RESEARCH ARTICLE
1765
Cholesterol requirement for cation-independent
mannose 6-phosphate receptor exit from
multivesicular late endosomes to the Golgi
Ishido Miwako1,2, Akitsugu Yamamoto3, Toshio Kitamura4, Kuniaki Nagayama1 and Masato Ohashi1,*
1Dept
2Dept
3Dept
4Dept
of Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan
of Biophysics, Faculty of Science, Kyoto University, Kyoto 606-8501, Japan
of Physiology, Kansai Medical University, Osaka 570-8506, Japan
of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
*Author for correspondence (e-mail: [email protected])
Accepted 11 February 2001
Journal of Cell Science 114, 1765-1776 © The Company of Biologists Ltd
SUMMARY
The regulation of endocytic traffic of receptors has central
importance in the fine tuning of cell activities. Here, we
provide evidence that cholesterol is required for the exit of
cation-independent mannose 6-phosphate receptor (CIMPR) from the endosomal carrier vesicle/multivesicular
bodies (ECV/MVBs) to the Golgi. A previously established
Chinese hamster ovary cell mutant, LEX2, exhibits
arrested ECV/MVBs in which CI-MPR and lysosomal
glycoprotein-B (lgp-B) are accumulated. The abnormal
accumulation of CI-MPR within the ECV/MVBs in LEX2
cells was corrected in a post-translational manner by the
supplementation of medium with cholesterol. Furthermore,
it was shown that, by expression cloning using LEX2
mutant, the introduction of the NAD(P)H steroid
dehydrogenase-like protein, an enzyme involved in the later
stage of cholesterol biosynthesis, allows the exit of CI-MPR
from the MVBs to the Golgi and reduces the number of
arrested ECV/MVBs in LEX2 cells. The recovery of the exit
transport of CI-MPR from the ECV/MVBs was associated
with the restoration of the normal cellular free cholesterol
level and segregation between CI-MPR and lgp-B, both of
which had been localized at the internal small vesicles of
the arrested ECV/MVBs. By contrast, the restoration of
cholesterol failed to correct the defective processing of
endocytosed LDL to a degradative compartment in LEX2
cells. These results suggest that cholesterol is required for
ECV/MVB reorganization that drives the sorting/transport
of materials destined for the Golgi out of the pathways
towards lysosomes.
INTRODUCTION
In addition, cholesterol might modulate the late endocytic
traffic connected to MVBs. Evidence has been provided that
sterol enrichment in a late endosome might retard clearance of
bulk-endocytosed material from the late endosome (Neufeld et
al., 1999). Cation-independent mannose 6-phosphate receptor
(CI-MPR) transports mannose 6-phosphate-containing
lysosome proteins from the trans-Golgi network (TGN) to
endosomes and cycles back to the TGN for reuse (Kornfeld,
1992). It also serves as a suppressor of IGF2 proliferative
action by internalizing and transporting IGF2 (Kornfeld, 1992).
In the genetic disease Niemann-Pick type C (NPC), cholesterol
accumulates in late endosomes and the exit of CI-MPR from
the late endosomes is impaired (Kobayashi et al., 1999).
Because the accumulation of cholesterol in late endosomes
appeared to precede that of CI-MPR caused by drugs
mimicking the Niemann-Pick cells’ defects, it was suggested
that an abnormally elevated cholesterol level in late endosomes
led to the retention of CI-MPR (Kobayashi et al., 1999;
Mukherjee and Maxfield, 1999).
In this study, we provide evidence that the normal level of
cellular free cholesterol is required for the exit of CI-MPR from
the late endosomal MVBs. We have previously established a
Chinese hamster ovary (CHO) cell mutant, LEX2, that exhibits
The eukaryotic cell uses the membrane traffic system as a
device to organize the flow of materials and signal transduction
(Ceresa and Schmid, 2000; Leof, 2000). Protein-protein and
protein-lipid interactions are key components that comprise
physicochemical bases for the membrane traffic system. As a
result of the interactions, intracellular organelles exhibit their
characteristic structures that appear appropriate for their
specific task. One of such structures observed in the endocytic
system is the multivesicular body (MVB), so called because it
contains a number of internal vesicles. The importance of
MVBs has been implicated in several physiological processes
such as axonal transport and exosome biogenesis (Denzer et
al., 2000; Gu and Gruenberg, 1999; Parton et al., 1992; Thery
et al., 1999). The unique construction of MVBs has attracted
much attention concerning their structure-function
relationships, which remain to be established.
The role for cholesterol has recently been implicated in
several membrane traffic systems, including putative raftmediated events and transport through the endocytic pathways
(Grimmer et al., 2000; Keller and Simons, 1998; Puri et al.,
1999; Rodal et al., 1999; Subtil et al., 1999; Thiele et al., 2000).
Key words: Cholesterol, Multi-vesicular body, Sorting, NAD(P)H
steroid dehydrogenase-like protein, Endocytosis, Expression cloning
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JOURNAL OF CELL SCIENCE 114 (9)
arrested multivesicular endosomes much like the endosomal
transport intermediates, previously referred to as endosomal
carrier vesicles (ECVs) or maturing endosomes, in which CIMPR and lysosomal glycoprotein-B (lgp-B) are accumulated
(Ohashi et al., 2000). LEX2 exhibits defective lysosomal
degradation of LDL, but normal levels of endocytic uptake of
LDL and normal recycling of LDL receptors (Ohashi et al.,
2000). Thus, LEX2 mutant should be a good tool for dissecting
the mechanisms of sorting and exporting events at the late
endocytic MVBs. We found that the abnormal accumulation of
CI-MPR within the MVBs in LEX2 cells can be corrected in
a post-translational manner by the supplementation of medium
with cholesterol. Furthermore, by expression cloning using
LEX2 mutant, we show that the introduction of the NAD(P)H
steroid dehydrogenase-like protein (NSDHL), an enzyme
involved in the later stage of cholesterol biosynthesis (Liu et
al., 1999), allows the exit of CI-MPR from the MVBs to the
Golgi and lowers the number of these arrested large MVBs in
LEX2 cells. The recovery of exit transport of CI-MPR from
the MVBs was associated with the restoration of the normal
cellular free cholesterol level and segregation between CI-MPR
and lgp-B, both of which had been localized at the internal
small vesicles of the arrested ECV/MVBs. By contrast, the
restoration of cholesterol failed to correct the defective
processing of endocytosed LDL to a degradative compartment
in LEX2 cells. These results suggest that cholesterol is required
for ECV/MVB reorganization that drives the sorting/transport
of materials destined for the Golgi out of the pathways towards
lysosomes.
MATERIALS AND METHODS
Cell incubation
Wild-type and mutant CHO cells were cultured in Ham’s F12 medium
supplemented with 5% FCS, 100 U/ml penicillin and 10 µg/ml
streptomycin (FCS/F12). For microscopy, cells were grown on glass
coverslips. Cells were washed with Hanks’ balanced salt solution and
then incubated as described below (see Results). All experiments were
carried out before cells reached confluency.
When added to culture medium as a suspension, cholesterol (Sigma
grade: 99+%) was first dissolved in ethanol as a 33 mM stock solution
and the stock cholesterol was rapidly injected into medium with
vigorous vortexing (0.15% final ethanol). The control medium
contained 0.15% ethanol without cholesterol. MβCD-cholesterol
(cholesterol-water soluble, ~40 mg cholesterol per gram solid),
cycloheximide and puromycin·2HCl were purchased from Sigma.
