Effects of Mutations of ABCA1 in the First Extracellular Domain on Subcellular Trafficking and ATP Binding/Hydrolysis*,

Arowu R. TanakaDagger , Sumiko Abe-Dohmae||, Tomohiro OhnishiDagger , Ryo AokiDagger , Gaku MorinagaDagger , Kei-ichiro Okuhira||, Yuika IkedaDagger , Fumi Kano, Michinori MatsuoDagger , Noriyuki KiokaDagger , Teruo AmachiDagger , Masayuki Murata, Shinji Yokoyama||, and Kazumitsu UedaDagger **

From the Dagger  Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan, || Biochemistry, Cell Biology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan, and the  Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki 444-8585, Japan

Received for publication, July 10, 2002, and in revised form, December 5, 2002

    ABSTRACT
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INTRODUCTION
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ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha -32P]ATP was stimulated by adding ortho-vanadate in the presence of Mn2+ as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
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Cholesterol is not catabolized in the peripheral cells and therefore mostly released and transported to the liver for conversion to bile acids to maintain cholesterol homeostasis. The same pathway may also remove cholesterol that has pathologically accumulated in the cells such as an initial stage of atherosclerosis. Assembly of high density lipoprotein (HDL)1 particles by helical apolipoproteins with cellular lipid has been recognized as one of the major mechanisms for cellular cholesterol release (1, 2). The importance of this active cholesterol-releasing pathway in regulating cholesterol homeostasis became apparent by the finding that it is impaired in the cells from patients with Tangier disease, a genetic deficiency of circulating HDL (3, 4). Mutations were identified in ATP-binding cassette transporter A1 (ABCA1) of the Tangier disease (TD) patients (5-7), but the molecular mechanism of ABCA1 in the apolipoprotein-mediated HDL assembly remains unclear. Although direct interaction between ABCA1 and apoA-I at the cell surface has been suggested on the basis of chemical cross-linking experiments (8, 9), an indirect role of ABCA1 in the apoA-I binding to the cell was also proposed by a model that ABCA1 induces phosphatidylserine exofacial flopping to generate the microenvironment required for the docking of apoA-I at the cell surface (10). The predominant substrates of the ABCA1-mediated lipid release reaction are still to be determined for the HDL assembly reaction (11, 12).

More than 30 mutations have been mapped in the ABCA1 gene in patients with familial hypoalphalipoproteinemia (FHA) and TD (5-7, 13-15). Many mutations have been identified in the putative first extracellular domain (ECD1) and the first nucleotide binding fold (NBF1) of ABCA1. We and Fitzgerald et al. (16-18) recently demonstrated that ECD1 exists in the extracellular space by introducing an epitope tag into ABCA1 ECD1 and by analyzing glycosylation of the truncated form of ABCA1. To investigate the mechanistic background for these mutations to cause the dysfunction of ABCA1, we characterized the function and subcellular localization of ABCA1-GFP and its TD mutants stably expressed in HEK293 cells. Three TD mutants (R587W, W590S, Q597R), clustered in ECD1, were examined in the present report. Immunostaining and confocal microscopic analysis showed that ABCA1 is mainly localized to the plasma membrane (PM), where ECD1 is expected to be exposed to the outside of the cell, but also in intracellular compartments to a substantial extent. The TD mutations in ECD1 resulted in a distinct influence on the function and subcellular localization of ABCA1. All three mutants were functionally impaired for the apoA-I-mediated HDL assembly. On the other hand, the two mutants R587W and Q597R were only partially or scarcely localized to the PM, whereas W590S was localized to the PM as efficiently as the wild type. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant.

    EXPERIMENTAL PROCEDURES
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Materials-- Anti-GFP antibody was purchased from Santa Cruz Biotechnology. All other chemicals were obtained from Sigma, Wako Pure Chemical Industries, or Nacalai Tesque.

Generation of an Antibody to ABCA1 ECD1-- The putative extracellular domain ECD1, amino acids 45-639 of human ABCA1, was expressed as a C terminus His tag fusion protein in Escherichia coli BL21(DE3) and purified by Ni2+ chromatography (Qiagen). A rat polyclonal antibody, generated using this His tag-fused ECD1, specifically interacted with human ABCA1 stably or transiently expressed in HEK293 cells in Western blotting (data not shown) and immunostaining (see Fig. 1).

