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Originally published In Press as doi:10.1074/jbc.M601072200 on May 18, 2006

J. Biol. Chem., Vol. 281, Issue 29, 20283-20290, July 21, 2006
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Transition from Dimers to Higher Oligomeric Forms Occurs during the ATPase Cycle of the ABCA1 Transporter*

Doriane Trompier1, Mélanie Alibert, Suzel Davanture, Yannick Hamon, Michel Pierres, and Giovanna Chimini2

From the Centre d'Immunologie de Marseille-Luminy INSERM CNRS UniversitédelaMéditerranée, Parc Scientifique de Luminy, 13288 Marseille Cedex 09 France

Received for publication, February 3, 2006 , and in revised form, May 4, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fluorescence resonance energy transfer and native PAGE analytical techniques were employed to assess the quaternary structure of ABCA1, an ATP binding cassette transporter playing a crucial role in cellular lipid handling. These experimental approaches support the conclusion that ABCA1 is associated in dimeric structures that undergo transition into higher order structures, i.e. tetramers, during the ATP catalytic cycle. Our data hence underline molecular assembly as a crucial parameter in ABCA1 function and the advantage of native PAGE as analytical tool for intractable membrane proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We explored the quaternary structure of ABCA1, a prototype of the A subclass of mammalian ATP binding cassette (ABC)3 transporters and a key player in reverse cholesterol transport (1). At the plasma membrane of peripheral cells, namely macrophages, ABCA1 controls the docking of nascent apoproteins and their lipidation, leading to the formation of high density lipoprotein particles. ABCA1 also elicits an outward flip of phosphatidylserine at the plasma membrane (2). Whether the flip and docking are concomitant events or follow a hierarchical order is still unknown; however, both are required for the ABCA1-induced cellular effluxes of phospholipids (3). Although compelling evidence argues for a direct receptor-ligand interaction between ABCA1 and the apoA-I lipid acceptor, some unusual elements suggest that the lipid environment may contribute significantly to the interaction of the acceptor with the cell surface (4, 5). In this context, an assessment of the quaternary assembly of the transporter may help clarify the issue and, indeed, it has been recently suggested that tetrameric ABCA1 complexes are the minimal functional structure required for lipidation of apoA-I particles (6).

Understanding the rules of ABCA1 molecular assembly bears an obvious medical interest. In fact, mutations in ABCA1 sequence may lead to Tangier disease, a genetic disorder of lipid metabolism with autosomic recessive transmission, but also to familial high density lipoprotein deficiency, a nosographically distinct syndrome transmitted as a dominant trait (7-9). The need for oligomerization to achieve complete function may underlie such discrepancy and explain an exquisite sensitivity to mutations in the primary sequence of the transporter. However, it has to be pointed out that the assessment of quaternary structure of full-length ABC transporters has been a controversial issue (10). To date, in the case of P-glycoprotein (11-13) and cystic fibrosis transmembrane conductance regulator (CFTR) (14-20), the indications of oligomeric assembly discretely distributed in specific cell types are numerous but still not consensual and largely dependent on the experimental setup.

To rule out methodological issues, we developed an analysis of the membrane arrangement of the ABCA1 transporter based on the combined use of multiple approaches, i.e. living cell fluorescence resonance energy transfer (FRET), velocity sucrose gradient centrifugation, and native PAGE. All of them converge in indicating that ABCA1 at equilibrium is predominantly associated in homodimeric structures, in both ABCA1-expressing cellular systems and transfected cells. In addition, we took advantage of a panel of ABCA1 variants, previously characterized for their functional activity and intracellular trafficking (2,3), to demonstrate that transitions from dimers to higher oligomeric forms occur during the ATP catalytic cycle.

In conclusion, we provide here the first mechanistic data about the ABCA1 transporter function. Our results introduce the notion of molecular assembly as a parameter in the assessment of ABCA1 physiological function. Whether this is a distinctive feature of ABCA1 or concerns other transporters of the A subclass deserves further investigation. Finally, we propose the use of analytical techniques such as native PAGE as effective alternatives to structural studies with intractable membrane proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids, Cellular Transfections, and Assessments of ABCA1 Function—Plasmids containing ABCA1, either wild type or variants or chimeras with fluorescent proteins, were generated in pBI vector (Clontech, Erembodegem, Belgium) as described previously (3). Shifts from the original plasmids containing EGFP-encoding sequences to ECFP or EYFP sequences were obtained either by subcloning or by PCR-driven incorporation of variant sequences with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla CA) according to the manufacturer's instructions. All constructs were validated by sequencing. Transient transfections were performed on 60% confluent monolayers of HeLa Tet-Off cells with a total of 5 µg of plasmid DNA mixed to EXGEN 500 (Euromedex, Mundolsheim, France) according to the manufacturer's instructions and as described previously (3). The mix was left in contact with cell monolayers for 18 h, and cells were seeded according to experimental needs 24 h after transfection. Transfection efficiency, monitored when appropriate by flow cytometry on a FACScalibur (BD Biosciences) 24 h after transfection, was consistently at around 40%. In the case of ECFP chimeras, transfection efficiency was monitored visually by confocal microscopy examination. Conventional confocal microscopy was performed by visualization on x, y, and z axes on an Axiovert 200 inverted microscope (Zeiss, Oberkochen, Germany) equipped with a Zeiss LSM 510 scanning module. In the case of multiple simultaneous transfections, the total amount of DNA was kept invariant (5 µg), and the ratio between the DNA species varied as mentioned. Cells were further analyzed for FRET or biochemical and functional assays at 60 h after transfection. Functional assays for ABCA1 expression, i.e. binding of annexin V and apoA-I, were performed at 60 h after transfection as described previously (21, 22).

