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• W1993012149 abstract "ATP-binding cassette transporter A1 (ABCA1) is known to mediate cholesterol efflux to lipid-poor apolipoprotein A-I. In addition, ABCA1 has been shown to influence functions of the plasma membrane, such as endocytosis and phagocytosis. Here, we report that ABCA1 expression results in a significant redistribution of cholesterol and sphingomyelin from rafts to non-rafts. Caveolin, a raft/caveolae marker also redistributes from punctate caveolae-like structures to the general area of the plasma membrane upon ABCA1 expression. Furthermore, we observed significant reduction of Akt activation in ABCA1-expressing cells, consistent with raft disruption. Cholesterol content in the plasma membrane is, however, not altered. Moreover, we provide evidence that a non-functional ABCA1 with mutation in an ATP-binding domain, A937V, fails to redistribute cholesterol, sphingomyelin, or caveolin. A937V also fails to influence Akt activation. Finally, we show that apolipoprotein A-I preferentially associates with non-raft membranes in ABCA1-expressing cells. Our results thus demonstrate that ABCA1 causes a change in overall lipid packing of the plasma membrane, likely through its ATPase-related functions. Such reorganization by ABCA1 effectively expands the non-raft membrane fractions and, consequentially, pre-conditions cells for cholesterol efflux. ATP-binding cassette transporter A1 (ABCA1) is known to mediate cholesterol efflux to lipid-poor apolipoprotein A-I. In addition, ABCA1 has been shown to influence functions of the plasma membrane, such as endocytosis and phagocytosis. Here, we report that ABCA1 expression results in a significant redistribution of cholesterol and sphingomyelin from rafts to non-rafts. Caveolin, a raft/caveolae marker also redistributes from punctate caveolae-like structures to the general area of the plasma membrane upon ABCA1 expression. Furthermore, we observed significant reduction of Akt activation in ABCA1-expressing cells, consistent with raft disruption. Cholesterol content in the plasma membrane is, however, not altered. Moreover, we provide evidence that a non-functional ABCA1 with mutation in an ATP-binding domain, A937V, fails to redistribute cholesterol, sphingomyelin, or caveolin. A937V also fails to influence Akt activation. Finally, we show that apolipoprotein A-I preferentially associates with non-raft membranes in ABCA1-expressing cells. Our results thus demonstrate that ABCA1 causes a change in overall lipid packing of the plasma membrane, likely through its ATPase-related functions. Such reorganization by ABCA1 effectively expands the non-raft membrane fractions and, consequentially, pre-conditions cells for cholesterol efflux. Apolipoprotein A-I (apoA-I) 5The abbreviations used are: apoA-I, apolipoprotein A-I; ABCA1, ATP-binding cassette transporter A1; MCD, methyl-β-cyclodextrin; YFP, yellow fluorescent protein; GFP, green fluorescent protein; EGF, epidermal growth factor; DiIC18, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate; BHK, baby hamster kidney; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; wt, wild type. -mediated lipid efflux is one of the earliest events in reverse cholesterol transport, a process that generates high density lipoprotein and transports excess cholesterol from the peripheral tissues, including the arterial wall, to the liver for biliary secretion. This process is absent in Tangier disease, due to mutations in ABCA1 (1Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J. Hayden M.R. Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1509) Google Scholar). Without a functional ABCA1, apoA-I is rapidly catabolized, leading to cholesterol accumulation in peripheral tissues and low plasma high density lipoprotein. ABCA1 therefore plays a key role in cholesterol efflux to lipid-poor lipoproteins, such as apoA-I. There has been considerable debate as to whether ABCA1 mediates apoA-I acquisition of phospholipids and cholesterol separately or simultaneously. In a “two-step model,” lipid-poor apoA-I firstly forms a high affinity complex with ABCA1 (2Fitzgerald M.L. Morris A.L. Rhee J.S. Andersson L.P. Mendez A.J. Freeman M.W. J. Biol. Chem. 2002; 277: 33178-33187Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 3Oram J.F. Lawn R.M. Garvin M.R. Wade D.P. J. Biol. Chem. 2000; 275: 34508-34511Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 4Wang N. Silver D.L. Costet P. Tall A.R. J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), which facilitates apoA-I association with phospholipid-rich domains. Second, this phospholipid-primed apoA-I acquires cholesterol from cholesterol-rich domains, a process thought to be independent of ABCA1. On the other hand, Smith et al. (5Smith J.D. Le Goff W. Settle M. Brubaker G. Waelde C. Horwitz A. Oda M.N. J. Lipid Res. 2004; 45: 635-644Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) reported that ABCA1 mediates concurrent efflux of phospholipid and cholesterol. These contrasting models demonstrate that the mechanism by which ABCA1 mediates lipid efflux to apoA-I has yet to be clarified. Interestingly, the plasma membrane bilayer is thought to contain a mosaic of tightly packed lipid microdomains termed “lipid rafts.” Commonly defined based on their insolubility in non-ionic detergents, these microdomains contain high concentrations of cholesterol, sphingolipids, caveolin, and many proteins involved in cell signaling. In terms of apoA-I-mediated efflux, these microdomains represent a conceptually attractive “in situ” reservoir of cholesterol in the plasma membrane. Despite their enrichment in cholesterol, however, these domains do not seem to play a major role in efflux, because apoA-I was shown to preferentially acquire cholesterol from the loosely packed, “non-raft” microdomains (6Drobnik W. Borsukova H. Bottcher A. Pfeiffer A. Liebisch G. Schutz G.J. Schindler H. Schmitz G. Traffic. 2002; 3: 268-278Crossref PubMed Scopus (141) Google Scholar, 7Mendez A.J. Lin G. Wade D.P. Lawn R.M. Oram J.F. J. Biol. Chem. 2001; 276: 3158-3166Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). ABCA1 itself also appears to be localized in Triton X-100-soluble fractions (non-rafts) (7Mendez A.J. Lin G. Wade D.P. Lawn R.M. Oram J.F. J. Biol. Chem. 2001; 276: 3158-3166Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). It is far from clear whether non-rafts are required for ABCA1-mediated cholesterol efflux. Indeed, recent work performed by Duong and colleagues (8Duong P.T. Collins H.L. Nickel M. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Lipid Res. 2006; 47: 832-843Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) further revealed a need for a better understanding of the relative contribution of these microdomains to the ABCA1-mediated formation of nascent high density lipoprotein particles. Aside from cholesterol efflux, ABCA1 has been linked to several functions on the plasma membrane, such as phagocytosis, endocytosis, and microvesiculation (9Alder-Baerens N. Muller P. Pohl A. Korte T. Hamon Y. Chimini G. Pomorski T. Herrmann A. J. Biol. Chem. 2005; 280: 26321-26329Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Hamon Y. Broccardo C. Chambenoit O. Luciani M.F. Toti F. Chaslin S. Freyssinet J.M. Devaux P.F. McNeish J. Marguet D. Chimini G. Nat. Cell Biol. 2000; 2: 399-406Crossref PubMed Scopus (467) Google Scholar, 11Luciani M.F. Chimini G. EMBO J. 1996; 15: 226-235Crossref PubMed Scopus (253) Google Scholar, 12Zha X. Genest Jr., J. McPherson R. J. Biol. Chem. 2001; 276: 39476-39483Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). These findings collectively imply that ABCA1 exerts significant influence on the plasma membrane. It is however unclear whether ABCA1 influences raft/non-raft partition and, consequently, how this may affect apoA-I-mediated cholesterol efflux. An attractive concept would be that ABCA1 actively disrupts the raft microdomains and makes cholesterol more readily available, thus facilitating cholesterol-apoA-I interaction and efflux. To test this possibility, we analyzed in detail the impact of ABCA1 expression on membrane