlyliberated from [3H]CL-LDL was absent from the PM, but waspresent in various endocytic compartment(s), including thosecontaining AL.

Identification of the AL Compartment(s) by Fluorescence Mi-croscopy of Intact Cells—The results shown in Figs. 4 and 5suggest that AL resides mainly in the membrane compart-ment(s) with buoyant density. For a different approach to iden-tify the AL-containing compartment(s), we performed fluores-cence microscopy with intact cells. CT43 cells were transientlytransfected with the NPC1-GFP construct and viewed under afluorescence microscope. The AL signal (red) was identifiedusing the anti-AL antibodies as the primary antigen in indirectimmunofluorescence. The NPC1-GFP signal (green) was iden-tified by the intrinsic fluorescence from GFP. The results showthat little NPC1-GFP signal colocalized with the AL signal(Fig. 6A). In a separate experiment, the two signals wereviewed under a confocal laser scanning microscope. The resultsclearly show that these signals did not colocalize with eachother (Fig. 6B).

Previously, Neufeld et al. (31) reported that the NPC1 pro-tein resides mainly in a compartment that contains LAMP2, amarker for the late endosome and lysosome; the NPC1-contain-ing compartment(s) does not contain CI-MPR. The CI-MPRprotein shuttles between the TGN and various endocytic com-partments (32). Consistent with the finding of Neufeld et al., wefound that NPC1-GFP significantly overlapped with theLAMP2 protein and LysoTracker (Fig. 7A, upper and middlepanels), but did not significantly overlap with the CI-MPRsignal except in the TGN region (lower panels). As expected, theAL signal did not colocalize with the LysoTracker signal or theLAMP2 signal (Fig. 7B, upper and middle panels). We alsofound that the AL signal (green) did not significantly colocalizewith any of the following organelle markers: EEA1 (an earlyendosomal marker), syntaxin-6 (a TGN marker), and caveo-lin-1 (a caveola marker) (Fig. 7B, lower panels). In addition, wefound that the AL signal did not colocalize with the flotillin-1signal (another caveola marker) or the GM130 signal (a Golgimarker) (data not shown).

FIG. 5. Monitoring the early fate of [3H]cholesterol using Op-tiprep gradients. A, AL and various subcellular organelle markerswere subjected to immunoblot analyses. EEA1 (for the early endosome),Na�/K�-ATPase (for the PM), Rab9 (for the late endosome), and syn-taxin-6 (for the TGN) were immunoblotted as described under “Exper-imental Procedures.” The results shown are the blots made using CT43cells grown in medium B for 48 h. The same results were obtained using25RA cells. Under the cell homogenization conditions described, the PMfractions of the 25RA and CT43 cells were consistently found as a broadpeak, ranging from fractions 11 to 19 (results of three independentexperiments). B–D, 25RA and CT43 cells were pulse-labeled with[3H]CL-LDL for 5 h at 14 °C and chased at 37 °C for 0 min (B), 30min (C), or 2 h (D); the post-nuclear cell homogenates were fractionatedon an Optiprep gradient and analyzed as described under “Experimen-tal Procedures.” Results are representative of two independentexperiments.

FIG. 6. Localization of NPC1 and AL in intact cells visualizedby fluorescence microscopy. A, CT43 cells transfected with NPC1-GFP were immunostained with anti-AL antibodies, followed by Alexa568-conjugated anti-rabbit secondary IgGs, and then viewed under afluorescence microscope. AL staining (left panel) and NPC1-GFP (mid-dle panel) were merged to produce the image shown in the right panel.One of the two cells shown is positive for NPC1-GFP expression. B, thesamples were viewed under a confocal laser scanning microscope. Adifferential interference contrast (DIC) image of two neighboring cells isshown in the first panel; both cells are positive for NPC1-GFP expres-sion (green; third panel). AL staining (red) is shown in the second panel.The merged image (fourth panel) contains the AL and NPC1-GFPimages. A control experiment showed that, in the absence of the pri-mary anti-AL antibodies, the secondary fluorescent IgG alone did notshow any fluorescent signal (data not shown).

