From the Centre d'Immunologie INSERM-CNRS de
Marseille Luminy, Parc Scientifique de Luminy 13288 Marseille, France,
§ Ecole Normale Supérieure de Physique de Marseille,
Institut Fresnel, Domaine Universitaire Saint Jérôme
13397 Marseille, France, and the ¶ Department of Biochemistry,
University of Gent, 9000 Gent, Belgium
Received for publication, November 10, 2000, and in revised form, January 9, 2001
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ABSTRACT |
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The identification of defects in ABCA1 as
the molecular basis of Tangier disease has highlighted its
crucial role in the loading with phospholipids and cholesterol of
nascent apolipoprotein particles. Indeed the expression of ABCA1
affects apolipoprotein A-I (apoA-I)-mediated removal of lipids from
cell membranes, and the possible role of ABCA1 as an apoA-I surface
receptor has been recently suggested. In the present study, we have
investigated the role of the ABCA1 transporter as an apoA-I receptor
with the analysis of a panel of transfectants expressing functional or
mutant forms of ABCA1. We provide experimental evidence that the forced
expression of a functional ABCA1 transporter confers surface competence
for apoA-I binding. This, however, appears to be dependent on ABCA1 function. Structurally intact but ATPase-deficient forms of the transporter fail to elicit a specific cell association of the ligand.
In addition the diffusion parameters of membrane-associated apoA-I indicate an interaction with membrane lipids rather than proteins. These results do not support a direct molecular interaction between ABCA1 and apoA-I, but rather suggest that the ABCA1-induced modification of the lipid distribution in the membrane, evidenced by
the phosphatidylserine exofacial flopping, generates a biophysical microenvironment required for the docking of apoA-I at the cell surface.
The removal of cellular lipids is promoted by high density
lipoproteins (HDL),1 the
plasma shuttle mediating reverse cholesterol transport from peripheral
tissues to the liver for further uptake and metabolism (1). However,
whether the interaction of the lipid-poor apoA-I particle, protein core
of the nascent HDL, with cell membranes is mediated by a specific
receptor and how its loading with phospholipids and cholesterol occurs
is still a matter of debate (2). The recent discovery that a defective
ABCA1 transporter leads to Tangier disease (3-9) has directly
implicated this transmembrane protein in the active release of cellular
lipids and prompted an investigation into its role as a candidate
apoA-I receptor (10, 11). Indeed a correlation between the cAMP-induced
cell surface apoA-I binding and the expression of ABCA1 in
macrophage-like cell lines has been reported (12). Very recently, in
addition, a direct molecular interaction between ABCA1 and apoA-I at
the cell surface has been proposed on the basis of chemical
cross-linking experiments (10, 11). To gain further insight into this
issue, we developed an apoA-I binding assay based on the use of a
fluorochrome-conjugated ligand. The analysis of apoA-I binding to a
panel of transfectants expressing either functionally intact or
defective ABCA1 proteins (13) led us to exclude that the transporter
behaves as a bona fide receptor for apoA-I. Indeed, whereas
surface binding increases with the expression of a functional ABCA1,
the expression of structurally intact but functionally impaired ABCA1
proteins fails to elicit specific binding. Considering that, as
previously demonstrated, ABCA1 promotes the transbilayer redistribution
of phospholipids at the plasma membrane (13), we propose that ABCA1
favors the specific docking of apoA-I at the cell surface by providing
a distinctive spatial arrangement of phospholipid species in the outer
membrane leaflet. This model is supported by: (i) the colinear increase
of apoA-I binding and exofacial PS exposure as a function of ABCA1
expression, and (ii) the mobility parameters of membrane-bound apoA-I.
Indeed, the values of translational diffusion coefficients, assessed by
fluorescence correlation spectroscopy (FCS) are consistent with the
molecular interaction of apoA-I with rapidly diffusing lipids rather
than membrane-anchored receptors (14-16).
Cell Culture--
RAW 264.7 cells (ATCC, Rockville, MD) were
routinely maintained in culture in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 1 mM sodium
pyruvate, 1 mM penicillin/streptomycin. ABCA1·EGFP,
mutant ABCA1·EGFP, and control transfectants were obtained as
described (13) and maintained under hygromycin selection (0.2 mg/ml).
cAMP stimulation was performed for 24 h at 37 °C in the
presence of 0.3 mM cpt-cAMP (Sigma-Aldrich).