Microscopy
Immunofluorescence microscopy was carried out essentially as
described previously (Ohashi et al., 1999). Antibodies, except for antisyntaxin 6 antibody, were as described previously (Ohashi et al.,
2000). Mouse monoclonal anti-syntaxin 6 antibody was purchased
from Transduction Laboratories.
Cells were fixed with 3% paraformaldehyde in PBS for 15 minutes
and were then permeabilized with 50 µg/ml digitonin in PBS for 10
minutes. The cells were washed, blocked with 0.1% gelatin in PBS
for 30 minutes and processed for indirect immunofluorescence using
corresponding primary and secondary antibodies diluted in 0.1%
gelatin/PBS. The samples were mounted in SlowFade Light
(MolecularProbes) and were observed under a Carl Zeiss LSM 510
laser scanning microscope equipped with a PlanAPOCHROMAT ×63
or Plan-NEOFLUAR ×100 objective with an optical section set at <0.8
µm. Fluorescence intensity distribution was kept from saturating the
range of the detector by manipulating laser intensities and the gain of
the detector.
The semiquantification of colocalization in double fluorescence
microscopy based on a fluorescence intensity analysis was performed
essentially as described previously (Ohashi et al., 1999; Ohashi et al.,
2000). A parameter, Col(A/B), indicating localization of fluorescence
A within compartments positive for fluorescence B in a cell, was
defined as the percentage of fluorescence A included in region B, the
region of compartments positive for fluorescence B. Region B was
created, using Photoshop, from fluorescence B image data by
selecting areas showing fluorescence above a background level. The
background fluorescence level was chosen as the value limiting a
group of pixels that exhibit the lowest continuous fluorescence
intensity distribution (using Photoshop). The parameter Col(A/B) was
calculated by integrating fluorescence intensity included in region B
on the cell by cell basis, using the NIH Image program.
Conventional electron microscopy was performed as described
previously (Ohashi et al., 1999). Immunoelectron microscopy was
performed as described previously with permeabilization either by
freezing and thawing (for CI-MPR detection; Ohashi et al., 2000) or
by using saponin (for lgp-B detection; Ohashi et al., 1999).
Cellular free cholesterol determination
Subconfluent cells in a 100 mm dish were washed with ice-cold PBS
five times on ice. The cells were then scraped with a rubber policeman
on ice, collected by centrifugation at 4°C and suspended in PBS (130
µl) at 4°C. An aliquot (100 µl) was used to determine free cholesterol
in the cells using cholesterol E-test (WAKO, Osaka, Japan) according
to the manufacturer’s instructions, in a 1 ml assay in the presence of
0.1% Triton X-100. After color development, the assay mixtures were
clarified by centrifugation (20 000 g, 10 minutes), followed by
absorbance measurement. In a preliminary experiment, we confirmed
that this assay does not detect lanosterol, a close precursor to
cholesterol, using a lanosterol standard solution prepared as described
elsewhere (Bae et al., 1999). The rest of the cell samples were used
to determine protein by Bio-Rad Protein Assay using bovine serum
albumin as a standard, with prior solubilization of samples with 0.5
M NaOH. The cholesterol level was normalized to protein.
Retrovirus-mediated expression cloning
LEX2 cells expressing mouse ecotropic retroviral receptor (LEX2VR
cells) were prepared as follows: A 2.3 Kb BamHI (blunt-ended)
EcoRI cDNA fragment encoding the mouse ecotropic receptor was
excised from the pM5neo that harbours this cDNA (Baker et al., 1992)
and inserted into the XhoI (blunt-ended) EcoRI site of pCI-neo
expression vector (Promega). The resultant construct was used to
transfect LEX2 mutant cells using LipofectAmine (GIBCO/BRL).
The transfected cells were selected with G418 (500 µg/ml) and used
as a pool.
A packaging cell line Phoenix Eco for ecotropic retroviruses
(ATCC Inventory No. SD 3444) was provided courtesy of Garry P.
Nolan (Stanford University) and was maintained as described
elsewhere (Pear et al., 1997).
Retrovirus was produced for library expression as described
previously (Kitamura et al., 1995). A human brain retroviral cDNA
library in pLIB vector was obtained from Clontech. The following
procedures were carried out in two sets. The library DNA (8 µg/dish)
was transfected into Phoenix Eco cells (plated 1 day before the
transfection at 5.2×106/100 mm dish) using LipofectAmine
(GIBCO/BRL) and virus stocks were recovered. For the infection of
LEX2VR cells with retroviruses, LEX2VR cells (6.6×105/150 mm
dish) were seeded the night before infection and incubated with the
infection cocktail, which contains viruses (15 ml/dish), for 5 hours in
the presence of 10 µg/ml polybrene. Subsequently, 15 ml fresh
FCS/F12 was added to the culture and incubation continued. The
medium was changed to fresh FCS/F12 at 1 day of infection. At 2
days of infection, the cells were replated into LPDS/F12 (selection
Cholesterol role in CI-MPR exit from MVB
medium) at 5×106/150 mm dish. The selection medium was refreshed
every 2 days until surviving colonies became visible. Three colonies
survived and were cloned, expanded and stored frozen.
To recover integrated cDNAs, genomic DNA (0.1 µg) isolated from
each clone was subjected to Expand High Fidelity PCR (Boehringer)
using upstream and downstream retroviral primers for pLIB vector
(Clontech). The resultant PCR fragments were either sequenced
directly, or TA-subcloned before sequencing.
To retransduce PCR fragments into LEX2VR cells by retrovirusmediated gene transfer, the TA-cloned fragments were subcloned into
pLIB vectors using the SalI-NotI sites.
LDL degradation analysis
RET-LDL was prepared as described previously (Ohashi et al., 2000).
The degradation of RET-LDL was quantified, based on fluorescence
resonance energy transfer (RET) measurement by analytical flow
cytometry, using a FACSCalibur system (Beckton Dickinson), as
described previously (Ohashi et al., 1992). The disintegration of RETLDL was expressed as parameter r, as described (Ohashi et al., 1992).
The value r is estimated essentially independently of the amount of
endocytic uptake of RET-LDL by cells (Ohashi et al., 1992).
RESULTS
Cholesterol induces CI-MPR exit from MVBs to the
Golgi in LEX2 mutant cells
In wild-type CHO cells, we previously observed CI-MPR
localized mainly in a Golgi-like distribution closely associated
with syntaxin 6 (Ohashi et al., 2000) (Fig. 1Ag,h), known to
be present primarily in the trans-Golgi network (TGN) (Bock
et al., 1997). In LEX2 mutant cells, CI-MPR was localized in
the arrested endosomal MVBs positive for lgp-B/cathepsin D,
yet away from the syntaxin 6-positive TGN-related structures
(Ohashi et al., 2000). These observations were made upon our
routine 1-day pretreatment of cells with Ham’s F12 medium
supplemented with 5% lipoprotein deficient serum
(LPDS/F12) (Fig. 1Ab,f), in order to induce the maximum
expression of LDL receptor by depleting cellular cholesterol
(Goldstein et al., 1983). This preincubation was carried out
because our primary criterion for a mutant phenotype was
reduced low-density lipoprotein (LDL) degradation, and thus
we needed to analyze the endocytic processing of LDL in
mutant cells (Ohashi et al., 2000; Ohashi et al., 1992).
