From the Lipid Metabolism Unit, Massachusetts General
Hospital and Harvard Medical School, Boston, Massachusetts 02114 and
the § University of Miami School of Medicine, Diabetes
Research Institute, R-134, Miami, Florida 33101
Received for publication, January 18, 2001, and in revised form, January 29, 2001
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ABSTRACT |
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Mutations in the ATP-binding cassette transporter
A1 (ABCA1) transporter are associated with Tangier disease and a defect in cellular cholesterol efflux. The amino terminus of the ABCA1 transporter has two putative in-frame translation initiation sites, 60 amino acids apart. A cluster of hydrophobic amino acids form a
potentially cleavable signal sequence in this 60-residue extension. We
investigated the functional role of this extension and found that it
was required for stable protein expression of transporter constructs
containing any downstream transmembrane domains. The extension directed
transporter translocation across the ER membrane with an orientation
that resulted in glycosylation of amino acids immediately distal to the
signal sequence. Neither the native signal sequence nor a green
fluorescent protein tag, fused at the amino terminus, was cleaved from
ABCA1. The green fluorescent protein fusion protein had efflux activity
comparable with wild type ABCA1 and demonstrated a predominantly plasma
membrane distribution in transfected cells. These data establish a
requirement for the upstream 60 amino acids of ABCA1. This region
contains an uncleaved signal anchor sequence that positions the amino
terminus in a type II orientation leading to the extracellular
presentation of an ~600-amino acid loop in which loss-of-function
mutations cluster in Tangier disease patients.
Cholesterol is an integral membrane constituent and a
precursor in multiple metabolic pathways, including steroid hormone synthesis and bile acid production. Since elevated levels of
unesterified cholesterol are toxic to cells (1), intricate mechanisms
have evolved to regulate both the endogenous synthesis of cholesterol and the uptake and metabolism of dietary cholesterol (2). Furthermore, it is now clear that cells can also reduce cholesterol accumulation through an active efflux process (3).
Perhaps the clearest demonstration of the importance of the active
efflux pathway in regulating cholesterol homeostasis is Tangier
disease, a rare autosomal recessive disorder that is characterized by
nearly absent levels of circulating high density lipoproteins and accumulation of cholesterol esters in peripheral tissues (4, 5). At
the cellular level, Tangier fibroblasts have impaired cholesterol
efflux in response to apolipoprotein stimulation while retaining their
ability to promote cholesterol efflux by passive diffusion to whole
lipoproteins and other artificial acceptors (6-8). Genetic mapping of
Tangier cohorts has shown that mutations in the ABC transporter,
ABCA1,1 segregate with a low
or absent high density lipoprotein phenotype and a defect in
cholesterol efflux (9-13). Conversely, overexpression of ABCA1 in cell
culture systems stimulates the efflux of cholesterol to apolipoprotein
A-I (14, 15). These findings tightly link ABCA1 function to the active
efflux of cellular cholesterol.
ABC transporters represent one of the largest gene families identified
to date, with representation in both prokaryotic and eukaryotic
organisms (16). The ABC transporters are polytopic membrane proteins
that couple ATP hydrolysis to the movement of a variety of molecules,
both small and large, across lipid bilayers. ABCA1 is a full ABC
transporter, with one open reading frame encoding two six-transmembrane
domains and two ATP binding cassettes. Like CFTR, the cystic fibrosis
transporter, ABCA1 has a large central region rich in polar and charged
residues similar to the CFTR regulatory domain, but it is unique in
that this domain is interrupted by a stretch of highly hydrophobic
amino acids (17). CFTR, like several other members of the ABC class,
lacks an obvious signal sequence at its amino terminus, indicating that
its first transmembrane domain probably performs the task of initially
anchoring the protein to the endoplasmic reticulum during protein
synthesis. The initial description of the mouse and human ABCA1
cDNAs suggested that a hydrophobic domain, located ~600 amino
acids from the amino terminus, would also serve this signal anchor
function (17, 18).
Recent work, however, has uncovered an in-frame, upstream methionine
that if used as the translation initiation start site would result in a
60-amino acid NH2-terminal extension of the protein
(19-21). This extension contains a potentially cleavable signal
sequence with cleavage predicted to occur between amino acids 45 and 46 (19). Wang et al. have recently reported that this extension
is important for efflux activity (15). Here we have investigated the
role that the amino-terminal extension plays in the cell biology of
ABCA1. We found that it was critical for stable expression of ABCA1, an
observation that could not be accounted for solely by the more
favorable context for translation initiation in which the upstream
methionine resides. Rather, the hydrophobic amino acids in the
extension appear to function as a topologically important signal anchor
sequence whose cleavage is not required for efflux activity. The signal
anchor sequence inserts transporter constructs into the endoplasmic
reticular membrane with the NH2 terminus of the protein on
the cytosolic face of the membrane. This orientation places the largest
putative amino acid loop of the transporter in the exoplasmic space. It
is within this loop, previously proposed to be an intracellular domain,
that a large number of loss-of-function mutations in Tangier disease
patients are clustered. Finally, we provide evidence that the signal
anchor sequence is not cleaved from the transporter, permitting us to tag the amino terminus of the protein with GFP and to visualize the
transporter's cellular localization by confocal fluorescent microscopy.
