© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Regulation of Acyl-Coenzyme A:Cholesterol Acyltransferase (ACAT) Synthesis, Degradation, and Translocation by High-Density Lipoprotein2 at a Low Concentration
From the Department of Medicine, Baylor College of Medicine, and The Methodist Hospital, Houston, Tex.
Correspondence to Henry J. Pownall, PhD, Department of Medicine, Baylor College of Medicine, MS A-601, 6565 Fannin St, Houston, TX 77030. E-mail hpownall{at}bcm.tmc.edu
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Abstract—Although plasma HDL2 cholesterol concentration stands in inverse relation to risk for atherosclerotic disease, little is known about the mechanism of the apparent cardioprotection. In mouse P388D1 macrophages, HDL2 at a low concentration (
40 µg/mL)
inhibits macrophage acyl-coenzyme A:cholesterol
acyltransferase (ACAT), the enzyme that catalyzes esterification of
intracellular cholesterol. The effects of
HDL2 on ACAT synthesis, degradation, and
intracellular translocation were investigated in mouse P388D1
macrophages. HDL2 at a low concentration
enhanced ACAT synthesis but not total ACAT mass. Immunocytochemical
studies showed that in the absence of lipoproteins, ACAT associated
primarily with the perinuclear region of the cell. The addition of
HDL2, however, induced the transfer of ACAT to
vesicular structures and the cell periphery adjacent to the plasma
membrane. Subfractionation combined with immunoprecipitation
complemented these observations and showed that
HDL2 promoted the transfer of ACAT to the plasma
membrane fraction. Brefeldin A, which inhibits vesicular protein
transport from the endoplasmic reticulum to the Golgi compartment in
mammalian cells, blocked ACAT translocation and partially restored ACAT
activity. These results suggest that HDL2 is an
initiating factor in a signal transduction pathway that leads to
intracellular ACAT translocation and inactivation.
Key Words: macrophage • lipid metabolism • cholesteryl ester • vesicular transport • ACAT
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Introduction |
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Plasma HDL cholesterol concentration has a strong inverse relation to coronary artery disease incidence.1 2 HDL is an acceptor of macrophage cholesterol,3A and our recent studies of macrophages suggest that low-concentration HDL2 (40 µg/mL) inhibits acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity.3B Recruitment into the artery wall of blood-borne monocytes, which can differentiate into macrophage foam cells, is an early step in atherogenesis.4 5 6 7 8 Lipoprotein-derived free cholesterol (FC) can transfer to the plasma membrane9 and then to the rough endoplasmic reticulum (ER10 ), where it is converted to CE by ACAT.11 12
Human ACAT, which was first cloned by Chang et al,13 is located in the ER.14 15 In ACAT knockout mice, there was no detectable CE in the adrenal gland and no ACAT activity within peritoneal macrophages.16 In C57BL/6J mice fed an atherogenic diet15 and in cholesterol-loaded macrophages in vitro,17 ACAT activity was upregulated. ACAT mRNA levels did not appear to be altered under these conditions, however. Thus, ACAT activity appears to be regulated posttranscriptionally. ACAT contains 5 leucine heptad domains that mediate multimer formation and 2 myristoylation sites that could control attachment to membranes.13
ACAT inhibition by HDL2 at a low concentration could be due to a direct effect on the enzyme, a change in substrate availability, or an altered distribution within the cell. In the present study, we determined whether ACAT synthesis, degradation, and intracellular localization are controlled by HDL2 at a low concentration.
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Methods |
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Materials
RPMI-1640, gentamicin, and glutamine were from Gibco Laboratories. Defined FBS and other chemicals were purchased from Sigma Chemical Co unless otherwise indicated. [3H]Acetate and [14C]oleate were from Amersham Pharmacia Biotech, Inc, and [35S]methionine was from ICN Biomedicals Inc. Vectors were from Invitrogen. Murine P388D1 cells and ACAT c1 gene sequences were from Drs David P. Via and Ta-Yuan Chang, respectively. HDL2 was isolated from normal human plasma through flotation between d=1.063 g/mL and d=1.12 g/mL, concentrated, sterilized, divided into aliquots, sealed under argon, stored at 4°C, and used within 1 month.
Cell Culture
P388D1 cells were maintained in RPMI-1640 medium that
contained 10% FBS and 0.1 g/L gentamicin. Before each experiment,
cells were pretreated for 24 hours with serum-free RPMI-1640 containing
5% BSA. Cells were incubated with HDL2 for 4
hours in an atmosphere of 95% air and 5%
CO2.