Unexpectedly, we found that when LEX2 cells were pretreated
with F12 medium supplemented with 5% fetal calf serum
(FCS/F12) instead of LPDS/F12, CI-MPR was observed in a
Golgi-type distribution (Fig. 1Aa,e), much like that seen in
wild-type cells (Fig. 1Ac,g), closely associated with syntaxin
6 (Fig. 1Ae) but distinct from lgp-B (Fig. 1Aa). The shift of
CI-MPR distribution by LPDS/F12 in LEX2 cells was also
clear in semiquantification of the image data (Fig. 1Ai,k). In
addition, CI-MPR redistribution caused by LPDS/F12,
although smaller, was observed in wild-type cells. Upon
LPDS/F12 incubation, the colocalization of CI-MPR and
syntaxin 6 decreased (Fig. 1Ag,h,l), whereas change in the
colocalization of CI-MPR and lgp-B was marginal (Fig.
1Ac,d,j).
The effect of the LPDS/F12 treatment on CI-MPR
accumulation could have resulted from the depletion of any
lipoproteins absent in LPDS. Supplementation of LPDS/F12
with total lipoprotein (20 µg cholesterol/ml) resulted in CIMPR in the Golgi distribution in LEX2 cells (not shown).
1767
Further, the addition of pure cholesterol (20 µg/ml), as a
suspension or methyl-β-cyclodextrin (MβCD)-cholesterol
complex (cholesterol-water soluble, 5 µg cholesterol/ml), to
the LPDS/F12 pretreatment led to CI-MPR in the Golgi
distribution in LEX2 cells (Fig. 1Ba-d). Thus, cholesterol from
lipoproteins is required in LEX2 mutant cells for the Golgi
distribution of CI-MPR. Because LDL degradation in LEX2
cells is slow but not entirely absent (not shown) (Ohashi et al.,
2000), LEX2 cells would have acquired cholesterol through
some lipoprotein degradation and/or through cholesterol
transfer from the lipoprotein fraction (Fielding and Fielding,
1997). Although MβCD-cholesterol at 5 µg cholesterol/ml
was appropriate for long-term cell incubation (>3 days),
higher concentrations of MβCD-cholesterol (e.g. 20 µg
cholesterol/ml) in pretreating LPDS/F12 caused cell roundingup (after ~1 day) and eventually cell death (after ~2 days),
consistent with earlier reports suggesting that the build-up of
cellular free cholesterol causes cell toxicity (Brown and
Goldstein, 1980; Warner et al., 1995). However, in the
rounded-up cells that had been treated with MβCD-cholesterol
(10 or 20 µg cholesterol/ml) for 1 day, the CI-MPR was
observed in the Golgi pattern separated from lgp-B (not
shown). By contrast, the Golgi distribution caused by the
cholesterol suspension was partial and might reflect inefficient
cholesterol delivery to intracellular compartments from the
cholesterol suspension, rather than from the total lipoprotein
fraction or MβCD-cholesterol. Thus, we tested cholesterol
suspensions at higher concentrations (e.g. 130 µg/ml);
however, this resulted in cytotoxic effects.
We next examined if the cholesterol deprivation from
medium arrests CI-MPR exit from ECV/MVBs. For this, the
time course of CI-MPR redistribution in LEX2 cells upon
treatment with LPDS/F12 was examined. Because CI-MPR
cycles between late endosomes and the Golgi, the distribution
between these compartments should be determined by the
steady-state equilibrium of transport between these two. Thus,
it would be possible, for example, that the CI-MPR
accumulation in ECV/MVBs and the disappearance from
the Golgi, observed upon LPDS/F12 incubation, reflects
accelerated export of CI-MPR from the Golgi, rather than its
arrest within ECV/MVBs. If the exit from ECV/MVBs is
inhibited, the accumulation of CI-MPR in lgp-B-positive
vesicles should precede the CI-MPR displacement from the
syntaxin 6-positive Golgi. The accumulation of CI-MPR in
lgp-B-positive vesicles was already recognized after a 12 hour
incubation in LPDS/F12 (Fig. 2a,c); at this time, however, the
CI-MPR displacement from the syntaxin 6-positive Golgi was
hardly observed (Fig. 2b,d). Although the localization of CIMPR in lgp-B-positive vesicles gradually increased in a linear
manner (Fig. 2a,c), the reduction in the co-localization of CIMPR with syntaxin 6 was slower at the earlier phase of the
incubation (up to ~20 hours), but became faster at the later
phase (~1 day). Thus, the results suggest that cholesterol
depletion first causes the arrest of the exit of CI-MPR from the
late endosomal MVBs, thereby resulting in the displacement
of CI-MPR from the Golgi owing to a shift towards a new
steady state.
We also examined whether the accumulated CI-MPR in
MVBs can be directed back to the Golgi in LEX2 cells. LEX2
cells were treated first with LPDS/F12 to allow CI-MPR
to distribute in the MVBs, and then with LPDS/F12
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JOURNAL OF CELL SCIENCE 114 (9)
Fig. 1. Cholesterol is required for
the Golgi localization of CI-MPR
in LEX2 mutant cells. (A) FCS
supports the Golgi localization of
CI-MPR in LEX2 mutant cells.
LEX2 (a,b,e,f) and wild-type
(c,d,g,h) cells were incubated with
FCS/F12 (a,c,e,g) or LPDS/F12
(b,d,f,h) for 1 day at 37°C. The
cells were then processed for
double immunofluorescence
confocal microscopy. (a-d) Double
localization of CI-MPR (green)
and lgp-B (red). Arrows indicate
the colocalization of CI-MPR and
lgp-B. (e-h) Double localization of
CI-MPR (green) and syntaxin 6
(red). Arrows indicate CI-MPRpositive, syntaxin 6-negative
structures. The arrowhead in h
indicates the colocalization of CIMPR and syntaxin 6. Bar, 10 µm.
(i-l) The semiquantification of the
colocalization data in a-h is
provided in the corresponding
graphs. The data are the
means±s.e.m. (n=10). (B) Pure
cholesterol or MβCD-cholesterol
complex supports the Golgi
localization of CI-MPR in LEX2
mutant cells. LEX2 cells were
incubated with 5% LPDS/F12
containing cholesterol (20 µg/ml;
CL) as a suspension or MβCDcholesterol (5 µg/ml; MβCD-CL)
for 26 hours at 37°C, and were
then processed for double
immunofluorescence confocal
microscopy for CI-MPR and lgp-B
(a,b) or CI-MPR and syntaxin 6 (c,d). The semiquantification of these colocalization data is shown. The data are the means±s.e.m. (n=10). For
comparison, the colocalization data upon the parallel FCS/F12 and LPDS/F12 incubations are indicated as tick marks on the ordinate.
supplemented with MβCD-cholesterol (20 µg cholesterol/ml)
for 3 hours. This incubation with cholesterol redirected CIMPR from the MVBs to the Golgi (Fig. 3a,b,e-h, −), whereas
MβCD alone at the same concentration failed to exert the effect
(not shown). A higher concentration (400 µg cholesterol/ml)
of MβCD-cholesterol caused rounding up of cells in 3 hours
and eventual cell death. However, MβCD-cholesterol (400 µg
cholesterol/ml) incubation for 3 hours still induced the
redirection of CI-MPR to the Golgi pattern, distinct from the
lgp-positive structures in the round-up LEX2 cells (not shown).
When FCS/F12 medium was used, 7 hours were required for
CI-MPR to fully recover the Golgi distribution (not shown). In
addition, the suspension of pure cholesterol had the ability to
significantly redirect CI-MPR to the Golgi, although the
redirection was again partial (not shown).