Immunostaining and Fluorescence Microscopy-- Cells were grown on a 35-mm glass-base dish (Iwaki). The cells were incubated with primary antibodies in phosphate-buffered saline containing 5% skim milk. After being washed, these cells were incubated with the fluorescent-labeled secondary antibodies. The cells were directly viewed with ×100 Plan-NEOFLUAR oil immersion objective on a Zeiss confocal microscope LSM510.

DNA Construction-- DNA fragments (XhoI-BclI) containing each missense TD mutation (R587W, W590S, or Q597R) were generated using the polymerase chain reaction method with R587W (XhoI) primer (5'-GTCCTCGAGCTGACCCCTTTGAGGACATGTGGTACGTC-3'), W590S (XhoI) primer (5'-GTCCTCGAGCTGACCCCTTTGAGGACATGCGGTACGTCTCGGGGGGCTTC-3'), or Q597 (XhoI) primer (5'-GTCCTCGAGCTGACCCCTTTGAGGACATGCGGTACGTCTGGGGGGGCTTCGCCTACTTGCGGGATGTGGTG-3'), where the mutated nucleotide is underlined, and BclI primer (5'-CGATGCCCTTGATGATCACAGCCACTGAG-3'). The DNA fragment was replaced with the XhoI-BclI fragment of human ABCA1 (16).

Glycosylation of ABCA1-GFP Protein-- Endoglycosidase H (Endo H) and peptide N-glycosidase F (PNGaseF) (New England Biolabs, Beverly, MA) digestions were done as described by the manufacturer. In brief, 10 µg of membrane proteins from HEK293 cells stably expressing the wild-type, R587W, W590S, or Q597R ABCA1-GFP were treated with 500 units of Endo H or 0.3 units of PNGaseF for 1 h at 37 °C. The deglycosylated proteins were separated by SDS-PAGE (7.5%) and analyzed by immunoblotting by using the anti-GFP antibody.

Cellular Lipid Release Assay-- Cells were subcultured in 6-well plates (TPP, 92406) at a density of 1.0 × 106 cells in a 1/1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (DF) supplemented with 10% (v/v) fetal calf serum. After incubation for 48 h, the cells were washed with Dulbecco's phosphate-buffered saline and incubated in the same medium supplemented with 0.1% bovine serum albumin and 10 µg/ml apoA-I. The lipid content in the medium was determined after a 24-h incubation as described previously (19). To compare lipid release from HEK293 cells transiently expressing ABCA1-GFP, GFP fluorescence of transfected cells was measured with a FL600 fluorescent plate reader (Bio-Tek Instruments, Inc.) (19), and expression levels of the wild-type and mutant ABCA1-GFP were normalized with GFP fluorescence. Expression levels of the wild-type and mutant ABCA1-GFP were in a range of ±20%.

Vanadate-induced Nucleotide Trapping in ABCA1 with 8-Azido-[alpha -32P]ATP-- A membrane fraction (20-30 µg) was prepared from HEK293 cells stably expressing the wild-type or W590S ABCA1-GFP. It was incubated with 15 µM 8-azido-[alpha -32P]ATP, 2 mM ouabain, 0.1 mM EGTA, and 40 mM Tris-Cl, pH 7.5, in a total volume of 6 µl for 15 min at 37 °C in the presence or absence of 1 mM ortho-vanadate and 3 mM MgSO4 or MnCl2. The reaction was stopped by adding 500 µl of ice-cold 40 mM Tris-Cl buffer containing 0.1 mM EGTA and 1 mM MgSO4 or MnCl2. The supernatant containing unbound ATP was removed from the membrane pellet after centrifugation (15,000 × g, 5 min, 2 °C), and this procedure was repeated once more. The pellets were resuspended in 8 µl of TE buffer containing 1 mM MgSO4 or MnCl2 and irradiated for 5 min (at 254 nm, 8.2 mW/cm2) on ice. The sample was analyzed by autoradiogram after electrophoresis in a 7% SDS-polyacrylamide gel. Experiments were done in triplicate.

    RESULTS
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Subcelluar Localization of ABCA1-GFP-- In our previous report, we have shown that the hemagglutinin epitope inserted between residues 207 and 208 of human ABCA1 was recognized by the anti-hemagglutinin antibody from the outside of cells (16). To confirm the extracellular localization of the hydrophilic domain containing residue 207 (ECD1), non-permeabilized HEK293 cells were incubated with a rat polyclonal antibody against the protein corresponding to amino acids 45-639 of human ABCA1 for immunostaining. ABCA1-GFP, which was apparently on the cell surface, was visualized by the antibody, whereas the protein in the intracellular compartments was not (Fig. 1). The results suggested that ABCA1-GFP was localized to the PM and the intracellular compartments, and the ECD1 domain of ABCA1 on the PM was exposed to outside of the cells.


