Distinct endosomal compartments in early trafficking of low density lipoprotein-derived cholesterol.

We previously studied the early trafficking of low density lipoprotein (LDL)-derived cholesterol in mutant Chinese hamster ovary cells defective in Niemann-Pick type C1 (NPC1) using cyclodextrin (CD) to monitor the arrival of cholesterol from the cell interior to the plasma membrane (PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. (2000) J. Biol. Chem. 275, 4013-4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool(s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incubation with CD (>30 min) caused cholesterol in internal membrane compartment(s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD (5-10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liberating cholesterol from cholesteryl ester in LDL. Biochemical and microscopic evidence indicates that most of the AL is present in endocytic compartment(s) distinct from the late endosome/lysosome. Our results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum.

endocytotic vesicles. After endocytosis, LDL enters the endosomal/lysosomal system, where cholesteryl ester, a major lipid found in LDL, is hydrolyzed by the enzyme acid lipase (AL) (1). Mutations in AL cause cholesteryl ester to eventually accumulate in the lysosome (2,3). After the hydrolytic action by AL, the transport of LDL-derived cholesterol from the endosome/lysosome to the plasma membrane (PM) or to the endoplasmic reticulum for re-esterification requires the protein named Niemann-Pick type C1 (NPC1). Mutations in NPC1 cause unesterified cholesterol and other lipids to accumulate in the late endosome and lysosome. Despite significant advances, the events that led to eventual accumulation of cholesterol in the late endosome/lysosome remain unclear. To delineate the early trafficking events of LDL-derived cholesterol, we previously performed pulse-chase experiments using [ 3

H]cholesteryl linoleate-labeled LDL ([ 3 H]CL-LDL) in
Chinese hamster ovary (CHO) mutant cells defective in the npc1 locus, CT43, along with their parental cells, 25RA (4). To monitor the arrival of [ 3 H]cholesterol at the PM, we utilized a cyclodextrin (CD)-based intact cell assay. CD is a water-soluble molecule that has a high affinity for cholesterol and has been widely used to monitor the arrival of cholesterol at the PM from the cell interior (5)(6)(7)(8) H]cholesterol that accumulates in the aberrant endosome/lysosome is resistant to extraction by CD. Based on this CD sensitivity test, the early pool(s) was assumed to be the PM (4). These results led us to hypothesize that, in NPC1 cells, cholesterol liberated from cholesteryl ester in LDL first moves to the PM independent of NPC1 and then moves back to the cell interior and accumulates in the aberrant late endosome/lysosome. Using a similar CD-based assay, other investigators independently reached the same conclusion (7).
The original CD-based assay used by us and by others involved continuous incubation of cells with CD for 30 min or longer. Thus, it is possible that prolonged incubation of cells with CD may cause redistribution of cellular cholesterol, so cholesterol originally residing in internal membranes moves to the PM and becomes extractable by CD. Recently, Haynes et al. (9) showed that, in CHO cells, depending on the incubation time used (ranging from 30 s to 20 min), CD is capable of extracting cellular cholesterol from two or three kinetically distinct pools; rearrangement of cholesterol between these pools could occur under various treatments. In this work, we further investigated the early trafficking events of LDL-derived cholesterol. To follow the fate of newly hydrolyzed cholesterol more precisely, we redesigned the procedures for the * This work was supported by National Institutes of Health Grant HL 36709 (to T.-Y. C.). The Herbert C. Englert Cell Analysis Laboratory was established by equipment grants from the Fannie E. Rippel Foundation and the National Institutes of Health Shared Instrument Program, and its operation is supported in part by NCI Core Grant CA 23108 from the National Institutes of Health (to the Norris Cotton Cancer Center). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Dartmouth Medical School, HB 7200, Hanover, NH 03755. Tel.: 603-650-1622; Fax: 603-650-1128; E-mail: Ta.Yuan.Chang@dartmouth. edu. 1  pulse-chase experiment and the CD treatment. We also performed biochemical and immunofluorescence experiments to define the hydrolytic compartment(s) involved in producing LDL-derived cholesterol. A model, revised from the one previously proposed by this laboratory (4), describing the early itinerary of LDL-derived cholesterol in the context of the endocytic pathway is presented.
