From the Laboratory of Cellular Biochemistry,
Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan,
Biochemistry, Cell
Biology and Metabolism, Nagoya City University Graduate School of
Medical Sciences, Nagoya 467-8601, Japan, and the ¶ Center for
Integrative Bioscience, Okazaki National Research Institutes,
Okazaki 444-8585, Japan
Received for publication, July 10, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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ABCA1 mediates release of cellular cholesterol
and phospholipid to form high density lipoprotein (HDL). The three
different mutants in the first extracellular domain of human ABCA1
associated with Tangier disease, R587W, W590S, and Q597R, were examined
for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in
HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that
ABCA1-GFP was mainly localized to the plasma membrane (PM) but also
substantially in intracellular compartments. All three mutant
ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and
Q597R were associated with impaired processing of oligosaccharide from
high mannose type to complex type and failed to be localized to the PM,
whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the
dysfunction in the W590S mutant. Photoaffinity labeling of W590S with
8-azido-[ Cholesterol is not catabolized in the peripheral cells and
therefore mostly released and transported to the liver for conversion to bile acids to maintain cholesterol homeostasis. The same pathway may
also remove cholesterol that has pathologically accumulated in the
cells such as an initial stage of atherosclerosis. Assembly of high
density lipoprotein (HDL)1
particles by helical apolipoproteins with cellular lipid has been
recognized as one of the major mechanisms for cellular cholesterol release (1, 2). The importance of this active cholesterol-releasing pathway in regulating cholesterol homeostasis became apparent by the
finding that it is impaired in the cells from patients with Tangier
disease, a genetic deficiency of circulating HDL (3, 4). Mutations were
identified in ATP-binding cassette transporter A1 (ABCA1) of the
Tangier disease (TD) patients (5-7), but the molecular mechanism of
ABCA1 in the apolipoprotein-mediated HDL assembly remains unclear.
Although direct interaction between ABCA1 and apoA-I at the cell
surface has been suggested on the basis of chemical cross-linking
experiments (8, 9), an indirect role of ABCA1 in the apoA-I binding to
the cell was also proposed by a model that ABCA1 induces
phosphatidylserine exofacial flopping to generate the microenvironment
required for the docking of apoA-I at the cell surface (10). The
predominant substrates of the ABCA1-mediated lipid release reaction are
still to be determined for the HDL assembly reaction (11, 12).
More than 30 mutations have been mapped in the ABCA1 gene in
patients with familial hypoalphalipoproteinemia (FHA) and TD (5-7, 13-15). Many mutations have been identified in the putative first extracellular domain (ECD1) and the first nucleotide binding fold
(NBF1) of ABCA1. We and Fitzgerald et al. (16-18) recently demonstrated that ECD1 exists in the extracellular space by introducing an epitope tag into ABCA1 ECD1 and by analyzing glycosylation of the
truncated form of ABCA1. To investigate the mechanistic background for
these mutations to cause the dysfunction of ABCA1, we characterized the
function and subcellular localization of ABCA1-GFP and its TD mutants
stably expressed in HEK293 cells. Three TD mutants (R587W, W590S,
Q597R), clustered in ECD1, were examined in the present report.
Immunostaining and confocal microscopic analysis showed that ABCA1 is
mainly localized to the plasma membrane (PM), where ECD1 is expected to
be exposed to the outside of the cell, but also in intracellular
compartments to a substantial extent. The TD mutations in ECD1 resulted
in a distinct influence on the function and subcellular localization of
ABCA1. All three mutants were functionally impaired for the
apoA-I-mediated HDL assembly. On the other hand, the two mutants R587W
and Q597R were only partially or scarcely localized to the PM, whereas
W590S was localized to the PM as efficiently as the wild type.
Vanadate-induced nucleotide trapping was examined to elucidate the
mechanism for the dysfunction in the W590S mutant.
Materials--
Anti-GFP antibody was purchased from Santa Cruz
Biotechnology. All other chemicals were obtained from Sigma, Wako Pure
Chemical Industries, or Nacalai Tesque.