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Fig. 1.   Immunofluorescence confocal microscopy analysis of HEK293 cells stably expressing ABCA1-GFP. GFP, GFP fluorescence of HEK293 cell stably expressing ABCA1-GFP. Anti-ECD1, immunofluorescent observation with anti-ECD1 antibody and anti-rat IgG-Alexa594. Merge, overlaid GFP and Alexa594 fluorescence.

Effects of ECD1 Mutations on Subcellular Localization of ABCA1-GFP-- Many mutations in patients with TD and FHA have been identified in ECD1 of ABCA1, and three mutations (R587W, W590S, Q597R) cluster in the vicinity between amino acids 587 and 597 (20)(Fig. 2A). To study the role of ECD1 domain in the HDL assembly function of ABCA1, we introduced these three TD mutations in ECD1 into ABCA1-GFP and transiently or stably expressed in HEK293. The expression levels of mutant ABCA1-GFP stably expressed in HEK293 cells were comparable with that of the wild-type ABCA1-GFP as shown later in Figs. 3 and 5. Confocal microscopic examination revealed that R587W and Q597R appeared to be localized mainly in the ER and not to the PM (Fig. 2B). In contrast, W590S was localized to the PM as much as the wild-type ABCA1-GFP was, although more was found with intracellular vesicles than with the wild type (Fig. 2B). Immunostaining with the antibody against ECD1 confirmed the proper orientation of W590S (Fig. 2C).


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Fig. 2.   Effects of ECD1 mutations on subcellular localization of ABCA1-GFP. A, a putative secondary structure of ABCA1 and localization of Tangier Disease mutations R587W, W590S, and Q597R in ECD1. B, GFP fluorescence of HEK293 cells stably expressing the wild-type (WT) ABCA1-GFP and three TD mutants R587W, W590S, and Q597R ABCA1-GFP. C, immunofluorescent observation of W590S ABCA1-GFP with anti-ECD1 antibody and anti-rat IgG-Alexa594.


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Fig. 3.   Glycosylation of ABCA1-GFP. The wild-type (WT), R587W, W590S, and Q597R ABCA1-GFP were treated with none (-), Endo H (H), or PNGaseF (F) and separated with 7% SDS-PAGE. Western blotting was done with anti-GFP antibody.

Glycosylation of ABCA1-GFP-- Glycosylation of the wild-type ABCA1-GFP and its mutants R587W, W590S, and Q597R was examined by the treatment with PNGaseF and Endo H (Fig. 3A). Endo H cleaves two proximal N-acetylglucosamine residues of the high mannose type but not of the complex type, whereas PNGaseF cleaves sugar chains of both the high mannose and complex types. The treatment with PNGaseF increased the electrophoretic mobilities of 280-kDa ABCA1 to produce the 250-kDa protein, the deglycosylated form of ABCA1-GFP. ABCA1 with TD mutations, R587W and Q597R ABCA1-GFP, was sensitive to Endo H to produce the deglycosylated form of ABCA1-GFP, whereas the wild-type ABCA1-GFP was little digested by Endo H. These results indicated that R587W and Q597R ABCA1-GFP did not contain complex oligosaccharides and supported the confocal microscopy observation, which suggested the localization of these two TD mutants in the ER or the cis-Golgi complex. On the other hand, W590S ABCA1-GFP was resistant to Endo H, indicating that it does not contain high mannose oligosaccharides but contains complex oligosaccharides and reached the trans-Golgi complex.

