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INTRODUCTION |
Plasma levels of high density lipoprotein
(HDL)1 cholesterol are
negatively correlated with the risk of developing atherosclerosis, the
leading cause of death in western, industrialized countries (1, 2). The
role of HDL in cholesterol metabolism includes the delivery of
cholesterol esters to steroidogenic tissues (3, 4) and the transfer of
cholesterol from peripheral tissues to the liver in a process termed
reverse cholesterol transport (5, 6). Reverse cholesterol transport
requires the extraction of cholesterol from extrahepatic cells by HDL
and the subsequent delivery of cholesterol esters to hepatocytes. The
mechanism for the delivery of cholesterol esters from HDL to cells is
described as selective uptake, because the uptake of cholesterol ester
is independent of HDL internalization (7, 8). Selective uptake of
cholesterol ester from HDL is characterized by the initial movement of
cholesterol ester into a reversible, plasma membrane pool and the
subsequent internalization to a nonreversible, intracellular pool (9,
10).
The mechanisms of cholesterol and cholesterol ester exchange between
the cell surface and HDL are not well understood. Receptor-independent and receptor-dependent hypotheses have been proposed to
explain the transfer of cholesterol and cholesterol ester between the cell surface and HDL (6). In the receptor-independent model, diffusion
is thought to account for both the uptake of cholesterol esters and the
efflux of free cholesterol. In contrast, HDL-binding proteins, such as
class B, type I scavenger receptor (SR-BI) and class B, type II
scavenger receptor, can mediate the selective uptake of cholesterol
esters from HDL (11-13).
SR-BI, appears to be a physiological HDL receptor. Several studies
support this assertion. First, SR-BI binds HDL and mediates the
selective uptake of cholesterol esters (11, 13, 14). Second, SR-BI
mediates the efflux of cholesterol from cells to HDL (15, 16). Third,
the expression of SR-BI is greatest in tissues that selectively take up
cholesterol esters from HDL, including liver, adrenal, testis, and
ovary (11, 14). In mice, overexpression of SR-BI in hepatic tissue
causes a dramatic decline in plasma HDL and an increase in biliary
cholesterol (17). Finally, disruption of the SR-BI gene leads to an
increase in plasma cholesterol concentrations in mice (18).
Collectively, these data indicate that SR-BI is an HDL receptor
involved in cholesterol homeostasis.
SR-BI has been localized to caveolae (13, 19), which are cholesterol-
and sphingomyelin-rich microdomains in the plasma membrane (20-22).
Caveolae appear to be directly involved in cellular cholesterol
homeostasis. Newly synthesized cholesterol translocates from the
endoplasmic reticulum (ER) to caveolae before diffusing into the bulk
plasma membrane (23). Caveolae mediate the efflux of free cholesterol
derived either from de novo synthesis or low density
lipoproteins (24). In addition, caveola morphology is dependent on the
presence of free cholesterol. Depletion of membrane cholesterol causes
invaginated caveolae to flatten within the plane of the membrane (25,
26). Caveolin is a cholesterol-binding protein associated with the
caveola coat (21, 27). The subcellular distribution of caveolin is, in
part, controlled by caveola cholesterol. Oxidation of caveola
cholesterol by cholesterol oxidase causes caveolin to translocate to
the ER (22). Thus, both caveola proteins and morphology are dependent
on cholesterol.
SR-BI mediates the selective uptake of cholesterol esters from HDL and
is localized to caveolae (13, 19). Therefore, we hypothesized that
caveolae are acceptors for HDL-derived cholesterol ethers. We further
speculated that caveolae constitute a reversible pool of cholesterol
ethers within the plasma membrane. The present study demonstrates that
caveolae are acceptors for HDL-derived cholesterol ethers, and that
caveolae constitute a reversible plasma membrane pool of cholesterol ethers.