micro-organizations. We observed that ABCA1 disrupts microdomains (rafts) and redistributes cholesterol/sphingomyelin to non-raft domains. ABCA1 also disrupts caveolae formation. Consequently, ABCA1 expression impairs EGF-induced Akt activation, a process known to be sensitive to raft integrity (13Zhuang L. Lin J. Lu M.L. Solomon K.R. Freeman M.R. Cancer Res. 2002; 62: 2227-2231PubMed Google Scholar). We also provide evidence that this membrane reorganization is dependent on a functional nucleotide binding domain of ABCA1, suggesting involvement of ATPase-related functions in membrane reorganization. Furthermore, we observed preferential association of apoA-I with the non-raft fraction of the plasma membrane. Our results thus provide a potential mechanism by which ABCA1 facilitates apoA-I cell association and cholesterol efflux by disrupting the tightly packed lipid raft microdomains. Material and Reagents—Baby hamster kidney (BHK) cells stably expressing either an empty vector (mock) or ABCA1 under the control of a mifepristone-inducible Gene-switch promoter were prepared as described previously (14Vaughan A.M. Oram J.F. J. Lipid Res. 2003; 44: 1373-1380Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). To facilitate morphological analysis, we further subcloned ABCA1 or mutant cells and chose the colonies that express equal levels of ABCA1 among individual cells. Cell culture media and reagents were from Invitrogen. Mifepristone, methyl-β-cyclodextrin, and Triton X-100 were purchased from Sigma. A cholesterol kit was from VWR International (West Chester, PA). Polyclonal antibody against ABCA1 was from Novus Biological Inc. (Littleton, CO). Anti-caveolin antibodies were from BD Transduction Laboratories. Polyclonal anti-Akt antibody (C-20) was from Santa Cruz Biotechnology (Santa Cruz, CA), and polyclonal anti-phospho-Akt (Ser-473) was from Cell Signaling Technology (Beverly, MA). Fluorescent secondary antibodies and DiIC18 were purchased from Molecular Probes (Eugene, OR). YFP-caveolin is a C-terminally YFP-tagged caveolin 1 and was reported to traffic similarly to caveolin-GFP that transports to caveolae identically to untagged caveolin (15Pol A. Martin S. Fernandez M.A. Ingelmo-Torres M. Ferguson C. Enrich C. Parton R.G. Mol. Biol. Cell. 2005; 16: 2091-2105Crossref PubMed Scopus (171) Google Scholar). YFP-caveolin was obtained originally from Dr. Robert G. Parton (University of Queensland, Brisbane, Australia). Cell Culture—BHK cells were maintained in DMEM plus 10% fetal calf serum at 37 °C in a 5% CO2 incubator. Incubating cells for 18-20 h in DMEM with 1 mg/ml BSA and 10 nm mifepristone induced ABCA1 expression. Mock or mutant cells were treated identically as controls. Endogenous Caveolin-1 Distribution—ABCA1- and mock-expressing BHK cells were grown to near confluence in glass bottom dishes and incubated for 18-20 h in DMEM containing 1 mg/ml BSA and 10 nm mifepristone. Endogenous caveolin-1 was visualized using a plasma membrane-specific mouse monoclonal antibody against caveolin-1 followed by incubation with an Alexa-488 goat anti-mouse secondary antibody. Confocal fluorescent images of the basal membrane were taken using a Nikon TE300 inverted fluorescent microscope with a 60× (numerical aperture, 1.4) objective and the 488 nm line of an argon ion laser. Cholesterol Mass Determination—Cellular lipids were extracted by organic solvent and dried under N2. The dry samples were directly resuspended by vigorous vortexing in 500 μl of the Free Cholesterol E reagent supplied with the cholesterol mass determination kit (Wako Chemicals, Richmond, VA), incubated for 10 min at 37 °C, and the absorbance was determined at 600 nm. The Triton X-100-extractable cholesterol fraction was calculated as % cholesterol as follows: (Triton X-100-soluble fraction)/(Triton X-100-soluble fraction + Triton X-100-insoluble fraction). Triton X-100 Extraction—Mifepristone-induced 75% confluent BHK cells grown in 6-well plates were washed twice with ice-cold PBS and chilled on ice for 30 min in DMEM containing 10 mm Hepes, pH 7.4. The medium was then