Imaging FRET Measurements—FRET was measured by the method of acceptor photobleaching or donor dequenching (23-25). This method relies on both efficient bleaching of the acceptor (EYFP) and minimal fading of donor fluorescence (ECFP) during image acquisition. FRET assessments were performed with a Zeiss LSM 510 scanning module on an Axiovert 200 inverted microscope (Zeiss) equipped with a 25 milliwatt argon/2 laser beam and a polychromatic multichannel detector (META detector) to spectrally resolve emission spectra. ECFP and EYFP were illuminated respectively with the 458 (60% intensity of the acoustico-optical tunable filter) and 514 nm (6% intensity of the AOTF) laser lines. A x63 oil immersion objective lens was used with a pinhole diameter settled to 1.00 Airy units. To maximize selectivity, four-dimensional stacks were acquired in spectral mode with wavelength series at 10-nm intervals recorded for every time series. EYFP and ECFP images were subsequently reconstituted by linear unmixing of the wavelength series according to the spectra experimentally recorded on cells transfected with either ECFP- or EYFP-bearing constructs. Regions of interest (ROI) corresponding to membrane colocalization of the two fluorochromes were selected visually on images acquired on double-transfected cells. To minimize the time required for bleaching and thus lateral drifting of the samples, 2-3 ROI of similar and regular shape were selected per bleaching step. ROI were bleached at 514 nm (100% at acoustico-optical tunable filter) by 400 reiterations as calibrated previously to efficiently bleach EYFP signal to background levels, without interference at the ECFP emission wavelength. ECFP and EYFP spectra were previously calibrated on cells expressing only ECFP or EYFP chimeras (26). Bleaching time varied from 150 to 200 s. A set of five images was acquired before and after bleaching in spectral mode and reconstituted by linear unmixing to visually check both bleaching efficiency and stability of the sample. FRET efficiency was calculated according to the formula Ef = Ipost - Ipre x 100/Ipost on numerical values tabulated by the LSM software. Background levels, measured as pixel values outside cells or in the cytoplasmic region, were computed in each experimental set and never exceeded 20% of signal.

Generation of Monoclonal Antibodies—A rat monoclonal antibody against a synthetic peptide (Schafer-N, Copenhagen, Denmark) corresponding to residues 215-233 in the mouse ABCA1 sequence (Swiss-Prot entry P41233 [GenBank] ) was generated by repeated immunization of LOU/c rats with 50 µg of ovalbumin-conjugated peptide. Upon fusion of rat splenocytes with the non-secreting mouse myeloma X63.Ag8.653, hybridomas were selected for reactivity by enzyme-linked immunosorbent assay on Nunc Maxisorp plates coated with both native and ovalbumin-conjugated peptide. Hybridoma 891.3 was selected on the basis of the specific reactivity of its supernatant against the ABCA1 protein assessed by flow cytofluorometry (not shown) and Western blotting (see Fig. 2A).

Velocity Gradient Centrifugation—Mouse macrophage RAW 264.7 cells were treated overnight in 300 µM pCPT-cAMP (Sigma, Saint Quentin, France) to induce ABCA1 over-expression as described (27). Cells were rinsed with phosphate-buffered saline and then solubilized at 4 °C in 150 mM NaCl and 50 mM TrisHCl, pH 7.4, in the presence of 1% SDS, 0.2% Triton X-100 (Sigma), 0.09% Nonidet P-40 (Roche Diagnostics, Meylan, France), 1% {alpha}-DM (Anatrace, Maumee, OH), or 1% beta-DM (Sigma) and supplemented with a protease inhibitor mixture (Roche Diagnostics) and 1 mM phenylmethylsulfonyl fluoride. After ultracentrifugation at 100,000 x g for 20 min at 4 °C, protein concentration in the cleared post-nuclear supernatant was determined by the Bradford-based protein assay (Bio-Rad). One ml of supernatant normalized at a protein content of 1 mg/ml was then layered on top of an 11-ml linear, continuous 10-40% or 5-30% sucrose gradient, containing the appropriate detergents, and spun at 260,000 x g for 12 h. At equilibrium, linearity was confirmed by refractometric quantification on blank gradients, whereas for loaded gradients, 24 x 500-µl fractions were collected from the top, resolved by 5.5% SDS-PAGE, and blotted onto nitrocellulose membrane and analyzed for reactivity with ABCA1 mAb 891.3 following standard procedures (3). Molecular mass markers thyroglobulin (669 kDa), catalase (232 kDa), aldolase (158 kDa) (Amersham Biosciences, Saclay, France), and urease (hexamer 545 kDa, trimer, 272 kDa) (Sigma) were diluted in 1 ml of the appropriate detergent buffer and floated as described above. Peak migration of each species was determined by A280 nm measurements of all fractions.