Cell Lines and Cell Culture-25RA is a CHO cell line that is resistant to the cytotoxicity of 25-hydroxycholesterol (15) and that contains a gain-of-function mutation in SCAP (SREBP cleavage-activating protein) (16). The CT43 mutant cell line was isolated as one of the cholesterol trafficking mutants from mutagenized 25RA cells (17). It contains the same gain-of-function mutation in SCAP. In addition, it contains a premature translational termination mutation near the 3Ј-end of the npc1 coding sequence, producing a nonfunctional truncated NPC1 protein (4). CHO cells were maintained in medium A (Ham's F-12 medium plus 10% FBS and 10 g/ml gentamycin) as monolayers at 37°C with 5% CO 2 . When medium B (Ham's F-12 medium supplemented with 5% delipidated FBS plus 35 M oleic acid and 10 g/ml gentamycin) was used at lower temperatures (18°C or lower), Ham's F-12 medium (titrated to pH 7.4 without sodium bicarbonate) was used, and cells were placed in a water bath without CO 2 . A human fibroblast (Hf) cell line derived from an NPC patient (No. 93.22) was the generous gift of Dr. Peter Pentchev (National Institutes of Health). Hf cell lines isolated from patients with Wolman's disease (GM00863A and GM01606A) and with mucolipidosis II (I-cell disease) (GM02013D) were from the NIGMS Human Genetic Cell Repository (Camden, NJ). Hepatocyte-like HepG2 and monocytic THP-1 cells were obtained from American Type Culture Collection (Manassas, VA). Hf and HepG2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 100 units/ml penicillin/streptomycin at 37°C with 10% CO 2 . THP-1 cells were maintained under the same conditions, except that RPMI 1640 medium was used instead. Prior to the experiment, THP-1 was treated with 100 nM phorbol 12-myristate 13-acetate and 100 nM 1␣,25dihydroxyvitamin D 3 (both from Sigma) for at least 72 h to induce differentiation (18).
LDL-derived Cholesterol Trafficking Assays-[ 3 H]CL-LDL with specific radioactivity of ϳ5 ϫ 10 4 cpm/g of protein was prepared as previously described (19). Cells were plated in 6-or 12-well dishes as previously described (4,20). Prior to each experiment, the cells were cultured for 2 days in medium B (to deplete stored cholesterol within the cell). Cells were chilled on ice, labeled with 30 g/ml [ 3 H]CL-LDL in medium B for 5 h at 18°C, and washed once with 1% bovine serum albumin-containing cold phosphate-buffered saline (PBS) at 4°C and three 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 the chase period, rapid metabolism of [ 3 H]CL-LDL occurs in a time-dependent manner. To obtain reproducible results, we found that it was essential to use healthy cells grown at late log phase for plating and to control the temperature and pH of the growth media in a precise manner. To control the temperature, we incubated tissue culture plates or dishes on platforms covered with water in a constant temperature water bath. To control the pH in a precise manner, we used media devoid of sodium bicarbonate and titrated to pH 7.4 within 1 week before usage. An acyl-CoA:cholesterol O-acyltransferase inhibitor (2 M F12511) was included whenever cells were incubated at 37°C. F12511 was previously shown to inhibit acyl-CoA:cholesterol O-acyltransferase activity at the submicromolar level (21). Labeled cellular lipids were extracted and analyzed by TLC as described (17) Isolation of the PM-To isolate the PM from the cells, we employed the 30% Percoll gradient procedure essentially as described (20). All procedures were performed at 4°C. Briefly, after the pulse-chase experiment, cells in two 150-mm dishes were collected. The cells were scraped 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 steel tissue grinder (Dura-Grind, Wheaton). The post-nuclear supernatant was loaded onto a 30% Percoll gradient. After centrifugation at 84,000 ϫ g for 30 min, 25 fractions were collected from the top. The PM fractions usually corresponded to fractions 9 and 10, as evidenced by a visible white membrane band; this band showed high enrichment in Na ϩ /K ϩ -ATPase and caveolin-1 protein (20). In addition, we performed biotinylation of PM proteins in intact cells at 4°C for 10 min using sulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce), which showed that only fractions 9 and 10 were highly enriched in the biotinylated proteins (data not shown). The 3 H-labeled lipids were extracted using chloroform/methanol and analyzed by TLC as previously described (20).