Effects of ECD1 Mutations on apoA-I-mediated Cholesterol Release-- To analyze the functional consequences of these mutations, apoA-I-mediated release of cholesterol and choline-phospholipid was examined from the stable transformants (Fig. 4, A and B). The wild-type ABCA1-GFP exported 0.14 ± 0.01 and 1.14 ± 0.07 µg of cholesterol from cells grown in 6-well plates in the absence and presence of apoA-I, respectively, and 1.99 ± 0.25 and 5.88 ± 0.13 µg of choline-phospholipids, respectively, to generate HDL in the medium by reducing about 15% of cell cholesterol. The R587W mutation resulted in the apoA-I-mediated release of cholesterol and choline-phospholipids to 24 and 23% of the wild-type ABCA1-GFP, respectively. The Q597R mutant almost completely lost this activity. The results were apparently consistent with the inefficient localization to the cell surface of ABCA1-GFP with these mutations. The apoA-I-mediated release of cholesterol and choline-phospholipids from HEK293 expressing W590S ABCA1-GFP were 7.4 and 13%, respectively, of those expressing the wild-type ABCA1-GFP, although W590S ABCA1-GFP was localized to the PM as efficiently as the wild type. The apoA-I-mediated release of cellular cholesterol and choline-phospholipids was also examined with HEK293 transiently expressing the wild-type and mutant ABCA1-GFPs (Fig. 4, C and D). The results were similar to those observed with the stable transformants shown in Fig. 4, A and B.


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Fig. 4.   Effects of ECD1 mutations on apoA-I-mediated cholesterol and phospholipid transport. Cholesterol (A) and choline-phospholipid (B) content in the medium in a 6-well plate containing HEK293 cells stably expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP were measured after a 24-h incubation in the presence (black bars) or absence (white bars) of 10 µg/ml apoA-I. The relative amount of cholesterol (C) and choline-phospholipid (D) in the medium in a 6-well plate containing HEK293 cells transiently expressing the wild-type (WT), R587W (RW), W590S (WS), and Q597R (QR) ABCA1-GFP was measured after a 24-h incubation in the presence of 10 µg/ml apoA-I. The expression levels of mutants were normalized with the GFP fluorescence of cells. Lipid release from HEK293 cells transiently expressing ABCA1-GFP subtracted by that from non-transformed HEK293 cells was represented as 100% in C and D.

Interaction of ABCA1-GFP with 8-Azido-[alpha -32P]ATP-- To elucidate the mechanism for the loss of function of ABCA1 in the W590S mutant, we examined the interaction of ABCA1-GFP with ATP. Among membrane proteins of the cells expressing the wild-type ABCA1-GFP, a 280-kDa protein was specifically photoaffinity-labeled with ATP (Fig. 5A, lane 7), whereas no protein was labeled in the untransfected HEK293 cells (lane 5). The mobility of the photoaffinity-labeled membrane protein in SDS-PAGE was identical to that of the wild-type ABCA1-GFP visualized by Western blotting (Fig. 5B, lane 1). The photoaffinity labeling was negative when the samples were incubated in the absence of Mg2+ (Fig. 5A, lane 3), indicating that 8-azido-[alpha -32P] ATP tightly binds to ABCA1 in the presence of Mg2+.


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Fig. 5.   Vanadate-induced trapping in ABCA1-GFP. A, photoaffinity labeling of ABCA1-GFP with 8-azido-[alpha -32P]ATP. Membranes (20 µg) prepared from HEK293 cells stably expressing the wild-type (WT) ABCA1-GFP (lanes 3, 4, 7, 8, 11, and 12) or from untransfected HEK293 cells (HEK) (lanes 1, 2, 5, 6, 9, and 10) were incubated with 15 µM 8-azido-[alpha -32P]ATP in the absence or presence of 1 mM ortho-vanadate (Vi) and 3 mM MgSO4 (lanes 5-8) or MnCl2 (lanes 9-12) for 15 min at 37 °C. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ligands and analyzed as described under "Experimental Procedures." B, immunoblots of membranes prepared from HEK293 cells stably expressing the wild-type (4 µg, lane 1) or W590S (6 µg, lane 2) ABCA1-GFP or from untransfected HEK293 cells (6 µg, lane 3). Proteins were separated on a 7% SDS-polyacrylamide gel and reacted with monoclonal antibody against GFP. C, membranes prepared from HEK293 cells stably expressing the wild type (WT) (20 µg, lanes 1 and 2) or W590S (30 µg, lanes 3 and 4) were incubated with 15 µM 8-azido-[alpha -32P]ATP in the absence or presence of 1 mM ortho-vanadate (Vi) and 3 mM MnCl2 for 15 min at 37 °C. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ligands and analyzed as described under "Experimental Procedures."

Multidrug transporters, MDR1 (ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2), are known to trap Mg-ADP in the presence of ortho-vanadate, an analog of phosphate, and form a stable inhibitory intermediate during the ATP hydrolysis cycle. Photoaffinity labeling of these proteins with 8-azido-[alpha -32P] ATP is therefore stimulated when the membrane containing these proteins reacts with the nucleotide in the presence of ortho-vanadate (21-24). We thus expected that ortho-vanadate would stimulate photoaffinity labeling of ABCA1. However, no increase of photoaffinity labeling of ABCA1 was observed (lane 8) in comparison with that in the absence of ortho-vanadate (lane 7).