Turbidimetric Assay--
Fluorescent-labeled apoA-I was tested
versus native apoA-I for its ability to interact with
dimyristoyphosphatidylcholine (DMPC) vesicles and analyzed by
monitoring the decrease in optical density at 325 nm as a function of
temperature as described (17, 18)
Lipids Effluxes--
Cells were labeled for 72 h in
Dulbecco's modified Eagle's medium containing 1% fetal calf serum,
1.5 µCi/ml [14C]cholesterol, and 10 µCi/ml
[3H]choline chloride (both from Amersham Pharmacia
Biotech). Cells were then incubated for 24 h in Dulbecco's
modified Eagle's medium, 1% fetal calf serum with or without 0.3 mM cpt-cAMP. Cells were then washed in phosphate-buffered
saline, 0.5% bovine serum albumin, and effluxes were performed for
16 h in a 0.5% bovine serum albumin medium with or without 10 µg/ml apoA-I. Medium was separated from cells, and lipids were
extracted with chloroform and methanol (19). Radioactivity in the
medium and cells was determined by liquid scintillation counting. The
percentage of efflux is expressed as the number of counts in the medium
divided by the total number of counts. Each value is the average of
four points.
Immunoprecipitation--
Immunoprecipitation analysis was
performed on 107 RAW 264.7 cells labeled overnight with 300 µCi/ml 35S protein labeling mix (PerkinElmer Life
Sciences). Immunoprecipitation of ABCA1 with an ABCA1 antiserum (Ab16,
1:500) was performed as described (20).
ApoA-I Binding--
Recombinant apoA-I, carrying an N-terminal
histidine tag was expressed in Escherichia coli and purified
as described (17). This recombinant protein shows physicochemical
properties similar to that of the native protein (21). ApoA-I was
conjugated to the fluorochrome with the fluorolinkTM Cy5
monofunctional dye 5-pack (PA25001, Amersham Pharmacia Biotech). For
all experiments the labeled apoA-I (apoA-I/Cy5) was diluted to 100 µg/ml in binding buffer (1.8 mM CaCl2, 1 mM MgCl2, 5 mM KCl, 150 mM NaCl, 10 mM HEPES, pH 7.4), and aggregates
were removed by ultracentrifugation for 30 min at 100,000 × g. Binding was performed in the presence of 10 µg/ml of
apoA-I/Cy5 (or as indicated for saturation experiments) for 1 h at
4 °C on 5 × 105 cells detached by mild
trypsinization (0.005% in phosphate-buffered saline). At the end of
the incubation period, cells were rapidly washed prior to fixation with
1% paraformaldehyde.
Annexin V Binding--
Annexin V (ann-V/Cy5) labeled as
described (22), was diluted at 4 µg/ml in binding buffer, and
aggregates were removed by ultracentrifugation for 30 min at
100,000 × g. Binding was performed in binding buffer
for 10 min at 4 °C, in the presence of a final annexin V/Cy5
concentration of 4 µg/ml. The mean of the relative fluorescence
intensity (RFI) for annexin V/Cy5 was calculated for each subset of
given EGFP RFI.
FACS Analysis--
Single or dual-channel flow cytometric
recordings were performed on a FACScalibur (Becton Dickinson) and
analyzed by Flowjow software (Tree Star Inc., San Carlos, CA). For each
type of cells, the same settings were kept for all experiments. Cells
were manually subdivided in function of their ABCA1 expression
reflected by EGFP RFI. Binding data are calculated from the mean of Cy5
RFI on the selected cell populations. cAMP or ABCA1-induced binding was
calculated as the point to point difference between cAMP-treated and
-untreated RAW cells or the EGFP-positive and -negative cells. Saturation curves were fitted according to the equation,
y = A + B(1
Quantitation of cell surface-associated ABCA1 was calculated by the
Optimas software (Media Cybernetics, Silver Spring, MD) on confocal
images (Leica TCS4D) as the ratio between total cell RFI and
membrane-associated RFI visually gated on single cells and on multiple sections.
