Distinct Endosomal Compartments in Early Trafficking of Low Density Lipoprotein-derived Cholesterol*
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 and the
Division of Human Genetics, Children's Hospital
Medical Center, Cincinnati, Ohio 45229
Received for publication, January 17, 2003
, and in revised form, April 10, 2003.
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ABSTRACT
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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,
40134021). 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
(510 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.
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INTRODUCTION
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In mammalian cells, low density lipoprotein
(LDL)1 binds to its
receptor at the cell surface and is recruited into clathrin-coated 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
[3H]cholesteryl linoleate-labeled LDL ([3H]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
[3H]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
(58).
Our results show that [3H]cholesterol, newly released from the
hydrolysis of [3H]CL-LDL, emerges in the early pool(s) in a manner
unaffected by the npc1 mutation. Subsequently (within 2 h), in the
parental cells, [3H]cholesterol is distributed to the PM and the
endoplasmic reticulum. In CT43 cells, [3H]cholesterol accumulates
in the characteristic aberrant endosome/lysosome. [3H]Cholesterol
that is present in the early pool(s) is extractable by CD, whereas
[3H]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
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.
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EXPERIMENTAL PROCEDURES
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MaterialsFetal bovine serum (FBS), protease inhibitor
mixture, Nonidet P-40, 2-hydroxypropyl-
-cyclodextrin, monoclonal
antibody against dinitrophenyl (DNP), paraformaldehyde, and human apoA-I were
purchased from Sigma. The acyl-CoA:cholesterol O-acyltransferase inhibitor
F12511
[GenBank]
was a gift of Pierre Fabre Research (Castres, Cedex, France). Percoll
and [1,2,6,7-3H]CL were from Amersham Biosciences. Optiprep
(Nycomed) was from Axis-Shield. FuGENE 6 transfection reagent was from Roche
Applied Science. The ProLong antifade kit, Alexa 488- or Alexa 568-conjugated
goat anti-rabbit or anti-mouse IgG, LysoTracker Red (DND-99),
3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP),
and Zenon rabbit IgG labeling kits were from Molecular Probes, Inc..
Monoclonal antibodies against EEA1, caveolin-1, and syntaxin-6 were from BD
Biosciences. Monoclonal antibody against Na+/K+-ATPase
was from Upstate Biotechnology, Inc. Monoclonal antibody against hamster LAMP2
(lysosomal-associated membrane
protein-2) was from the Developmental Studies Hybridoma
Bank maintained by the University of Iowa. Rabbit polyclonal antibodies
against AL were produced as described
(10). Monoclonal antibody
against vacuolar ATPase (V-ATPase) was a generous gift from Professor Satoshi
Sato (Kyoto University, Kyoto, Japan); this antibody (OSW2) is directed
against the 100116-kDa subunit of the V0 domain of V-ATPase
and has been shown to specifically recognize the vacuolar type proton pump
(11). Rabbit polyclonal
antibodies against the cation-independent mannose 6-phosphate receptor
(CI-MPR) and against Rab9 were generous gifts from Professor Suzanne Pfeffer
(Stanford University) (12).
Delipidated FBS was prepared as described
(13). LDL (density of
1.0191.063 g/ml) was prepared from fresh human plasma by sequential
flotation as previously described
(14). High density lipoprotein
(HDL; density of 1.0631.21 g/ml) was prepared by the same flotation
method and purified by heparin affinity chromatography.
Cell Lines and Cell Culture25RA 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 3'-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 µg/ml
gentamycin) as monolayers at 37 °C with 5% CO2. When medium B
(Ham's F-12 medium supplemented with 5% delipidated FBS plus 35
µM oleic acid and 10 µg/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 CO2. 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% CO2. 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
,25-dihydroxyvitamin D3 (both from Sigma) for at least 72 h
to induce differentiation
(18).
LDL-derived Cholesterol Trafficking
Assays[3H]CL-LDL with specific radioactivity of
5
x 104 cpm/µg 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 µg/ml
[3H]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 [3H]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 µM F12511
[GenBank]
) was included whenever
cells were incubated at 37 °C. F12511
[GenBank]
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); the percent hydrolysis
was calculated as [3H]cholesterol counts divided by the sum of
[3H]CL and [3H]cholesterol counts. For cholesterol
efflux experiments, cells were incubated with 4%
2-hydroxypropyl-
-cyclodextrin (CD) in medium B in the presence of the
acyl-CoA:cholesterol O-acyltransferase inhibitor at 37 °C for the
indicated times. The labeled lipids were extracted and analyzed as described
(4,
17). The percent cholesterol
efflux was calculated as [3H]cholesterol counts in the medium
divided by the sum of [3H]CL counts in the cell and
[3H]cholesterol counts in the cell and in the medium.
Isolation of the PMTo 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 x 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+/K+-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 3H-labeled lipids were
extracted using chloroform/methanol and analyzed by TLC as previously
described (20).
11% Percoll Gradient AnalysesAll 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 x g for 40 min using a
Beckman Model Ti-70.1 rotor. 10 fractions were collected from the top. >80%
of the PM marker (Na+/K+-ATPase) was concentrated in
fractions 1 and 2, whereas >80% of the late endosomal/lysosomal markers
(LAMP1/LAMP2) were concentrated in fractions 9 and 10 as previously described
(4). The 3H-labeled
lipids were extracted using chloroform/methanol and analyzed by TLC as
previously described (20).
Optiprep Gradient AnalysesThe 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
post-nuclear supernatant (1 ml) was placed onto 9 ml of a linear 520%
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 3H-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 x g for 90 min or at 150,000
x 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 1x
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 marker), 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 x g for 30 min to remove the
Percoll particles. Each fraction was then quantitated for its fluorescence at
Ex577 nm/Em590 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 non-fluorescent detergent Thesit
(Roche Applied Science) at 0.2%, and the solubilized material was
ultracentrifuged at 150,000 x g for 6 h. The fluorescent signal
present in the supernatant was quantitated in the fluorometer at Ex488
nm/Em507 nm.
Construction and Transfection of GFP-tagged NPC1The
construct encoding 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 FuGENE 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 23 days of transfection for imaging
analysis and within 4 days for Percoll gradient analysis.
Fluorescence MicroscopyCells 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
LysoTracker Red in the medium at 37 °C for 2 h prior to the experiment.
For DAMP staining, intact cells were incubated with 50 µM 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 -20
°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 ProLong antifade medium onto the glass
slides before image processing. Samples were viewed and photographed using a
Zeiss Axiophot microscope with a x63 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
LysoTracker Red/Alexa 568, respectively. The images were processed using
MetaView Version 4.5 software (Universal Imaging Corp., Downing-town, 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.
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RESULTS
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Early Trafficking of LDL-derived Cholesterol Probed with a Long Versus
Short Incubation with CDWe grew CT43 and 25RA cells in
cholesterol-free medium for 2 days and pulse-labeled them with
[3H]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
± 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
(0120 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).