ACAT Fusion Antigen Preparation
ACAT cDNA was subcloned from pcDNA/neo/ACAT c1 (8.0
kb) into the expression vector PRsetB (2.9 kb), by which the ACAT gene
(bp 1486 to 2455, corresponding to ACAT amino acid residues 31 to 353)
was engineered downstream of the T7 promoter and ATG
His6 track, using
BamHI and
EcoRI sites. In-frame insertion
of the ACAT C1 gene into the new construct was verified by DNA
sequencing. The PRsetB/ACAT vector was transfected into fresh,
competent JM109 cells,18 and
protein expression was induced by the addition of M13/T7 phages (5
pfu/cell) and isopropyl
-D-thiogalactopyranoside
(1 mmol/L). The ACAT fusion protein was isolated from the
inclusion bodies through SDS-PAGE (15% acrylamide) and
collected via electroelution.
ACAT IgG Purification by Affinity
Chromatography
After 4 inoculations with the fusion protein, rabbit
serum proteins were precipitated with saturated ammonium sulfate (pH
7.4) and purified with Econo-Pac Serum IgG columns (Bio-Rad). An ACAT
affinity gel was prepared with Affi-Gel 10 (Bio-Rad) and the ACAT
fusion protein. Antisera were applied to the column, and nonspecific
rabbit IgGs were eluted with 1.2 mol/L NaCl and 50 mmol/L
phosphate buffer (pH 7.4). ACAT IgGs were eluted in 4.5 mol/L guanidine
hydrochloride, dialyzed against PBS at 4°C, sterilized, and stored in
PBS.
ACAT Turnover
Cells were incubated for 18 hours with
[35S]methionine with or without 40 µg/mL
HDL2 (50 µCi/60-mm dish) in methionine- and
cysteine-free medium. At various times, cells were harvested and washed
in PBS and then lysed in 0.5 mL/dish of extraction buffer (1
mmol/L PMSF, 50 mmol/L Tris, 150 mmol/L NaCl, 1% Nonidet
P-40, pH 9.0); 500 µg of each sample was used for
immunoprecipitation. The lysate was precleaned with 2 µg/mL preimmune
rabbit IgG (Sigma Chemical Co) at 4°C for 4 hours. Protein–preimmune
IgG complexes were removed by the addition of 5% by volume 20%
protein A–agarose beads (Sigma Chemical Co). The supernatant was
continuously incubated with 2 µg/mL ACAT IgG at 4°C overnight.
Immune complexes were removed; centrifuged into a pellet;
washed 3 times in 50 mmol/L Tris, 100 mmol/L NaCl, 1%
Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS, pH 8.0; washed in
PBS; dissolved in SDS-PAGE sample buffer; and separated on 12%
SDS-PAGE. Proteins were transferred to a supported nitrocellulose
membrane (Bio-Rad), which was exposed to a CS screen (Bio-Rad) for 48
hours. Images were scanned and analyzed with a Molecular Imager
(Bio-Rad).
To study ACAT turnover, cells were pretreated for 18 hours with [35S]methionine (50 µCi/60-mm dish) in a methionine- and cysteine-free medium. Cells were washed and incubated in serum-free medium; protein synthesis was terminated with 5 µg/mL cycloheximide for 2 hours. Incubation was continued with 5 µg/mL cycloheximide with or without 40 µg/mL HDL2 for various times. To quantify ACAT mass, cells were labeled with [35S]methionine (25 µCi/100-mm dish) in serum-free medium for 18 hours. Cells were then washed and incubated for 4 hours with 40 µg/mL HDL2 in the fresh labeling medium (serum free, 5% BSA, and 25 µCi [35S]methionine). ACAT turnover and total mass were determined by measuring the amount of immunoprecipitatable ACAT-associated radioactivity.
Immunocytochemistry and
Immunofluorescence
Quiescent P388D1 cells were cultured overnight on
coverslips in serum-free RPMI supplemented with 5 µg/mL BSA. Cells
were incubated for 4 hours with or without 40 µg/mL
HDL2. Some cultures also contained 2 µg/mL
brefeldin A. After a PBS wash, cell monolayers were fixed in 4%
paraformaldehyde at room temperature for 30 minutes.
Cells were then permeabilized with 0.2% Triton X-100
at room temperature for 10 minutes, washed in Tris-buffered saline
(TBS), and blocked with 1% BSA-TBS for 4 hours.