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EXPERIMENTAL PROCEDURES |
Materials--
Ham's F-12 medium, Geneticin (G418 sulfate),
fetal calf serum, L-glutamine, trypsin-EDTA,
penicillin-streptomycin, and OptiPrep were purchased from Life
Technologies, Inc.. Percoll, polyvinylidene difluoride membrane and
Tween 20 were purchased from Sigma. Bradford reagent was purchased from
Bio-Rad. Rabbit IgG directed against caveolin-1 was obtained from
Transduction Laboratories (Lexington, KY). Mouse IgG directed against
the human transferrin receptor was supplied by Zymed
Laboratories Inc. (San Francisco, CA). Horseradish peroxidase-conjugated IgGs were supplied by Cappel (West Chester, PA).
Super Signal chemiluminescent substrate was purchased from Pierce.
1
,2
-(n)-[3H]Cholesteryl-oleoyl ether (47 Ci/mmol) was supplied by Amersham Pharmacia Biotech.
125I-Na (1 mCi/ml) was purchased from DuPont NEN.
Buffers--
Sample buffer (5 ×) consisted of 0.31 M Tris, pH 6.8, 2.5% (w/v) SDS, 50% (v/v) glycerol, and
0.125% (w/v) bromphenol blue. Tris-buffered saline consisted of 20 mM Tris, pH 7.6, and 137 mM NaCl. Blotting
buffer consisted of Tris-buffered saline plus 0.5% Tween 20 and 5%
dry milk. Wash buffer consisted of Tris-buffered saline plus 0.5%
Tween 20 and 0.2% dry milk. Tris-saline consisted of 50 mM
Tris, pH 7.4, and 150 mM NaCl.
Cell Culture--
ldlA-7 (a low density lipoprotein
receptor-negative Chinese hamster ovary (CHO) cell line) cells were
generously provided by Dr. Monty Kreiger (Massachusetts Institute of
Technology, Cambridge, MA). CHO lines were cultured in Ham's F-12
medium containing 5% fetal bovine serum, 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin. The human SR-BI (hSR-BI) cDNA was cloned into the
pCMV5 expression vector (28) and transfected into ldlA-7 cells with the
MBS transfection kit (Stratagene, La Jolla, CA). Lines expressing
hSR-BI were selected and maintained in medium containing 0.5 mg/ml G418
sulfate. For the analysis of cell associated cholesterol ether (CE),
cells were seeded (2.5 × 105 cells/well) in six-well
plates on day 0. Cells were fed on day 1, and uptake into reversible
and nonreversible pools was determined at confluence on day 2. For cell
fractionation studies, cells were seeded (5 × 105
cells/plate) in 10-cm dishes on day 0. Cells were confluent on days
4-5.
Lipoprotein Isolation and Radiolabeling--
HDL (d
1.063-1.21 g/ml) was isolated from fresh human plasma by density
gradient ultracentrifugation as described previously (29). The
HDL3 subfraction (d 1.13-1.18 g/ml) was
isolated from other HDL subfractions using a density gradient
fractionator (ISCO). [1
,2
-3H]Cholesteryl-oleoyl
ether was incorporated into HDL3 as described previously
(30). The specific activity of [3H]CE-HDL ranged from 32 to 35 dpm/ng of cholesterol. HDL3 apolipoproteins were
iodinated by the iodine monochloride method (31) to a specific activity
of 400-600 cpm/ng of protein.
Uptake and Ligand Binding Assays--
The selective uptake of
cholesterol ether from HDL into cells was determined using
nonhydrolyzable [3H]CE (9). Confluent ldlA-7 and hSR-BI
(ldlA-7 cells expressing hSR-BI) cells were rinsed twice with PBS
(37 °C). Ham's F-12 medium containing 5% human
lipoprotein-deficient serum and 10 µg/ml [3H]CE-HDL was
added to the cells for the indicated times. After incubation, uptake
was terminated by aspirating the medium and washing the cell monolayers
four times with Tris-saline (4 °C). The cells were dissolved in 1 M NaOH, and the amount of radiation was determined by
scintillation counting. To determine the quantity of CE within the
reversible plasma membrane pool, medium (efflux medium) containing 100 µg/ml unlabeled HDL was added for 120 min at 37 °C (9). The efflux
medium was collected, and [3H]CE was quantified by liquid
scintillation counting. Radiolabel present in the efflux medium was
designated the reversible pool (9). Cells were then dissolved in 1 N NaOH. Total cellular protein was determined by Bradford
assay, and the radiolabel remaining was designated the nonreversible
pool (9). 125I-HDL binding experiments were conducted in
the same manner as the uptake assays.