Generation of an Antibody to ABCA1 ECD1--
The putative
extracellular domain ECD1, amino acids 45-639 of human ABCA1,
was expressed as a C terminus His tag fusion protein in
Escherichia coli BL21(DE3) and purified by Ni2+
chromatography (Qiagen). A rat polyclonal antibody, generated using
this His tag-fused ECD1, specifically interacted with human ABCA1
stably or transiently expressed in HEK293 cells in Western blotting
(data not shown) and immunostaining (see Fig. 1).
Immunostaining and Fluorescence Microscopy--
Cells were grown
on a 35-mm glass-base dish (Iwaki). The cells were incubated with
primary antibodies in phosphate-buffered saline containing 5%
skim milk. After being washed, these cells were incubated with the
fluorescent-labeled secondary antibodies. The cells were directly
viewed with ×100 Plan-NEOFLUAR oil immersion objective on a Zeiss
confocal microscope LSM510.
DNA Construction--
DNA fragments
(XhoI-BclI) containing each missense TD mutation
(R587W, W590S, or Q597R) were generated using the polymerase chain
reaction method with R587W (XhoI) primer
(5'-GTCCTCGAGCTGACCCCTTTGAGGACATGTGGTACGTC-3'), W590S (XhoI) primer
(5'-GTCCTCGAGCTGACCCCTTTGAGGACATGCGGTACGTCTCGGGGGGCTTC-3'), or Q597 (XhoI) primer
(5'-GTCCTCGAGCTGACCCCTTTGAGGACATGCGGTACGTCTGGGGGGGCTTCGCCTACTTGCGGGATGTGGTG-3'), where the mutated nucleotide is underlined, and BclI
primer (5'-CGATGCCCTTGATGATCACAGCCACTGAG-3'). The DNA fragment was
replaced with the XhoI-BclI fragment of human ABCA1
(16).
Glycosylation of ABCA1-GFP Protein--
Endoglycosidase H (Endo
H) and peptide N-glycosidase F (PNGaseF) (New England
Biolabs, Beverly, MA) digestions were done as described by the
manufacturer. In brief, 10 µg of membrane proteins from HEK293 cells
stably expressing the wild-type, R587W, W590S, or Q597R ABCA1-GFP were
treated with 500 units of Endo H or 0.3 units of PNGaseF for 1 h
at 37 °C. The deglycosylated proteins were separated by SDS-PAGE
(7.5%) and analyzed by immunoblotting by using the anti-GFP antibody.
Cellular Lipid Release Assay--
Cells were subcultured
in 6-well plates (TPP, 92406) at a density of 1.0 × 106 cells in a 1/1 mixture of Dulbecco's modified Eagle's
medium and Ham's F12 medium (DF) supplemented with 10% (v/v) fetal
calf serum. After incubation for 48 h, the cells were washed with
Dulbecco's phosphate-buffered saline and incubated in the same medium
supplemented with 0.1% bovine serum albumin and 10 µg/ml apoA-I. The
lipid content in the medium was determined after a 24-h incubation as described previously (19). To compare lipid release from HEK293 cells
transiently expressing ABCA1-GFP, GFP fluorescence of transfected cells
was measured with a FL600 fluorescent plate reader (Bio-Tek Instruments, Inc.) (19), and expression levels of the wild-type and
mutant ABCA1-GFP were normalized with GFP fluorescence. Expression levels of the wild-type and mutant ABCA1-GFP were in a range of ±20%.
Vanadate-induced Nucleotide Trapping in ABCA1 with
8-Azido-[ Subcelluar Localization of ABCA1-GFP--
In our previous report,
we have shown that the hemagglutinin epitope inserted between residues
207 and 208 of human ABCA1 was recognized by the anti-hemagglutinin
antibody from the outside of cells (16). To confirm the extracellular
localization of the hydrophilic domain containing residue 207 (ECD1), non-permeabilized HEK293 cells were incubated with a rat
polyclonal antibody against the protein corresponding to amino acids
45-639 of human ABCA1 for immunostaining. ABCA1-GFP, which was
apparently on the cell surface, was visualized by the antibody, whereas
the protein in the intracellular compartments was not (Fig.