For immunocytochemistry, cells were incubated overnight at 4°C with ACAT IgG (1 µg/mL). The goat anti-rabbit IgGs (biotinylated, 1:1000 dilution; Elite ABC Kit; Vector Laboratories Intl Corp) were used as the second antibody to blot the cell monolayer at room temperature for 4 hours. After 3 washes with 0.25% Tween 20-TBS, cells were incubated overnight with a 1:2000 dilution of avidin-peroxidase conjugate; 3-amino-9-ethylcarbazole was used to detect the peroxidase activity after a 30-minute incubation. Cells were counterstained in 50% hematoxylin for 30 to 60 seconds. After washing in water and drying, the coverslip was mounted on a slide (Gel/Mount; Biomeda Corp).
For immunofluorescence studies, cells were incubated overnight at 4°C with rabbit anti-ACAT IgG (0.5 µg/mL) and with mouse anti-KDEL IgG (1:400 dilution; StressGen Biotechnologies Corp), which was used to label the ER. The cell monolayer was incubated for 2 hours with Texas Red–conjugated goat anti-rabbit IgG (Vector Laboratories Inc) and fluorescein-conjugated goat anti-mouse F(ab)'2 (Immunotech), respectively, at 1:1000 and 1:50 dilutions. After a PBS wash and drying, the coverslip was mounted on the slide (Gel/Mount; Biomeda Corp) and examined by deconvolution fluorescence microscopy with a Zeiss Axiovert S100 TV microscope and DeltaVision Restoration Microscopy System (Applied Precision, Inc). A Z series of focal planes were digitally imaged and deconvolved with the DeltaVision constrained iterative algorithm to generate high-resolution images.
Common and distinct immunolocalization of ACAT with KDEL sequences was determined on images obtained by fluorescence microscopy. ACAT P388D1 cells were plated on coverslips, treated with or with HDL2 and brefeldin A, and immunostained with either ACAT or KDEL antibodies. Specimens were examined with a Nikon OPTIPHOT microscope with a UFX-IIA photomicrographic attachment. Images were captured and recorded with the Image-Pro Plus Software Package (Media Cybernetics), and fields at 40x were used for quantitative analysis with IP Laboratory Spectrum (Signal Analytic Corporation). Signals derived from ACAT and KDEL stains were separately segmented, and the sizes of areas that covered qualified segments were measured with this program. Ratios (R) of ACAT to KDEL in 50 cells were expressed as mean±SD values, and statistical significance among groups was evaluated with ANOVA; complete superposition of ACAT and KDEL localization corresponds to R=1.
Effect of Brefeldin A on ACAT Activity
Cellular ACAT activity was determined by measuring
the rate of cholesteryl [14C]oleate
formation from
[14C]oleate.19
The serum-free RPMI-1640 medium used for pulsing consisted of 40
µg/mL HDL2, 100 µmol/L
[1-14C]oleate–BSA complex, and 2 µg/mL
brefeldin A. Cells were incubated in the pulsing medium for 4 hours at
either 37°C or 4°C, followed by a 30-minute chase in the serum-free
medium at 37°C. Cellular lipids were separated by thin-layer
chromatography and quantified by liquid scintillation
counting.
Subfractionation and Analysis of
Cellular Organelles
P388D1 cells were pretreated for 18 hours with
[35S]methionine (50 µCi/60-mm dish);
harvested in 5 mmol/L Tris·HCl, 10 mmol/L KCl, 2
mmol/L CaCl2, and 1 mmol/L PMSF, pH 7.4;
and lysed via sonication. Sucrose was added to a final concentration of
0.25 mol/L. The homogenate was subjected to sequential
centrifugation. Nuclei, mitochondria, plasma membranes,
and microsomes, respectively, were collected at
1200g (10 minutes),
3000g (10 minutes),
10 000g (20 minutes), and
100 000g (18 hours). Each
fraction was washed in PBS, and 500 µg protein from each fraction was
used for the immunoprecipitation. Aliquots were used to identify
fractions corresponding to the nucleus, ER, and plasma membrane. The
respective markers for these organelles were DNA, RNA, and 5'
nucleotidase activity.