To compare cell-associated 125I-HDL with
[3H]CE-HDL, [3H]CE uptake was expressed as
apparent HDL protein uptake, assuming that [3H]CE uptake
resulted from whole HDL particle uptake as described by Knetch and
Pittman (9).
SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
Cellular fractions were dissolved in 0.015% (w/v)
deoxycholate, concentrated by precipitation with 7% (w/v)
trichloroacetic acid, and washed in acetone (32). Pellets were
suspended in 1 × sample buffer plus 1.2% (v/v)
-mercaptoethanol and heated to 95 °C for 5 min immediately before
loading. Proteins were separated on a 12.5% polyacrylamide gel at 50 mA (constant current) and subsequently transferred to a polyvinylidene
difluoride membrane at 50 V (constant voltage) for 2 h. Membranes
were blocked with blotting buffer for 60 min at 22 °C. Primary
antibodies were diluted in blotting buffer and incubated with blocked
membranes for 60 min at 22 °C. Membranes were washed four times for
10 min in wash buffer. Horseradish peroxidase-conjugated IgGs directed
against the appropriate host IgG were diluted and incubated with
membranes as described for primary antibodies. Membranes were washed
four times for 10 min in wash buffer and visualized using chemiluminescence.
Isolation of Caveolae--
Caveolae were isolated as described
previously (32, 33). This method generates a highly purified fraction
of caveolae that is enriched in caveolin and free of bulk plasma
membrane markers such as transferrin receptor and integrins (32).
Twenty to 30 µg of purified caveolae are generally isolated from CHO cells with this method. The yield of caveolae was estimated by immunoblot analysis. Two, 5 and 10% of the caveola and plasma membrane
fractions were compared. The signal intensities varied linearly with
the percent of each fraction loaded. The estimated yield of plasma
membrane caveolae in the present study was 53% ± 8%.
Statistical Analysis--
Least squares analysis of variance was
used to evaluate the data with respect to cell fraction, time, and
their interaction using the analysis of variance procedure of STASTICA
(Statsoft, Tulsa, OK). When appropriate, fractions were compared within
a given time using the Tukey's honestly significant difference test. Means were considered different at p < 0.01.
 |
RESULTS |
SR-BI-dependent CE Uptake into Reversible and
Nonreversible Pools--
Selective uptake of HDL cholesterol ethers
involves the initial movement of CE into a reversible plasma membrane
pool and the subsequent movement into an internal, nonreversible pool
(9). We determined whether SR-BI mediates the selective uptake of CE into a reversible pool. SR-BI-negative CHO cells (ldlA-7) and ldlA-7
cells expressing hSR-BI were incubated in the presence of
[3H]CE-HDL (10 µg/ml) for various times at 37 °C.
The cells were washed to remove unbound [3H]CE-HDL and
then incubated with excess (100 µg/ml) unlabeled HDL (efflux medium)
for 120 min at 37 °C. The [3H]CE in the efflux medium
was designated the reversible pool (9). The remaining, cell-associated
CE was designated the nonreversible pool. Fig.
1A indicates that after 7.5 min the reversible CE pool was 5.0-fold greater in hSR-BI cells than in
ldlA-7 cells (p < 0.01;
versus
).
Similarly, the nonreversible CE pool was 8.4-fold greater in hSR-BI
cells than in ldlA-7 cells (p < 0.01; Fig.
1A,
versus
). The nonreversible CE pool
increased linearly (p < 0.01) in hSR-BI cells. In
contrast, the reversible CE pool did not increase (104 ± 11 ng of
CE/mg of cell protein) between 30 and 60 min.

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Fig. 1.