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The results shown in Figs. 6 and 7 show that, in CHO cells(25RA and CT43), the AL compartment(s) was distinct from thelate endosome/lysosome. To determine whether this findingapplies to other cell types, we used confocal microscopy toexamine the degree of overlap between the LysoTracker signal(red) and the AL signal (green) in three different human cells:Hf cells, hepatocyte-like HepG2 cells, and macrophage-likeTHP-1 cells (after phorbol ester treatment) (Fig. 8, A–C). In Hfcells, we found that a certain overlap between the AL andLysoTracker signals did exist in normal and NPC1 cells; thedegree of overlap varied from one cell to another. To provide asemiquantitative estimation, we examined numerous fieldsfrom 10 individual Hf cells and estimated that the percentageof the AL signal overlapping the LysoTracker signal averaged�20%. Representative results are shown in Fig. 8A. In HepG2cells and phorbol ester-treated THP-1 cells, there was little

detectable overlap between the LysoTracker and AL signals(Fig. 8, B and C).

AL is active only at acidic pH (33). To test whether the ALcompartment(s) is acidic, we used a monoclonal antibodyagainst the 100–116-kDa subunit of the V0 domain of humanV-ATPase (11) to examine its possible colocalization with AL inHf cells. V-ATPase is a key protein responsible for keepingvarious endocytic compartments acidic (by pumping protonsacross the membranes) (11, 34). The results show that, onaverage, �70% of the AL signal colocalized with the V-ATPasesignal. Typical results are shown in Fig. 9A (upper and lowerpanels). Additional experiments showed that no AL signal wasdetectable in Hf cells with Wolman’s disease, a disease in whichAL is deficient (data not shown). To further test whether ALresides mainly in acidic compartment(s), we used the com-pound DAMP. As first shown by Anderson et al. (26), DAMPreadily diffuses into the cells and is concentrated inside variousacidic organelles; its presence can be detected by immuno-staining using antibodies against the DNP group. This methodis capable of detecting weakly acidic compartments (27). Asshown in Fig. 9B, in HepG2 cells, a significant portion (�50%)of the AL-positive signals colocalized with the DAMP-positivesignals. Similar results were obtained in Hf and THP-1 cells(data not shown). These results strengthen the interpretationthat AL resides mainly in an acidic environment.

AL is one of the numerous acid hydrolases that containmannose 6-phosphate residues. Based on this motif, the MPRprotein directs the hydrolases from the TGN to various endo-cytic compartments (32). We therefore tested the localization of

FIG. 7. Possible colocalization of NPC1 and AL proteins withvarious organelle markers examined by fluorescence micros-copy. A: CT43 cells transfected with NPC1-GFP (green) were stainedwith LysoTracker (red), LAMP2 (red), or CI-MPR (red) and then viewedby fluorescence microscopy. In the merged picture (right panels), yellowrepresents significant overlap between the red and green signals. B:upper panels, 25RA cells were double-stained with LysoTracker (red)and AL (green). Middle panels, similarly, CT43 cells were double-stained with LAMP2 (red) and AL (green). Samples were viewed undera fluorescence microscope. Lower panels, CT43 cells were stained withAL (green) and with one of the organelle markers (red) as indicated:EEA1, syntaxin-6 (Synt6), and caveolin-1 (Cav-1). Samples were viewedunder a fluorescence microscope. Only the merged pictures are shown.For results shown in B, the same results were obtained for both 25RAand CT43 cells.

FIG. 8. Colocalization of AL with late endosomal/lysosomalmarkers in various human cell types. Cells as indicated were dou-ble-stained with LysoTracker (red) and AL (green). A, normal and NPC1Hf cells; B, HepG2 cells; C, THP-1 cells treated with 100 nM phorbol12-myristate 13-acetate and 100 nM 1�,25-dihydroxyvitamin D3 for 3days. The images shown in A–C are representatives of a large numberof photographs obtained with a confocal microscope equipped withdifferential interference contrast (DIC) capability; the differential in-terference contrast images are shown in the left panels. In the mergedpictures, yellow demonstrates significant overlap between the red andgreen signals.