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Specificity of Polyclonal ACAT Antibody
To confirm the specificity of ACAT IgG, preimmune IgG and ACAT IgG were used to immunoprecipitate the P388D1 cell lysate. Preimmune IgG nonspecifically precipitated 43-, 40-, 32-, 17-, and 11-kDa proteins. Anti-ACAT IgG precipitated 65-, 46-, and 17-kDa proteins. The 46-kDa protein represents
90% of the
intensity and corresponds to the mouse ACAT-1 protein, which migrates
abnormally in
SDS-PAGE.16
Effect of HDL2 on
Cellular ACAT Turnover
The rates of ACAT synthesis in the presence and absence
of HDL2 at a low concentration (40 µg/mL) were
estimated from the rates of incorporation of
[35S]methionine into immunoprecipitatable
ACAT that was subjected to SDS-PAGE and analyzed for
radioactivity
(Figure 1A
). At 15, 30, and 240 minutes,
HDL2 (40 µg/mL) increased synthesis of the
46-kDa ACAT by 80%, 35%, and 20%, respectively. Synthesis of the
65-kDa ACAT was also stimulated by HDL2 but
accounted for <10% of newly synthesized ACAT. Synthesis of a
nonspecific 32-kDa protein that was associated with preimmune IgG was
unchanged.
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Studies of ACAT degradation showed that 20% of the 65-
and 46-kDa ACAT proteins were degraded in serum-free medium after a
30-minute incubation
(Figure 1B
). No significant additional ACAT degradation (46
and 65 kDa) was observed during the next 210 minutes. The nonspecific
32-kDa protein, however, was degraded by 17% and 90% of the initial
amount after 30-minute and 4-hour incubations, respectively.
Incubations with HDL2 (40 µg/mL) did not
affect the rates of degradation of the 46- and 65-kDa ACAT proteins and
the nonspecific 32-kDa protein. These results suggest that ACAT
proteins are more resistant to intracellular degradation than
the nonspecific 32-kDa protein. HDL2 did not
significantly change either the 46-kDa or the 65-kDa ACAT protein after
a 4-hour incubation
(Figure 1C).
Effect of HDL2
and Brefeldin A on Subcellular ACAT Location
Staining of proteins in P388D1 macrophages with
preimmune serum was negative
(Figure 2A). Cells exposed to an ACAT-specific antibody
showed prominent ACAT-positive regions, which were distinguished by a
pinkish brown color. In the absence of HDL2, the
ACAT-positive regions were perinuclear
(Figure 2B). In the presence of HDL2,
ACAT-positive regions were diffuse and shifted from the perinuclear
region to the cell periphery
(Figure 2D). Some ACAT-positive staining within punctate
rings, whose size and shape were consistent with vesicles
(Figure 2E), was observed only in the presence of
low-concentration HDL2. The translocation of
ACAT from 1 part of the cell to another in response to
HDL2 was supported by tests with brefeldin A,
which inhibits vesicular protein transport from the ER to the Golgi
compartment.20 21 22 23
In the absence of HDL2, the specific brown stain
denoting the location of ACAT was more concentrated near the nucleus in
brefeldin A–treated cells
(Figure 2C) than in untreated cells
(Figure 2B). When cultures were treated
simultaneously with brefeldin A and
HDL2, however, the ACAT was confined to the
perinuclear region
(Figure 2F). Thus, brefeldin A blocked the
HDL2-mediated translocation of ACAT away from
the perinuclear region.
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Subcellular ACAT distribution was also investigated with
dual immunofluorescence microscopy
(Figure 3). In control cells, regions of green and red
fluorescence due to labeling of KDEL and ACAT were confined to
a common perinuclear region
(Figures 3A and 3B). The superposition of the
fluorescence from KDEL and ACAT labels, seen as a yellow image
in control cells
(Figure 3C), confirms that both labels were confined to the
same region of the cell (R=1.11±0.06).
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After incubation with low-concentration
HDL2, the fluorescent label associated
with KDEL remained associated with the perinuclear region
(Figure 3D). In contrast, the fluorescent label
associated with ACAT was more diffusely distributed and extended beyond
the ER network
(Figures 3E and 3F). Quantitative analysis showed that
the ratio of the ACAT-positive to KDEL-positive areas was higher in the
presence of HDL2 (R=1.51±0.06; +36%;
P<0.0004). Thus, the
incubation of low-concentration HDL2 with P388D1
macrophages stimulates the transport of a fraction of the ACAT
from the ER to the cell periphery. However, in the presence of
brefeldin A and HDL2
(Figures 3G–3I), ACAT and KDEL were confined to the
perinuclear region (R=0.99±0.01). Thus, brefeldin A blocks the
HDL2-mediated transport of ACAT to the cell
periphery.
Effect of HDL2 and
Brefeldin A on ACAT Activity
HDL2 (40 µg/mL) inhibited
cholesterol esterification (-50%;
P<0.001;
Table),
a process that was reversed with brefeldin A. Brefeldin A did not
affect FC or phospholipid syntheses
(Table).