SR-BI mediates selective uptake of
[3H]cholesterol ether into reversible and nonreversible
pools. A, low density lipoprotein receptor-negative CHO
(ldlA-7, and ) cells and ldlA-7 cells expressing hSR-BI ( and
) were incubated with [3H]CE-HDL (10 µg/ml,
37 °C) to determine whether selective uptake of CE into CHO cells
included uptake into reversible ( and ) and nonreversible ( and ) pools. After incubation with labeled HDL, cells were washed
and incubated with unlabeled HDL (100 µg/ml) for 120 min. The medium
was collected, and the [3H]CE present was designated the
reversible pool. The labeled CE that remained cell associated was
designated the nonreversible pool. B, the amount of
[3H]CE in the reversible ( ) and nonreversible ( )
pools was examined for up to 240 min. Values obtained from control
ldlA-7 cells were subtracted from hSR-BI cells to determine
SR-BI-specific uptake. Representative data are from one of two
independent experiments. Values are mean ± S.D.;
n = 3. Rev., reversible;
Non-Rev., nonreversible.
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To ensure that the reversible CE pool reached equilibrium,
SR-BI-dependent uptake was examined for up to 4 h.
SR-BI-specific uptake was calculated by subtracting the values obtained
for ldlA-7 cells from those obtained for hSR-BI cells (11). Fig.
1B shows that SR-BI-dependent uptake of CE into
the nonreversible (
) pool increased linearly (p < 0.01) and did not saturate under these conditions. In contrast, the
reversible (Fig. 1B,
) CE pool reached equilibrium by 60 min and remained constant for up to 4 h.
We next determined whether the reversible CE pool was integral to the
membrane or caused by displacement of bound [3H]CE-HDL
with unlabeled HDL. Cells expressing hSR-BI were incubated with
125I-labeled HDL and then processed as described for Fig.
1. 125I-Labeled HDL allows the tracking of the lipoprotein,
whereas [3H]CE tracks cholesterol ethers (11). The
selective uptake of [3H]CE was expressed as apparent
particle uptake (see "Experimental Procedures") into the reversible
and nonreversible CE pools (9). As shown in Fig.
2, after 7.5 min the uptake of
[3H]CE into the nonreversible (
and
) CE pool was
4-fold greater than that observed for 125I-labeled HDL
(54 ± 12.3 versus 12 ± 1.3 ng/mg of cell
protein). Likewise, the [3H]CE present in the reversible
(Fig. 2,
and
) CE pool was 20-fold greater than the HDL protein
(68.1 ± 8.2 versus 3.6 ± 0.8 ng/mg of cell
protein). These data demonstrate that SR-BI mediates the selective
uptake of HDL CE into both reversible and nonreversible pools.

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Fig. 2.
SR-BI mediated selective uptake of
[3H]cholesterol ether into a reversible pool is not
attributable to exchange of bound HDL. hSR-BI cells were incubated
with [3H]CE-HDL (10 µg/ml, and ) or
125I-HDL (10 µg/ml, and ) for up to 60 min
(37 °C) and chased with unlabeled HDL (100 µg/ml). The amount of
each label present in the reversible ( and ) and nonreversible
( and ) pools was quantified. [3H]CE present in the
reversible and nonreversible pools is expressed as apparent particle
uptake (HDL protein). Values are mean ± S.D.; n = 3.
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Caveolae Are HDL Cholesterol Ether Acceptors--
Confocal
microscopy experiments have demonstrated that the majority of SR-BI
expressed in CHO cells co-localizes with caveolin, a marker protein for
caveolae (19). We therefore hypothesized that caveolae are acceptors
for HDL-derived cholesterol ethers. To test this hypothesis, hSR-BI
cells were incubated with [3H]CE-HDL for various times
and then fractionated into cytosol, total plasma membranes (which
includes caveola membranes), and intracellular membranes
(e.g. ER and Golgi). Isolation of caveola membrane (CM) from
the plasma membrane (PM) showed that >80% of the [3H]CE
within the plasma membrane was associated with caveolae at 7.5 min
(Fig. 3A), indicating that
initially, <20% of selective uptake occurred outside of caveolae.