The MβCD-cholesterol-induced redirection of CI-MPR was
observed in the presence of puromycin (100 µM; Fig. 3c,d,eh, puro) or cycloheximide (70 µM) (Fig. 3e-h, cyclo) at
concentrations where protein synthesis is totally blocked (Cole
et al., 1998; Zhang et al., 1997). However, without cholesterol,
each of the protein synthesis inhibitors failed to exert the effect
(not shown). The significance of these observations is twofold:
First, they suggest that CI-MPR in the Golgi reflects a true
redistribution of CI-MPR from the MVBs, rather than the
appearance of newly synthesized CI-MPR in the Golgi.
Second, they suggest that the effect of cholesterol is posttranslational. Cholesterol is known to modulate the
transcription of genes encoding enzymes of cholesterol
biosynthesis and uptake from plasma lipoprotein, thereby
effecting the feedback regulation for cholesterol homeostasis
(Brown and Goldstein, 1999). However, the observation
precludes the possibility that the effect of cholesterol was
exerted through such a transcriptional feedback regulation
effected by specific modulation of protein synthesis. Rather,
the result is more consistent with the idea that cholesterol
directly affected membrane transport.
Expression of 3β-hydroxysteroid dehydrogenase in
LEX2 mutant cells induces CI-MPR exit from MVBs
To identify cDNA that can correct the arrest of CI-MPR within
ECV/MVBs, we exploited the well-established, retrovirusmediated cDNA expression cloning technique based on
Moloney murine leukemia virus (Kitamura et al., 1995). The
retroviruses offer one of the best utilities for delivering genes
Cholesterol role in CI-MPR exit from MVB
a
MPR/lgp
60
40
20
0
MPR/syn6
60
40
20
0
10
c
80
20
Time (hr)
lgp/MPR
60
40
20
0
0
10
d
80
Colocalization (%)
0
Colocalization (%)
b
80
Colocalization (%)
Colocalization (%)
80
1769
20
Time (hr)
syn6/MPR
60
40
20
0
0
10
20
Time (hr)
0
10
Time (hr)
20
Fig. 2. The LPDS incubation leads to an arrest of CI-MPR within
lgp-B-positive structures in LEX2 cells. LEX2 cells were incubated
with LPDS/F12 for an indicated time at 37°C. The cells were then
processed for double immunofluorescence confocal microscopy for
CI-MPR and lgp-B (a,c), or for CI-MPR and syntaxin 6 (b,d). The
colocalization was semiquantified and expressed as the means±s.e.m.
(n=10).
to target cells at high efficiency, in a manner suitable for
various gene transfer purposes, including gene therapy,
allowing for long-term, stable expression of introduced genetic
elements. Moreover, when used as a murine ecotropic vector
system, this expression technique is safe to handle because the
virus does not infect humans. Because the ecotropic virus
cannot infect CHO cells, owing to the cells’ lack of the virus
receptor, we first produced LEX2 cells that express murine
ecotropic retrovirus receptor (LEX2VR cells) by transfecting
LEX2 cells with a receptor expression construct. A control
experiment using an enhanced green fluorescent protein
reporter construct showed that ~20% LEX2VR cells were
transfected under the configuration presently employed.
LEX2 cells were found to be cholesterol auxotrophs:
Whereas wild-type cells can grow freely in LPDS/F12, LEX2
cells stopped growing and started to die after culture in
LPDS/F12 for more than 2 days (not shown). Supplementation
of LPDS/F12 with pure cholesterol rescued LEX2 cells from
death (not shown). Thus the cholesterol requirement and CIMPR distribution in the Golgi were correlated in LEX2 cells.
Expecting to isolate cDNAs that can, when introduced into
LEX2, redirect the CI-MPR from the endosomal MVBs to the
Golgi, we transfected LEX2 cells with human brain library
cDNA by retrovirus-mediated gene transfer and isolated
transfectants that could grow in LPDS/F12. Three survived
clones were isolated (revertants 1, 2 and 3), and CI-MPR in
these revertants showed the Golgi distribution (not shown).
From these revertants, genomic DNAs were extracted and the
integrated library cDNAs were recovered by PCR using
retroviral primers. In agarose gel electrophoresis, 1.7 kbp bands
that appeared common to all three revertants were observed
(Fig. 4Aa, arrow). These 1.7 kb PCR fragments were partially
Fig. 3. Cholesterol relocates CI-MPR from MVBs to the Golgi.
LEX2 cells were first subjected to a 26 hour LPDS/F12 incubation to
allow CI-MPR to accumulate in the MVBs. The cells were then
incubated with LPDS/F12 containing MβCD-cholesterol (20 µg/ml)
for 3 hours at 37°C in the absence (−) or presence of either
puromycin (puro; 100 µM) or cycloheximide (cyclo; 70 µM), and
processed for double immunofluorescence confocal microscopy.
(The puromycin/MβCD-cholesterol incubation included
preincubation with 100 µM puromycin without MβCD-cholesterol
for 20 minutes at 37°C before the MβCD-cholesterol was added).
(a-d) Images show double localization of (a,c) CI-MPR (green) and
lgp-B (red), and of (b,d) CI-MPR (green) and syntaxin 6 (red). Bar,
10 µm. (e-h) The semiquantification of the colocalization between
CI-MPR and lgp-B (e,f) or between CI-MPR and syntaxin 6 (g,h)
after the above incubations (−, puro, cyclo). The data are the
means±s.e.m. (n=10). For comparison, the colocalization data upon
26 hour FCS/F12 and LPDS/f12 incubations are indicated as tick
marks on the ordinate.
sequenced and a database search revealed that all these cDNAs
contained the predicted coding region for a human gene
encoding an NAD(P)H steroid dehydrogenase-like protein
(NSDHL) (Levin et al., 1996). The coding regions of two TAcloned cDNA fragments (LIB2 and LIB3, derived from
revertant 2 and 3, respectively) were sequenced completely.
LIB3 encoded the predicted amino acid sequence of normal
human NSDHL with a non-mutating DNA base substitution
(326T→G), and LIB2 encoded the protein with a single amino
acid mutation (R281H, 1036G→A base change) and two nonmutating DNA base substitutions (326T→G, 695A→G),
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JOURNAL OF CELL SCIENCE 114 (9)
Fig. 4. Introduction of NSDHL cDNA corrects
the cholesterol auxotrophy and the altered CIMPR distribution in LEX2 cells. (Aa) LEX2VR
cells were transfected with human brain library
cDNA by retrovirus-mediated gene transfer.
From the transfectants, three revertant colonies
(revertants 1-3) recovered from cholesterol
auxotrophy and were isolated. Genomic DNAs
were extracted from these revertants and
integrated library cDNAs recovered by PCR
using retroviral primers. A genomic DNA
prepared from LEX2VR cells was used as the
PCR control. The PCR products were analyzed
by 0.8% agarose gel electrophoresis. Lanes 1-3,
revertants 1-3; lane 4, control; M, 1 kbp DNA
extension ladder (tick marks indicate 7.1, 6.1,
5.1/5.0, 4.1, 3.1, 2.0, 1.6, 1.0 and 0.5 kbp from
the top). The arrow indicates the 1.7 kbp PCR
products that appear common to revertants 1-3;
these products were identified by sequencing as
cDNAs encoding human NSDHL. (b) NSDHL
corrects cholesterol auxotrophy of LEX2VR
cells. PCR fragments (1.7 kbp) from revertants
2 and 3 (LIB2 and LIB3, respectively) were
recloned into pLIB vectors (pLIB2 and pLIB3)
and reintroduced into LEX2VR cells by
retrovirus-mediated gene transfer: LEX2VR
cells were plated at 1×105 cells/60 mm dish 18
hours before transfection. The cells were then
treated with a 2 ml transfection cocktail
containing viruses produced by packaging cells
transfected with pLIB2 or pLIB3, or not
transfected (Mock). After 2 days, the cells were
passaged at one tenth into 60 mm dishes and
cultured in LPDS/F12 selective medium. After
4 days of culture, the surviving cell colonies were stained with Coomassie Brilliant Blue, as shown. (B) NSDHL corrects the altered CI-MPR
distribution in LEX2VR cells. LEX2VR cells were transfected with pLIB2 or pLIB3 by retrovirus-mediated gene transfer, and transfected cells
(LEX2LIB2 and LEX2LIB3) were selected by culturing in LPDS/F12. LEX2VR (a), LEX2LIB2 (b) and LEX2LIB3 (c) cells were cultured in
LPDS/F12 for 1 day at 37°C, and then immunofluorescently double-stained for CI-MPR (green) and lgp-B (red). Arrows indicate dotty
colocalization of CI-MPR and lgp-B (a). The arrowhead in c indicates perinuclear CI-MPR-positive, lgp-B-negative staining. Bar, 10 µm.