11% Percoll Gradient Analyses-All procedures were performed at 4°C. The fractionation method was performed as described previously (4,22). Briefly, after the pulse-chase experiment, cells from one 150-mm dish were 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 same stainless steel tissue grinder described above. To minimize breakage of membrane vesicles, 250 mM sucrose was included in the buffer. To increase recovery, the pellet was resuspended in buffer and homogenized a second time. The combined post-nuclear supernatant from cells was loaded onto 11% Percoll and centrifuged at 20,000 ϫ g for 40 min using a Beckman Model Ti-70.1 rotor. 10 fractions were collected from the top. Ͼ80% of the PM marker (Na ϩ /K ϩ -ATPase) was concentrated in fractions 1 and 2, whereas Ͼ80% of the late endosomal/lysosomal markers (LAMP1/LAMP2) were concentrated in fractions 9 and 10 as previously described (4). The 3 H-labeled lipids were extracted using chloroform/ methanol and analyzed by TLC as previously described (20).
Optiprep Gradient Analyses-The procedure was based on a previously described method (23) with modifications. Cells grown in one 150-mm dish were homogenized at 4°C as described above. The postnuclear supernatant (1 ml) was placed onto 9 ml of a linear 5-20% Optiprep gradient prepared in homogenization buffer at 4°C. Gradients were centrifuged at 27,000 rpm for 20 h at 4°C using a Beckman SW 41 rotor. 20 fractions (0.5 ml each) were carefully collected from the top. Immunoblot analyses were performed using antibodies against individual organelle markers as indicated. The 3 H-labeled lipids were extracted using chloroform/methanol and analyzed by TLC as previously described (20).
Immunoblot and Spectrofluorometric Analyses of Percoll Fractions-For immunoblot analysis, each Percoll fraction was ultracentrifuged either at 100,000 ϫ g for 90 min or at 150,000 ϫ g for 30 min to remove the Percoll particles. Afterward, the samples (located on top of the Percoll particles) were carefully collected using Pasteur pipettes. Proteins present in these fractions were concentrated by chloroform/methanol precipitation (24). The precipitated proteins were dissolved in lysis buffer (100 mM Tris (pH 8.0), 0.2 M NaCl, 1% Nonidet P-40, 1 mM EDTA, and 1ϫ protease inhibitor mixture), separated on SDS-polyacrylamide gel, and immunoblotted with polyclonal anti-AL antibodies (1:1000). To quantitate the LysoTracker signal (a late endosomal/lysosomal mark-er), we used a highly sensitive fluorometer to measure the fluorescence intensities present in various Percoll fractions. The method is briefly described as follows. Cells were incubated with 100 nM LysoTracker Red for 2 h at 37°C and then fractionated on a Percoll gradient at 4°C. The Percoll fractions were ultracentrifuged at 150,000 ϫ g for 30 min to remove the Percoll particles. Each fraction was then quantitated for its fluorescence at Ex 577 nm /Em 590 nm using a PC1 photon counting spectrofluorometer from ISS Inc. (Champaign, IL). For detection of the green fluorescent protein (GFP) signal in GFP-transfected or NPC1-GFP-transfected cells, a modified method was needed (because Percoll particles exhibited autofluorescent signals that strongly interfered with the GFP signal). Each Percoll fraction was solubilized with the nonfluorescent detergent Thesit (Roche Applied Science) at 0.2%, and the solubilized material was ultracentrifuged at 150,000 ϫ g for 6 h. The fluorescent signal present in the supernatant was quantitated in the fluorometer at Ex 488 nm /Em 507 nm .
Construction and Transfection of GFP-tagged NPC1-The construct encoding mouse NPC1 protein fused with GFP was created and subcloned 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 experiments showed that expression of NPC1-GFP, but not GFP alone, completely rescued the cholesterol accumulation defect in CT43 cells, indicating that the NPC1-GFP fusion protein is functional (25). Transfected cells were used within 2-3 days of transfection for imaging analysis and within 4 days for Percoll gradient analysis.