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CI-MPR against that of AL in Hf cells. The results show that,on average, �60% of the AL signal colocalized with the CI-MPRsignal. Typical results are shown in Fig. 9C (upper and middlepanels). To demonstrate the specificity of the signals elicited bythe anti-AL and anti-CI-MPR antibodies, we performed thecolocalization experiments in Hf cells from mucolipidosis II(I-cell disease) patients. In cells with I-cell disease, lysosomalenzymes including AL are released and secreted into the ex-tracellular milieu due to lack of the covalently modified man-nose 6-phosphate residues (32). Our results show that, there

was very little AL signal found inside the I-cell Hf cells; also,the CI-MPR signal had a distribution pattern resembling thatof a TGN marker (Fig. 9C, lower panels).

DISCUSSION

The results presented here suggest that, under the conditionsused, the following scenario occurs. Upon warming up at 37 °C,[3H]cholesterol is abundantly released from [3H]CL-LDL withina 30–60-min chase time. It is first present in certain early com-partment(s) in a manner independent of NPC1. [3H]Cholesterolin the early compartment(s) is resistant to a short incubationwith CD (5–10 min), but is sensitive to a longer incubation withCD (30 min or longer). After the 45–60-min chase time, in CT43cells, [3H]cholesterol enters and accumulates in the late compart-ment(s) and becomes resistant to both short and long CD incu-bations. During the chase period, continuous inclusion of CDinduces the [3H]cholesterol efflux from the early compartment(s)and prevents its entry into the late compartment(s). Once enter-ing the late compartment(s), [3H]cholesterol in CT43 cells be-comes resistant to CD even after a prolonged incubation time (60min). The late compartment(s) probably consists mainly of theaberrant late endosome/lysosome characteristic of all the NPC1cells. The early compartment(s) appears mainly in buoyant den-sity fractions on Percoll gradients, whereas the late compart-ment(s) is concentrated in heavy density fractions. In contrast towhat we previously believed, the early compartment(s) containslittle PM for the following reasons. During the first 30 min of thechase period, [3H]cholesterol was resistant to a short incubationwith CD (Fig. 1E). Also, within the first 75 min, the [3H]choles-terol content steadily increased in the isolated PM fraction of25RA cells; its increase was hardly seen in that of CT43 cells (Fig.2C). In addition, when HDL or apoA-I was used, significant effluxof [3H]cholesterol from [3H]CL-LDL occurred only in 25RA cells,but not in CT43 cells (Fig. 2, A and B).

Our results are consistent with early studies describing thefunction of NPC1, i.e. transport of LDL-derived cholesterol tothe PM requires NPC1 (6, 35). While this manuscript wasunder review, Wojtanik and Liscum (36) reported that LDL-derived cholesterol moves directly to the NPC1-containing com-partment(s) without passing through the PM first. Building onthe same model, our work demonstrates that the free choles-terol liberated from cholesteryl ester of LDL is present in earlyintracellular compartment(s) before it is transported to theNPC1-containing compartment. The early compartment(s)may consist of various internal membrane vesicles/organelles.One of the early compartments contains AL. The AL compart-

FIG. 9. Localization of AL in human cells. A, normal and NPC1 Hfcells were double-immunostained with V-ATPase (red) and AL (green).B, HepG2 cells were incubated with 50 �M DAMP for 30 min at 37 °Cand detected using the monoclonal anti-DNP antibody for immuno-staining. Similar results (not shown) were obtained in human fibro-blasts and THP-1 cells. C, normal, NPC1, and I-cell disease Hf cellswere immunostained with antibodies against CI-MPR (red) and AL(green) using the Zenon rabbit IgG labeling kits. The images shown inA–C are representatives of a large number of photographs obtained byconfocal microscopy. In the merged pictures, yellow demonstrates sig-nificant overlap between the red and green signals.

FIG. 10. Model describing the early trafficking events of LDL-derived cholesterol in the context of the endocytic pathway. LE,late endosome. See “Discussion” for details.

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ment(s) contains V-ATPase, a protein that maintains an acidicpH in various endocytic compartments, and CI-MPR, a proteinthat sorts lysosomal enzymes from the TGN to various endo-cytic compartments. Because the majority of the AL signal doesnot colocalize with markers for the TGN, for the early endo-some, or for the late endosome/lysosome, we suggest that ALresides mainly in acidic compartment(s) between the earlyendosome and the late endosome (Fig. 10).