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FIG. 1. Early trafficking of LDL-derived cholesterol after a long
versus short incubation with CD. AD, 25RA and
CT43 cells were pulse-labeled with [3H]CL-LDL for 5 h at 18 °C;
chased at 37 °C for 0 min (A), 30 min (B), 60 min
(C), or 120 min (D); and incubated with 4%
2-hydroxypropyl- -CD for 5120 min at 37 °C. For the 0-min time
point in A, CD was added at 18 °C for 10 min. Values are the
averages of triplicate dishes; results are representative of two independent
experiments. E and F, 25RA and CT43 cells were pulse-labeled
as described above and chased at 37 °C and then incubated with 4% CD for
10 min. The chase time as indicated includes the 10-min CD incubation time.
Values are the averages of duplicate dishes; results are representative of
three independent experiments. Error bars indicate S.E.
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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 [3H]cholesterol at 4
°C using the [3H]cholesterol/liposome method
(4) and then treated the
labeled cells with CD for 10 min at 37 °C. We found that 8090% 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
[3H]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-IBecause 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
[3H]CL-LDL-labeled cells were continuously incubated with HDL,
[3H]cholesterol was slowly but steadily removed in 25RA cells; in
contrast, this removal did not occur in CT43 cells
(Fig. 2A). Similarly,
when [3H]CL-LDL-labeled cells were incubated with apoA-I,
significant cholesterol efflux occurred in 25RA cells, but not in CT43 cells
(Fig. 2B).