(d-f) The semiquantification of these colocalization data after the LPDS/F12 incubation (LPDS) is provided together with data upon FCS/F12
incubation (FCS). The data are the means±s.e.m. (n=10).
compared with the sequence reported by Levin et al. (GenBank
accession number U47105). The nucleotide sequence
discrepancies might have derived either from the original library
or from mutation during retrovirus preparation and cDNA
recovery; however, these possibilities have not been examined.
The LIB2 and LIB3 cDNA fragments were re-cloned into a
pLIB vector, and the reintroduction of the resultant constructs
into LEX2VR cells by retrovirus-mediated gene transfer
resulted in the recovery of LEX2 growth in LPDS/F12 (Fig.
4Ab). These cells stably transfected with LIB2 and LIB3 were
selected in LPDS/F12 (LEX2LIB2 and LEX2LIB3 cells,
respectively). Whereas LEX2VR cells showed the same
abnormal CI-MPR distribution as the parental LEX2 cells (Fig.
4Ba,d), LEX2LIB2 and LEX2LIB3 cells exhibited CI-MPR in
a lgp-B-negative, perinuclear Golgi distribution (Fig. 4Bb,c,e,f)
even after the LPDS/F12 pretreatment. Thus, LIB2 and LIB3
could correct the abnormal CI-MPR arrest in LEX2VR cells.
Murine Nsdhl, sharing an 82.9% predicted amino acid
identity with human NSDHL, has recently been implicated in
cholesterol biosynthesis as a C-3 sterol dehydrogenase
involved in the complex series of reactions that result in the
sequential removal of the two C-4 methyl groups from the
sterol backbone of the cholesterol precursor lanosterol (Liu et
al., 1999). Thus, it is most likely that the enhancement of
cholesterol synthesis by the expression of this enzyme restored
CI-MPR exit from the endosomal MVBs in LEX2 cells.
Cellular cholesterol level correlates with CI-MPR
distribution
To examine if the observed changes in CI-MPR transport are
indeed associated with changes in cellular cholesterol levels,
free cholesterol was determined in wild-type, LEX2 and LEX2
transfected with NSDHL (LEX2LIB2 and LEX2LIB3). Under
our normal culture conditions using FCS/F12, wild-type cells
typically contained ~25 µg free cholesterol/mg cell protein
(Fig. 5A, FCS). Under the same conditions, LEX2, LEX2LIB2
and LEX2LIB3 cells contained levels of free cholesterol
comparable with those of wild-type cells (~25 to 30 µg/mg
protein; Fig. 5A, FCS). CHO cells can grow continuously in
the absence of exogenously added cholesterol, because the
feedback regulation of the endogenous cholesterol biosynthetic
pathway elevates cholesterol synthesis under cholesterol
B
Free cholesterol ( g/mg protein)
A
Free cholesterol ( g/mg protein)
Cholesterol role in CI-MPR exit from MVB
40
WT
LEX2
LEX2LIB2
LEX2LIB3
30
20
10
0
FCS
30
LPDS
MPR/lgp
20
10
0
0
10
20
Time (hr)
Fig. 5. Cellular cholesterol level correlates with CI-MPR
distribution. (A) Wild-type, LEX2, LEX2LIB2 and LEX2LIB3 cells
were incubated in FCS/F12 (FCS) or LPDS/F12 (LPDS) for 24 hours
at 37°C. Cellular free cholesterol was determined and expressed
normalized to protein. (B) The time course of cholesterol depletion
upon LPDS/F12 incubation in LEX2 cells at 37°C. Cellular free
cholesterol was determined and expressed normalized to protein. The
data are the means±s.e.m. (n=3).
starvation (Brown and Goldstein, 1999; Chang et al., 1997).
Indeed, when wild-type cells were cultured under cholesterol
starvation in LPDS/F12 for 24 hours, only a small reduction in
cellular free cholesterol (down to ~20 µg cholesterol/mg
protein) was observed (Fig. 5A, LPDS, WT). By contrast, upon
incubation in LPDS/F12, the cholesterol level in LEX2 cells
reduced to ~10-15 µg cholesterol/mg protein (Fig. 5A, LPDS,
LEX2). This cholesterol hyper-reduction in LEX2 cells was
relieved by the expression of NSDHL: after the LPDS/F12
incubation, LEX2LIB2 and LEX2LIB3 cells contained levels
of free cholesterol comparable with those observed in wildtype cells (Fig. 5A, LPDS, LEX2LIB2, LEX2LIB3). Thus,
NSDHL indeed restored free cholesterol in LEX2 cells to the
wild-type level, and cellular cholesterol levels are correlated
with the CI-MPR distribution in LEX2 cells.
The cholesterol reduction during LPDS/F12 incubation in
LEX2 cells was gradual, starting soon after the change of
medium from FCS/F12 to LPDS/F12, and reached a plateau at
~18 hours (Fig. 5B). The gradual reduction is consistent with the
idea that the gradual accumulation of CI-MPR in the MVBs (Fig.
2) results directly from cholesterol depletion in LEX2 cells.
These results corroborate the notion that cholesterol is required
for the exit of the receptor from the late endosomal MVBs.
Cholesterol induces MVB consumption along with
cargo sorting in LEX2 cells
The abnormal LEX2 MVBs were thought to result from the
inhibition of the consumption of ECV/MVBs, owing to defects
1771
in sorting and exit transport from ECV/MVBs to both the TGN
and lysosomes (Ohashi et al., 2000). To examine if the
cholesterol-induced CI-MPR transport to the Golgi in LEX2
cells is coupled with the consumption of MVBs, the frequency
of large MVBs (diameter >300 nm, filled with internal vesicles;
Fig. 6Aa) was analyzed by counting them in EM sections (Fig.
6Ab). As previously observed in LEX2 cells (Ohashi et al.,
2000), the number of large MVBs was significantly elevated in
LEX2VR cells (as the control) over wild-type cells upon 24
hour LPDS/F12 incubation (Fig. 6Ab, P<0.05 by Student’s ttest). However, LEX2VR cells cultured in FCS/F12 showed a
significantly lower number of large MVBs than in LPDS/F12
(Fig. 6Ab, P<0.05). Furthermore, upon LPDS/F12 incubation,
the number of large MVBs was significantly lower in
LEX2LIB2 and LEX2LIB3 cells, than in LEX2VR cells (Fig.
6Ab, P<0.05). Thus, the transport of CI-MPR was
accompanied by a reduced frequency of large MVBs.