These results establish the importance of the upstream 60 amino acids
for protein expression and indicate that the putative signal anchor
sequence contained within the extension does indeed function to
correctly orient the transporter in the membrane. The orientation
imposed by the use of this signal anchor sequence is different from
that which has been proposed in previous models of ABCA1. Thus, our
results necessitate a reconsideration of the transporter's membrane topology.
Reagents--
The following reagents were purchased from the
indicated suppliers: LipofectAMINE-2000 (Life Technologies, Inc.),
Ni2+-nitrilotriacetic acid-agarose (Qiagen),
N-glycosidase F (New England BioLabs, MA).
DNA Constructs--
ABCA1 cDNAs were constructed by ligation
of reverse transcriptase-polymerase chain reaction-generated fragments
amplified from normal human fibroblast RNA. The resulting clones were
sequenced in their entirety on both strands and found to be identical
to previously published wild type human cDNA sequences except at four codons that have been reported to represent common human polymorphisms (G for A at 2649 (Met for Ile at amino acid 883), T for C at 4943 (Leu for Pro at amino acid 1648), A for G at 5921 (Lys
for Arg at amino acid 1974), T for C at 6503 (Leu for Pro at amino acid
2168) (12, 22, 23). For purposes of comparison with the older
literature on human ABCA1, 60 amino acids or 60 nucleotides must be
subtracted from our sequence numbers in order to match designations
taken from clones lacking the full first exon; the older numbering
designation used a putative start site of translation at the codon
beginning with nucleotide 121 (12, 22). The nucleotide and amino acid
numbering scheme used in this paper designates the upstream initiator
methionine as the first amino acid, and the codon that encodes it
represents nucleotide positions 1-3.
Wild type ABCA1 cDNAs, starting at nucleotide position 1 or 115, were cloned into the expression plasmid, pcDNA1 (Invitrogen, Carlsbad, CA). Modifications in these clones were made using standard recombinant DNA methods in order to generate proteins with alterations in their NH2 and COOH termini. The proteins used in these
studies are depicted in Fig. 1A and are as follows: 1)
full-length ABCA1 (wild type (WT) ABCA1), ABCA1 initiating at
Met-1 and ending with amino acid 2261; 2) epitope-tagged ABCA1 (c1),
full-length ABCA1 to which a 9-amino acid epitope tag (TETSQVAPA),
derived from bovine rhodopsin, was added at the C terminus of the
protein (this tag is recognized by the ID4 monoclonal antibody)
(24-26); 3) ABCA1 initiating at Met-61 (c2) containing the rhodopsin
epitope tag; 4) ABCA1 truncated after the first 630 aa (c3), tagged
with the C-terminal rhodopsin epitope; 5) ABCA1 initiating at Met-61
and truncated at aa 630 (c4), tagged with the C-terminal rhodopsin epitope; 6 and 7) c5 and c6, derivatives of c3 and c4, respectively, to
which the amino acids representing the putative first six-transmembrane cassette (aa 631-867) were added upstream of the rhodopsin epitope; and 8) GFP-ABC, an enhanced green fluorescent protein fused in-frame to
WT ABCA1 at the alanine at amino acid position 2 (the ABCA1 initiator
methionine was deleted).
For antibody production, DNA sequences representing amino acids
2071-2261 of ABCA1 were ligated into the pQE-30
NH2-6-histidine plasmid (Qiagen). All constructs were
verified by restriction analysis with sequencing on both strands of
regions that had been generated by polymerase chain reaction.
Construct Expression and Polypeptide Purification--
COS-7 or
HEK293 cells were transfected with LipofectAMINE-2000 as recommended by
the manufacturer in 10% Dulbecco's modified Eagle's medium with
DNA-LipofectAMINE complexes formed in serum-free Opti-MEM I
(Life Technologies, Inc.). 24-48 h after transfection, cells were used
in efflux assays or harvested for Western or Northern blotting as
described below. The 6-His-tagged ABCA1 polypeptide was expressed in
XL1-Blue bacteria and purified by denaturing Ni2+
chromatography as recommended by the manufacturer (Qiagen).
Antibodies and Immunological Methods--
Samples were subjected
to SDS-PAGE, and separated proteins were transferred to nitrocellulose.
Membranes were blocked in 1× PBS, 5% nonfat dry milk, 1% bovine
serum albumin, and probed with anti-ABCA1 sera (1:1000) in blocking
buffer. Bound antibody was detected with an anti-rabbit IgG-horseradish
peroxidase antibody and enhanced chemiluminescence (Super Signal;
Pierce) followed by exposure to x-ray film, or alternatively, images
were captured by digital photography (FluorChem Imaging System, Alpha
Innotech Corp, San Leandro, CA).