Fluorescence Microscopy-Cells were grown on glass coverslips in 6-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. For DAMP staining, intact cells were incubated with 50 M DAMP for 30 min at 37°C (26, 27); its signal was detected with monoclonal antibody against 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, washed three times again, and permeabilized either with methanol (chilled at Ϫ20°C) for 1 min or with 1% Triton X-100 in PBS at room temperature for 10 min. After three more washes, the cells were blocked with 10% goat serum in PBS for 30 min at room temperature and incubated with predetermined concentrations of various primary antibodies in the blocking medium for 1 h. When anti-V-ATPase antibody was used as the primary antibody, the incubation time was for 20 min only. Antibody 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). For double labeling studies using rabbit anti-AL and rabbit anti-CI-MPR antibodies, 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 then washed 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 microscope with a ϫ63 objective equipped with a CCD camera (DEI-750, Optronics Engineering, Goleta, CA). Fluorescein isothiocyanate and rhodamine filters were used to visualize GFP/Alexa 488 and Lyso-Tracker Red/Alexa 568, respectively. The images were processed using MetaView Version 4.5 software (Universal Imaging Corp., Downingtown, PA). In selective experiments as indicated, the samples were also viewed under a Bio-Rad MRC-1024 krypton/argon laser scanning confocal microscope. The images were constructed using LaserSharp software and further processed using Adobe Photoshop Version 5.02.

RESULTS
Early Trafficking of LDL-derived Cholesterol Probed with a Long Versus Short Incubation with CD-We grew CT43 and 25RA cells in cholesterol-free medium for 2 days and pulse- labeled them with [ 3 H]CL-LDL for 5 h at 18°C. At this temperature, LDL was internalized, but accumulated in pre-lysosomal compartments without significant hydrolysis of CL. When the temperature was increased to 37°C, CL in LDL was rapidly hydrolyzed to free cholesterol and transported to designated locations (4). In numerous experiments, we found that the half-time of hydrolysis averaged 25 Ϯ 5 min in both cell types; a typical result is shown in Fig. 1F. During the warm-up period (i.e. immediately after the labeling), if cells were continuously incubated with CD for various time periods as indicated (0 -120 min), 25RA and CT43 cells showed the same degree of cholesterol efflux toward CD; the efflux significantly increased from 30 min on (Fig. 1A). When cells were chased at 37°C for 30 min before adding CD for up to 120 min, a slight defect in cholesterol efflux (starting at 15 min after adding CD) occurred in CT43 cells (Fig. 1B). In a separate experiment, a slight efflux defect in CT43 cells was also found in cells chased for 15 min before CD treatment (data not shown). In contrast, a severe efflux defect in CT43 cells occurred when cells were chased at 37°C for 60 min (Fig. 1C) or for 120 min (Fig. 1D) before adding CD. In a separate experiment, a severe efflux defect in CT43 cells was also shown in cells chased for 45 min before CD treatment (data not shown).
We next compared cholesterol effluxes in 25RA and CT43 cells using a procedure that involves a short incubation with CD (28). In a control experiment, we labeled the PM of 25RA and CT43 cells with [ 3 H]cholesterol at 4°C using the [ 3 H]cholesterol/liposome method (4) and then treated the labeled cells with CD for 10 min at 37°C. We found that 80 -90% of the cellular label could be removed (data not shown). Based on this finding, we redesigned the pulse-chase protocol as follows. Cells were labeled with [ 3 H]CL-LDL at 18°C, chased at 37°C for various time periods as indicated, and incubated with CD for 10 min; the percent cholesterol efflux was then monitored. The results show that, after the 40-min chase time, cholesterol efflux increased steadily with time in 25RA cells (Fig. 1E). In contrast, such an increase hardly occurred in CT43 cells. In a separate experiment, results similar to those shown in Fig. 1E were obtained when the CD incubation was reduced to 5 min (data not shown).
LDL-derived Cholesterol Efflux in 25RA and CT43 Cells in Response to HDL or ApoA-I-Because CD may induce an alteration in cellular cholesterol distribution, we examined the cholesterol efflux in 25RA and CT43 cells in response to two physiologically relevant acceptors, HDL and apoA-I. Both acceptors sequester cholesterol slowly from the PM of cells (29). When [ 3 H]CL-LDL-labeled cells were continuously incubated with HDL, [ 3 H]cholesterol was slowly but steadily removed in 25RA cells; in contrast, this removal did not occur in CT43 cells ( Fig. 2A). Similarly, when [ 3 H]CL-LDL-labeled cells were incubated with apoA-I, significant cholesterol efflux occurred in 25RA cells, but not in CT43 cells (Fig. 2B).