Vanadate did not induce nucleotide trapping in MRP6 (ABCC6) in the presence of Mg2+, but it did with Ni2+ ions (25). Therefore, we examined photoaffinity labeling of ABCA1-GFP in the presence of other metal ions. Significant stimulation was observed with wild-type ABCA1-GFP by ortho-vanadate in the presence of Mn2+ (Fig. 5A, lanes 11 and 12). These results suggested that Mn-ATP was hydrolyzed at NBFs of ABCA1, and a stable inhibitory complex ABCA1-MnADP-Vi was formed during the ATP hydrolysis cycle.

To determine whether the W590S mutation affects the ATP hydrolysis cycle of ABCA1, vanadate-induced nucleotide trapping in W590S ABCA1-GFP was examined in the presence of Mn2+ (Fig. 5C). Membrane proteins from HEK293 cells expressing a similar amount of wild-type ABCA1 or W590S ABCA1-GFP (Fig. 5B) were incubated with 8-azido-[alpha -32P]ATP in the absence or presence of ortho-vanadate. The photoaffinity labeling of W590S ABCA1-GFP was stimulated by adding ortho-vanadate in the presence of Mn2+ as much as in the presence of the wild type.

    DISCUSSION
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In this work, we described the influence of three clustered mutations in ECD1 associated with TD and FHA on the subcellular localization of ABCA1, apoA-I-mediated HDL assembly, apoA-I binding, and vanadate-induced nucleotide trapping. Immunostaining of ABCA1-GFP stably expressed in HEK293 cells revealed that ABCA1-GFP apparently resided on the cell surface as well as in intracellular compartments in agreement with previous reports (18, 26-28). Although the three mutations all reduced apoA-I-mediated lipid release and subsequent HDL assembly from HEK293 cells expressing ABCA1-GFP, whether transiently or stably, the mutants demonstrated differential behavior with respect to their subcellular localization. ABCA1-GFP with a R587W or Q597R mutation appeared to be impaired with intracellular trafficking and predominantly localized in the ER. On the other hand, W590S ABCA1-GFP was mainly localized to the PM as much as the wild-type ABCA1 was. The sensitivity to Endo H of the mutant ABCA1s was consistent with the their apparent impairment of intracellular trafficking. R587W and Q597 ABCA1-GFP contained high mannose oligosaccharides, indicating that they do not reach the trans-Golgi complex. In contrast, W590S ABCA1-GFP contained complex-type oligosaccharides as the wild-type does.

When the cells were treated with monensin, which prevents the delivery of protein from endosomes to the cell surface, after inhibiting protein synthesis by treatment with cycloheximide, ABCA1-GFP on the cell surface decreased, and the vesicular localization increased instead (see supplementary data, Fig. 1).2 When the cells were treated with brefeldin A, which blocks vesicular transport from the ER to the Golgi and to the cell surface along with the secretary pathway (29), the newly synthesized ABCA1-GFP was accumulated in the fused Golgi-ER, the amount of ABCA1-GFP on the PM was reduced, and the vesicles containing ABCA1-GFP were observed (see supplementary data 1).2 ABCA1-GFP was co-localized partly with Vti1b, a marker for the Golgi, with EEA1, a marker for early endosomes, and with lysotracker, a marker for acidic compartments (see supplementary data 2).2 These results suggested that newly synthesized ABCA1-GFP was first delivered to the PM through the ER and the Golgi and then shuttled rapidly between the PM and the intracellular vesicles, mainly the early endosomes. R587W and Q597R ABCA1-GFP appeared to be retained in the ER. It has been reported that one amino acid (Phe-508) deletion of the cystic fibrosis transmembrane conductance regulator (CFTR), the major mutation in cystic fibrosis patients that causes misfolding of the cystic fibrosis transmembrane conductance regulator, is degraded by the proteasome pathway before exiting from the ER (30). This region (R587 to Q597) in ECD1 would be critical for proper folding of ABCA1 and would probably affect the intracellular translocation process, whereas the W590S mutation does not. Fitzgerald et al. (17) reported that R587W or Q597R mutation did not affect the PM localization but disrupted the direct interaction with ApoA-I. This supports a major conformational alteration of ECD1 by these mutations. The reason for the discrepancy of subcellular localization is unknown between their results with the mutant ABCA1 transiently expressed in a high amount in HEK293 and ours studied with the mutant ABCA1-GFP in the stable transformants with modest expression.