Metabolic Labeling and Immunoprecipitation--
36 h after
transfection, cells were washed twice with warm PBS and incubated in
methionine-free Dulbecco's modified Eagle's medium for 15 min at
37 °C. [35S]methionine (Tran35S-label;
ICN) was then added to a final concentration of 80 µCi/ml, and the
cells were further incubated for 2.5 h at 37 °C followed by a
chase in complete media for 2.5 h. After labeling, cells were chilled on ice, washed twice with cold PBS, and lysed in lysis
buffer. For deglycosylation assays, immunoprecipitated proteins were
heated for 10 min at 100 °C, brought to 50 mM sodium
phosphate, pH 7.5, and 1% Nonidet P-40, and incubated with 1500 units
of N-glycosidase F for 1 h at 37 °C, as recommended
by the manufacturer (New England BioLabs). After SDS-PAGE,
immunoprecipitated proteins were detected using a PhosphorImaging
STORM860 system (Molecular Dynamics, Inc., Sunnyvale, CA). Relative
molecular weights of the c3 and c4 polypeptides were determined using
an exponential equation (y = 270.069 *
10 Preparation of Cell Membranes and Sucrose Density
Dradients--
Cells were scraped into PBS and collected by low speed
centrifugation. Cell pellets were resuspended in lysis buffer and
homogenized as described above. The suspension was centrifuged at
800 × g for 5 min to remove nuclei and unbroken cells.
The resulting supernatant was centrifuged at 15,000 × g for 10 min. The 15,000 × g pellet was
resuspended in 1.0 ml of lysis buffer using 10 strokes of the Dounce
homogenizer with the loose fitting pestle. The 15,000 × g supernatant was centrifuged at 100,000 × g for 60 min, and the resulting pellet was resuspended in
buffer as above. 0.7 ml of the 15,000 × g membrane
suspension was applied to the top of a 10-ml linear 15-40% sucrose
gradient formed on top of a 0.5-ml 70% sucrose cushion and centrifuged
at 160,000 × g for 18 h at 4 °C in an SW41Ti
rotor. Fractions were displaced from the bottom of tube with 70%
sucrose, and 1-ml fractions were collected. Fractions were stored at
Confocal Microscopy--
293 cells grown on glass coverslips
were transfected with the GFP-ABCA1 construct or unfused GFP. 36 h
after transfection, cells were washed with PBS and fixed in 2%
paraformaldehyde-PBS for 5 min. Mounted coverslips were analyzed on a
Leica TCS SP confocal scanning laser microscope using a × 63 oil
immersion lens. Images of 0.5-µm sections were captured using
the Leica TCS-NT software package.
Specificity of the Anti-ABCA1 Antibody Used for Transporter
Analysis--
A rabbit polyclonal antibody to the C terminus of human
ABCA1 was generated using a 6-histidine fusion protein representing amino acids 2071-2261 as described under "Experimental Procedures" (Fig. 1B). The polypeptide
proved to be immunogenic, inducing a high titer antisera that was
specific for ABCA1 by three criteria. First, in normal human
fibroblasts, it recognized a polypeptide running at ~240-250 kDa
that was inducible by cholesterol (Fig. 1C and data not
shown). Second, since ABCA1 is a member of a large family of homologous
transporters, we tested the specificity of the antisera in fibroblasts
taken from a patient with Tangier disease whose ABCA1 gene contains a
homozygous frameshift mutation. This patient's deletion of two base
pairs from exon 22 would result in termination of ABCA1 proximal to the
sequences contained in the polypeptide used to generate the antisera
(11, 21). As expected, a lysate of cultured fibroblasts from this
patient (TD2, Fig. 1C), loaded with cholesterol,
showed no immunoreactivity for ABCA1. The expected ~250-kDa protein
was readily detected in lysates of fibroblasts taken from normal
individuals (Nl) as well as from another patient with
Tangier disease (TD1, Fig. 1C) whose ABCA1 gene
contains a point mutation that has previously been shown not to ablate
protein expression (28). Finally, in 293 cells transfected with
full-length ABCA1, a band of the expected molecular weight was
produced, while mock-transfected cells had no immunoreactive band (Fig.
1D).
The Upstream 60 Amino Acids of ABCA1 Are Required for Polytopic
Protein Expression in Transfected Cells--
To investigate the
importance of the putative first 60 amino acids of ABCA1, we
constructed several cDNAs encoding proteins that either did or did
not contain these residues (Fig. 1A, c1 and c2,
respectively). These cDNAs were C-terminally tagged with a
rhodopsin epitope sequence, cloned into an expression vector, and then
transfected into 293 cells. Expression of the constructs was first
compared by radiolabeling cellular proteins followed by
immunoprecipitation with the ID4 monoclonal antibody that recognizes the rhodopsin epitope (Fig.
2A). Compared with cells
expressing the full-length construct (c1), ABCA1 lacking the 60-amino
acid extension (c2) produced little or no protein. These
immunoprecipitation results were confirmed using the anti-ABCA1
antisera in immunoblots of 293 cells transfected with the c1 and c2
constructs (Fig. 2B). Similar to the immunoprecipitation
data, the construct initiating at the downstream methionine 61 yielded
little or no signal by immunoblot, whereas the full-length construct
produced a robust band. Since Northern analysis showed comparable
levels of ABCA1 mRNA generated by both the c1 and c2 constructs
(Fig. 2C), the lack of protein expression by the c2
construct could not be due to a poorer transfection efficiency or
transcription rate of the DNA encoding the truncated protein.