1). The results suggested that ABCA1-GFP
was localized to the PM and the intracellular compartments, and the
ECD1 domain of ABCA1 on the PM was exposed to outside of the cells.
Effects of ECD1 Mutations on Subcellular Localization of
ABCA1-GFP--
Many mutations in patients with TD and FHA have been
identified in ECD1 of ABCA1, and three mutations (R587W, W590S, Q597R) cluster in the vicinity between amino acids 587 and 597 (20)(Fig. 2A). To study the role of ECD1
domain in the HDL assembly function of ABCA1, we introduced these three
TD mutations in ECD1 into ABCA1-GFP and transiently or stably expressed
in HEK293. The expression levels of mutant ABCA1-GFP stably expressed
in HEK293 cells were comparable with that of the wild-type ABCA1-GFP as
shown later in Figs. 3 and 5. Confocal
microscopic examination revealed that R587W and Q597R appeared to be
localized mainly in the ER and not to the PM (Fig. 2B). In
contrast, W590S was localized to the PM as much as the wild-type
ABCA1-GFP was, although more was found with intracellular vesicles than
with the wild type (Fig. 2B). Immunostaining with the
antibody against ECD1 confirmed the proper orientation of W590S (Fig.
2C).
Glycosylation of ABCA1-GFP--
Glycosylation of the wild-type
ABCA1-GFP and its mutants R587W, W590S, and Q597R was examined by the
treatment with PNGaseF and Endo H (Fig. 3A). Endo H cleaves
two proximal N-acetylglucosamine residues of the high
mannose type but not of the complex type, whereas PNGaseF cleaves sugar
chains of both the high mannose and complex types. The treatment with
PNGaseF increased the electrophoretic mobilities of 280-kDa ABCA1 to
produce the 250-kDa protein, the deglycosylated form of ABCA1-GFP.
ABCA1 with TD mutations, R587W and Q597R ABCA1-GFP, was sensitive to
Endo H to produce the deglycosylated form of ABCA1-GFP, whereas the
wild-type ABCA1-GFP was little digested by Endo H. These results
indicated that R587W and Q597R ABCA1-GFP did not contain complex
oligosaccharides and supported the confocal microscopy observation,
which suggested the localization of these two TD mutants in the ER or
the cis-Golgi complex. On the other hand, W590S ABCA1-GFP was resistant
to Endo H, indicating that it does not contain high mannose
oligosaccharides but contains complex oligosaccharides and reached the
trans-Golgi complex.
Effects of ECD1 Mutations on apoA-I-mediated Cholesterol
Release--
To analyze the functional consequences of these
mutations, apoA-I-mediated release of cholesterol and
choline-phospholipid was examined from the stable transformants (Fig.
4, A and B). The
wild-type ABCA1-GFP exported 0.14 ± 0.01 and 1.14 ± 0.07 µg of cholesterol from cells grown in 6-well plates in the absence and presence of apoA-I, respectively, and 1.99 ± 0.25 and
5.88 ± 0.13 µg of choline-phospholipids, respectively, to
generate HDL in the medium by reducing about 15% of cell cholesterol.
The R587W mutation resulted in the apoA-I-mediated release of
cholesterol and choline-phospholipids to 24 and 23% of the wild-type
ABCA1-GFP, respectively. The Q597R mutant almost completely lost this
activity. The results were apparently consistent with the inefficient
localization to the cell surface of ABCA1-GFP with these mutations. The
apoA-I-mediated release of cholesterol and choline-phospholipids from
HEK293 expressing W590S ABCA1-GFP were 7.4 and 13%, respectively, of
those expressing the wild-type ABCA1-GFP, although W590S ABCA1-GFP was
localized to the PM as efficiently as the wild type. The
apoA-I-mediated release of cellular cholesterol and
choline-phospholipids was also examined with HEK293 transiently
expressing the wild-type and mutant ABCA1-GFPs (Fig. 4, C
and D). The results were similar to those observed with the
stable transformants shown in Fig. 4, A and
B.