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FIG. 2. A and B, cholesterol efflux in 25RA and CT43 cells
mediated by 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 1.0 mg/ml HDL for various times as indicated (A) or with 10
µg/ml apoA-I for 6 h (B). C, appearance of
[3H]cholesterol in the PM of 25RA and CT43 cells. Cells were
pulse-labeled with [3H]CL-LDL for 5 h at 18 °C and chased at 37
°C for the indicated times. The PM was isolated as described under
"Experimental Procedures." Cholesterol enrichment in the PM is
expressed as relative percentages, calculated as the
[3H]cholesterol counts in the PM fractions divided by the sum of
the [3H]CL and [3H]cholesterol counts in the
post-nuclear supernatants. Results are representative of two independent
experiments. BSA, bovine serum albumin.
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LDL-derived [3H]Cholesterol Present in Isolated PM Fractions
of 25RA and CT43 CellsWe next used the procedure first described
by Graham (30) to isolate the
PM from cells labeled with [3H]CL-LDL. The PM isolated by this
method contained minimal contamination from internal membranes. Using this
procedure, we monitored the [3H]cholesterol content in the PM after
various chase times. The results show that the [3H]cholesterol
content in the PM increased significantly with time, reaching a maximum at
75 min in 25RA cells, whereas such an increase was hardly observed in the
CT43 cells (Fig.
2C).
Percoll Gradient Analyses of Various Membrane Fractions Containing
LDL-derived [3H]CholesterolTo detect the presence of
[3H]cholesterol in various membrane fractions, we performed Percoll
gradient centrifugation using cell homogenates prepared from labeled cells.
The Percoll fractions consisted of 10 fractions of increasing density, with
light fractions (fractions 14) enriched in the PM and early endosome
and with heavy fractions (fractions 9 and 10) enriched in the late endosome
and lysosome (4,
22). For labeling, we pulsed
cells with [3H]CL-LDL for 5 h at 18 °C. As shown in
Fig. 3A, when the
chase time was 0 min, [3H]CL was predominantly present in the
lighter fractions (fractions 14). At the 30-min chase time, a
significant decrease in [3H]CL occurred in both cell types;
concomitantly, a significant increase in the [3H]cholesterol counts
occurred in both cell types (Fig.
3B). The cholesterol counts were distributed in the
lighter fractions (fractions 14), medium density fractions (fractions
58), and heavy fractions (fractions 9 and 10). Importantly, the
[3H]cholesterol distribution patterns in 25RA and CT43 cells were
similar at the 30-min chase time point. In contrast, when the cells were
chased for a longer time period (for 1 h) in the absence of CD, a significant
difference in the [3H]cholesterol distribution was seen between
25RA and CT43 cells (Fig.
3C, solid bars): in 25RA cells,
[3H]cholesterol was distributed in the lighter fractions (fractions
14), medium density fractions (fractions 58), and heavy
fractions (fractions 9 and 10), whereas in CT43 cells,
[3H]cholesterol accumulated mainly in the heavy fractions
(fractions 9 and 10). If CD was included during the 1-h chase period,
[3H]cholesterol present in the heavy fractions (fractions 9 and 10)
and light fractions (fractions 14) significantly decreased, resulting
in a blurring of the difference in the [3H]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,
[3H]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).

