ATP-binding Cassette Transporter A1 Contains an NH2-terminal Signal Anchor Sequence That Translocates the Protein's First Hydrophilic Domain to the Exoplasmic Space*

Michael L. FitzgeraldDagger , Armando J. Mendez§, Kathryn J. MooreDagger , Lorna P. AnderssonDagger , Hess A. Panjeton§, and Mason W. FreemanDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 * 10-1.029x, r2 = 0.986) derived from a standard curve of unstained protein molecular weight markers (Sigma).

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 -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).

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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.


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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.

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.


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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."

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.


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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.

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.


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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.

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.


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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).

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.


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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.

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).


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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

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).


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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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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