We investigated the localization of CI-MPR and lgp-B
during the cholesterol-dependent MVB modulation by
immunoelectron microscopy. As was expected from the
fluorescence microscopic observations, after FCS/F12
incubation, CI-MPR was observed primarily on nonmultivesicular membranes at the peripheral Golgi (Fig. 6B).
Also as expected, after incubation in LPDS/F12 for 26 hours,
CI-MPR largely disappeared from the Golgi region and was
observed at the internal vesicles of the MVBs in LEX2 cells
(Fig. 6C). Large vacuoles observed in LEX2 cells in LPDS/F12
were much less positive for CI-MPR than the MVBs (not
shown; Ohashi et al., 2000). By contrast, within the LEX2
MVBs, lgp-B was localized both on the internal membrane
structures and on the limiting membranes (Fig. 6D). Large
vacuoles peripherally positive for lgp-B were also observed in
LEX2 cells (Fig. 6D). In LEX2LIB3 cells, where LEX2
cholesterol deficiency was rescued by NSDHL expression, lgpB was localized mainly on the membranes of single membranedelimited compartments of various sizes, including large
vacuoles, and to a lesser extent on electron-opaque material
within the lumen of these compartments (Fig. 6E). These
results suggest that cholesterol induced sorting of CI-MPR
from the internal membranes of MVBs into transport vesicles
to the Golgi, away from materials such as lgp-B destined for
later non-multivesicular endocytic compartments.
Within MVBs that remained in LEX2LIB3 cells, CI-MPR
and lgp-B were still observed at the internal vesicles (not
shown), suggesting that the disappearance of MVBs and
segregation of CI-MPR from lgp-B are coupled events. Taken
together, these results indicate that cholesterol induces MVB
reorganization coupled with cargo sorting in LEX2 cells.
Cholesterol deficiency associated with the CI-MPR
arrest is not the factor that commonly causes the
defective LDL degradation in group B mutants
To assess the role of cholesterol in processing from
ECV/MVBs to lysosomes, we examined if cholesterol or
NSDHL expression could correct the defective LDL
degradation in LEX2 cells. A fluorescent LDL (RET-LDL),
specifically designed for detection of intracellular LDL
degradation by flow cytometry (Ohashi et al., 1992), was used.
This method allows the detection of the early phase lysosomal
degradation of LDL that is essentially independent of the level
of LDL uptake (Ohashi et al., 1992). Cells were allowed to
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JOURNAL OF CELL SCIENCE 114 (9)
Fig. 6. Cholesterol induces MVB reorganization in LEX2 cells. (A) Introduction of NSDHL cDNA reduces the frequency of large MVBs in
LEX2 cells. Wild-type (WT), LEX2VR, LEX2LIB2 and LEX2LIB3 cells were cultured in FCS/F12 (FCS) or LPDS/F12 (LPDS) as indicated
for 26 hours at 37°C, and observed by conventional electron microscopy. (a) Large MVBs (>300 nm, filled with internal vesicles) were counted.
The mean number±s.e.m. (n=30) of large MVBs per cell section that show a cross-section of the nucleus is shown in b. (B,C) Immunoelectron
microscopic localization of CI-MPR in LEX2 cells after incubation for 26 hours in FCS/F12 (B) or in LPDS/F12 (C). Arrows indicate positive
staining. (D-F) Immunoelectron microscopic localization of lgp-B in LEX2 (D), LEX2LIB3 (E) and wild-type (F) cells after incubation for 26
hours in LPDS/F12. Arrows and arrowheads indicate staining on internal structures and peripheral membranes, respectively, of membrane
compartments. G, Golgi apparatus; n, nucleus; mb, multivesicular body; v, vacuole. Bar, 1 µm.
endocytose RET-LDL for 10 minutes, chased for 20 minutes,
and analyzed for RET-LDL degradation by analytical flow
cytometry (Fig. 7). Surprisingly, neither cholesterol (Fig. 7A)
nor stable NSDHL transfection (Fig. 7B, LEX2LIB2,
LEX2LIB3) was sufficient to correct defective RET-LDL
degradation in LEX2 cells. Because LEX2 cells that were
stably transfected with NSDHL contained cholesterol levels
comparable with those in wild-type cells (Fig. 5A), the
cholesterol restoration by NSDHL failed to recover the RETLDL degradation in LEX2 cells. Hence, cholesterol deficiency
was not the factor that had caused the defective LDL
degradation in LEX2 cells. Because LEX2LIB2 and
LEX2LIB3 cells are stably transfected with NSDHL, it is
unlikely that longer incubation with exogenous cholesterol will
recover RET-LDL degradation.
Further, the endosome/lysosome morphology of LEX2 cells
was not completely corrected by cholesterol restoration by
NSDHL transfection. Lysosomal glycoprotein exists both in a
membrane-associated form and in the lysosome internal matrix
possibly as aggregates of a soluble form (Bou-Gharios et al.,
1991; Jadot et al., 1996). Consistent with this, in wild-type cells,
lgp-B was localized both on delimiting membranes and in
internal electron-opaque materials of late endosomes/lysosomes
(Fig. 6F). However, these late endosomes/lysosomes in wildtype cells were only of small sizes, making a clear contrast with
the lgp-positive single membrane-delimited compartments such
as larger vacuoles observed in LEX2LIB3 cells (Fig. 6E).
To address this point further, we analyzed EX cells, another
member of our previously established mutants (Ohashi et al.,
2000). EX cells fall into the same complementation group as
LEX2 cells by RET-LDL degradation test (Ohashi et al., 2000).
However, EX cells showed considerably different phenotypes
from those of LEX2 cells: well-elaborated MVBs were not
prominent in EX cells (Ohashi et al., 2000). Furthermore, even
upon cultivation of EX cells in LPDS/F12, CI-MPR was mainly
localized in the Golgi, rather than in late endosomes (Fig. 8A).
Compartments positive for lgp-B in EX cells observed upon
LPDS/F12 incubation were smaller (dots) than in LEX2 cells,
Cholesterol role in CI-MPR exit from MVB
A
B
1.0
+ -/+
-
Cholesterol
+ -/+
0.2
0.0
2
X2
LI
B3
-
(3)
LI
B
0.0
(3)
LE
(6)
0.2
0.4
VR
0.4
0.6
LE
X2
0.6
0.8
T
(6)
X2
0.8
W
(3)
1.0
LEX2
LE
(3)
RET-LDL disintegration, r
RET-LDL disintegration, r
WT
1773
Fig. 7. Defective RET-LDL degradation in LEX2 cells was not
corrected by cholesterol or NSDHL. (A) Cholesterol does not
promote RET-LDL degradation in LEX2 cells. Wild-type (WT) and
LEX2 cells were incubated in LPDS/F12 without (−) or with (+)
cholesterol (20 µg/ml) as a suspension for 24 hours at 37°C, as
indicated. Some cells treated without cholesterol were further
incubated in LPDS/F12 containing cholesterol (20 µg/ml) as a
suspension for 10 hours at 37°C (−/+); these cells were made to
endocytose RET-LDL (10 µg/ml) in a pulse (10 minutes)-chase (20
minutes) protocol, and were analyzed for RET-LDL degradation by
analytical flow cytometry. The mean of calculated value r, indicating
RET-LDL degradation, is shown. Bars indicate s.e.m. The number of
experiments is indicated in parentheses. (B) Introduction of NSDHL
does not promote RET-LDL degradation in LEX2 cells. Wild-type
(WT), LEX2VR, LEX2LIB2 and LEX2LIB3 cells were cultured in
LPDS/F12 for 24 hours at 37°C, and processed for flow cytometric
analysis for RET-LDL degradation in a pulse (10 minutes)-chase (20
minutes) protocol. The mean of calculated value r, indicating RETLDL degradation, is shown. Bars indicate s.e.m. (n=4).
scattered over the cytoplasm, and largely CI-MPR-negative
(Fig. 8Aa,c). If the normal cholesterol levels are associated with
CI-MPR localization to the Golgi, the cholesterol level in EX
cells should be normal. Indeed, EX cells showed a level of free
cholesterol comparable with that in wild-type cells, irrespective
of incubation in FCS/F12 or LPDS/F12 (Fig. 8B), and they
grew normally in LPDS/F12 (not shown). Thus, whereas the
CI-MPR arrest can be attributed to cholesterol deficiency, the
defective RET-LDL degradation common to group B mutants
cannot. These results demonstrate that cholesterol deficiency is
not the factor that commonly causes the defective LDL
degradation in group B mutants.