W590S ABCA1-GFP was localized to the PM as much as the wild-type ABCA1-GFP when expressed in HEK293. However, apoA-I-mediated release of cellular cholesterol and choline-phospholipid was severely impaired. To elucidate the reason for this functional impairment in the W590S mutant, nucleotide interaction was examined with the wild-type and W590S ABCA1-GFP by using 8-azido-[alpha -32P]ATP. The wild-type ABCA1-GFP was photoaffinity-labeled in the presence of Mg2+, but no vanadate-induced nucleotide trapping was observed, being consistent with human ABCA1 expressed in Sf9 insect cells (31). Other transporter-type ABC proteins, such as MDR1 (ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2), trap Mg-ADP in the presence of ortho-vanadate and form a stable inhibitory intermediate during the ATP hydrolysis cycle (21-24) so that ABCA1, showing no obvious vanadate-induced nucleotide trapping, was proposed not to be an active transporter but a regulator in apoA-I-dependent cholesterol release (31). However, vanadate-induced nucleotide trapping was demonstrated to be positive with ABCA1 in the presence of Mn2+ in this study. Vanadate-induced nucleotide trapping did not occur in the presence of Mg2+ but can be detected with Ni2+ ions in MRP6 (ABCC6) (25), and it was detected in ABCG2 with Co2+ ions (32). MRP6 and ABCG2 have been shown to function as active transporters for an anionic cyclic pentapeptide BQ-123 (33) and anticancer drugs (34), respectively. These results suggest that ABCA1 may function as an active transporter in apoA-I-dependent cholesterol release.

W590S ABCA1-GFP showed vanadate-induced nucleotide trapping in the presence of Mn2+. This suggests that the first catalytic reaction to form a stable inhibitory complex ABCA1-MnADP-Vi is not impaired by the mutation. It has been reported that apoA-I does not properly interact with ATP hydrolysis mutants of ABCA1 (10) and that apoA-I can be interacted with ABCA1-W590S as with the wild-type ABCA1 (17, 35). These results suggest that W590S ABCA1-GFP possesses, at least, minimum ATPase activity, which supports apoA-I binding. W590S mutation may impair a step after the interaction with apoA-I, such as proper loading of phospholipid and/or cholesterol or proper release of apoA-I after phospholipid/cholesterol loading.

More than 30 mutations have been mapped in the ABCA1 gene in patients with FHA and TD (5-7, 13-15). One subgroup of the mutations is suggested to be associated with splenomegaly, and the other may be associated with coronary heart disease (20). Many mutations have been located in ECD1 of ABCA1, and three missense mutations cluster in the vicinity between amino acids 587 and 597 in ECD1. Interestingly, clinical manifestations of these mutations are apparently different (20): R587W is associated with coronary heart disease, whereas W590S is associated with splenomegaly. In this study, we demonstrated that the defect of HDL assembly in R587W and Q597R is due to the impaired localization of ABCA1 to the PM. Subcellular trafficking and vanadate-induced nucleotide trapping in the presence of Mn2+ were not impaired in ABCA1-GFP containing the W590S mutation so that W590S seems to have a different type of functional defect. Further characterization of TD mutations for the function of ABCA1 would facilitate understanding of the molecular mechanism for cellular cholesterol release and its homeostasis.

    ACKNOWLEDGEMENT

We thank Kyowa Hakko Kogyo Co. Ltd. for generating anti-ECD1 polyclonal antibody.

    FOOTNOTES

* This work was supported by Grant-in-aid for Scientific Research 10217205 on Priority Areas "ABC Proteins" from the Ministry of Education, Science, Sports, and Culture of Japan and by the Nakajima Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplementary data showing a figure showing the trafficking of ABCA1-GFP in HEK293 cells and a figure showing the characterization of ABCA1-GFP vesicles.

These authors contributed equally to the work.

** To whom correspondence should be addressed. Tel.: 81-75-753-6105; Fax: 81-75-753-6104; E-mail: uedak@kais.kyoto-u.ac.jp.

Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M206885200

    ABBREVIATIONS

The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP-binding cassette transporter A1; ECD1, the first extracellular domain; NBF, nucleotide binding fold; TD, Tangier disease; FHA, familial hypoalphalipoproteinemia; PM, plasma membrane; ER, endoplasmic reticulum; Endo H, endoglycosidase H; PNGaseF, N-glycosidase F; GFP, green fluorescent protein.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

1. Hara, H., and Yokoyama, S. (1991) J. Biol. Chem. 266, 3080-3086[Abstract/Free Full Text]
2. Yokoyama, S. (2000) Biochim. Biophys. Acta 1529, 231-244[Medline] [Order article via Infotrieve]
3. Francis, G. A., Knopp, R. H., and Oram, J. F. (1995) J. Clin. Invest. 96, 78-87[Medline] [Order article via Infotrieve]
4. Remaley, A. T., Schumacher, U. K., Stonik, J. A., Farsi, B. D., Nazih, H., and Brewer, H. B., Jr. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 1813-1821[Abstract/Free Full Text]
5. Brooks-Wilson, A., Marcil, M., Clee, S., Zhang, L., Roomp, K., van Dam, M., Yu, L., Brewer, C., Collins, J., Molhuizen, H., Loubser, O., Ouelette, B., Fichter, K., Ashbourne-Excoffon, K., Sensen, C., Scherer, S., Mott, S., Denis, M., Martindale, D., Frohlich, J., Morgan, K., Koop, B., Pimstone, S., Kastelein, J., and Hayden, M. (1999) Nat. Genet. 22, 336-345[CrossRef][Medline] [Order article via Infotrieve]
6. Bodzioch, M., Orso, E., Klucken, J., Langmann, T., Bottcher, A., Diederich, W., Drobnik, W., Barlage, S., Buchler, C., Porsch-Ozcurumez, M., Kaminski, W., Hahmann, H., Oette, K., Rothe, G., Aslanidis, C., Lackner, K., and Schmitz, G. (1999) Nat. Genet. 22, 347-351[CrossRef][Medline] [Order article via Infotrieve]
7. Rust, S., Rosier, M., Funke, H., Real, J., Amoura, Z., Piette, J., Deleuze, J., Brewer, H., Duverger, N., Denefle, P., and Assmann, G. (1999) Nat. Genet. 22, 352-355[CrossRef][Medline] [Order article via Infotrieve]
8. Oram, J., Lawn, R., Garvin, M., and Wade, D. (2000) J. Biol. Chem. 275, 34508-34511[Abstract/Free Full Text]
9. Wang, N., Silver, D., Costet, P., and Tall, A. (2000) J. Biol. Chem. 275, 33053-33058[Abstract/Free Full Text]
10. Chambenoit, O., Hamon, Y., Marguet, D., Rigneault, H., Rosseneu, M., and Chimini, G. (2001) J. Biol. Chem. 276, 9955-9960[Abstract/Free Full Text]
11. Arakawa, R., Abe-Dohmae, S., Asai, M., Ito, J.-I., and Yokoyama, S. (2000) J. Lipid Res. 41, 1952-1962[Abstract/Free Full Text]
12. Fielding, P. E., Nagao, K., Hakamata, H., Chimini, G., and Fielding, C. J. (2000) Biochemistry 39, 14113-14120[CrossRef][Medline] [Order article via Infotrieve]
13. Lawn, R. M., Wade, D. P., Garvin, M. R., Wang, X., Schwartz, K., Porter, J. G., Seilhamer, J. J., Vaughan, A. M., and Oram, J. F. (1999) J. Clin. Invest. 104, R25-R31[Medline] [Order article via Infotrieve]
14. Remaley, A. T., Rust, S., Rosier, M., Knapper, C., Naudin, L., Broccardo, C., Peterson, K. M., Koch, C., Arnould, I., Prades, C., Duverger, N., Funke, H., Assman, G., Dinger, M., Dean, M., Chimni, G., Santamarina-Fojo, S., Fredrickson, D. S., Denefle, P., and Brewer, H. B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12685-12690[Abstract/Free Full Text]
15. Brousseau, M. E., Schaefer, E. J., Dupuis, J., Eustace, B., Van Eerdewegh, P., Goldkamp, A. L., Thurston, L. M., FitzGerald, M. G., Yasek-McKenna, D., O'Neill, G., Eberhart, G. P., Weiffenbach, B., Ordovas, J. M., Freeman, M. W., Brown, R. H., Jr., and Gu, J. Z. (2000) J. Lipid Res. 41, 433-441[Abstract/Free Full Text]