Fluorescence Correlation Spectroscopy--
FCS measurements were
carried out with a ConfoCor2 module/LSM510 confocal microscope (Carl
Zeiss SA, LePecq, France). For FCS excitation, 488- and 633-nm laser
lines were used to illuminate a × 40 C-apochromat objective. In
the image plane, a 70- and a 90-µm pinhole for the 488- and 633-laser
line, respectively, defined the confocal volume. After the laser focal
spot had been positioned on the top of the cell, a set of five FCS
measurements of 10 s was recorded on that location and averaged
for determination of the diffusion coefficients. Measurements were
stepped in 1-µm increment from a z-stack of previously
recorded EGFP images of an individual cell. FCS measurements were
performed on AG cells after an incubation of 5 min at room temperature
in the presence of 40 nM apoA-I/Cy5. The apparatus was
calibrated by measuring the known three-dimensional diffusion of
rhodamine-6G in solution (D Fluorescence-based Assay for ApoA-I Binding--
We previously
reported that the forced expression of an ABCA1·EGFP chimera is able
to induce an increased cellular release of choline-containing
phospholipids and cholesterol to the specific acceptor apoA-I (13). In
the same experimental system, we also observed that a reduction of
lipid effluxes tracks the progressive silencing of ABCA1 expression
induced by tetracycline (not shown). To better characterize the
functional effects of the graded ABCA1 expression in transfected cells,
easily monitored by FACS analysis, we set out to develop a
fluorescence-based assay of apoA-I binding. The assay was first
validated by checking whether the fluorochrome conjugation altered the
physiological properties of apoA-I. No significant difference in the
behavior of labeled versus unlabeled apoA-I was detectable
by a standard turbidimetric assay for phospholipid binding (Fig.
1A) nor by a classical
phospholipid and cholesterol efflux assay from cAMP-treated RAW cells
(Fig. 1B).
The flow cytometric analysis of apoA-I cellular binding was then
performed on unstimulated RAW cells and showed a very low level of
cell-associated fluorescence (Fig.
2A). In agreement with
previously reported data (12, 25), the cell-associated fluorescence
homogeneously increased after cAMP treatment (24 h at 37°, 0.3 mM), as shown by the right shift of the mean RFI (3.4 ± 0.4-fold increase over unstimulated cells, n = 4).
As expected the cAMP-induced binding could be competed by a 50-fold
molar excess of unlabeled apolipoprotein. The increase in apoA-I
surface binding was paralleled by an increased synthesis of ABCA1
protein as detected by immunoprecipitation from metabolically labeled RAW cells (Fig. 2B). This is likely to result from a
cAMP-mediated transcriptional activation of the ABCA1 gene,
as suggested by Refs. 11, 25, and 26. The saturation curves measured in the presence of increasing amounts of labeled ligand on cAMP-stimulated and -unstimulated RAW cells allowed the estimation of the parameters of
specific cAMP-induced apoA-I binding in our assay
(Kd of 1.44 ± 0.12 µg/ml/5.1 ± 0.4 × 10 ApoA-I Surface Binding Requires Functional ABCA1--
To elucidate
the link between ABCA1 expression and apoA-I binding, we carried out a
set of experiments on macrophages derived from ABCA1-null
animals (9, 13), which showed a 2.1 ± 0.2-fold decrease in apoA-I
surface labeling versus cells from wild-type controls
(n = 2, not shown) and on AG cells, i.e.
HeLa cells expressing a functional chimeric ABCA1 transporter under the
control of a tetracycline-sensitive promoter (13). The chimera consists
of a C-terminal fusion of EGFP to the 2261 amino acid full-length mouse
ABCA1 transporter (GenBankTM/EBI accession number X75926;
nucleotides 84-6869, Ref. 28). By means of dual-channel flow
cytometric recordings, we analyzed the behavior of apoA-I surface
binding in these cells as a function of ABCA1 expression (Fig.