LDL-derived [ 3 H]Cholesterol Present in Isolated PM
Fractions of 25RA and CT43 Cells-We next used the procedure first described by Graham (30) to isolate the PM from cells labeled with [ 3 H]CL-LDL. The PM isolated by this method contained minimal contamination from internal membranes. Using this procedure, we monitored the [ 3 H]cholesterol content in the PM after various chase times. The results show that the [ 3 H]cholesterol content in the PM increased significantly with time, reaching a maximum at ϳ75 min in 25RA cells, whereas such an increase was hardly observed in the CT43 cells (Fig. 2C).  [5][6][7][8], and heavy fractions (fractions 9 and 10), whereas in CT43 cells, [ 3 H]cholesterol accumulated mainly in the heavy fractions (fractions 9 and 10). If CD was included during the 1-h chase period, [ 3 H]cholesterol present in the heavy fractions (fractions 9 and 10) and light fractions (fractions 1-4) significantly decreased, resulting in a blurring of the difference in the [ 3 H]cholesterol distribution between 25RA and CT43 cells (Fig. 3C, hatched bars). In a separate experiment, the chase time was increased to 2 h. We found that, in the absence of CD, [ 3 H]cholesterol continued to accumulate in the heavy fractions (fractions 9 and 10) in CT43 cells (Fig. 3D,  solid bars); if CD was included during the last hour of the 2-h chase time, the cholesterol that accumulated in the heavy fractions of CT43 cells was resistant to extraction by CD (Fig.  3D, compare hatched and solid bars). Fig. 3

suggest that [ 3 H]cholesterol newly liberated from [ 3 H]CL-LDL may be present in multiple membrane fractions (Percoll fractions 1-8) before it
is sequestered in the late endosome/lysosome (Percoll fractions 9 and 10). Because more than one membrane compartment may be present in any of the Percoll fractions, the identities of these early fractions could not be positively determined at present. On the other hand, hydrolysis of cholesteryl ester in LDL requires the action of the enzyme AL. Therefore, the compartment(s) that contains AL is involved during the early trafficking of [ 3 H]cholesterol liberated from [ 3 H]CL-LDL. In the literature, the localization of AL has been assumed to be in lysosomes (1); however, direct evidence is lacking. We thus focused our effort to identify the compartment(s) containing AL. We used the specific antibodies against AL (10) to perform immunoblot analysis on various Percoll fractions. These antibodies identified a single 41-kDa protein band. The results show that, in both 25RA and CT43 cells, all of the AL-positive signals were distributed in the buoyant fractions (fractions 1-3); no detectable signal could be found in either the heavy or medium density fractions. Representative results are shown in Fig. 4A. A control experiment showed that LysoTracker, a marker for the late endosome and lysosome, was predominantly found in the heavy fractions (fractions 9 and 10) (Fig. 4B). In another experiment, the NPC1-GFP fusion protein expressed in CT43 cells was also predominantly found in the heavy fractions (Fig.  4C); a control experiment showed that GFP alone expressed in CT43 cells was predominantly localized in the buoyant fractions (Fig. 4C). Figs. 3 and 4 demonstrate that, on a 11% Percoll gradient, the AL-containing membranes were located in light fractions (fractions 1-3). However, because the PM fractions were also enriched in these fractions, one could not determine whether [ 3 H]cholesterol newly liberated from [ 3 H]CL-LDL was present in the AL compartment(s) or in the PM. To clarify this issue, we used another subcellular fractionation method that separates the PM from the endosomal compartments using the Optiprep gradient procedure first developed by Sheff et al. (23). As shown in Fig. 5A, immunoblot analyses demonstrated that both AL and the early endosomal marker EEA1 were predominantly located in early fractions (fractions 3-5), whereas the PM marker Na ϩ /K ϩ -ATPase was broadly enriched in heavier fractions (fractions [11][12][13][14][15][16][17][18][19]. The late endosomal marker Rab9 was located mainly in fractions 7 and 8; the trans-Golgi network (TGN) marker syntaxin-6 was located mainly in fractions 9 and 10. Next, we pulse-labeled 25RA and CT43 cells with [ 3 H]CL-LDL as described above, chased the cells at 37°C for various times as indicated (0 min to 2 h), and analyzed [ 3 H]cholesterol present in cell homogenates after Optiprep gradient fractionation. At zero time, as expected, little [ 3 H]cholesterol was present in various fractions in both cell types (Fig. 5B). A control experiment showed that, at zero time, the unhydrolyzed [ 3 H]CL was located as a broad peak in fractions 1-7 in both cell types (data not shown). When cells were chased for 30 min, the [ 3 H]cholesterol fractions emerged and were seen as a broad peak (fractions 4 -11) that centered at fraction 7 in both cell homogenates (Fig. 5C). When cells were chased for 2 h, [ 3 H]cholesterol fractions continued to accumulate as a broad