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Effect of HDL2 on ACAT
Distribution Among Organelles
Cells were incubated with and without
HDL2 for 4 hours. The subcellular organelles
were isolated from cell lysate via centrifugation and
identified
(Figures 4A and 4B). The ACAT associated with each fraction
was identified via immunoprecipitation
(Figure 4C). In the absence of HDL2,
the 46-kDa ACAT was confined exclusively to the fractions containing ER
and nuclei. After a 4-hour incubation with HDL2,
however, a band corresponding to the 46-kDa ACAT appeared in the plasma
membrane fraction but not in the nuclear fraction. Like the 46-kDa
ACAT, the 65-kDa ACAT was concentrated in the ER with a small amount
being translocated to the plasma membrane fraction in response to
incubation with HDL2. The ACAT antibody also
precipitated proteins of 86 and 35 kDa from all
fractions.
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Discussion |
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HDL may be cardioprotective through reverse cholesterol transport, whereby HDL accepts FC from peripheral tissue and transports it to the liver for degradation or recycling.24 25 Our previous studies in murine P388D1 macrophages showed that incubation with HDL2 at low concentrations was associated with reduced esterification of cellular cholesterol and enhanced FC efflux.3B The present study was designed to identify the mechanism(s) by which low-concentration HDL2 inhibits ACAT activity.
Specificity of Polyclonal ACAT Antibody
ACAT is highly polymorphic. According to radiation
inactivation
experiments,26 27
the functional size of ACAT is 170 to 200 kDa. Western blot
analysis with our polyclonal ACAT antibody raised against an
ACAT fusion protein (57% of full-length ACAT) revealed 46- and
65-kDa ACAT-positive proteins in murine P388D1 macrophages. The
65-kDa ACAT was a minor component and represented only
10% of total ACAT. The appearance of 46-kDa ACAT protein was
independent of the time (30 minutes to 48 hours) between protein
electrophoresis and of the addition of the protease
inhibitor PMSF to the assay solution (data not shown).
Thus, it is also probable that the 46-kDa species, which is the
dominant form of ACAT in P388D1 macrophages, was not formed
through postculture proteolysis. This conclusion is consistent
with the data of others. A polyclonal ACAT antibody (DM10) reacted with
a 46-kDa mouse
ACAT.14 16
Quantitative disappearance of 46-kDa ACAT protein was observed in
heterozygotic and homozygotic ACAT knockout
mice.16 The discrepancy
between the calculated and observed sizes of ACAT proteins may be due
to the unusually hydrophobicity of ACAT or to limitations of the
antibody.13 14 In
ACAT-transfected Sf9 cells, DM9 antibodies recognized a 56-kDa (minor)
and a 50-kDa (major) ACAT
protein,28 whereas DM10
antibody detected only the 50-kDa
ACAT.14 Although ACAT gene
expression products exist in multiple isoforms, the 46- and 65-kDa
ACAT species are nearly always found, with the former being more
prominent in Western blots.
Chemical modification of rabbit ACAT revealed 2 species distinguished by different apparent Ki values toward diethylpyrocarbonate.29 30 The human ACAT gene has 2 promoters that could produce different ACAT mRNAs.31 Multiple ACAT transcripts occur in a variety of mouse15 and rabbit32 tissues, indicating that the ACAT gene may encode different isoforms. According to our data, P388D1 macrophages express ACAT species that are either different isoforms or the same protein with a structural modification. Most of our conclusions are based on changes in the more abundant 46-kDa ACAT.
Translational Regulation of ACAT by
HDL2 at Low Concentrations
HDL2 at a low concentration
induced synthesis of 46-kDa ACAT protein synthesis (80% more than
control) in the first 15 minutes
(Figure 1A). After a 4-hour incubation, the rate of ACAT
synthesis was only 20% higher in the
HDL2-treated macrophages than in
control. Similar results were observed with the 65-kDa ACAT, suggesting
that the expression of both species is regulated by the same mechanism.
These results suggest that the induction of ACAT synthesis is an early
cellular response to low-concentration HDL2.