Native PAGE Fractionation and Analysis—Monolayers of HeLa cells (60 h after transfection with the indicated plasmid), cAMP-induced RAW 264.7 cells, or thioglycollate peritoneal elicited macrophages (PEM) were washed in phosphate-buffered saline before solubilization in 1% {alpha}-DM in 150 mM NaCl and 50 mM TrisHCl, pH 7.4, for 30 min at 4 °C. Homogenates were then spun at 4 °C at 100 000 x g for 20 min, and protein concentrations in the cleared post-nuclear supernatant were determined. 5-20 µg of proteins were loaded on a Deriphat/PAGE system adapted from Peter and Thornber (28). Samples were fractionated on 3-8% polyacrylamide gradient gels overlaid with a 3% stacking gel in 48 mM glycine, 12 mM TrisHCl, pH 8.3. Running buffer contained 12 mM TrisHCl, pH 8.3, and 96 mM glycine. 0.1% Deriphat 160 (Cognis Care Chemicals, St. Fargeau Pthierry, France) was included in the upper reservoir.

After electrophoresis at 4 °C at 50 volts for 12 h, proteins were electrically transferred to nitrocellulose membrane at 4 °C in 25 mM Tris HCl, 192 mM glycine, and 20% (v/v) methanol. The migration of native protein standards (Amersham Bio-sciences) was analyzed by Ponceau S staining. Membranes were subsequently probed with the 891.3 mAb or anti-GFP mAb (Roche Diagnostics) followed by incubation with the appropriate secondary antibody and revealed by ECL detection reagent (Amersham Biosciences). Densitometric evaluation of the intensity of bands was performed with the software AIDA 2.11 on scanned autoradiographic images.


Figure 1
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  		FIGURE 1.
Imaging FRET establishes physical proximity between the C-terminal (C-ter) extremities of the ABCA1 transporter. Fluorescence resonance energy transfer was measured on HeLa cells transfected with various combinations of ABCA1 EYFP and ECFP chimeras. The fluorescent protein graft was positioned either in the C-terminal (C-ter) or in the N-terminal (N-ter) extremity of the transporter (see "Results"). Although in all three combinations, the efficiency of transfer was insensitive to acceptor density, measured as relative fluorescence intensity (RFI) of EYFP (A and not shown), only in the case of C-terminal pairs (B as opposed to C and D), the efficiency of transfer was sensitive to variations in acceptor: donor ratio, measured as ratio between RFI for EYFP and ECFP, respectively. Mean values ± S.E. are shown in the graph and summarized in Table 1. Measurements were obtained as detailed under "Experimental Procedures" from more than 10 individual transfection experiments.