DISCUSSION
The results presented here have clearly indicated that cholesterol
is required for the exit of CI-MPR from the ECV/MVBs. After
the incubation of LEX2 cells in cholesterol-free medium for ~1
day, the cellular free cholesterol level was reduced by ~50% (Fig.
5), and CI-MPR accumulated in ECV/MVBs. The time course
of the cholesterol depletion paralleled the arrest of CI-MPR
within ECV/MVBs (Fig. 2; Fig. 5). Supplementation of medium
with cholesterol allowed the transport of the arrested CI-MPR
from the ECV/MVBs to the Golgi in less than three hours (Fig.
3). In addition, the stable expression of NSDHL in LEX2 cells
Fig. 8. Cholesterol deficiency associated with the CI-MPR arrest is
not the factor that commonly caused the defective LDL degradation
in group B mutants. (A) CI-MPR is localized at the Golgi upon
LPDS/F12 incubation in EX cells. EX cells were cultured in
LPDS/F12 for 1 day at 37°C, and immunofluorescently doublestained for CI-MPR and lgp-B (a and c, respectively), or CI-MPR
and syntaxin 6 (b and d, respectively). The arrows in a and c indicate
perinuclear CI-MPR-positive, lgp-B-negative staining. The
arrowheads in a and c indicate lgp-B-positive, CI-MPR-negative,
punctate staining scattered in the cytoplasm. Arrows in b and d
indicate the perinuclear colocalization of CI-MPR and syntaxin 6.
Bar, 10 µm. Semiquantification of these colocalization data is
provided in e and f. The data are the means±s.e.m. (n=10). (B) EX
cells contained levels of free cholesterol comparable with those
observed in wild-type cells. EX cells were incubated in FCS/F12
(FCS) or LPDS/F12 (LPDS) for 24 hours at 37°C. Cellular free
cholesterol was determined and expressed normalized to protein. The
data are the means±s.e.m. (n=3). For comparison, the data from wildtype and LEX2 cells upon the same incubation are indicated as tick
marks on the ordinates.
restored free cholesterol to the wild-type level, and allowed the
exit of CI-MPR from ECV/MVBs. These results suggest that the
exit of CI-MPR from ECV/MVBs can be controlled in a sensitive
manner by free cholesterol levels ranging from 50-100% of the
wild-type level. A recent paper by Sandvig et al. (Grimmer et al.,
2000) described that the TGN-specific sulphation of a modified
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JOURNAL OF CELL SCIENCE 114 (9)
cation-dependent MPR (CD-MPR) was inhibited after removal
or addition of cholesterol in HeLa cells, suggesting that the
transport of CD-MPR to the Golgi is regulated by cholesterol.
Their biochemical finding nicely complements our present
conclusion that cholesterol is required for the exit transport of CIMPR, the other of the two known MPRs, from ECV/MVBs. It
has been known for some time that the CI-MPR distribution
between the TGN and late endosomes varies substantially among
different cell types. The sensitive regulation of CI-MPR traffic
would provide an explanation for the variation in the distribution
of this receptor in various cell types, which may reflect
differences in the regulation and requirements of free cholesterol
in specific cell types.
A cholesterol requirement has previously been implicated in
clathrin-mediated endocytosis (Rodal et al., 1999; Subtil et al.,
1999). However, LDL uptake in LEX2 cells is comparable with
that observed in wild-type cells (~80% of the wild-type value
during a 10-minute pulse) under the cholesterol depleting
conditions (Ohashi et al., 2000). Some explanations for the
insensitivity of the LDL uptake to cholesterol depletion in LEX2
cells would be possible. In previous studies (Rodal et al., 1999;
Subtil et al., 1999), MβCD was used to acutely extract plasma
membrane cholesterol in 15-30 minutes. The slow cholesterol
depletion in LEX2 cells in LPDS/F12 (Fig. 5B) perhaps does not
lead to the same biophysical changes in the plasma membrane
bilayer that was proposed to have caused the defective clathrinmediated endocytosis under the acute cholesterol extraction
(Subtil et al., 1999). Alternatively, LDL used for the uptake assay
might have provided a small amount of cholesterol required for
endocytosis via cholesterol transfer, for immediate use at the
plasma membrane (Fielding and Fielding, 1997).
Although some specific protein-protein interactions that
would operate on CI-MPR sorting/transport at late endosomes
have been well documented (Diaz and Pfeffer, 1998; Krise et
al., 2000; Orsel et al., 2000), it is still largely unknown how CIMPR is sorted from lysosome-destined molecules, packaged
and transported out of ECV/MVBs. It is generally thought that
MVB contents, including internal vesicles, are delivered to the
hydrolytic environment of late endosome/lysosomes and then
degraded, resulting in disappearance of the MVB morphology
(Futter et al., 1996; Odorizzi et al., 1998). However, the fact that
cholesterol reduced the number of MVBs without promoting
degradative endocytic processing in LEX2 cells suggests that
the consumption of MVBs, at least in part, can be attributed to
the sorting and/or transport of materials such as receptors and
cholesterol to the Golgi, rather than the degradation of MVB
contents. Thus, an interesting possibility is that MVB structure
reorganization is a requirement for CI-MPR sorting/transport
out of ECV/MVBs. Because CI-MPR was detected mostly in
the internal small vesicles within the arrested ECV/MVBs in
LEX2 cells, and lgp-B was found in both internal vesicles and
the limiting membranes, cholesterol would be indispensable for
such MVB reorganization that drives segregation between CIMPR and lysosome-destined molecules, both of which under
low cholesterol are observed at the internal small vesicles of
ECV/MVBs (Fig. 9). On a more general note, the coupling
between MVB structure reorganization and cargo sorting/
transport supports an emerging concept whereby late
endosomal MVBs function as a sorting and distributing device
(Kobayashi et al., 1999; Kobayashi et al., 1998).