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FIG. 3. Early trafficking of LDL-derived transport analyzed by Percoll gradient
centrifugation. A and B, 25RA and CT43 cells were
pulse-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
gradient analysis. [3H]CL (A) and
[3H]cholesterol (B) in each Percoll fraction were analyzed
according to the procedures described under "Experimental
Procedures." Within each cell type, to normalize variation in total
3H counts recovered from different samples, the values reported
were normalized so 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 chased at 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, the values reported were normalized so that the
sum of the counts in cellular cholesterol and cholesterol in the medium was
the same for different samples. 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 were analyzed as described for C. Results are
representative of two independent experiments.
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Identification of the AL Compartment(s) in Percoll
FractionsThe results shown in
Fig. 3 suggest that
[3H]cholesterol newly liberated from [3H]CL-LDL may be
present in multiple membrane fractions (Percoll fractions 18) 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
[3H]cholesterol liberated from [3H]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 13); 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).

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FIG. 4. Distribution of AL and NPC1 on Percoll gradients. A,
immunoblot analysis of AL. Each Percoll fraction from 25RA cells grown in
medium B at 37 °C was analyzed by SDS-PAGE and immunoblotted for AL as
described under "Experimental Procedures." A single band at
41 kDa was detected in the light fractions. The results shown are
representative of two independent experiments. The same results were obtained
when CT43 cells were used for analysis. B, distribution of
LysoTracker. C, distribution of GFP or NPC1-GFP in Percoll fractions
from CT43 cells transfected with GFP alone or with NPC1-GFP. In B and
C, the fluorescent signal in each Percoll fraction was quantitated
using the fluorometer as described under "Experimental
Procedures."
|
|
Monitoring the Early Fate of [3H]Cholesterol Using Optiprep
GradientsThe results shown in Figs.
3 and
4 demonstrate that, on a 11%
Percoll gradient, the AL-containing membranes were located in light fractions
(fractions 13). However, because the PM fractions were also enriched in
these fractions, one could not determine whether [3H]cholesterol
newly liberated from [3H]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 35), whereas the PM
marker Na+/K+-ATPase was broadly enriched in heavier
fractions (fractions 1119). 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 [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 homogenates after Optiprep gradient
fractionation. At zero time, as expected, little [3H]cholesterol
was present in various fractions in both cell types
(Fig. 5B). A control
experiment showed that, at zero time, the unhydrolyzed [3H]CL was
located as a broad peak in fractions 17 in both cell types (data not
shown). When cells were chased for 30 min, the [3H]cholesterol
fractions emerged and were seen as a broad peak (fractions 411) that
centered at fraction 7 in both cell homogenates
(Fig. 5C). When cells
were chased for 2 h, [3H]cholesterol fractions continued to
accumulate as a broad peak (fractions 512) that centered at fraction 9
in CT43 cells. In contrast, in 25RA cells, a significant portion of
[3H]cholesterol was redistributed to various regions across the
entire gradient, including the heavier fractions where the PM was located
(Fig. 5D). These
results, along with the results shown in Figs.
24,
show that [3H]cholesterol newly liberated from
[3H]CL-LDL was absent from the PM, but was present in various
endocytic compartment(s), including those containing AL.

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FIG. 5. Monitoring the early fate of [3H]cholesterol using Optiprep
gradients. A, AL and various subcellular organelle markers were
subjected to immunoblot analyses. EEA1 (for the early endosome),
Na+/K+-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).
BD, 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), 30 min (C), or 2 h (D); the post-nuclear cell
homogenates were fractionated on an Optiprep gradient and analyzed as
described under "Experimental Procedures." Results are
representative of two independent experiments.
|
|
Identification of the AL Compartment(s) by Fluorescence Microscopy of
Intact CellsThe results shown in Figs.
4 and
5 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).

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FIG. 6. Localization of NPC1 and AL in intact cells visualized by fluorescence
microscopy. A, CT43 cells transfected with NPC1-GFP were
immunostained with anti-AL antibodies, followed by Alexa 568-conjugated
anti-rabbit secondary IgGs, and then viewed under a fluorescence microscope.
AL staining (left panel) and NPC1-GFP (middle 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, the samples were
viewed under a confocal laser scanning microscope. A differential interference
contrast (DIC) image of two neighboring cells is shown in the
first panel; both cells are positive for NPC1-GFP expression
(green; third panel). AL staining (red) is shown in
the second panel. The merged image (fourth panel) contains
the AL and NPC1-GFP images. A control experiment showed that, in the absence
of the primary anti-AL antibodies, the secondary fluorescent IgG alone did not
show any fluorescent signal (data not shown).
|
|
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).