 
Membrane Vesicle Preparations and Chemical Interference with ATP Hydrolysis—cAMP-treated RAW cells were collected by centrifugation and resuspended in 10 mM TrisHCl, pH 7.5, 250 mM sucrose, 0.2 mM MgCl2, and 1mM phenylmethylsulfonyl fluoride supplemented with a protease inhibitor mixture (Roche Diagnostics). Cells were homogenized with a ball-bearing homogenizer. Collection of membranes was performed according to the procedure described previously (29). The cell homogenate was adjusted to 1 mM EDTA and then diluted 5-fold with 10 mM TrisHCl, pH 7.5, and 25 mM sucrose and centrifuged at 1000 x g to remove nuclei and unbroken cells. The supernatant was overlaid on a 35% sucrose cushion in 10 mM TrisHCl, pH 7.5, 1 mM EDTA and centrifuged at 16,000 x g for 30 min. Membranes at the interface were collected, diluted 5-fold in 10 mM TrisHCl, pH 7.5, and 250 mM sucrose, and then centrifuged at 100,000 x g for 45 min. The pellet was resuspended in the same solution, protein concentration was determined, and membranes were stored in aliquots at 6 µg/µlat -80 °C. For chemical interference assays, 50 µg of membrane vesicles were treated with 40 mM TrisHCl, pH 7.5, with either 2 mM AMP-PNP or 2mM ATP{gamma}S in the presence of 6 mM MgCl2 or with 2 mM ATP in the presence BeFx (2 mM BeSO4 and 10 mM NaF). The mixture was incubated for 30 min at 37 °C, and after centrifugation (16,000 x g, 20 min, 4 °C), the pellet was solubilized in lysis buffer (1% {alpha}-DM, 150 mM NaCl, 50 mM TrisHCl, pH 7.4) for 30 min at 4 °C. Homogenates were then spun at 4 °C at 100,000 x g for 20 min, and protein concentration was determined before loading of samples (2 µg) on native PAGE. ATP and analogues were purchased from Sigma. To remove trace amounts of contaminating ATP, 5 mM AMP-PNP or ATP{gamma}S was treated with hexokinase as described previously (30). BeSO4 was from Fluka Chemie AG, Buchs, Switzerland.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FRET Analysis of ABCA1 Dimerization—To first assess the existence of ABCA1 supramolecular structures, we used FRET methodology, considering its minimal interference with native membrane architecture. We thus constructed chimeras between ABCA1 and the yellow and cyan variant of EGFP, a pair of acceptor donor fluorochromes particularly appropriate for FRET analysis. Two sets of chimeras were generated possessing the fluorescent moiety either at the N-terminal or at the C-terminal end of the transporter. They will be referred to as YABCA1 or CABCA1 for the N-terminal chimeras and ABCA1Y or ABCA1C for the C-terminal chimeras. Because they are localized in both cases on the cytosolic side of the membrane, the fluorescent graft was not expected to disturb the topological assembly of the transporter. Indeed, all the chimeras were equally expressed (data not shown and Ref. 3); they were also validated as neutral for both trafficking and function, as assessed, respectively, by confocal microscopy or the ability to elicit binding of apoA-I or annexin V at the surface of transfected cells (data not shown and Ref. 3). FRET was measured on cells simultaneously transfected with equimolar amounts of pairs of EYFP and ECFP chimeras in different combinations, i.e. C-terminal-tagged pairs, N-terminal-tagged pairs, or a mixture of N- and C-terminal-tagged molecules. Under microscopic visualization, ROI were selected, and transfer of energy was measured as an increase in the fluorescence intensity of the donor ECFP after acceptor photobleaching.

A set of five scans was recorded before and after bleaching, and variations in fluorescence intensity were monitored. As a control, the bleaching performed in cells transfected with either ABCA1C or ABCA1Y alone never resulted in a detectable increase in emission at donor wavelength (data not shown). Background values were assessed for each experiment on both cytosolic and extracellular ROI and were considered acceptable if lower than 20% of the fluorescence intensity of the donor or acceptor fluorochrome in a given preparation.

As shown in Fig. 1, significant FRET was measured in the cases of simultaneous transfections with both N-tagged pairs and C-tagged pairs and with equimolar mixtures of N and C grafts. In all three combinations, the efficiency of transfer was found to be independent of acceptor density, measured as intensity of EYFP fluorescence (Fig. 1A and not shown). However, the efficiency of transfer was found to be significantly sensitive to the acceptor:donor ratio only in the combination of C pairs (ABCA1C and ABCA1Y, Fig. 1, B-D, and Table 1 for average values of recorded FRET in the different combinations). This validated the transfer as due to the physical proximity rather than random molecular collision and indicated that the contact takes place at the C-terminal extremities of ABCA1. Consequently, this implied that the most likely orientation of the molecular cluster is a "snail-like" structure with a tail-to-tail orientation in the simplest dimeric structure.


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  		TABLE 1
FRET efficiency measured according to the formula Ef = Ipost - Ipre X 100/Ipost

Values are expressed as mean ± S.E. ABCA1 and its mutants were expressed as chimeras with EYFP or ECFP, indicated by Y or C added to the name of the mutant. n = number of individual assessments. A:D acceptor:donor ratio; N-ter, N-terminal; C-ter, C-terminal.

 
ABCA1 Dimers Exist on Naturally Expressing Cells—To further characterize these supramolecular structures, we turned our attention to the biochemical analysis of RAW 264.7 cells stimulated with cAMP, a well established condition for the induction of high expression levels of ABCA1 (27). We then analyzed the fractionation by velocity sucrose gradients of cell lysates exposed to detergents of different chemical characteristics. At floating equilibrium, 24 fractions were collected and analyzed by Western blotting for their reactivity with the 891.3 mAb, specifically recognizing the ABCA1 transporter as shown in Fig. 2A. Upon fractionation on 10-40% sucrose gradients in the presence of the denaturing detergent SDS, most ABCA1 was recovered in fractions 4-6, which thus correspond to the flotation of monomeric forms; however, upon solubilization with non-ionic detergents (from Triton X-100 to Nonidet P-40 and DM), ABCA1 was progressively recovered in fractions corresponding to higher molecular weights (Fig. 2B). To better evaluate the size of ABCA1-containing particles, we performed a 5-30% sucrose gradient and fractionated cell lysates obtained in the presence of either the {alpha} or the beta isoforms of DM (Fig. 2C). In this latter condition, ABCA1 was recovered in fractions corresponding to a molecular mass >360 kDa as evaluated by the parallel fractionation of native markers. On this basis, we concluded that, in native cells, high molecular forms of the transporter are present and are likely to correspond to dimeric associations in accordance to the previous results based on FRET analysis of transfected cells (Fig. 2, B and C).