3). The cell population was subdivided into EGFP-negative cells (i.e. cells that have lost the
expression of the transporter) and EGFP-positive cells by manually
gating below or above the threshold of autofluorescence. As shown in Fig. 3A, EGFP-negative AG cells demonstrated a very low
binding, comparable with that of nonstimulated RAW or of control
mock-transfected HeLa cells.
Conversely, the whole population of EGFP-positive (RFI > 4 in the
experiment shown) AG cells shows a significant increase in
apoA-I-specific binding (3.2 ± 1.4-fold increase in mean RFI over
negative cells, n = 10), similar to that induced by
cAMP treatment on RAW cells. The specific binding showed an apparent Kd of 24.3 ± 8 × 10 ApoA-I Interaction with the Cell Membrane--
Because the
expression of ABCA1 in transfectants is heterogeneous, as indicated by
the broad distribution of EGFP fluorescence, we manually gated at
discrete EGFP fluorescence intensity intervals to detail the behavior
of apoA-I binding as a function of the expression of ABCA1 (Fig.
4A). The correlation between
apoA-I (reflecting the density of surface binding sites) and EGFP
fluorescent intensity (reflecting total cellular content in ABCA1),
shown in Fig. 4B, suggests that the surface binding of
apoA-I is sensitive to the density of ABCA1 molecules at the cell
surface. The latter cannot be assessed directly in our system but can
be extrapolated from the total EGFP fluorescence. Indeed the digital
quantification of fluorescence distribution on confocal microscopy
recordings in low, medium, and high ABCA1-expressing cells showed that
a stable fraction (35 ± 5%, n = 16) of total
cell-associated fluorescence can be attributed to molecules at the
plasma membrane. The saturation curve for apoA-I (Fig. 4B)
allowed to estimate that increasing amounts of ABCA1 at the cell
surface affected maximum binding without altering binding affinity (in
the experiment shown, values are: Kd, 27 × 10
We then similarly tested the correlation between ABCA1 expression and
the exposure of PS at the outer membrane, another ABCA1-elicited phenotype strictly dependent on the activity of the ABCA1 transporter (13). By plotting annexin V binding versus EGFP
fluorescence, we detected again a bimodal behavior with a drop in the
slope at high ABCA1 expression levels (Fig. 4C,
To explore further whether apoA-I cell surface association is mediated
via the interaction with a membrane-anchored receptor, we measured the
mobility parameters of apoA-I and ABCA1·EGFP by FCS (Fig.
4D). This method allows the quantitation of the retardation in diffusion acquired by apoA-I after its interaction with the cell
membrane of ABCA1-expressing AG cells. The translational diffusion
coefficient D In this study, we have described the development of a
fluorescence-based assay for apoA-I binding, which we applied to the investigation of the ABCA1 transporter as a candidate apoA-I receptor. Based upon our results, we can conclude that the surface expression of
ABCA1 is essential to the generation of specific cellular binding sites, but we ruled out ABCA1 as a molecular receptor for lipid-free apolipoproteins.
We actually observed that the expression of similar amounts of
correctly folded but functionally impaired ABCA1 molecules did not
elicit any apoA-I binding and that increasing the number of expressed
ABCA1 molecules did not colinearly increase the number of apoA-I
binding sites. Several molecular interpretations are possible. The
ATPase-deficient forms of ABCA1 may fail to adopt the molecular
conformation required for apoA-I docking. This implies that alternative
conformations of the transporter exist during the ATP cycle at both
sites and that only one of the transition states is permissive for
binding. This is conceivable, because it is known that ATP
binding/hydrolysis is able to modify solvent accessibility of ABC
transporters and could account for the similar loss of activity of the
three mutant transporters (30). However, the
energy-dependent conformational change does not provide a satisfactory explanation for the lack of increase in apoA-I binding capacity at high expression of functional ABCA1. This rather suggests that even the presence of functional ABCA1 at the cell surface is not
sufficient to generate apoA-I binding sites.
We may, hence, envision that the activity of ABCA1 modulates the
accessibility to apoA-I of a partner receptor, yet to be identified.
Inactive forms of ABCA1 would not be able to interact with the
receptor, and the availability of the molecule in the recipient HeLa
cell may be rate-limiting and thus account for the observed saturation
of surface binding at high expression of functional ABCA1.