peak (fractions 5-12) that centered at fraction 9 in CT43 cells. In contrast, in 25RA cells, a significant suggest that AL resides mainly in the membrane compartment(s) with buoyant density. For a different approach to identify the AL-containing compartment(s), we performed fluorescence microscopy with intact cells. CT43 cells were transiently transfected with the NPC1-GFP construct and viewed under a fluorescence microscope. The AL signal (red) was identified using the anti-AL antibodies as the primary antigen in indirect immunofluorescence. The NPC1-GFP signal (green) was identified by the intrinsic fluorescence from GFP. The results show that little NPC1-GFP signal colocalized with the AL signal (Fig. 6A). In a separate experiment, the two signals were viewed under a confocal laser scanning microscope. The results clearly show that these signals did not colocalize with each other (Fig. 6B).

Monitoring the Early Fate of [ 3 H]Cholesterol Using Optiprep Gradients-The results shown in
Previously, Neufeld et al. (31) reported that the NPC1 protein resides mainly in a compartment that contains LAMP2, a marker for the late endosome and lysosome; the NPC1-containing compartment(s) does not contain CI-MPR. The CI-MPR protein shuttles between the TGN and various endocytic compartments (32). Consistent with the finding of Neufeld et al., we found that NPC1-GFP significantly overlapped with the LAMP2 protein and LysoTracker (Fig. 7A, upper and middle  panels), but did not significantly overlap with the CI-MPR signal except in the TGN region (lower panels). As expected, the AL signal did not colocalize with the LysoTracker signal or the LAMP2 signal (Fig. 7B, upper and middle panels). We also found that the AL signal (green) did not significantly colocalize with any of the following organelle markers: EEA1 (an early endosomal marker), syntaxin-6 (a TGN marker), and caveolin-1 (a caveola marker) (Fig. 7B, lower panels). In addition, we found that the AL signal did not colocalize with the flotillin-1 signal (another caveola marker) or the GM130 signal (a Golgi marker) (data not shown).

FIG. 5. Monitoring the early fate of [ 3 H]cholesterol using Optiprep gradients.
A, AL and various subcellular organelle markers were subjected to immunoblot analyses. EEA1 (for the early endosome), Na ϩ /K ϩ -ATPase (for the PM), Rab9 (for the late endosome), and syntaxin-6 (for the TGN) were immunoblotted as described under "Experimental Procedures." The results shown are the blots made using CT43 cells grown in medium B for 48 h. The same results were obtained using 25RA cells. Under the cell homogenization conditions described, the PM fractions of the 25RA and CT43 cells were consistently found as a broad peak, ranging from fractions 11 to 19 (results of three independent experiments). B-D, 25RA and CT43 cells were pulse-labeled with The results shown in Figs. 6 and 7 show that, in CHO cells (25RA and CT43), the AL compartment(s) was distinct from the late endosome/lysosome. To determine whether this finding applies to other cell types, we used confocal microscopy to examine 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-like THP-1 cells (after phorbol ester treatment) (Fig. 8, A-C). In Hf cells, we found that a certain overlap between the AL and LysoTracker signals did exist in normal and NPC1 cells; the degree of overlap varied from one cell to another. To provide a semiquantitative estimation, we examined numerous fields from 10 individual Hf cells and estimated that the percentage of the AL signal overlapping the LysoTracker signal averaged ϳ20%. Representative results are shown in Fig. 8A. In HepG2 cells 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 AL compartment(s) is acidic, we used a monoclonal antibody against the 100 -116-kDa subunit of the V 0 domain of human V-ATPase (11) to examine its possible colocalization with AL in Hf cells. V-ATPase is a key protein responsible for keeping various endocytic compartments acidic (by pumping protons across the membranes) (11,34). The results show that, on average, Ͼ70% of the AL signal colocalized with the V-ATPase signal. Typical results are shown in Fig. 9A (upper and lower  panels). Additional experiments showed that no AL signal was detectable in Hf cells with Wolman's disease, a disease in which AL is deficient (data not shown). To further test whether AL resides mainly in acidic compartment(s), we used the compound DAMP. As first shown by Anderson et al. (26), DAMP readily diffuses into the cells and is concentrated inside various acidic organelles; its presence can be detected by immunostaining using antibodies against the DNP group. This method is capable of detecting weakly acidic compartments (27). As shown in Fig. 9B, in HepG2 cells, a significant portion (Ͼ50%) of the AL-positive signals colocalized with the DAMP-positive signals. Similar results were obtained in Hf and THP-1 cells (data not shown). These results strengthen the interpretation that AL resides mainly in an acidic environment.