16. Tanaka, A., Ikeda, Y., Abe-Dohmae, S., Arakawa, R., Sadanami, K., Kidera, A., Nakagawa, S., Nagase, T., Aoki, R., Kioka, N., Amachi, T., Yokoyama, S., and Ueda, K. (2001) Biochem. Biophys. Res. Commun. 283, 1019-1025[CrossRef][Medline] [Order article via Infotrieve]
17. Fitzgerald, M. L., Morris, A. L., Rhee, J. S., Andersson, L. P., Mendez, A. J., and Freeman, M. W. (2002) J. Biol. Chem. 277, 33178-33187[Abstract/Free Full Text]
18. Fitzgerald, M. L., Mendez, A. J., Moore, K. J., Andersson, L. P., Panjeton, H. A., and Freeman, M. W. (2001) J. Biol. Chem. 276, 15137-15145[Abstract/Free Full Text]
19. Abe-Dohmae, S., Suzuki, S., Wada, Y., Hiroyuki Aburatani, E., Vance, D., and Yokoyama, S. (2000) Biochemistry 39, 11092-11099[CrossRef][Medline] [Order article via Infotrieve]
20. Schmitz, G., Kaminski, W. E., and Orso, E. (2000) Curr. Opin. Lipidol. 11, 493-501[CrossRef][Medline] [Order article via Infotrieve]
21. Taguchi, Y., Yoshida, A., Takada, Y., Komano, T., and Ueda, K. (1997) FEBS Lett. 401, 11-14[CrossRef][Medline] [Order article via Infotrieve]
22. Takada, Y., Yamada, K., Taguchi, Y., Kino, K., Matsuo, M., Tucker, S. J., Komano, T., Amachi, T., and Ueda, K. (1998) Biochim. Biophys. Acta 1373, 131-136[Medline] [Order article via Infotrieve]
23. Urbatsch, I. L., Sankaran, B., Weber, J., and Senior, A. E. (1995) J. Biol. Chem. 270, 19383-19390[Abstract/Free Full Text]
24. Hashimoto, K., Uchiumi, T., Konno, T., Ebihara, T., Nakamura, T., Wada, M., Sakisaka, S., Maniwa, F., Amachi, T., Ueda, K., and Kuwano, M. (2002) Hepatology 36, 1236-1245[CrossRef][Medline] [Order article via Infotrieve]
25. Cai, J., Daoud, R., Alqawi, O., Georges, E., Pelletier, J., and Gros, P. (2002) Biochemistry 41, 8058-8067[CrossRef][Medline] [Order article via Infotrieve]
26. Hamon, Y., Broccardo, C., Chambenoit, O., Luciani, M., Toti, F., Chaslin, S., Freyssinet, J., Devaux, P., McNeish, J., Marguet, D., and Chimini, G. (2000) Nat. Cell Biol. 2, 399-406[CrossRef][Medline] [Order article via Infotrieve]
27. Remaley, A. T., Stonik, J. A., Demosky, S. J., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Eggerman, T. L., Patterson, A. P., Duverger, N. J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) Biochem. Biophys. Res. Commun. 280, 818-823[CrossRef][Medline] [Order article via Infotrieve]
28. Neufeld, E. B., Remaley, A. T., Demosky, S. J., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590[Abstract/Free Full Text]
29. Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071-1080[Medline] [Order article via Infotrieve]
30. Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129-135[Medline] [Order article via Infotrieve]
31. Szakacs, G., Langmann, T., Ozvegy, C., Orso, E., Schmitz, G., Varadi, A., and Sarkadi, B. (2001) Biochem. Biophys. Res. Commun. 288, 1258-1264[CrossRef][Medline] [Order article via Infotrieve]
32. Ozvegy, C., Varadi, A., and Sarkadi, B. (2002) J. Biol. Chem.
33. Madon, J., Hagenbuch, B., Landmann, L., Meier, P. J., and Stieger, B. (2000) Mol. Pharmacol. 57, 634-641[Abstract/Free Full Text]
34. Litman, T., Druley, T. E., Stein, W. D., and Bates, S. E. (2001) Cell Mol. Life Sci. 58, 931-959[Medline] [Order article via Infotrieve]
35. Rigot, V., Hamon, Y., Chambenoit, O., Alibert, M., Duverger, N., and Chimini, G. (2002) J. Lipid Res. 43, 2077-2086[Abstract/Free Full Text]


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