One interpretation of the failure for cholesterol to recover the
ECV/MVB
TGN
cholesterol
vacuole
non-degradative
LDL
CI-MPR
lgp-B
group B
rab7+
more degradative
LEX1
lysosome
Fig. 9. A schematic model for cholesterol requirement at late
endosomes in CHO cells. Cholesterol is indispensable for
ECV/MVB reorganization, which operates to sort and transport CIMPR out of the endocytic pathway to the TGN. As a result of the
reorganization, the multivesicular morphology is diminished, leading
to vacuole generation. The vacuole is a non-degradative compartment
and is normally a transient structure that is rapidly processed into
later more degradative, rab7-positive endosomes (rab7+), which
interact with lysosomes. This model accommodates the phenotypes
of our CHO mutants in the endocytic pathway (Ohashi et al., 1999;
Ohashi et al., 2000) as follows. In LEX2 cells, the cholesterol
deficiency causes the retention of CI-MPR within ECV/MVBs. In
group B mutants (including LEX2), the process from the vacuole to
the more degradative endosomes is commonly defective as indicated
(group B). This is because a factor(s) (other than the normal
cholesterol level) required to drive this process are missing in group
B cells. Accordingly, in group B cells, both endocytosed materials
(including LDL) and the lysosomal proteins (including lgp-B) that
normally transit this vacuole before being targeted to lysosomes are
accumulated in the vacuoles. Because LEX2 belongs to group B but
is specifically defective in processing not only to lysosomes but also
to the TGN, CI-MPR, LDL and lgp-B are accumulated in
ECV/MVBs in LEX2 cells. Note that, owing to the defective
processing towards lysosomes, the multivesicular morphology of
ECV/MVBs is not diminished by the lysosomal degradation of the
internal membranes. As a result, well-elaborated ECV/MVBs that
contain CI-MPR, LDL and lgp-B are accumulated in LEX2 cells
(Ohashi et al., 2000) under low cholesterol conditions. By contrast,
another previously established mutant, LEX1, is defective in the
interaction between the rab7+ late endosomes and lysosomes (Ohashi
et al., 1999; Ohashi et al., 2000) as indicated (LEX1).
RET-LDL degradation in LEX2 cells is that cholesterol does not
correct the defective processing of endocytosed ligand further
into hydrolytic late endosome/lysosome stages (Fig. 9). It is
conceivable that the defective step shared by group B mutants is
this processing into hydrolytic stages (Fig. 9), because
insufficient cholesterol is not the factor that commonly caused
defective degradation of RET-LDL in group B mutants. In this
Cholesterol role in CI-MPR exit from MVB
context, vacuoles of various sizes observed in LEX2, LEX2LIB3
(Fig. 6D,E) and other group B mutants (Ohashi et al., 2000) may
represent an aberrant non-degradative intermediate connected
to more degradative late endosome/lysosomes (Fig. 9).
Identification of the common group B defective gene might
specify the requirement for processing from the ECV/MVBs
into more hydrolytic late endosomes, and might clarify how late
endocytic sorting and transport are regulated. In particular, it
might clarify whether the divergent pathways originating from
the MVBs, one circulating back to the Golgi and the other for
degradation, are subject to concerted regulation, whereas
cholesterol requirement at present appears to discriminate
between these two.
The interpretation for cholesterol requirement depicted in
Fig. 9 would also explain why, in wild-type cells, the increase
in the colocalization between CI-MPR and lgp-B upon
LPDS/F12 incubation appeared smaller compared with the
decrease in colocalization between CI-MPR and syntaxin 6
(Fig. 1Aj,l). Because processing towards lysosomes is normal
in wild-type cells, lgp-B is distributed predominantly in
hydrolytic late endosomes and lysosomes, rather than in
ECV/MVBs. If not transported out of the endocytic pathway
to the Golgi, owing to reduced cholesterol, CI-MPR would
proceed to these degradative late endosomes/lysosomes, where
eventually it would get degraded. The increased colocalization
of CI-MPR and lgp-B would then become marginal.
It has been suggested that an abnormally elevated level of
cholesterol in late endosomes leads to CI-MPR retention in the
late endosomes, because the accumulation of cholesterol in late
endosomes appeared to precede that of CI-MPR caused by drugs
mimicking the Niemann-Pick cells’ defects (Kobayashi et al.,
1999). In the present results, the administration of MβCDcholesterol at high concentrations that eventually caused
cytotoxicity still induced the redirection of CI-MPR from lgppositive compartments in LEX2 cells. This observation appears
consistent with the notion that cholesterol acts solely in a
positive manner on the machinery for CI-MPR exit transport,
rather than acting as an inhibitory agent. However, the possibility
cannot be ruled out that the specific arrest and the build-up of
cholesterol within late endosomes causes CI-MPR accumulation
because, in LEX2 cells, there is no reason for MβCD-cholesterol
to cause a cholesterol arrest specifically in endosomes whereas,
in NPC cells, cholesterol is stuck in late endosomes.
Nevertheless, a different kind of explanation would also be
possible for the late endosomal accumulation of CI-MPR in
NPC cells. The cholesterol requirement for CI-MPR exit from
the ECV/MVBs to the Golgi provides some implications for
the role of the product of a gene responsible for NPC disease,
NPC1. NPC1 is predicted to be a polytopic membranespanning protein with a putative sterol-sensing domain and a
terminal di-leucine motif that targets proteins to endocytic
compartments (Carstea et al., 1997). Although the role of the
NPC1 has yet to be established, the characteristics of NPC1
protein led to a suggestion that NPC1 functions as a sensor of
the endosomal cholesterol content (Kobayashi et al., 1999;
Neufeld et al., 1999). Cholesterol transport out of the endocytic
pathway has been suggested to be vesicular transport (Neufeld
et al., 1999; Underwood et al., 1998), and an NPC1 role was
implicated in bulk endocytic traffic (Neufeld et al., 1999).
Thus, it is possible that the cholesterol requirement for the exit
of CI-MPR is also mediated by interaction between cholesterol
1775
and late-endosomal NPC1, which would turn on the sorting and
vesicular exporting machinery for cholesterol as well as for CIMPR. If so, the malfunction of NPC1 would make the
cholesterol-NPC1 switching system turn off, and would
thereby cause the accumulation of both cholesterol and CIMPR within the MVBs, as observed in NPC cells (Kobayashi
et al., 1999). Such cooperative roles for cholesterol and NPC1
in sorting events and exit transport from the MVBs might be
comparable with the proposed roles for cholesterol and
synaptophysin in membrane curvature reorganization in
synaptic vesicle biogenesis (Thiele et al., 2000).
Finally, our results indicate that NSDHL activity can
potentially regulate CI-MPR transport from late endocytic
MVBs to the Golgi in a sensitive manner. The defects of this
enzyme cause human embryonic developmental disorder,
CHILD syndrome (Konig et al., 2000), and two mouse
embryonic disorders, bare patches (Bpa) and striated (Str) (Liu
et al., 1999). Interestingly, the functional importance of CIMPR in embryonic development has been demonstrated in
mutant mice (Lau et al., 1994; Ludwig et al., 1996; Wang et
al., 1994). In addition, the functions of other developmentally
important receptors, such as the sonic hedgehog receptor,
Patched and ErbB tyrosine kinases, have recently been
proposed to be associated with late endosomal membrane
trafficking (Incardona et al., 2000; Lenferink et al., 1998). Ptc1 (a member of Patched) and NPC1, both with a putative sterol
sensing domain, were found to colocalize in intracellular
vesicles, presumably of late endocytic nature (Incardona et al.,
2000). Thus, it is tempting to propose the existence of an
integration mechanism of developmental signaling mediated
by cholesterol and growth factor receptors, at the level of
endosomal MVBs. Our mutants in the endocytic pathway,
including LEX2, might prove to be useful tools for addressing
this interesting possibility of late endosomal signal integration.
We thank Keiji Imoto, Akihiko Nakano and Mikio Furuse for
helpful advice, Bruce Granger and Takashi Ueno for antibodies,
Tamotsu Yoshimori for continuous support, and Hiroshi Okawara and
Masahiro Takagi for maintaining the cell-culture facilities. This work
was supported in part by grants-in-aid from the Ministry of Education,
Science, Sports and Culture of Japan.
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