Translation Initiation Can Occur at Met-61--
Since the
nucleotide sequences surrounding the downstream methionine at position
61 have a suboptimal context for translation initiation (29-31), a
relatively trivial explanation for the above finding would be that
constructs dependent on initiation at Met-61 are poorly translated
(19). To test the ability of Met-61 to serve as a translational
initiator, a construct was generated which truncated ABCA1 just
proximal to its first putative transmembrane domain. Although
translation initiation at Met-61 did not support significant expression
of the full transporter, it was able to drive expression of the 570-aa
segment of ABCA1 that is proximal to this putative first transmembrane
domain (Fig. 3A, mock
versus c4, performed in duplicate). The expression of c4 was
next compared with a construct that was otherwise identical (c3) but
included the 60-aa extension (Fig. 3B, compare c4
versus c3). Expression of c4 was somewhat decreased compared
with c3, but this difference could not solely account for the
difference in expression of the full transporters shown in Fig. 2.
However, when the proximal six-transmembrane cassette (6-TM1) was added
back to these truncation mutants, expression of the half-transporter
was markedly diminished in the construct initiating at Met-61 (Fig.
3B, compare c6 versus c5). These data indicate
that the first 60 amino acids of ABCA1 provide a function that is
critical to expression of the polytopic membrane protein. The marked
size difference and heterogeneity of the band representing c3
(molecular mass of ~90 kDa) when compared with the c4 band (~60
kDa) could not be explained by their predicted polypeptide molecular
masses (72.2 versus 64.9 kDa, respectively), suggesting that c3 was undergoing a post-translational modification. Since previous models of the topology of ABCA1 had suggested that all
but one of the potential N-glycosylation sites in these
polypeptides were intracellular and therefore would not be
glycosylated, we next examined the post-translational modifications of
these proteins.
The 60-aa Extension Induces the Translocation and Glycosylation of
Residues Proximal to the First Putative Transmembrane
Domain--
There are 12 potential N-glycosylation sites
between the putative signal sequence and amino acid 630, where the c3
and c4 constructs were truncated. To examine glycosylation, ABCA1
constructs were transfected into COS-7 cells, and
[35S]methionine-labeled proteins were immunoprecipitated
with the ID4 monoclonal antibody. The precipitated proteins were then
either treated or not treated with the glycoamidase enzyme,
N-glycosidase F, and separated by SDS-PAGE. The c4
polypeptide, which initiates at Met-61, ran as a narrow band whose
migration was unaffected by glycoamidase treatment (Fig.
3C). In contrast, glycoamidase treatment of the c3
polypeptide significantly increased its migration rate and resolution.
The c5 polypeptide, which contains the putative transmembrane domains
of the half-transporter, also had its migration dramatically increased
by deglycosylation, whereas the c6 protein, lacking the signal
sequence, was barely detectable in this assay (Fig. 3B and
data not shown). These findings indicate that ABCA1 constructs that
contain distal transmembrane domains require the upstream signal
sequence for stable protein expression and cannot use the downstream
transmembrane domains as surrogates for signal anchor activity. In the
absence of this signal sequence, protein detection is dramatically
compromised, presumably due to protein degradation resulting from
misfolding following the failure to insert into the membrane of the
endoplasmic reticulum. These results establish that the 60-amino acid
extension of ABCA1 contains a functional signal sequence that directs
the translocation of distal sequences across the endoplasmic reticulum.
This translocation results in the N-glycosylation of
asparagine residues located in the region from amino acid 98 (the first
potential glycosylated asparagine) to amino acid 630, the residue at
which the constructs used were terminated.
Signal Sequence Cleavage Is Not Required for Efflux
Activity--
Several of the computer algorithms used to predict
signal sequence structure suggested that the ABCA1 sequence would be
cleaved between residues 45 and 46 (19, 21). Fig. 3B
provides conflicting evidence as to the cleavage of the signal
sequence. The molecular weight difference between polypeptides c3 and
c4 is determined by the number of residues remaining in the 60-amino
acid extension following translocation and signal cleavage, should the
latter occur. If no cleavage occurs, the entire 60-amino acid extension would be present with the increase in size of c3 over c4 calculated to
be 7.3 kDa. With cleavage after residue 45, c3 would only be 15 amino
acids larger than c4, a size difference calculated to be 1.8 kDa. The
relative molecular weights of the deglycosylated c3 and c4 proteins
were determined by comparison with unstained molecular weight markers
using SDS-PAGE (methods and data not shown). The difference between c3
and c4 was found to be 6.4 kDa (c3, 69.7 kDa versus the
expected 72.3 kDa; c4, 63.3 kDa versus the expected 64.9 kDa) and indicated no signal cleavage. In contrast, the c5 protein was
noted to yield a doublet on SDS-PAGE (Fig. 3, B and
C), suggesting the possibility that the faster migrating band might arise from signal cleavage of the polypeptide following membrane translocation.
To examine this issue further, we tested whether secretion of the c3
protein occurred. Lacking the distal transmembrane segments of the
transporter contained in the c5 construct, the c3 polypeptide's cleavage by a signal peptidase would be predicted to yield a soluble protein within the ER lumen whose secretion could potentially be
detected in media taken from transfected cells. Fig.