Alternatively, the specific docking of apoA-I may not be mediated by
interaction with a unique protein receptor, but rather rely on a
meticulous molecular arrangement of lipids at the cell surface
(31-33). Only the latter would support the interaction of the
apolipoprotein with the membrane and the removal of phospholipids and
cholesterol which follows. According to this model, apoA-I binding
should be considered a consequence of the already demonstrated ability
of the transporter to modulate the transbilayer arrangement of lipids
(13). The fact that a saturable behavior is also observed when plotting
the expression of ABCA1 versus the exofacial PS exposure
supports this hypothesis. In the latter case saturation is not
surprising. It is conceivable that a cell tolerates only a limited
amount of PS on the outer leaflet and that above this threshold a
feedback response counteracts further membrane modifications
potentially dramatic for cell viability. From a molecular standpoint,
this may correspond to an overactivation of the aminophospholipid
translocase, or other enzymatic activities able to flip inward the
excess PS residues (34). The safety feedback loop will thus
concomitantly buffer further increases both of PS flop and apoA-I
binding. This hypothesis, schematized in Fig.
5, is reinforced by the diffusion
parameters of both membrane-bound apoA-I and ABCA1·EGFP as assessed
by fluorescence correlation spectroscopy. The translational diffusion
coefficient (D
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
e
X/C) and
Kd and Bmax values were
calculated from these (23). Bmax values are
given in arbitrary units (AU).
= 2.8 × 10
6
cm2/sec
1). Data fitting was performed with a
least squares algorithm. The apoA-I autocorrelation function (G
) was
fitted taking into account the isomerization state of Cy5 dye, the free
Brownian motion for unbound molecules, and two-dimensional motion for
membrane-bound molecules (15). The ABCA1·EGFP autocorrelation
function was fitted using the anomalous diffusion model (24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Fluorochrome conjugation does not alters
apoA-I function. Fluorochrome conjugation of apoA-I does not alter
its properties for binding phospholipids in a standard turbidimetric
assay (A) or for extracting choline-containing phospholipids
and cholesterol from cAMP-activated RAW cells (B). Specific
effluxes are: choline 3.9 ± 0.4% for unlabeled apoA-I and
3.1 ± 0.23% for apoA-I/Cy5, and cholesterol 11 ± 0.8% for
unlabeled apoA-I versus 9.6 ± 0.8% for apoA-I Cy5,
n = 2, for each experimental value averaged from
quadruplicates.
8 M, n = 3)
(Fig. 2C; Refs. 25, 27).
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Fig. 2.
Fluorescence-based assay for apoA-I
binding. ApoA-I binding on unstimulated or cAMP-stimulated RAW
cells shows a 3.4 ± 0.4-fold (n = 4) increase of
mean RFI after cAMP treatment (A), which is accompanied by
an increased synthesis of ABCA1 as detected by immunoprecipitation
(B, Ref. 12). Binding parameters in the presence of the
indicated amount of labeled apoA-I are shown in C. ,
binding on unstimulated RAW cells;
, cAMP-induced binding;
,
total binding on stimulated cells. cAMP stimulation was carried out for
24 h in the presence of 0.3 mM cAMP. In this
representative experiment of three, values are Kd,
4.6 × 10
8 M and
Bmax, 43 ± 9 AU.
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Fig. 3.
ApoA-I binding is exclusively induced by a
functional ABCA1 transporter. A, dot plots
of dual-channel flow cytometric analysis on AG and mock-transfected
control cells show that the presence of ABCA1 molecules (EGFP channel)
elicits apoA-I binding (Cy5 channel). The histogram panels show the
behavior of Cy5 RFI on the gated cell populations. A right shift of
mean RFI in ABCA1 positive versus negative or
mock-transfected cells is detectable. Gates are shown on the dot
panels. B, binding parameters of ABCA1-induced apoA-I
cell association as measured in the presence of the indicated
increasing amounts of labeled apoA-I. , EGFP-negative;
,
EGFP-positive cells;
, ABCA1-induced binding. In the representative
experiment (of 3) values are: Kd 21.5 × 10
8 M, Bmax 65.9 ± 5 AU. C, dual-channel cytometric analysis on
transfectants expressing mutants form of ABCA1 shows that the function
of ABCA1 is essential to elicit apoA-I binding. Dot plots
indicate the lack of increase in Cy5 RFI even at high values of EGFP
RFI. Histogram plots recorded on Cy5 channel further demonstrate that
the mean Cy5 RFI is indistinguishable from that of mock-transfected or
EGFP-negative cells. Gates are shown on the dot plots.