replaced with 1 ml/well DMEM/Hepes in the presence or absence 1% Triton X-100 and further incubated on ice for 30 min. The medium was then collected, and the first wash with 500 μl of ice-cold PBS was combined with the medium. Lipids were then extracted overnight at 4 °C by adding 4 volumes of Folch solution (chloroform/methanol (2:1)). The organic phase was collected and evaporated under N2 atmosphere. Lipids were also extracted twice from cells in 1 ml/well of a 3:2 hexane:isopropanol solution, and the organic phase was evaporated under N2 atmosphere as described previously. For labeling free and esterified cholesterol, cells were labeled for 48 h with 1 μCi/ml [3H]cholesterol. After Triton X-100 treatment, tritiated samples were separated by high-performance thin layer chromatography (Whatman) using a 80:20:6.7:1 hexane:ether:methanol:acetic acid mixture as the elution system. For 3H-labeled phospholipids, half-confluent cells were labeled for 24 h with 1 μCi/ml [3H]choline and the elution system was composed of chloroform:methanol:acetic acid:formic acid:water (35:15:6:2: 1). Spots corresponding to cholesteryl ester, free cholesterol, phosphatidylcholine, and sphingomyelin were scraped and counted in a β-scintillation counter. Cholesterol Efflux—BHK cells were incubated in normal growing media (DMEM plus 10% fetal calf serum) with 0.5 μCi/ml [3H]cholesterol for 2 days to label cellular cholesterol to equilibrium. Cells were then switched to DMEM with 1 mg/ml BSA and 10 nm mifepristone for 18-20 h. To measure cholesterol efflux, cells were incubated with 10 μg/ml apoA-I for 4 h at 37 °C. Medium was collected, centrifuged to remove detached cells, and counted for 3H. Cells were lysed with NaOH (0.5 n) overnight and counted for 3H. Cholesterol efflux was calculated as the percentage of total [3H]cholesterol released into the medium. For methyl-β-cyclodextrin (MCD)-induced cholesterol efflux, cells were also labeled with 0.5 μCi/ml [3H]cholesterol for 2 days, followed by 18-20 h of incubation with 1 mg/ml BSA and 10 nm mifepristone. Cells were then incubated with 5 mm MCD either at 37 °C for 1 min or on ice for the indicated time. Medium was collected, centrifuged, and counted for [3H]cholesterol. Cell-associated [3H]cholesterol was measured from NaOH lysates as mentioned above. Efflux was again calculated as the percentage of total [3H]cholesterol released into the medium. Fluorescent Microscopy—For immunofluorescent staining of ABCA1, mutant, and mock-expressing BHK cells were incubated for 18-20 h in DMEM containing BSA and 10 nm mifepristone. Cells were then fixed with 4% paraformaldehyde and permeabilized with 0.1 mg/ml saponin. ABCA1 was visualized by a primary polyclonal antibody against ABCA1 followed by an Alexa-488 goat anti-rabbit secondary antibody. Confocal fluorescent images were taken using a Nikon TE2000 inverted fluorescent microscope with C1 confocal attachment and a 60× (numerical aperture, 1.4) objective. Images from ABCA1 and mock cells were taken under identical conditions. For caveolin distribution, ABCA1, mutant, and mock-expressing BHK cells were transiently transfected with YFP-caveolin using Lipofectamine 2000. Cells were then incubated overnight in DMEM/BSA plus mifepristone prior to fluorescent microscopic observations. For caveolin and ABCA1 co-localization experiments, an Alexa-594 secondary antibody and the 594 nm line of a HeNe laser were used. Filipin staining of cellular free cholesterol was performed as described previously (16Mukherjee S. Zha X. Tabas I. Maxfield F.R. Biophys. J. 1998; 75: 1915-1925Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). DiIC18 Cold Triton X-100 Extractability—Cold Triton X-100 extractability of surface-bound DiIC18 on the plasma membrane was performed as described previously (17Hao M. Mukherjee S. Maxfield F.