AL is one of the numerous acid hydrolases that contain mannose 6-phosphate residues. Based on this motif, the MPR protein directs the hydrolases from the TGN to various endocytic compartments (32). We therefore tested the localization of FIG. 7. Possible colocalization of NPC1 and AL proteins with various organelle markers examined by fluorescence microscopy. A: CT43 cells transfected with NPC1-GFP (green) were stained with LysoTracker (red), LAMP2 (red), or CI-MPR (red) and then viewed by fluorescence microscopy. In the merged picture (right panels), yellow represents 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 doublestained with LAMP2 (red) and AL (green). Samples were viewed under a fluorescence microscope. Lower panels, CT43 cells were stained with AL (green) and with one of the organelle markers (red) as indicated: EEA1, syntaxin-6 (Synt6), and caveolin-1 (Cav-1). Samples were viewed under a fluorescence microscope. Only the merged pictures are shown. For results shown in B, the same results were obtained for both 25RA and CT43 cells. The images shown in A-C are representatives of a large number of photographs obtained with a confocal microscope equipped with differential interference contrast (DIC) capability; the differential interference contrast images are shown in the left panels. In the merged pictures, yellow demonstrates significant overlap between the red and green signals.
CI-MPR against that of AL in Hf cells. The results show that, on average, Ͼ60% of the AL signal colocalized with the CI-MPR signal. Typical results are shown in Fig. 9C (upper and middle  panels). To demonstrate the specificity of the signals elicited by the anti-AL and anti-CI-MPR antibodies, we performed the colocalization experiments in Hf cells from mucolipidosis II (I-cell disease) patients. In cells with I-cell disease, lysosomal enzymes including AL are released and secreted into the extracellular milieu due to lack of the covalently modified mannose 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 that of a TGN marker (Fig. 9C, lower panels). DISCUSSION The results presented here suggest that, under the conditions used, the following scenario occurs. Upon warming up at 37°C, [ 3 H]cholesterol is abundantly released from [ 3 H]CL-LDL within a 30-60-min chase time. It is first present in certain early compartment(s) in a manner independent of NPC1. [ 3 H]Cholesterol in the early compartment(s) is resistant to a short incubation with CD (5-10 min), but is sensitive to a longer incubation with CD (30 min or longer). After the 45-60-min chase time, in CT43 cells, [ 3 H]cholesterol enters and accumulates in the late compartment(s) and becomes resistant to both short and long CD incubations. During the chase period, continuous inclusion of CD induces the [ 3 H]cholesterol efflux from the early compartment(s) and prevents its entry into the late compartment(s). Once entering the late compartment(s), [ 3 H]cholesterol in CT43 cells becomes resistant to CD even after a prolonged incubation time (60 min). The late compartment(s) probably consists mainly of the aberrant late endosome/lysosome characteristic of all the NPC1 cells. The early compartment(s) appears mainly in buoyant density fractions on Percoll gradients, whereas the late compartment(s) is concentrated in heavy density fractions. In contrast to what we previously believed, the early compartment(s) contains little PM for the following reasons. During the first 30 min of the chase period, [ 3 H]cholesterol was resistant to a short incubation with CD (Fig. 1E). Also, within the first 75 min, the [ 3 H]cholesterol content steadily increased in the isolated PM fraction of 25RA cells; its increase was hardly seen in that of CT43 cells (Fig.  2C). In addition, when HDL or apoA-I was used, significant efflux of [ 3 H]cholesterol from [ 3 H]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 the function of NPC1, i.e. transport of LDL-derived cholesterol to the PM requires NPC1 (6,35). While this manuscript was under review, Wojtanik and Liscum (36) reported that LDLderived cholesterol moves directly to the NPC1-containing compartment(s) without passing through the PM first. Building on the same model, our work demonstrates that the free