To further refine these results, we assessed ABCA1 oligomeric structures by native PAGE (Fig. 2D). In cAMP-stimulated RAW cells, ABCA1 was detected as a major band migrating at a molecular mass >440 kDa, consistent with a dimer. In transfected cells, the ABCA1-reactive band migrated at a slightly higher molecular mass, consistent with the presence of the EYFP graft (Fig. 2D, right panel). The dimeric structures in both RAW and transfected cells were confirmed by the resolution at 1% {alpha}-DM of a lower band migrating at 250 kDa, thus compatible with a monomer (Fig. 2D and not shown). Altogether, these results confirm that at equilibrium, ABCA1 is predominantly associated in dimeric forms in both cellular systems. Densitometric evaluation indicated that the dimers accounted for 80-90% of the detectable transporter, assuming equivalent reactivity of the antibody. However, in lysates of mouse PEM, a major physiological ABCA1 expression site (31), increased amounts of monomers were detected (Fig. 2D). These accounted for 25-40% of the total ABCA1 signal. Since the amount of ABCA1 at the plasma membrane is higher in cAMP-stimulated RAW cells than in PEM,4 this observation suggests that dimeric association may be influenced by either the intracellular localization of the transporter or its functional state.

ABCA1 Variants and Oligomerization—In the aim of identifying the cellular dimerization compartment and the impact of reduced function on the dimerization potential of the transporter, we analyzed by native PAGE the behavior of naturally occurring ABCA1 variants. We selected two sets of variants. The first took advantage of the Q597R and {Delta}L693 mutations, previously shown to be massively retained in the endoplasmic reticulum (3), whereas the second set comprised mutants that, although correctly expressed and trafficked to the plasma membrane, showed reduced ability to elicit the functional phenotypes associated with ABCA1 expression in transfected cells.

The analysis of lysates from cells transfected with EGFP-tagged Q597R or {Delta}L693 mutants either alone or in combination with wild type ungrafted ABCA1 transporter indicated that three molecular species of dimers are detectable (Fig. 3A). The expected lower form, due to homodimers of the wild type transporter, was exclusively detected by the 891.3 antibody, whereas the higher forms also reacted with the anti-GFP antibody and therefore corresponded to hetero- and homodimeric associations of the EGFP-grafted variant transporters. This indicates that both variants are able to dimerize. However, the simultaneous expression of mutant and wild type forms of ABCA1 did not modify the trafficking behavior of individual forms as shown by the confocal analysis of double-transfected cells (Fig. 3, B and C). This indicated that the dimerization occurs in the endoplasmic reticulum and that dimers containing the variants are transient and unproductive structures because they were unable to rescue the trafficking defect.

We next analyzed whether ABCA1 dysfunction was associated with an altered ability to dimerize as assessed by both FRET and native PAGE. We chose to analyze W590S and C1477R, two Tangier-associated mutations previously characterized and known to differentially affect the ABCA1-induced binding of apoA-I and annexin V (3, 32). In both cases, FRET measurements on cells transfected with appropriately tagged constructs supported dimerization since an efficiency of the energy transfer similar to that of wild type and similarly sensitive to variations in acceptor-to-donor ratios was detected (Fig. 4, A and B, and Table 1). However, upon fractionation on native PAGE, the lysates of cells transfected with W590S or C1477R showed a higher molecular mass band migrating at >800 kDa and accounting for 9 and 27%, respectively, of the total ABCA1 signal (Fig. 4C, upper panel), in the absence of any modification of expression levels as shown by the SDS-PAGE analysis (Fig. 4C, lower panel). On the basis of its apparent molecular weight, this band is presumably a tetramer, and its intensity roughly correlates to the impaired apoA-I docking ability of the analyzed variants (3). These data clearly indicate that molecular forms other than dimers occur during the functional cycle of the transporter and suggest that the rate of transition between these states may be determinant for the overall ABCA1 function.