During a 4-hour incubation, low-concentration
HDL2 did not affect the rate of degradation of
either 46- or 65-kDa ACAT
(Figure 1B) or the total ACAT pool size
(Figure 1C). Taken together, the data in
Figure 1 suggest that the amount of new ACAT formed during
the 4-hour incubation is small compared with the size of a large, and
apparently quite stable, existing ACAT pool. Therefore, even the 80%
and 20% increases in ACAT synthesis observed at 15 and 240 minutes,
respectively, in the presence of low-concentration
HDL2 do not contribute significantly to the
overall ACAT pool size. Differences in ACAT activity and cellular CE
content are probably not due to differences in cellular ACAT content.
This conclusion is not unprecedented:
macrophages17 and
hepatoblastoma cells33
possess higher ACAT activities than controls without upregulation of
ACAT mRNA and protein mass.
Intracellular Translocation of ACAT in Response
to Low-Concentration HDL2
HDL2 at low concentrations
inhibited ACAT activity and cellular
CE.3B Our biochemical
evidence supports the hypothesis that low-concentration
HDL2 induces the vesicular transfer of ACAT from
the ER to the cell periphery. Brefeldin A, which blocks vesicular
protein transport, reversed the HDL2-mediated
ACAT inhibition
(Table).
Because our observations were made with intact cells, the
HDL2-mediated decrease in ACAT activity could be
due to a decrease in the availability of the substrate
cholesterol or an increase in cellular phospholipid, which
is the major cell membrane component that binds
cholesterol. However, cholesterol and
phospholipid syntheses in cells incubated with
HDL2 alone or with HDL2
plus brefeldin A were the same. Most evidence suggests that brefeldin A
does not affect intracellular transport of
cholesterol34 35
or
phosphatidylethanolamine.36
Thus, brefeldin A must directly affect ACAT translocation and not the
translocation or cellular concentration of its substrates.
Using antibody DM10 and
immunofluorescence methodology, Chang et
al14 found ACAT mainly in
the nuclear envelope and the ER. Because of the contiguity between the
nuclear membrane and the ER, and the similar pattern of ACAT
distribution between the nucleus and ER, some ACAT must be associated
with the nuclear membrane or 1 or more perinuclear
sites.37 We found
essentially the same subcellular distribution of ACAT in the absence of
HDL2
(Figures 2B and 3A–3C). When incubated with low-concentration
HDL2, however, ACAT occurred within punctate
rings consistent with vesicles
(Figure 2E) and at the cell periphery beyond the ER network
(Figures 2D and 3F). A similar effect has been observed in
cultured human macrophages in which ACAT was confined to
tubular rough ER, but in response to acetylated-LDL, it
appeared in small vesicles that were positive for a marker for
ER.38 ACAT translocation to
the cell periphery in response to low-concentration
HDL2, however, was blocked by brefeldin A
(Figures 2F and 3I).
Subcellular fractionation studies showed an accumulation of
ACAT in the fraction that contains the ER even after incubation with
HDL2. However, the subcellular fractions that
contained the ER also contained vesicular bodies, which according to
the immunocytochemical findings are ACAT rich
(Figure 2D). In the presence of low-concentration
HDL2, some ACAT was associated with the plasma
membrane fraction. The amount was small, however, and the limitations
of this technique preclude a definitive assignment.
We propose that the incubation of P388D1 macrophages with low-concentration HDL2 induces vesicular transport of ACAT from the ER and nuclear envelope to the cell periphery. The attendant inhibition of ACAT, which increases the intracellular cholesterol pool size, would complement the role of HDL2 as a cholesterol acceptor in the reverse cholesterol transport pathway. The HDL2-mediated translocation of ACAT from the ER to the cell periphery is temporally associated with and may be mechanistically linked to reduced ACAT activity, reduced cellular CE content, and enhanced FC efflux. The initiating event could be the binding of HDL2 to a specific cell surface receptor that is part of a signal transduction pathway that leads to intracellular ACAT translocation and inactivation. Translocation could lead to inactivation via several mechanisms. ACAT substrates acyl-CoA and cholesterol may be less accessible to the enzyme. This seems unlikely. Most intracellular membranes contain cholesterol, with the plasma membranes, in particular, being cholesterol rich.39 Although not all acyl-CoA is concentrated in membranes, it can be rapidly transported to membranes via spontaneous transfer.40 Second, ACAT may require a cofactor that resides exclusively in the ER. Third, ACAT may exist in active and inactive structures according to its location in the ER or cell periphery, respectively. Future studies should focus on the latter possibilities and on the structure and function of the 65- and 46-kDa ACAT proteins.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-30914 and HL-56865. Dr Li was a student in the Cardiovascular Sciences Graduate Program of The DeBakey Heart Center.
Received March 15, 2000; accepted September 22, 2000.
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