D, saturation curves of ApoA-I binding on MM cells,
expressing the ATPase-deficient form of ABCA1 show absence of specific
binding even at high levels of expression (assessed by EGFP RFI).
,
EGFP-negative;
, EGFP-positive cells.
8
M, 6.9 ± 2.4 µg/ml, n = 3 (Fig.
3B). In contrast to the macrophage cell line, no
modification of binding was observed after 24 h of cAMP incubation
or cholesterol loading of AG and mock-transfected cells (not shown).
This indicates that both stimuli, at least under our experimental
conditions, do not promote post-translational activation of the
transporter, and this indirectly confirms previous reports locating its
action to the transcriptional level. In addition, the lack of induction
of apoA-I binding on mock-transfected HeLa cells supports a
cell-restricted sensitivity of ABCA1 regulatory sequences to both the
cAMP and cholesterol-mediated activation (27). To establish whether the
physical presence of ABCA1 at the cell surface was sufficient to elicit
apoA-I binding, we measured the interaction of labeled apoA-I to
transfectants expressing mutant forms of ABCA1 (KM, MK, and MM). As
already described, the mutations harbored by these proteins hamper ATP
binding/hydrolysis at either or both the nucleotide-binding cassettes
of the transporter without altering its folding or its intracellular
routing (13). No specific apoA-I binding was detectable despite the
presence of equivalent amounts of transporter at the cell surface, as
indicated by similar intensity of EGFP fluorescence in the cell lines
tested (Fig. 3, C and D). These data thus clearly
indicate that an intact function of ABCA1 is essential to generate
apoA-I binding competence at the plasma membrane.
8 M and Bmax,
28 ± 2.6 for ABCA1+; 21 × 10
8 M
and Bmax, 78 ± 6.7 for ABCA++; 23 × 10
8 M and Bmax,
114 ± 6 for ABCA1+++). By plotting the values of mean RFI for
apoA-I (or the Bmax values) as a function of
ABCA1 expression (Fig. 4C,
), we observed that the
correlation coefficient between the two parameters decreased sharply at
high levels of ABCA1 expression. This apparent saturation indicates
that the availability of ABCA1 molecules at the cell surface is not the sole parameter affecting the binding of the apolipoprotein.
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Fig. 4.
ApoA-I interaction at the cell surface.
ABCA1-induced apoA-I docking at the cell surface is saturable.
A, dot plots of dual-channel flow cytometric
recordings on AG cells. Gates corresponding to three arbitrarily chosen
ABCA1 expression levels (+, ++, and +++) are shown. In the experiment
shown these are ABCA1+ EGFP RFI: >5 and <10; ABCA1++ EGFP RFI: >11
and < 50; ABCA1+++ EGFP RFI: >50. B, analysis of the
effect of increased ABCA1 expression on apoA-I binding parameters shows
that the binding affinity is unchanged, whereas
Bmax estimations increase as a function of ABCA1
expression. Kd 27 × 10 8
M and Bmax 28 ± 2.6 for
ABCA1+; 21 × 10
8 M for ABCA1++ and
Bmax 78 ± 6.7; 23 × 10
8 M and Bmax
114 ± 6 for ABCA1+++. Bmax values are in
arbitrary units. C, dual plot showing a saturable and
parallel behavior of surface PS exposure and apoA-I binding at high
level of ABCA1 expression. Values are expressed as mean of RFI for each
channel. A single experiment out of three is shown. D,
fluorescence correlation spectroscopy measurements. The diffusion
parameters of membrane-bound apoA-I (
) and ABCA1·EGFP (
) are
not consistent with a molecular interaction between the two partners.