4 demonstrates that we were unable to
detect any secreted, rhodopsin-tagged c3. In contrast, the expression
of another rhodopsin-tagged type II receptor, engineered to contain a
cleavable signal sequence in place of its transmembrane anchor (SR-A),
was readily detected after immunoprecipitation with the ID4 antibody
(26). These results provide further evidence that the signal sequence
of ABCA1 is not cleaved. If not cleaved, it would thus serve as a
transmembrane anchor of the amino terminus of the protein. Variable
post-translational modifications in the c5 protein, perhaps arising as
a result of the protein overexpression achievable with transfected
cDNAs, could account for the c5 doublet visualized in Fig. 3, but
the significance of this finding remains unresolved at present.
Since we were interested in tagging the NH2 terminus of the
ABCA1 protein with a green fluorescent protein, it was important to
examine whether any protein cleavage might separate GFP from downstream
sequences, rendering the fusion protein useless as a surrogate for
ABCA1. 293 cells were transfected with GFP-ABCA1 or WT ABCA1 constructs
(Fig. 1A) and labeled with [35S]methionine.
Immunoprecipitation of the labeled proteins with a monoclonal antibody
directed against GFP, in cells transfected with the GFP-ABCA1
construct, precipitated a protein with the expected size of the
chimeric protein (Fig. 5A).
This protein was not detected with a nonspecific antibody (IgG) or with
the GFP antibody in cells transfected with wild type ABCA1,
establishing the specificity of the immunoreaction. We therefore
performed additional immunoprecipitations to determine if any cleaved
GFP could be detected in cells transfected with the chimera. Lysates from cells transfected with native GFP contained the expected 27-kDa
protein precipitable with the anti-GFP monoclonal antibody, whereas
cells transfected with GFP-ABCA1 did not. (Fig. 5B). To confirm that GFP was not cleaved from GFP-ABCA1, the migration of wild
type ABCA1 and GFP-ABCA1 was analyzed on denaturing SDS-PAGE gradient
gels. After transfer to nitrocellulose and detection by immunoblotting
with the polyclonal anti-ABCA1 antibody, the cells transfected with the
chimeric transporter contained only a single immunoreactive protein
(Fig. 5C). Since the molecular weight of this protein was
greater than that of the wild type ABCA1, these results confirmed the
lack of cleavage of a GFP-containing fragment from the fusion
protein.
GFP-ABCA1 Retains Cholesterol Efflux Function--
The inclusion
of an uncleaved GFP tag at the amino terminus of ABCA1 could alter the
transporter's cellular localization and processing. To test the
functional significance of that modification, we compared the efflux
activity of the GFP chimera to that of wild type ABCA1. 293 cells were
transfected with empty vector or the ABCA1 constructs, labeled with
[3H]cholesterol for 24 h, and then incubated with or
without apoA-I for 4 h, as previously described (15). Compared
with mock-transfected cells, the ABCA1 construct significantly
stimulated efflux in an apoA-I-dependent manner (Fig.
6). The GFP chimera's efflux activity
was equal to or greater than that of the wild type ABCA1. These results
indicate that ABCA1-mediated cholesterol efflux activity does not
require signal sequence cleavage and that the GFP-ABCA1 chimera is a
functionally equivalent surrogate for the wild type protein in this
efflux assay.
GFP-ABC Co-localizes with Wild Type ABCA1 to the Plasma
Membrane--
To explore the cellular distribution of ABCA1 and the
GFP-ABCA1 chimera, we used cell fractionation methods to examination their localization. Initial fractionation of cell homogenates taken
from 293 cells transfected with WT ABCA1 and GFP-ABCA1 demonstrated that virtually all of the anti-ABCA1 immunoreactive protein was present
in membranes that pelleted at 15,000 × g (Fig.
7A). Membranes pelleted at
100,000 × g from the 15,000 × g
supernatant had little or no detectable ABCA1 immunoreactivity. The
distribution of WT ABCA1 and GFP-ABCA1, expressed in 293 cells, was
further examined by density gradient centrifugation of the 15,000 × g membrane fraction. The distribution of WT ABCA1 and the
GFP chimera were similar across the sucrose fractions (Fig. 7,
B and C). Measurement of free cholesterol in the
gradient fractions, used as a marker for plasma membranes, showed that
the peak cholesterol concentration coincided with the peak of ABCA1
immunoreactivity (Fig. 7D). Neither ABCA1 nor cholesterol
content coincided with the distribution of total membrane protein in
the fractions (Fig. 7E), suggesting co-localization to the
cell surface. While these data establish that ABCA is present in a
cholesterol-rich membrane fraction, having the expected density of
plasma membranes, further studies are needed to verify the identity of
the membrane fractions. Nevertheless, these results indicated that
fluorescent localization of the GFP-ABCA1 chimera could be used as a
marker of the cellular distribution of the wild type transporter.
293 cells were therefore transfected with native GFP, the GFP-ABCA1
chimera, or empty vector constructs. 36 h after transfection, cells were fixed in 2% paraformaldehyde and analyzed by confocal microscopy. Mock-transfected cells showed little or no intrinsic fluorescence (Fig. 8A)
compared with GFP-transfected cells (Fig. 8B). The latter
demonstrated an intense, continuous distribution of fluorescence
throughout the cytosol, whereas the GFP-ABCA1 chimera decorated
the cell membrane as well as apparent internal vesicular structures
(Fig. 8C).