Interaction of ABCA1-GFP with
8-Azido-[
Multidrug transporters, MDR1 (ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2),
are known to trap Mg-ADP in the presence of ortho-vanadate, an analog of phosphate, and form a stable inhibitory intermediate during the ATP hydrolysis cycle. Photoaffinity labeling of these proteins with 8-azido-[
Vanadate did not induce nucleotide trapping in MRP6 (ABCC6) in the
presence of Mg2+, but it did with Ni2+ ions
(25). Therefore, we examined photoaffinity labeling of ABCA1-GFP in the
presence of other metal ions. Significant stimulation was observed with
wild-type ABCA1-GFP by ortho-vanadate in the presence of
Mn2+ (Fig. 5A, lanes 11 and
12). These results suggested that Mn-ATP was
hydrolyzed at NBFs of ABCA1, and a stable inhibitory complex ABCA1-MnADP-Vi was formed during the ATP hydrolysis cycle.
To determine whether the W590S mutation affects the ATP hydrolysis
cycle of ABCA1, vanadate-induced nucleotide trapping in W590S ABCA1-GFP
was examined in the presence of Mn2+ (Fig. 5C).
Membrane proteins from HEK293 cells expressing a similar amount of
wild-type ABCA1 or W590S ABCA1-GFP (Fig. 5B) were incubated with 8-azido-[ In this work, we described the influence of three clustered
mutations in ECD1 associated with TD and FHA on the subcellular localization of ABCA1, apoA-I-mediated HDL assembly, apoA-I binding, and vanadate-induced nucleotide trapping. Immunostaining of ABCA1-GFP stably expressed in HEK293 cells revealed that ABCA1-GFP apparently resided on the cell surface as well as in intracellular compartments in
agreement with previous reports (18, 26-28). Although the three
mutations all reduced apoA-I-mediated lipid release and subsequent HDL assembly from HEK293 cells expressing ABCA1-GFP, whether
transiently or stably, the mutants demonstrated differential behavior
with respect to their subcellular localization. ABCA1-GFP with a R587W
or Q597R mutation appeared to be impaired with intracellular trafficking and predominantly localized in the ER. On the other hand,
W590S ABCA1-GFP was mainly localized to the PM as much as the wild-type
ABCA1 was. The sensitivity to Endo H of the mutant ABCA1s was
consistent with the their apparent impairment of intracellular trafficking. R587W and Q597 ABCA1-GFP contained high mannose
oligosaccharides, indicating that they do not reach the trans-Golgi
complex. In contrast, W590S ABCA1-GFP contained complex-type
oligosaccharides as the wild-type does.
When the cells were treated with monensin, which prevents the delivery
of protein from endosomes to the cell surface, after inhibiting protein
synthesis by treatment with cycloheximide, ABCA1-GFP on the cell
surface decreased, and the vesicular localization increased instead
(see supplementary data, Fig.
1).2 When the cells were
treated with brefeldin A, which blocks vesicular transport from the ER
to the Golgi and to the cell surface along with the secretary pathway
(29), the newly synthesized ABCA1-GFP was accumulated in the fused
Golgi-ER, the amount of ABCA1-GFP on the PM was reduced, and the
vesicles containing ABCA1-GFP were observed (see supplementary data
1).2 ABCA1-GFP was co-localized partly with Vti1b, a marker
for the Golgi, with EEA1, a marker for early endosomes, and with
lysotracker, a marker for acidic compartments (see supplementary data
2).2 These results suggested that newly synthesized
ABCA1-GFP was first delivered to the PM through the ER and the Golgi
and then shuttled rapidly between the PM and the intracellular
vesicles, mainly the early endosomes. R587W and Q597R ABCA1-GFP
appeared to be retained in the ER. It has been reported that one
amino acid (Phe-508) deletion of the cystic fibrosis
transmembrane conductance regulator (CFTR), the major mutation in
cystic fibrosis patients that causes misfolding of the cystic fibrosis
transmembrane conductance regulator, is degraded by the proteasome
pathway before exiting from the ER (30). This region (R587 to Q597) in
ECD1 would be critical for proper folding of ABCA1 and would probably
affect the intracellular translocation process, whereas the W590S
mutation does not. Fitzgerald et al. (17) reported that
R587W or Q597R mutation did not affect the PM localization but
disrupted the direct interaction with ApoA-I. This supports a major
conformational alteration of ECD1 by these mutations. The reason for
the discrepancy of subcellular localization is unknown between their
results with the mutant ABCA1 transiently expressed in a high amount in
HEK293 and ours studied with the mutant ABCA1-GFP in the stable
transformants with modest expression.