Figure 2
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  		FIGURE 2.
Biochemical assessment of ABCA1 dimerization. A, the specificity of monoclonal antibody 891.3 is demonstrated by its reactivity by Western blot on lysates from cAMP-stimulated RAW 264.7 cells, HeLa cells expressing ABCA1 EYFP (A1Y, left panel), and PEM (right panel, +/+) from wild type mice. No reactivity is detected in unstimulated RAW cells, ABCA7 EYFP (A7Y)-expressing, or mock-transfected (mock, left panel) HeLa cells. Similarly, reduction and loss of the signal follows the heterozygous (-/+) and homozygous (-/-) deletion of the abca1 gene (PEM, right panel). MW, molecular weight markers. B, velocity sucrose gradient centrifugation of cAMP-stimulated RAW cell lysates shows that the sedimentation of ABCA1-containing particles is dependent on the detergent used. Although in the presence of 1% SDS, denatured monomeric ABCA1 floats in fractions 4-5, the lysis in the presence of milder detergents progressively shifts the recovery of ABCA1 into denser fractions (fractions 6-7). C, flotation in the presence of {alpha}- or beta-DM evidences ABCA1 dimerization in cAMP-stimulated RAW cell lysates. Estimation of molecular weight is performed by the flotation of native protein markers (thyroglobulin, urease, and catalase) in parallel and equivalent gradients. D, native PAGE confirms that ABCA1 is predominantly assembled in dimers. Western blot analysis of cAMP-stimulated RAW cells lysed in the presence of increasing amounts of {alpha}-DM and fractionated on native PAGE indicates that ABCA1 is detected as a major band at ~500 kDa and consistent with dimeric assembly (left panel). As expected, the dimer is progressively denatured into monomer by increasing detergent concentrations. In the right panel, a comparison of relative amounts of dimers in lysates from cAMP-stimulated RAW cells, A1Y-transfected HeLa cells, and peritoneal elicited macrophages (PEM) is shown. Detection was performed exclusively with the 891.3 mAb.

 
ATP Catalytic Cycle and Oligomerization—To explore this issue, we analyzed whether transition forms occur as part of the ATP catalytic cycle, a basic functional feature of ABCA1 as an ABC transporter. We reasoned that a variant of ABCA1 with an impaired ability to perform the cycle, such as that provoked by the Lys to Met mutation in the Walker A motif, which locks the ATP-bound state, could allow the visualization of transition states. We preliminarily verified that ABCA1MM carrying the Lys to Met mutation at both nucleotide binding domains, previously shown to be efficiently expressed and trafficked (2, 33), retained the ability to dimerize as assessed by FRET. To this aim, we constructed ABCA1MM ECFP and EYFP grafts at the C-terminal end of the transporter and measured the efficiency of transfer between molecules upon transfection in HeLa cells, as described above. The FRET parameters measured for ABCA1MM were essentially superimposable to those of the wild type, in terms of both efficiency of energy transfer and sensitivity to variations in acceptor:donor ratio (Fig. 5A and Table 1). However, the ABCA1-reactive pattern determined by native PAGE was dramatically different from that of the wild type (Fig. 5B, left panel). Indeed, additional ABCA1-reactive bands were detected in the absence of any modification of the level of expression in transfected cells as detected by SDS-PAGE (Fig. 5B, lower left panel). On the basis of the apparent molecular weight, the banding pattern in native conditions is consistent with dimers, tetramers, and even higher supramolecular assemblies. Alternatively, major conformational changes inducing aberrant migration of the dimeric complexes under native conditions may be evoked. At any rate, this pattern indicated that ATP binding induces transition states that, when trapped by the MM mutant, can be efficiently detected by native PAGE. Interestingly, the mutants bearing the Lys to Met substitution at either the first or the second nucleotide binding domain showed an identical behavior on native PAGE, i.e. a banding pattern of much weaker intensity but essentially similar to that of the double mutant (Fig. 5B, upper left panel). This fits with a model of alternative and equivalent functioning of the two ATP binding sites since hydrolysis at either nucleotide binding domain suffices to release most of the changes trapped by the ABCA1MM mutant.


Figure 3
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  		FIGURE 3.
ABCA1 dimerization occurs in the endoplasmic reticulum. A, native PAGE profile of lysates from HeLa cells transfected with mixtures of wild type (wt) ABCA1 and the endoplasmic reticulum-retained variants evidences the presence of hetero- and homodimers. The mixtures contained equimolar amounts of ungrafted ABCA1 and of either Q597R or {Delta}L693 variants grafted with EGFP (Q597RG or {Delta}L693G). For comparison, the banding profile of ungrafted (A1WT) and EYFP-grafted (A1Y) ABCA1 is shown. Detection was performed as indicated by either the 891.3 or the anti-GFP mAb. MW, molecular weight markers. B and C, confocal analysis of cells transfected with equimolar mixtures of wild type ABCA1 grafted with ECFP (A1C) and either Q597R or {Delta}L693 grafted with EGFP indicate that the presence of wild type transporter in the dimers fails to rescue the trafficking defect of both variants.

 


Figure 4
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  		FIGURE 4.
Oligomerization of Tangier associated ABCA1 variants. A and B, imaging FRET of cells expressing appropriately tagged W590S or C1477R, two ABCA1 variants associated with Tangier disease and variably affecting the functional readouts associated with ABCA1 expression. Both can associate efficiently in dimers as demonstrated by FRET parameters superimposable to that of wild type transporter (Table 1). C, upper panel, native PAGE profiles of lysates from cells transfected with ABCA1Y, W590S, W590SY, or C1477RY indicate that the two variants associated in dimers. However, an additional band migrating at a size compatible with tetrameric assembly is detected. Of note, the intensity of the band is directly correlated to the loss of apoA-I binding ability associated with the variant (3). Lower panel, SDS-PAGE profiles of corresponding lysates indicate comparable expression levels for the various ABCA1 variants. Detection was performed by the anti-GFP mAb. MW, molecular weight markers.