Curves represent the autocorrelation function G(t) of apoA-I
and ABCA1·EGFP as indicated by arrows. The cross
correlation curve (CC) is also shown.
). This
suggests the possibility of a causal link between the two measured phenomena.
for free apoA-I was 2.4 × 10
7
cm2/sec
1 ± 0.6 on 13 independent
measurements and for membrane-bound D
= 1.6 × 10
8 cm2/sec
1 ± 1.0 measured on
10 independent cells. The latter values are close to those measured for
membrane lipids in fluid phase (15, 16, 29). The lateral diffusion
parameters of ABCA1 at the plasma membrane could not be fitted by
assuming a single population with uniform diffusion characteristics but
were consistently fitted with the model of anomalous diffusion. The
fitting of 13 different data sets from different cells allowed the
estimation of a diffusion coefficient
of 1.1 × 10
10 cm2/sec
± 0.3 with an
of 0.47 ± 0.06. The different diffusional
behavior of ABCA1·EGFP and apoA-I together with the lack of
significance of the cross-correlation curves indicates that the two
partners are not interacting in the time scale of the detection.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) of apo A-I suggests its molecular interaction with
lipids rather than with a protein receptor, which should theoretically
retard its mobility to a D
in the range of 10
9 to
10
10 cm2/sec
1 (15, 16, 29). On
the other hand, ABCA1·EGFP behaves according to an anomalous
diffusion model (24). This behavior has been previously reported for
membrane proteins, like IgE receptor and the LDL receptor (24) and
indicates obstruction in lateral diffusion likely to originate from the
interaction with other cellular components. In addition, the
cross-correlation analysis of diffusion parameters recorded for the
ligand and the candidate receptor excludes their interaction in the
time scale of the experimental detection.
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Fig. 5.
Mechanistic interpretation of ABCA1-induced
apoA-I docking. ABCA1 induces a local and transient modification
of the spatial arrangement of lipid species on the outer membrane
leaflet. This is witnessed by the exposure of PS but is likely to
result in a more complex and yet undetermined destabilization of
membrane lipid architecture. The ABCA1-promoted modification of
membrane environment is appropriate for apoA-I docking and the
following apoA-I-mediated extraction of phospholipids from the outer
leaflet. Cholesterol loading onto PL-loaded apoA-I will then follow
(2). A further step of lipid rearrangement across the bilayer may be
envisioned.
Our results are only apparently at odds with those of chemical
cross-linking reported in (10, 11), that rather emphasizes the spatial
proximity of ABCA1 and membrane-bound apoA-I even in the absence of
direct molecular interaction. We propose hence that the role played by
ABCA1 in promoting apoA-I binding is, in essence, a consequence of the
ABCA1-orchestrated modification of the biophysical properties of the
membrane. Whether the modification is homogeneously spread over the
cell surface or generates only locally and transiently a favorable
apoA-I docking environment remains still to be ascertained. The latter
case can be reasonably surmised on the basis of the reported
physical proximity of the two partners.
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ACKNOWLEDGEMENTS |
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We thank N. Duverger for discussion, P. Schwille and Carl Zeiss S. A. for help with fluorescence correlation spectroscopy and J. M. Freyssinet for recombinant annexin V.
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FOOTNOTES |
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* This work was supported by institutional funding from INSERM and CNRS and specific funding by the Association de Recherche pour le Cancer (ARC) and Ligue National Contre le Cancer (LNCC).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.
To whom correspondence should be addressed: Centre
d'Immunologie INSERM-CNRS de Marseille Luminy, Parc Scientifique de
Luminy Case 906, 13288 Marseille Cedex 09, France. Tel.: 33 4 91269404; Fax: 33 4 91269430; E-mail: chimini@ciml.univ-mrs.fr.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M010265200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; ABC, ATP binding cassette; ApoA-I, Apolipoprotein A-I; AU, arbitrary units; cpt-cAMP, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate; Cy5, cyanine 5; DMPC, dimyristoyphosphatidylcholine; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorter; FCS, fluorescence correlation spectroscopy; PS, phosphatidylserine; PL, phospholipids; RFI, relative fluorescence intensity.
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REFERENCES |
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