ABCA1 is a polytopic membrane protein involved in the efflux of
cholesterol and phospholipid to apoA-I. A potential 60-amino acid
extension of the human ABCA1 protein was recently identified, and Wang
et al. have demonstrated that this extension is important for efflux activity in 293 cells (15). No direct data were provided to
explain the requirement for that activity. In this work, we demonstrate
that this amino-terminal extension is required for stable expression of
full-length human ABCA1. Using a series of constructs initiating at
either Met-1 or at the first internal methionine (Met-61) of ABCA1, our
results indicate that both residues can serve as effective translation
initiation sites for a truncated, cytosolic ABCA1 mutant protein. Only
the upstream methionine, however, leads to the generation of stable
protein expression in constructs that contain any of the downstream
transmembrane domains of ABCA1. This finding suggested that the
hydrophobic residues contained within the amino-terminal extension were
serving as a signal sequence that directed translocation of the
transmembrane domains across the endoplasmic reticulum membrane. We
then directly demonstrated the translocation function of the signal
sequence residues in truncated ABCA1 mutants and showed that this
results in the N-glycosylation of residues immediately
distal to the signal sequence. This finding is inconsistent with the
topological models initially proposed for ABCA1 in which the first 580 (now 640) amino acids were modeled as a large intracellular domain.
Although our data contradict some prior models of ABCA1 topological
orientation (17, 32), it is entirely consistent with the behavior of
many signal anchor sequences embedded in similar charged amino acid
contexts (33). The predominance of positively charged residues
amino-terminal to the signal sequence and negatively charged residues
distal to it would predict that the NH2-terminal 25 amino
acids of the ABCA1 protein would face the cytosol, yielding a type II
orientation of the signal anchor sequence (Fig.
9). Our results, demonstrating
N-glycosylation of at least some of the 12 potentially
glycosylated asparagines immediately distal to the signal sequence,
indicates that the large protein loop between the signal sequence and
next putative transmembrane domain resides outside the cell. Indeed,
the ABCR transporter that is a close homologue of ABCA1 also contains
an amino-terminal hydrophobic domain and has been modeled with its
first major hydrophilic domain extracellular and glycosylated (34). An
analysis of the sequences of the currently known members of the ABCA
family of transporters suggests that they all contain the putative
signal anchor domain that we have demonstrated to be functional in
ABCA1.2 Thus, it seems likely
that this topological orientation will be a shared feature of the class
(35).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.029x, r2 = 0.986)
derived from a standard curve of unstained protein molecular weight
markers (Sigma).
70 °C. Equal volumes from each fraction were analyzed by
denaturing PAGE utilizing a Tris acetate-buffered 3-8% polyacrylamide gel (Invitrogen, Carlsbad, CA) under reducing conditions, according to
the manufacturer's procedures. After electrophoresis, proteins were
transferred to nitrocellulose and immunoblotted as described above.
Aliquots from each fraction were taken to measure total protein, by the
BCA method (Pierce), or free cholesterol (27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DNA constructs and anti- ABCA1
antibody specificity. A, ABCA1 constructs. Diagrammed
are the constructs used in the studies that follow: full-length ABCA1
(WT ABCA1), GFP fused to the NH2 terminus of full-length
ABCA1 (GFP-ABCA1), full-length ABCA1 tagged at the carboxyl terminus
with the rhodopsin epitope (c1), ABCA1 initiating at the first internal
methionine (Met-61) and carrying the rhodopsin epitope (c2), amino
acids 1-630 of ABCA1 tagged with the rhodopsin epitope (c3), amino
acids 61-630 of ABCA1 tagged with the rhodopsin epitope (c4), amino
acids 1-867 of ABCA1 tagged with the rhodopsin epitope (c5), and amino
acids 61-867 of ABCA1 tagged with the rhodopsin epitope (c6).
B, purified 6-His polypeptide encoding ABCA1 amino acids
2071-2261, which was injected into rabbits as described under
"Experimental Procedures" was immunoblotted with the resultant
ABCA1 anti-serum. C, anti-ABCA1 antibody specificity.
Immunoblot of normal (Nl) and Tangier fibroblasts induced
with cholesterol is shown; TD1 has a point mutation that maintains
expression, whereas TD2 has a two-base pair deletion that frameshifts
the protein proximal to the region recognized by the ABCA1 antiserum.
D, immunoblot of 293 cells mock-transfected with empty
vector (mock) or transfected with WT ABCA1.
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Fig. 2.
The NH2-terminal 60-amino acid
extension is required for efficient expression of full-length
ABCA1. A, 36 h after transfection with either
empty vector (mock) or the indicated ABCA1 constructs, COS-7 cells were
labeled with [35S]methionine. Cell lysates were generated
and normalized for cell associated counts. Immunoprecipitation of
proteins containing the rhodopsin epitope tag with the 1D4 monoclonal
reactive were performed as described under "Experimental
Procedures." Immunoprecipitated proteins were separated by SDS-PAGE
and visualized by phosphorimaging. B, 293 cells were
transfected as in A, and equal amounts of postnuclear
supernatant protein (10 µg) were separated by SDS-PAGE, transferred
to nitrocellulose, and analyzed by immunoblotting with the anti-ABCA1
antisera (1:1000 dilution). Bound antibody, detected with anti-rabbit
horseradish peroxidase, was visualized by ECL and autoradiography.