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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 double-stained 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 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, AC).
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
20%.
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).

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FIG. 8. Colocalization of AL with late endosomal/lysosomal markers in various
human cell types. Cells as indicated were double-stained with LysoTracker
(red) and AL (green). A, normal and NPC1 Hf cells;
B, HepG2 cells; C, THP-1 cells treated with 100
nM phorbol 12-myristate 13-acetate and 100 nM
1 ,25-dihydroxyvitamin D3 for 3 days. The images shown in
AC 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.
|
|
AL is active only at acidic pH
(33). To test whether the AL
compartment(s) is acidic, we used a monoclonal antibody against the
100116-kDa subunit of the V0 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, >70% 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 (>50%) 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.

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FIG. 9. Localization of AL in human cells. A, normal and NPC1 Hf
cells were double-immunostained with V-ATPase (red) and AL
(green). B, HepG2 cells were incubated with 50
µM DAMP for 30 min at 37 °C and detected using the
monoclonal anti-DNP antibody for immunostaining. Similar results (not shown)
were obtained in human fibroblasts and THP-1 cells. C, normal, NPC1,
and I-cell disease Hf cells were immunostained with antibodies against CI-MPR
(red) and AL (green) using the Zenon rabbit IgG labeling
kits. The images shown in AC are representatives of a large
number of photographs obtained by confocal microscopy. In the merged pictures,
yellow demonstrates significant overlap between the red and
green signals.
|
|
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 CI-MPR against that of AL in Hf cells. The results show that,
on average, >60% 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,
[3H]cholesterol is abundantly released from [3H]CL-LDL
within a 3060-min chase time. It is first present in certain early
compartment(s) in a manner independent of NPC1. [3H]Cholesterol in
the early compartment(s) is resistant to a short incubation with CD
(510 min), but is sensitive to a longer incubation with CD (30 min or
longer). After the 4560-min chase time, in CT43 cells,
[3H]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 [3H]cholesterol
efflux from the early compartment(s) and prevents its entry into the late
compartment(s). Once entering the late compartment(s),
[3H]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, [3H]cholesterol was resistant to a
short incubation with CD (Fig.
1E). Also, within the first 75 min, the
[3H]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
[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 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 LDL-derived
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 compartment(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).

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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.
|
|
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
(3739).
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.
 |
FOOTNOTES
|
---|
* 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{at}dartmouth.edu.
1 The abbreviations used are: LDL, low density lipoprotein; AL, acid lipase;
PM, plasma membrane; NPC1, Niemann-Pick type C1; CL, cholesteryl linoleate;
[3H]CL-LDL, [3H]cholesteryl linoleate-labeled low
density lipoprotein; CHO, Chinese hamster ovary; CD, cyclodextrin; FBS, fetal
bovine serum; DNP, dinitrophenyl; DAMP,
3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine;
V-ATPase, vacuolar ATPase; MPR, mannose 6-phosphate receptor; CI-MPR,
cation-independent mannose 6-phosphate receptor; HDL, high density
lipoprotein; Hf, human fibroblast; PBS, phosphate-buffered saline; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GFP, green
fluorescent protein; TGN, trans-Golgi network. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Cathy Chang, Naomi Sakashita, and other members of the Chang
Laboratory for assistance and helpful discussion over the course of this work;
Bob Maue for providing the mouse npc1-gfp cDNA; Henry Higgs
for instruction and help with the use of the spectrofluorometer; and Professor
Ira Mellman for advice on performing the Optiprep gradient experiment. We also
thank Helina Morgan for carefully editing the manuscript. Confocal microscopy
was performed with the help of Ken Orndorff and Alice Givan in the Herbert C.
Englert Cell Analysis Laboratory at the Dartmouth Medical School.
 |
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