Fig. 3B shows that at 7.5 min the specific activity of
[3H]CE (ng of [3H]CE/mg of total protein in
the subcellular fraction) was 12-fold greater in the caveolae fraction
than in the plasma membrane fraction (1733 ± 79 versus
142 ± 49 ng/mg of protein). Similar to the reversible pool of CE,
the amount of [3H]CE associated with caveolae saturated
by 30 min and remained constant for 4 h (data not shown). However,
the amount of [3H]CE associated with the plasma membrane
and intracellular membranes increased throughout the incubation period
(data not shown).

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Fig. 3.
Selective uptake of labeled
[3H]cholesterol ether into the plasma membrane occurs via
caveolae. hSR-BI cells were incubated with
[3H]CE-HDL (10 µg/ml, 37 °C), washed, and processed
to isolate caveolae. A, [3H]CE present in the
total plasma membrane ( ) and in caveolae ( ). B,
Concentration (ng of [3H]CE/mg of protein) of
[3H]CE in caveolae ( ) and total plasma membrane ( )
fractions. Representative data are from four independent experiments.
Values are mean ± S.D.; n = 2.
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Caveola membranes were analyzed by immunoblotting (Fig.
4A). The caveola marker
protein caveolin was present in the total PM fraction and was highly
enriched in the CM of the plasma membrane. SR-BI was also highly
enriched in the caveola fraction. The noncaveola proteins transferrin
receptor and clathrin were detected in the plasma membrane fraction but
not in the caveola fraction. The relative distribution of total protein
and SR-BI are shown in Fig. 4B. Although the caveola
fraction contained only 1.6% of the starting postnuclear supernatant
protein, it contained 60 ± 4% of the total plasma membrane SR-BI
as determined by immunoblot analysis of hSR-BI in the plasma membrane
and caveola membrane fractions (see "Experimental Procedures").

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Fig. 4.
Caveolin and SR-BI are highly enriched in the
caveola fraction. A, Representative immunoblots of
noncaveola proteins transferrin receptor and clathrin and caveola
proteins caveolin and SR-BI. 10 µg of protein were loaded in each
lane. B, mass of total proteins and relative amount of SR-BI
in each of the subcellular fractions. Twenty percent of each
subcellular fraction was resolved by SDS-polyacrylamide gel
electrophoresis before transfer to nylon and immunoblotting for SR-BI.
PNS, postnuclear supernatant; CYTO, cytosol;
IM, intracellular membranes. Values are mean ± S.D.;
n = 6.
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Caveola-associated CE Constitutes a Reversible CE
Pool--
SR-BI-dependent selective uptake of HDL
cholesterol ether involves uptake into a reversible plasma membrane
pool (Fig. 1). If the reversible pool corresponds to the caveola
compartment, then incubation with excess unlabeled HDL should result in
an efflux of caveola-associated CE to HDL. To test this, hSR-BI cells were incubated with [3H]CE-HDL (10 µg/ml, 37 °C, 60 min). The cells were then washed and incubated with unlabeled HDL (100 µg/ml, 37 °C) for 0, 60, or 120 min. Plasma membranes were
isolated, and caveolae were prepared as described. As shown in Fig.
5A, the [3H]CE
associated with caveolae declined during the efflux period, whereas the
[3H]CE in the plasma membrane remained constant,
indicating that caveola [3H]CE constituted the
reversible pool of CE.

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Fig. 5.
The caveola pool of
[3H]cholesterol ether is reversible. A, hSR-BI
cells were incubated with [3H]CE-HDL (10 µg/ml,
37 °C) for 60 min. Cells were washed and incubated with unlabeled
HDL (100 µg/ml, 37 °C) for 0, 60, or 120 min. Caveolae were
prepared as described. The amount of [3H]CE present in
each fraction was quantified by scintillation counting. B,
isolated total plasma membranes from hSR-BI cells were incubated with
[3H]CE-HDL for 60 min (37 °C). Labeled membranes were
incubated in the presence of unlabeled HDL for 0, 60, or 120 min.
(37 °C). Caveolae were prepared from plasma membranes as described.
The amount of [3H]CE present in each fraction was
quantified by scintillation counting. , total plasma membranes; ,
caveola membranes. Representative data are from two independent
experiments. Values are mean ± S.D.; n = 2.