C, 293 cells were transfected as above, and total RNA was
isolated, fractionated on 1% formaldehyde-agarose gels, and
transferred to nitrocellulose. ABCA1 mRNA was detected using a
32P-radiolabeled probe derived from ABCA1 cDNA
(top panel). The membrane was stripped, and total
RNA quantitation was demonstrated by hybridizaton with a radiolabeled
18 S ribosomal RNA oligonucleotide probe (lower
panel). Shown are phosphor images of the probed
membranes.
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Fig. 3.
Met-61 supports expression of a polypeptide
truncated proximal to the first putative transmembrane domain of ABCA1
but not a protein containing the first six-transmembrane cassette.
A, an ABCA1 construct initiating at Met-61 but lacking any
transmembrane domains is readily detected in transfected cells. COS-7
cells were transfected with empty vector (mock) or the c4
construct and labeled with [35S]methionine, and
immunoprecipitations were performed and analyzed as in Fig.
2A. B, the addition of the transmembrane domains
to constructs initiating at Met-61 leads to loss of protein detection.
COS-7 cells were transfected with empty vector (mock) or the
indicated ABCA1 constructs and labeled with
[35S]methionine, and immunoprecipitations were performed
and analyzed as in Fig. 2A. C, endoglycosidase
treatment removes sugar groups from ABCA1 constructs that contain the
signal sequence domain but not those initiating at Met-61. COS-7 cells
were transfected with the indicated constructs and labeled, lysed, and
immunoprecipitated as above. Prior to gel electrophoresis, the
indicated samples were treated with N-glycosidase F as
described under "Experimental Procedures."
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Fig. 4.
A lack of secreted c3 polypeptide indicates a
lack of signal sequence cleavage. COS-7 cells were transfected
with empty vector (mock), the c3 construct (c3), or a
secretable form of the scavenger receptor-A (SR-A) and labeled with
[35S]methionine. Immunoprecipitations were performed with
the ID4 monoclonal antibody to detect secreted proteins containing the
rhodopsin epitope. Precipitated proteins were separated by SDS-PAGE and
visualized by phosphorimaging.
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Fig. 5.
A GFP-ABCA1 fusion shows no evidence of
signal sequence cleavage. A, the GFP-ABCA1 chimera is
detectable with an anti-GFP antibody. 293 cells were transfected with
GFP-ABCA1 or WT ABCA1 as indicated and labeled with
[35S]methionine, and immunoprecipitations were performed
with the indicated antibodies, normal mouse IgG (IgG), or the GFP
monoclonal antibody (GFP) as described under "Experimental
Procedures." PhosphorImager analysis of precipitated proteins after
separation by 6% SDS-PAGE is depicted. B, monomeric GFP is
not cleaved from the GFP-ABCA1 chimera. 293 cells were transfected with
unfused GFP or GFP-ABCA1 and labeled with
[35S]methionine, and immunoprecipitations were performed
with the indicated antibodies as in A. Shown is a phosphor
image of precipitated proteins after separation by 15% SDS-PAGE.
C, the GFP-ABCA1 chimera migrates as a single protein of
larger molecular weight than WT ABCA1. 293 cells were transfected with
WT ABCA1 or GFP-ABCA1, and 10 µg of a postnuclear supernatant was
fractionated by 2-15% SDS-PAGE, transferred to nitrocellulose, and
probed for ABCA1 expression with the anti-ABCA1 antisera as in Fig.
2B.
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Fig. 6.
GFP-ABCA1 has apoA-I-stimulated efflux
activity comparable with WT ABCA1. 293 cells were transfected with
empty vector (mock), WT ABCA1, or GFP-ABCA1, and the
cholesterol pool was labeled with [3H]cholesterol.
36 h after transfection, cells were washed in media
containing 1 mg/ml fatty acid-free albumin and exposed to 10 µg/ml
apoA-I as indicated for 4 h. Cholesterol effluxed to the
media was determined by scintillation counting and expressed as
a percentage of total cholesterol (medium + cell-associated).
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Fig. 7.
Membrane distribution of wild type ABCA1 and
GFP-ABCA1 chimera. 293 cells were transfected with WT ABCA1 or
GFP-ABCA1 chimera, and 36 h after transfection cells were
collected and homogenized as described under "Experimental
Procedures." The homogenate was centrifuged at 1000 × g for 10 min to remove nuclei and unbroken cells, and the
resulting supernatant was centrifuged at 15,000 × g
for 10 min. The 15,000 × g supernatant was centrifuged
for 1 h at 100,000 × g. Membrane pellets were
resuspended in buffer. A, 10 µg of protein from the
15,000 × g (15K) and the 100,000 × g (100K) pellets were separated by PAGE and
transferred to nitrocellulose, and ABCA1 was detected by immunoblotting
as described under "Experimental Procedures." Molecular weight
markers are indicated on the left, and migration of ABCA1 is
indicated on the right. The resuspended 15,000 × g membranes were applied to the top of a 15-40% sucrose
gradient and centrifuged for 18 h at 160,000 × g,
and fractions were collected from the top of the tube. B,
aliquots from each fraction were analyzed for ABCA1 immunoreactivity
after PAGE and transfer to nitrocellulose as above. C,
relative abundance of ABCA1 immunoreactivity in each fraction
determined by densitometry of the bands in B. D,
distribution of free cholesterol mass in the gradient fractions.