W590S ABCA1-GFP was localized to the PM as much as the wild-type
ABCA1-GFP when expressed in HEK293. However, apoA-I-mediated release of
cellular cholesterol and choline-phospholipid was severely impaired. To
elucidate the reason for this functional impairment in the W590S
mutant, nucleotide interaction was examined with the wild-type and
W590S ABCA1-GFP by using 8-azido-[ W590S ABCA1-GFP showed vanadate-induced nucleotide trapping in the
presence of Mn2+. This suggests that the first catalytic
reaction to form a stable inhibitory complex ABCA1-MnADP-Vi is not
impaired by the mutation. It has been reported that apoA-I does not
properly interact with ATP hydrolysis mutants of ABCA1 (10) and that
apoA-I can be interacted with ABCA1-W590S as with the wild-type ABCA1
(17, 35). These results suggest that W590S ABCA1-GFP possesses, at least, minimum ATPase activity, which supports apoA-I binding. W590S
mutation may impair a step after the interaction with apoA-I, such as
proper loading of phospholipid and/or cholesterol or proper release of
apoA-I after phospholipid/cholesterol loading.
More than 30 mutations have been mapped in the ABCA1 gene in
patients with FHA and TD (5-7, 13-15). One subgroup of the mutations is suggested to be associated with splenomegaly, and the other may be
associated with coronary heart disease (20). Many mutations have been
located in ECD1 of ABCA1, and three missense mutations cluster in the
vicinity between amino acids 587 and 597 in ECD1. Interestingly,
clinical manifestations of these mutations are apparently different
(20): R587W is associated with coronary heart disease, whereas W590S is
associated with splenomegaly. In this study, we demonstrated that the
defect of HDL assembly in R587W and Q597R is due to the impaired
localization of ABCA1 to the PM. Subcellular trafficking and
vanadate-induced nucleotide trapping in the presence of
Mn2+ were not impaired in ABCA1-GFP containing the W590S
mutation so that W590S seems to have a different type of functional
defect. Further characterization of TD mutations for the function of
ABCA1 would facilitate understanding of the molecular mechanism for cellular cholesterol release and its homeostasis.
-32P]ATP was stimulated by
adding ortho-vanadate in the presence of Mn2+
as much as in the presence of wild-type ABCA1. These results suggest
that the defect of HDL assembly in R587W and Q597R is due to the
impaired localization to the PM, whereas W590S has a functional defect
other than the initial ATP binding and hydrolysis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP--
A membrane fraction (20-30
µg) was prepared from HEK293 cells stably expressing the wild-type or
W590S ABCA1-GFP. It was incubated with 15 µM
8-azido-[
-32P]ATP, 2 mM ouabain, 0.1 mM EGTA, and 40 mM Tris-Cl, pH 7.5, in a total
volume of 6 µl for 15 min at 37 °C in the presence or absence of 1 mM ortho-vanadate and 3 mM
MgSO4 or MnCl2. The reaction was stopped by
adding 500 µl of ice-cold 40 mM Tris-Cl buffer containing
0.1 mM EGTA and 1 mM MgSO4
or MnCl2. The supernatant containing unbound ATP was
removed from the membrane pellet after centrifugation (15,000 × g, 5 min, 2 °C), and this procedure was repeated once
more. The pellets were resuspended in 8 µl of TE buffer containing 1 mM MgSO4 or MnCl2 and irradiated
for 5 min (at 254 nm, 8.2 mW/cm2) on ice. The sample was
analyzed by autoradiogram after electrophoresis in a 7%
SDS-polyacrylamide gel. Experiments were done in triplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
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Fig. 1.