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Knetch and Pittman (9) demonstrated that isolated plasma membranes are
capable of selective uptake of HDL CE. They also established that the
reversible pool of HDL-derived cholesterol ethers can be effluxed from
isolated plasma membranes (9). To determined whether efflux of caveola
HDL CE occurred from isolated plasma membranes, the membranes were
labeled in vitro with [3H]CE-HDL for 60 min at
37 °C. Efflux was then determined after 0, 60, or 120 min of
incubation with excess unlabeled HDL (100 µg/ml). Caveolae were
isolated as in previous experiments. Interestingly, the incorporation
of [3H]CE into isolated plasma membranes appears to be
greater in vitro than in intact cells (Fig. 5, A
versus B). Consistent with efflux from whole cells,
[3H]CE declined in the caveola fraction during incubation
with unlabeled HDL (Fig. 5B). In contrast,
[3H]CE associated with the plasma membrane fraction did
not change during the efflux period.
The Reversible Caveola Pool of CE Is Internalized to a
Nonreversible Pool--
We next determined whether the
SR-BI-dependent reversible pool proceeded to a
nonreversible pool. Cells were incubated for 30 min with
[3H]CE-HDL and then washed and incubated in medium only
(no HDL) for up to 3 h. Efflux medium (100 µg/ml HDL) was then
added for 120 min, and the reversible and nonreversible pools were
determined. Fig. 6A shows that
the nonreversible pool tended to increase with time, whereas the
reversible pool decreased with time (p < 0.01, linear). The [3H]CE associated with the
SR-BI-dependent reversible pool moved to a nonreversible
pool.

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Fig. 6.
Movement of HDL-derived
[3H]cholesterol ether from the reversible pool to the
nonreversible pool in the absence of HDL. hSR-BI cells were
incubated with [3H]CE-HDL (10 µg/ml) for 30 min. Cells
were washed and incubated in the absence of HDL for up to 3 h.
A, after the incubation, the [3H]CE in the
reversible pool ( ) was quantified by incubating cells in the
presence of unlabeled HDL (100 µg/ml) for 120 min and counting
radiolabeled CE in the medium. The labeled CE that remained cell
associated was designated the nonreversible pool ( ). B,
after the incubation, caveolae were prepared as described. The amount
of [3H]CE present in each fraction was quantified by
scintillation counting. , total plasma membranes; , caveola
membranes; , intracellular membranes. Representative data are from
two independent experiments. Values are mean ± S.D.;
n = 2.
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If caveolae are the SR-BI-dependent reversible pool, then
[3H]CE associated with caveolae should decline during a
chase period. To test this, hSR-BI cells were incubated with
[3H]CE-HDL for 30 min and then washed and incubated in
the absence of any HDL for up to 3 h. The cells were then
subfractionated, and the amount of [3H]CE in each
fraction was quantified. The amount of [3H]CE declined in
the CM fraction and the total PM fraction but increased in the
intracellular membrane fraction (Fig. 6B).
 |
DISCUSSION |
The present study demonstrates that cholesterol ether from HDL is
initially transferred to caveolae and that the transfer of HDL
cholesterol ether to caveolae requires the expression of SR-BI. We
previously demonstrated that the majority of murine SR-BI co-localizes
with caveolin, a caveola marker protein (13, 19). In the present study,
~60% of total plasma membrane SR-BI co-purifies with caveolin. The
yield of SR-BI in the caveola fraction is similar to the yield of
caveolae based on immunoblots of caveolin (60%; Fig. 4;
versus 53%; "Experimental Procedures"). These results suggest that the incomplete recovery of SR-BI is attributable to the
incomplete recovery of caveolae, because caveolin present in the plasma
membrane is predominantly associated with caveola membranes, as shown
by Anderson and colleagues by immunoelectron microscopy (21). However,
some fraction of SR-BI (potentially up to 40%) may not be associated
with caveolae. Importantly, >80% of the [3H]CE is
associated with caveolae by 7.5 min of uptake. This strongly suggests
that caveolae are involved in the initial uptake of CE. The absolute
dependence of selective CE uptake on caveolae will require additional studies.