E, distribution of total protein and the density profile of
the sucrose gradients.
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Fig. 8.
GFP-ABCA1 decorates the cell membrane and
intracellular compartments. 293 cells were transfected with empty
vector (A), unfused GFP (B), or GFP-ABCA1
(C). 36 h after transfection, cells were washed and
briefly fixed in 2% paraformaldehyde and imaged using scanning laser
confocal microscopy. Shown are representative 0.5-µm
sections.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Proposed topological model for ABCA1.
This revised model for ABCA1's transmembrane structure utilizes data
presented in this paper that establish that the loop bordered by amino
acids ~44 and 640 is glycosylated and is therefore
extracellular. Secondary structure predictions of the transmembrane
boundaries of the other segments of the protein are conflicting, and we
have therefore left their sequences undefined. It is also unclear at
present if the large hydrophobic domain, within the central region
separating the two half transporters, traverses the plasma
membrane.
Our work failed to provide convincing evidence for cleavage of the ABCA1 signal sequence. We therefore propose that it serves as a signal anchor, tethering the amino terminus of the protein to the plasma membrane and serving as the first transmembrane domain of the protein. This topology would place residues ~45-639 outside of the cell and would make the original first putative transmembrane domain, extending from residue ~640 to ~660, the second transmembrane domain of the transporter. The direction of orientation of this second transmembrane span is thereby reversed from previous models and dictates further downstream alterations in the ABCA1 topology. Protein modeling has suggested the presence of six putative transmembrane domains in the region extending from amino acid 639 to 850 (17, 32). Since an even number of transmembrane domains within this region is incompatible with positioning of the ATP-binding cassette on the cytosolic face of the membrane in our model, one of these putative transmembrane domains must not span the membrane, or a seventh transmembrane domain must be introduced. Given the canonical six-transmembrane structure of ABC half-transporters, the most parsimonious explanation for the topology of ABCA1 is that the signal sequence constitutes the first of six transmembrane domains and that one of the six previously identified hydrophobic regions of the transporter's proximal half fails to traverse the membrane. Work is currently in progress in our laboratory to resolve these topological issues.
The failure to cleave sequences at the amino terminus of ABCA1 led us to tag the protein with green fluorescent protein. We demonstrate that this chimeric protein partitions into membrane fractions in a manner similar to the wild type ABCA1 and that it retains equivalent cholesterol efflux function when transfected into 293 cells. Confocal microscopy using the chimera demonstrated fluorescence at the cell surface as well as in apparent internal membrane organelles. Such a distribution has been previously described for ABCA1 when tagged at the carboxyl terminus with either GFP or the FLAG epitope (14, 15). Since there is a putative PDZ binding domain at the carboxyl terminus of ABCA1, whose functional significance has not yet been demonstrated (13), there may be advantages to tagging the NH2 terminus of the molecule for certain types of cell biologic experiments. Our data indicate that this can be done without apparent disruption in the protein's cellular localization or efflux function in transfected cells. Thus, the GFP-ABCA1 chimera should prove useful in future studies of ABCA1.
The demonstration that the signal sequence of ABCA1 can position
residues ~45-639 outside the cell is intriguing in light of the
known clustering of ABCA1 mutations in patients with Tangier disease.
Approximately one-third of the loss-of-function mutations reported to
date in Tangier disease patients have clustered in the region between
amino acids 277 and 635 (9-11, 13, 22, 23, 36, 37). If these residues
are extracellular as predicted by our work, instead of intracellular as
suggested in previous models, they could influence the binding of
acceptor apoproteins, such as apoA-I, to the transporter. Recent
cross-linking data demonstrating a direct interaction between ABCA1 and
apoA-I (15, 38) suggest that studies of ligand binding to ABCA1
constructs containing these mutations could elucidate the mechanism by
which these mutations produce disease and the nature of the apoA-I
interaction (39).
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ACKNOWLEDGEMENTS |
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We thank Brian Seed, Hank Kronenberg, and Harvey Lodish for helpful comments about the work and manuscript. We are grateful to Peter Herbert for his help in acquiring Tangier disease cellular materials.
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FOOTNOTES |
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* This work was supported by NHLBI, National Institutes of Health, Grants UO1-66678, HL45098, and HL56985 (to M. W. F) and HL53451 (to A. J. M.) and National Research Service Award HL10398 (to M. L. F.).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. Tel.: 617-726-5906; Fax: 617-726-2879; E-mail: freeman@molbio.mgh.harvard.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M000474200
2 M. W. Freeman and M. L. Fitzgerald, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ABCA1, ATP-binding cassette transporter A1; aa, amino acid; apoA-I, apolipoprotein A-I; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; WT, wild type.
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