 
To further support this finding and to unambiguously rule out any structural interference due to the mutation introduced in the transporter, we analyzed whether interfering with the catalytic cycle could trap transient assembly of the endogenously expressed transporter. To this aim, membrane vesicles were prepared from cAMP-stimulated RAW 264.7 cells and treated in conditions hampering ATP hydrolysis: either with non-hydrolyzable competitors of ATP (ATP{gamma}S or AMP-PNP) or with ATP + BeFx in the absence of Mg2+ ions (34). The subsequent analysis by native PAGE revealed, in all treated samples, the appearance of supradimeric forms (Fig. 5C, left panel). This definitely confirmed that, upon ATP binding, the transporter undergoes transition from dimeric to higher order molecular assemblies. Of note and as expected, the efficiency of the chemical treatments in inducing oligomerization is rather low when compared with the ABCA1MM mutant, as shown by the densitometric profiles (Fig. 5, B and C, right panels).

To strengthen the identification of the ATP-bound forms as tetramers rather than dimers undergoing conformational changes, we reasoned that titration of the ABCA1MM with increasing amounts of the wild type transporter should allow us to differentiate the two conditions (Fig. 5, D and E). The titration was performed by transfecting HeLa cells with 5µg of total DNA in which the proportion of wild type encoding plasmid was stepwise increased over that of ABCA1MMY. This induced the expected expression profile of the two forms of the transporter (Fig. 5D, right panel, SDS PAGE) and resulted, at rather low wild type titers, in a substantial rescue of wild type behavior, as assessed either by native PAGE or by annexin V and apoA-I binding (Fig. 5D, left panel, and E). These data definitely indicate an efficient intermolecular cooperation that is inconsistent with dimeric structures and can be accounted for only by considering interactions in higher order associations such as tetramers.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The combined use of biophysical approaches, such as FRET, and biochemical methods, such as native PAGE, allowed us to demonstrate that the ABCA1 transporter is predominantly present in cells as homodimers. These structures are equally present in naturally expressing cells such as cAMP-stimulated RAW 264.7 or in models of forced expression of the transporter. The assembly takes place in the endoplasmic reticulum, which is a rather common rule in the assembly of multimolecular channel structures (35). This point raises the question as to whether the transporter is already in a fully active configuration in this early biosynthetic compartment or whether it requires further post-translational regulations, such as phosphorylation or specific molecular interactions, to achieve full functional maturation.

The ABCA1 dimers, as determined by FRET, are oriented so that the C-terminal extremities of ABCA1 are in close proximity. A similar arrangement has been proposed for cystic fibrosis transmembrane conductance regulator, in which, in addition, molecular contacts through the PDZ region have been proposed (18, 36, 37). In the case of ABCA1, both a putative PDZ binding motif present at the C terminus and the VFVNFA sequence located at positions 2210-2215 have been identified as potential interaction sites (32, 38, 39). They may be required either to stabilize the dimeric interaction or to introduce additional molecular partners in the complex. Chemical interactions such as disulfide bridges between the numerous cysteines present in the extracellular loops of the transporter may also participate in dimer stabilization (40). However, they are probably part of a more complex stabilizing array since, at odds with the results of Denis et al. (6), we could not dissociate the dimeric structures by treatment with reducing agents (not shown). Consistently with their results, however, we evidenced that ABCA1 dimeric associations also prevail in the three cellular systems that we studied.