Immunofluorescence confocal microscopy
analysis of HEK293 cells stably expressing ABCA1-GFP.
GFP, GFP fluorescence of HEK293 cell stably expressing
ABCA1-GFP. Anti-ECD1, immunofluorescent observation with
anti-ECD1 antibody and anti-rat IgG-Alexa594. Merge,
overlaid GFP and Alexa594 fluorescence.
View larger version (56K):
[in a new window]
Fig. 2.
Effects of ECD1 mutations on subcellular
localization of ABCA1-GFP. A, a putative secondary
structure of ABCA1 and localization of Tangier Disease mutations R587W,
W590S, and Q597R in ECD1. B, GFP fluorescence of HEK293
cells stably expressing the wild-type (WT) ABCA1-GFP and
three TD mutants R587W, W590S, and Q597R ABCA1-GFP. C,
immunofluorescent observation of W590S ABCA1-GFP with anti-ECD1
antibody and anti-rat IgG-Alexa594.
View larger version (31K):
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Fig. 3.
Glycosylation of ABCA1-GFP. The
wild-type (WT), R587W, W590S, and Q597R ABCA1-GFP were
treated with none ( ), Endo H (H), or PNGaseF
(F) and separated with 7% SDS-PAGE. Western blotting was
done with anti-GFP antibody.
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Fig. 4.
Effects of ECD1 mutations on apoA-I-mediated
cholesterol and phospholipid transport. Cholesterol (A)
and choline-phospholipid (B) content in the medium in a
6-well plate containing HEK293 cells stably expressing the wild-type
(WT), R587W (RW), W590S (WS), and
Q597R (QR) ABCA1-GFP were measured after a 24-h incubation
in the presence (black bars) or absence (white
bars) of 10 µg/ml apoA-I. The relative amount of cholesterol
(C) and choline-phospholipid (D) in the medium in
a 6-well plate containing HEK293 cells transiently expressing the
wild-type (WT), R587W (RW), W590S
(WS), and Q597R (QR) ABCA1-GFP was measured after
a 24-h incubation in the presence of 10 µg/ml apoA-I. The expression
levels of mutants were normalized with the GFP fluorescence of cells.
Lipid release from HEK293 cells transiently expressing ABCA1-GFP
subtracted by that from non-transformed HEK293 cells was represented as
100% in C and D.
-32P]ATP--
To elucidate the mechanism for
the loss of function of ABCA1 in the W590S mutant, we examined the
interaction of ABCA1-GFP with ATP. Among membrane proteins of the cells
expressing the wild-type ABCA1-GFP, a 280-kDa protein was specifically
photoaffinity-labeled with ATP (Fig.
5A, lane 7),
whereas no protein was labeled in the untransfected HEK293 cells
(lane 5). The mobility of the photoaffinity-labeled membrane
protein in SDS-PAGE was identical to that of the wild-type ABCA1-GFP
visualized by Western blotting (Fig. 5B, lane 1).
The photoaffinity labeling was negative when the samples were incubated in the absence of Mg2+ (Fig. 5A, lane
3), indicating that 8-azido-[
-32P] ATP tightly
binds to ABCA1 in the presence of Mg2+.
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Fig. 5.
Vanadate-induced trapping in ABCA1-GFP.
A, photoaffinity labeling of ABCA1-GFP with
8-azido-[ -32P]ATP. Membranes (20 µg) prepared from
HEK293 cells stably expressing the wild-type (WT) ABCA1-GFP
(lanes 3, 4, 7, 8,
11, and 12) or from untransfected HEK293 cells
(HEK) (lanes 1, 2, 5,
6, 9, and 10) were incubated with 15 µM 8-azido-[
-32P]ATP in the absence or
presence of 1 mM ortho-vanadate (Vi)
and 3 mM MgSO4 (lanes 5-8) or
MnCl2 (lanes 9-12) for 15 min at 37 °C.