cholesterol liberated from cholesteryl ester of LDL is present in early intracellular compartment(s) before it is transported to the NPC1-containing compartment. The early compartment(s) may consist of various internal membrane vesicles/organelles. One of the early compartments contains AL. The AL compart- ment(s) contains V-ATPase, a protein that maintains an acidic pH in various endocytic compartments, and CI-MPR, a protein that sorts lysosomal enzymes from the TGN to various endocytic compartments. Because the majority of the AL signal does not colocalize with markers for the TGN, for the early endosome, or for the late endosome/lysosome, we suggest that AL resides mainly in acidic compartment(s) between the early endosome and the late endosome (Fig. 10).
Based on recent literature, the localization of "lysosomal" enzyme in "non-lysosomal" organelles is not surprising. First, it has been demonstrated that the sorting of lysosomal enzyme is differentially affected by mutations and isotypes of MPR (37)(38)(39). Mammalian cells possess two different types of MPRs, cation-dependent and cation-independent. These receptors sort overlapping yet distinct sets of lysosomal enzymes from the TGN to the endocytic organelles. There is heterogeneity of mannose 6-phosphate modification and recognition markers present in various lysosomal enzymes. This diversity can confer the differential sorting of lysosomal proteins in the endocytic pathway. Indeed, it has been reported that, in J774 macrophages, different cathepsin enzymes show differential localization: cathepsin H is highly enriched in early endosomal fractions; cathepsin S is in late endosomes; and cathepsins B and L are in classical lysosomes (40).
Based on enzyme activity measurements, Runquist and Havel (41) earlier showed that, in rat liver, a significant amount of AL in vitro is present in early and late endosomal fractions. Buton et al. (42) showed that, in mouse macrophages, cholesteryl ester in aggregated LDL is degraded by AL at a rate far exceeding that of protein degradation, suggesting that for aggregated LDL, the site of cholesteryl ester hydrolysis is functionally distinct from the conventional lysosomal pathway. Our current results extend these findings and demonstrate that, in the various cell types examined, AL resides mainly in an endocytic compartment distinct from the endosome/lysosome. To describe the early trafficking events of LDL-derived cholesterol in the context of the endocytic pathway, we propose the following model. LDL-derived cholesteryl ester is hydrolyzed in a non-lysosomal endocytic compartment containing AL. The liberated cholesterol is then delivered to the late endosomal compartment, where NPC1 mediates its transport to the PM or to the endoplasmic reticulum for esterification (Fig. 10).
To explain the heterogeneity observed in the degree of colocalization between the AL and LysoTracker signals in Hf cells, we speculate that the AL compartment may fuse with the late endosome; the fusion may occur at different rates in different cell types. Before fusion occurs, the two compartments would be found in close proximity. In Hf cells, the fusion event may occur at a considerably slower rate than in other cell types, thus accounting for some degree of apparent overlap between the AL signal and the late endosomal/lysosomal signal seen in Hf cells. Other possibilities can not be ruled out at present. For example, vesicular transport may account for the transit of cholesterol from the AL compartment to the late endosome; the cell type-specific difference described here may be attributed to the difference in the vesicular trafficking rate. It is also possible that, in Hf cells (and in other cell types yet to be examined), a certain portion of AL is physically present in the late endosome/lysosome, in addition to its presence in the non-lysosomal compartment described in our current work. The results presented in Fig. 3 suggest that, in addition to the AL compartment, LDL-derived cholesterol may traverse to other endocytic compartment(s) before it finally appears in the late endosome/ lysosome. Further biochemical investigation at the cellular level is required to elucidate the exact relationship of these compartments.