Figure 5
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  		FIGURE 5.
Functionally relevant oligomers occur as intermediate states during the ATP catalytic cycle. A, imaging FRET parameters of ECFP- and EYFP-grafted ABCA1MM mutants expressed in HeLa cells indicates an intact ability to dimerize since both the efficiency of intermolecular transfer and its sensitivity to variation in acceptor:donor ratios are superimposable to that of the wild type transporter. B, upon native PAGE fractionation (upper left panel), lysates of cells expressing the ABCA1MM (A1MM) mutant show additional ABCA1-reactive bands migrating at a molecular weight (MW) consistent with tetramers and even higher supramolecular forms. Interestingly, a similar, although much weaker, pattern is detected in cells expressing ABCA1 variants carrying the Lys to Met mutation in Walker A motif or either the first (A1MK) or the second (A1KM) nucleotide binding domain. Comparable levels of expression of the ABCA1 variants are evidenced by fractionation on SDS-PAGE (lower panel). The appearance of ABCA1 supradimeric forms is monitored by the densitometric tracing (right panel, arrows). C, induction of supradimeric forms of endogenously expressed ABCA1 by chemical interference with ATP hydrolysis. Membrane vesicles, prepared from cAMP-stimulated RAW cells, were treated as indicated before loading on native PAGE (left panel). The appearance of the ABCA1 supradimeric forms is monitored by the densitometric tracing (right panel, arrows). D, titrations of the non-functional ABCA1MM (A1MM) mutant with increasing amounts of wild type transporter (A1)(A1WT) lead, at low wild type titers (1:4), to an impressive reduction of trapped tetrameric forms upon fractionation of cell lysates by native PAGE (left panel). The proportion of ABCA1 wild type and ABCA1MMY is checked after fractionation on SDS-PAGE (right panel) followed by hybridization with anti-GFP mAb (revealing exclusively ABCA1MMY) and 891.3 mAb (detecting both wild type and mutant forms). The modification of native PAGE profiles is paralleled by a substantial rescue of functional activity of the transporter as assessed by apoA-I and annexin V binding (E). Values (expressed as mean of four individual experiments ± S.E.) are, in the case of annexin V, 15.68% ± 6 in the absence of ABCA1 wild type, 70.70% ± 5 for 1:4, 81.85% ± 6 at 2.5:2.5, and 91.48% ± 3 at 4:1, whereas for apoA-I binding, they are 16.13% ± 8 in the absence of ABCA1 wild type, 63.60% ± 7 at 1:4, 76.84% ± 8 at 2.5:2.5, and 92.46 ± 4 at 4:1.

 
By the use of functional variants of ABCA1, we could evidence that supradimeric assemblies take place; these assemblies are transient in nature but can be trapped experimentally by interfering with the ATP catalytic cycles, namely by stabilizing the ATP-bound state. The latter can be achieved either via engineered mutations in the ATP binding sites, allowing the binding of the nucleotide but hampering its hydrolysis (33), or via chemical interference with ATP hydrolysis on endogenously expressed transporter. Indeed, both upon transfection of the mutant ABCA1MM in HeLa cells and upon chemical treatment of membrane vesicles derived from cAMP-treated RAW 264.7 cells, we detected the formation of higher order molecular assemblies containing ABCA1. On the basis of titration experiments, we interpreted these structures as tetramers rather than dimers undergoing major conformational changes upon ATP binding.

The notion of transient molecular assembly during the cycle of ABCA1 is of extreme interest in that it introduces the parameter of molecular interactions as modulators of function. In addition, this finding stresses the advantage of native PAGE as a powerful, but largely underexploited, tool for the study of the quaternary structure of ABC transporters.

Collectively, the results presented here allow us to draw a model coupling molecular assembly of ABCA1 to its catalytic cycle as an ABC transporter and to its established function of lipid extruder. In the basal state, a fraction of ABCA1 dimers exists in open conformation, i.e. adequate to enter the cycle of nucleotide binding and hydrolysis. What controls this competence remains to be ascertained, but the partitioning of the transporter into different membrane domains (41, 42) could well be part of it. ATP binding then induces the association of ABCA1 dimers in higher order structures, which promptly dissociate upon ATP hydrolysis. The latter is also required to elicit the cellular phenotypes associated with expression of ABCA1: binding of apoA-I, phosphatidylserine exposure at the cell surface, and the subsequent efflux of phospholipids and cholesterol. Resetting of the transporter in the initial conformation takes place after effluxes of membrane phospholipids.

This scenario is largely extrapolated and certainly oversimplified. It provides nonetheless a useful start point for further investigation. Indeed, it is likely that other molecular partners acting as regulators of cycle entering, modulators, or functional effectors take part in the ABCA1 complex at the various stages. Their identification is crucial and will require further investigations oriented, for instance, to the proteomic analysis of ABCA1 complexes in opportunely reconstituted cellular systems.


   FOOTNOTES
 
* This work was supported in part by institutional grants from INSERM and CNRS and specific grants from the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Funded in part by a fellowship allocated by the Nouvelle Société Française d'Athérosclérose/Fournier Pharma. Back

2 To whom correspondence should be addressed: Centre d'Immunologie de Marseille-Luminy INSERM CNRS Université delaMéditerranée, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 09 France. Tel.: 33-4-91269404; Fax: 33-4-91269430; E-mail: chimini{at}ciml.univ-mrs.fr.

3 The abbreviations used are: ABC, ATP binding cassette; FRET, fluorescence resonance energy transfer; mAb, monoclonal antibody; ROI, regions of interest; DM, dodecyl maltopyranoside; PEM, peritoneal elicited macrophage; GFP, green fluorescent protein; EGFP, enhanced GFP; EYFP, enhanced yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein; AMP-PNP, adenosine 5'-(beta,{gamma}-imido)triphosphate; ATP{gamma}S, adenosine 5'-3'-(thiotriphosphate). Back

4 A. Zarubica and D. Trompier, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Roberto Bassi and Didier Marguet for enlightening discussions and are indebted to the Plateforme d'Imagerie Commune Scientifique Luminy imaging core facility for expert technical assistance. The technical assistance of Matthieu Pophillat is also acknowledged.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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