Proteins were photoaffinity-labeled with UV irradiation after removal
of unbound ligands and analyzed as described under
"Experimental Procedures." B, immunoblots of membranes
prepared from HEK293 cells stably expressing the wild-type (4 µg,
lane 1) or W590S (6 µg, lane 2) ABCA1-GFP or
from untransfected HEK293 cells (6 µg, lane 3). Proteins
were separated on a 7% SDS-polyacrylamide gel and reacted with
monoclonal antibody against GFP. C, membranes prepared from
HEK293 cells stably expressing the wild type (WT) (20 µg,
lanes 1 and 2) or W590S (30 µg, lanes
3 and 4) were incubated with 15 µM
8-azido-[
-32P]ATP in the absence or presence of 1 mM ortho-vanadate (Vi) and 3 mM MnCl2 for 15 min at 37 °C. Proteins were
photoaffinity-labeled with UV irradiation after removal of unbound
ligands and analyzed as described under "Experimental
Procedures."
-32P] ATP is therefore
stimulated when the membrane containing these proteins reacts with the
nucleotide in the presence of ortho-vanadate (21-24). We
thus expected that ortho-vanadate would stimulate
photoaffinity labeling of ABCA1. However, no increase of photoaffinity
labeling of ABCA1 was observed (lane 8) in comparison with
that in the absence of ortho-vanadate (lane
7).
-32P]ATP in the absence or presence of
ortho-vanadate. The photoaffinity labeling of W590S
ABCA1-GFP was stimulated by adding ortho-vanadate in the
presence of Mn2+ as much as in the presence of the wild type.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP. The
wild-type ABCA1-GFP was photoaffinity-labeled in the presence of
Mg2+, but no vanadate-induced nucleotide trapping was
observed, being consistent with human ABCA1 expressed in Sf9
insect cells (31). Other transporter-type ABC proteins, such as MDR1
(ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2), trap Mg-ADP in the presence of
ortho-vanadate and form a stable inhibitory intermediate
during the ATP hydrolysis cycle (21-24) so that ABCA1, showing no
obvious vanadate-induced nucleotide trapping, was proposed not to be an
active transporter but a regulator in apoA-I-dependent
cholesterol release (31). However, vanadate-induced nucleotide
trapping was demonstrated to be positive with ABCA1 in the presence of
Mn2+ in this study. Vanadate-induced nucleotide trapping
did not occur in the presence of Mg2+ but can be detected
with Ni2+ ions in MRP6 (ABCC6) (25), and it was detected in
ABCG2 with Co2+ ions (32). MRP6 and ABCG2 have been shown
to function as active transporters for an anionic cyclic pentapeptide
BQ-123 (33) and anticancer drugs (34), respectively. These results
suggest that ABCA1 may function as an active transporter in
apoA-I-dependent cholesterol release.
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ACKNOWLEDGEMENT |
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We thank Kyowa Hakko Kogyo Co. Ltd. for generating anti-ECD1 polyclonal antibody.
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FOOTNOTES |
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* This work was supported by Grant-in-aid for Scientific Research 10217205 on Priority Areas "ABC Proteins" from the Ministry of Education, Science, Sports, and Culture of Japan and by the Nakajima Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplementary data showing a figure
showing the trafficking of ABCA1-GFP in HEK293 cells and a figure
showing the characterization of ABCA1-GFP vesicles.
¶ These authors contributed equally to the work.
** To whom correspondence should be addressed. Tel.: 81-75-753-6105; Fax: 81-75-753-6104; E-mail: uedak@kais.kyoto-u.ac.jp.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M206885200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP-binding cassette transporter A1; ECD1, the first extracellular domain; NBF, nucleotide binding fold; TD, Tangier disease; FHA, familial hypoalphalipoproteinemia; PM, plasma membrane; ER, endoplasmic reticulum; Endo H, endoglycosidase H; PNGaseF, N-glycosidase F; GFP, green fluorescent protein.
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REFERENCES |
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