We also demonstrated in the present study that cholesterol ether
associated with caveolae constituted a reversible pool from which the
sterol could efflux back to HDL. Furthermore, caveola-associated cholesterol ether was chased into an internal, nonreversible pool. These findings are consistent with earlier work by Pittman and Knetch
(9), who used Hep G2 and adrenal cells to demonstrate that HDL
cholesterol ether was taken up into a small, reversible plasma membrane
pool before progressing to an internal, nonreversible pool. Both
hepatocytes and adrenal cells abundantly express SR-BI, and it is
therefore likely that the selective uptake observed in these earlier
studies was mediated, at least in part, by SR-BI (11, 34).
We used subcellular fractionation and pulse-chase methods to
demonstrate that cholesterol ether moved to an intracellular membrane
compartment. We have not identified the intracellular membrane
compartment. Once the cholesterol ether reached the intracellular membrane compartment, it could not be rapidly effluxed from these cells. These data show that cholesterol ether can be selectively internalized to an intracellular membrane compartment. Furthermore, these data suggest that internalization of caveola cholesterol ester
does not require its hydrolysis to unesterified, free cholesterol.
How does cholesterol ether traffick between caveolae and an
intracellular membrane compartment? Recent research on caveola function
offers two possible mechanisms. First, Schnitzer et al. (35)
has shown that endothelial cell caveolae contain vesicle-trafficking proteins such as SNAPs and SNAREs. Furthermore, this group has shown
that endothelial caveolae can invaginate and detach from the plasma
membrane proper. The detached caveolae are capable of translocating
through the cytosol and fusing with other membranes. Cholesterol ethers
could therefore be internalized and transported to intracellular
membranes in this manner. However, the ability of caveolae to
vesiculate is contentious and may not occur in other types of cells
(26, 36, 37). This mechanism would also internalize entire HDL
particles and therefore not be selective uptake. A second possible
mechanism for sterol trafficking was described by Uittenbogaard
et al. (33). We demonstrated that a protein chaperone
complex consisting of caveolin, HSP56, cyclophilin 40, and
cyclophilin-A transported newly synthesized cholesterol directly from
the ER to caveolae. This type of mechanism might also permit the uptake
of cholesterol ethers from caveolae without the internalization of
SR-BI and HDL. However, the caveolin-chaperone complex has not yet been
demonstrated to traffick from caveolae to the ER or to transport
cholesterol ethers.
We have shown that both SR-BI and an isoform, class B, type II
scavenger receptor, are localized to caveolae (38). In addition, another class B scavenger receptor, CD36, has also been localized to
caveolae (39, 40). Both SR-BI (19) and CD36 (41) are palmitoylated.
Protein acylation is one mechanism whereby proteins can be targeted to
caveolae. Shenoy-Scaria et al. (42), for example,
demonstrated that dual acylation targeted nonreceptor tyrosine kinases
to caveolae. Shaul et al. (43) demonstrated that endothelial
nitric oxide synthase required acylation for caveolae association. The
role of acylation in the targeting of SR-BI or CD36 to caveolae has not
been tested. Recently, Reaven et al. (44) published electron
micrographs showing the localization of SR-BI to microvillar channels
in luteal cells. The relationship between microvillar channels and
caveolae is unclear. Parton et al. (45, 46) demonstrated
that caveolae can form deep invaginations into the cytosol in skeleton
muscle myocytes. Microvillar channels may be caveolae or the functional
equivalent in luteal cells. Alternatively, it is possible that SR-BI is
not associated with caveolae in all types of cells.
SR-BI plays an important role in regulating cholesterol flow between
lipoproteins and cell membranes. We suggest the following pathway for
the selective uptake of HDL cholesterol ester: 1) HDL binds to SR-BI in
caveolae; 2) CE is transferred to caveolae; and 3) caveola-associated
CE translocates to an intracellular membrane compartment. However, our
data do not rule out the possibility that caveola-associated CE moves
into the bulk (noncaveola) plasma membrane before transport to an
intracellular compartment. Additional work is required to elucidate
these steps in CE transport and their potential regulatory mechanisms.