Based on recent literature, the localization of “lysosomal” en-zyme in “non-lysosomal” organelles is not surprising. First, it hasbeen demonstrated that the sorting of lysosomal enzyme is dif-ferentially affected by mutations and isotypes of MPR (37–39).Mammalian cells possess two different types of MPRs, cation-de-pendent and cation-independent. These receptors sort overlap-ping yet distinct sets of lysosomal enzymes from the TGN to theendocytic organelles. There is heterogeneity of mannose 6-phos-phate modification and recognition markers present in variouslysosomal enzymes. This diversity can confer the differentialsorting of lysosomal proteins in the endocytic pathway. Indeed, ithas been reported that, in J774 macrophages, different cathepsinenzymes show differential localization: cathepsin H is highlyenriched in early endosomal fractions; cathepsin S is in lateendosomes; and cathepsins B and L are in classical lysosomes(40).

Based on enzyme activity measurements, Runquist andHavel (41) earlier showed that, in rat liver, a significantamount of AL in vitro is present in early and late endosomalfractions. Buton et al. (42) showed that, in mouse macrophages,cholesteryl ester in aggregated LDL is degraded by AL at a ratefar exceeding that of protein degradation, suggesting that foraggregated LDL, the site of cholesteryl ester hydrolysis is func-tionally distinct from the conventional lysosomal pathway. Ourcurrent results extend these findings and demonstrate that, inthe various cell types examined, AL resides mainly in an en-docytic compartment distinct from the endosome/lysosome. Todescribe the early trafficking events of LDL-derived cholesterolin the context of the endocytic pathway, we propose the follow-ing model. LDL-derived cholesteryl ester is hydrolyzed in anon-lysosomal endocytic compartment containing AL. The lib-erated cholesterol is then delivered to the late endosomal com-partment, where NPC1 mediates its transport to the PM or tothe endoplasmic reticulum for esterification (Fig. 10).

To explain the heterogeneity observed in the degree of colo-calization between the AL and LysoTracker signals in Hf cells,we speculate that the AL compartment may fuse with the lateendosome; the fusion may occur at different rates in differentcell types. Before fusion occurs, the two compartments wouldbe found in close proximity. In Hf cells, the fusion event mayoccur at a considerably slower rate than in other cell types,thus accounting for some degree of apparent overlap betweenthe AL signal and the late endosomal/lysosomal signal seen inHf cells. Other possibilities can not be ruled out at present. Forexample, vesicular transport may account for the transit ofcholesterol from the AL compartment to the late endosome; thecell type-specific difference described here may be attributed tothe difference in the vesicular trafficking rate. It is also possi-ble that, in Hf cells (and in other cell types yet to be examined),a certain portion of AL is physically present in the late endo-some/lysosome, in addition to its presence in the non-lysosomalcompartment described in our current work. The results pre-sented in Fig. 3 suggest that, in addition to the AL compart-ment, LDL-derived cholesterol may traverse to other endocyticcompartment(s) before it finally appears in the late endosome/lysosome. Further biochemical investigation at the cellularlevel is required to elucidate the exact relationship of thesecompartments.

Acknowledgments—We thank Cathy Chang, Naomi Sakashita, andother members of the Chang Laboratory for assistance and helpfuldiscussion over the course of this work; Bob Maue for providing themouse npc1-gfp cDNA; Henry Higgs for instruction and help with theuse of the spectrofluorometer; and Professor Ira Mellman for advice onperforming the Optiprep gradient experiment. We also thank HelinaMorgan for carefully editing the manuscript. Confocal microscopy wasperformed with the help of Ken Orndorff and Alice Givan in the HerbertC. Englert Cell Analysis Laboratory at the Dartmouth Medical School.

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Shigeki Sugii, Patrick C. Reid, Nobutaka Ohgami, Hong Du and Ta-Yuan Chang Lipoprotein-derived Cholesterol

Distinct Endosomal Compartments in Early Trafficking of Low Density

doi: 10.1074/jbc.M300542200 originally published online April 28, 20032003, 278:27180-27189.J. Biol. Chem. 

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