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13072-13077Crossref PubMed Scopus (251) Google Scholar). Cells were transferred from growth medium to Medium 1 (150 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 2 g/liter glucose, and 20 mm Hepes, pH 7.4). Lipid analogs were transferred as monomers from fatty acid-free BSA carriers (18Mukherjee S. Soe T.T. Maxfield F.R. J. Cell Biol. 1999; 144: 1271-1284Crossref PubMed Scopus (309) Google Scholar). In some of the experiments, DiIC18 in ethanol was directly added to labeling medium at 37 °C. In particular, cells were labeled with DiIC18 for 20 s at 37 °C, rinsed with ice-cold Medium 1, and then incubated on ice with cold Triton X-100 (1%) for 30 min. Triton X-100-resistant membranes were visualized by imaging DiIC18 using a standard rhodamine filter set. Images were quantified by manually outlining each cell using the ImageJ software, and measuring fluorescent intensity from entire cell area. This is the fraction of DiIC18 remaining after Triton X-100 treatment (insoluble fraction, Fi). To extrapolate DiIC18 fluorescent intensity before Triton X-100 in the same cell, we randomly sampled several regions of interest from areas excluding any visible holes and determined average of fluorescent intensities from these regions of interest. We used this to calculate fluorescent intensity before Triton X-100 treatment from the entire cell area, or total DiIC18 (Ft). The percentage of DiIC18 extracted by Triton X-100 (Fs) in each individual cell could then be calculated as: Fs = (Ft - Fi) × 100/Ft. For each data point, more than 50 individual cells were measured and pooled to produce an average and standard error of the mean (S.E.). Cy3-Transferrin Labeling of EYFP-Caveolin-1-transfected Cells—Mock, ABCA1, and A937V mutant cells were grown in 35-mm glass-bottom dishes and transfected with EYFP-caveolin-1 as described above. All cell lines were then incubated in DMEM with 1 mg/ml BSA and 10 nm mifepristone for 18 h. The cells were placed on ice and washed with ice-cold PBS. To stain the transferrin receptor on the cell surface, the cells were incubated with 80 ng/ml of Cy3-transferrin in Medium 1 for 30 min on ice. The cells were then washed 2× with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min on ice. Cy3-transferrin and plasma membrane EYFP-caveolin-1 were visualized using a Nikon TE2000 inverted fluorescent microscope with a 60× (numerical aperture, 1.4) objective. Fluorescence images were taken using a cooled digital camera (Cascade 512B EM, Photometrics). Detergent-free Purification of Caveolae Membrane—The procedure used was based on an established protocol by Smart et al. (19Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar). Mock, ABCA1, and A937V mutant cells were grown to 80% confluence in 20-cm plates (five per cell line), and incubated in DMEM with 1 mg/ml BSA and 10 nm mifepristone for 18 h to induce ABCA1 expression. All of the following steps were carried out at 4 °C. Plates were washed twice with 3 ml of ice-cold buffer A (250 mm sucrose, 2.0 mm EDTA, 40 mm Tricine, pH 7.8). Cells were then scraped and collected in 3 ml of buffer A. Pelletting of cells was performed by centrifugation at 1000 × g, for 5 min, in a Beckman Coulter Allegra 6R centrifuge. The pellets were resuspended in 1 ml of buffer A and homogenized with 20 strokes of a Dounce homogenizer. Homogenates were transferred to 1.5-ml Eppendorf tubes and centrifuged at 1000 × g for 10 min to remove nuclei. After collection of supernatants, the pellets were resuspended in 1 ml of buffer A, and homogenization and centrifugation steps were repeated as described. Postnuclear supernatants from both homogenizations were combined (2 ml total) and layered onto 23 ml of 30% Percoll, and then centrifuged in a Ti-70 rotor at 86,000 × gmax for 30 min, using a Beckman Coulter Optima L-90K Ultracentrifuge. The Percoll gradient was then collected in 2-ml fractions, including the 2-ml plasma membrane band. The plasma membrane fractions were transferred to glass test tubes and sonicated, on ice, three times in succession for 10 s on setting 3 of a Branson Sonifier 450. The sonicated plasma membrane fractions were then combined with 1.84 ml of 50% Optiprep and 160 μl of buffer A, and placed in the bottom of SW41 tubes. After vortexing, a 6-ml continuous 20-10% Optiprep gradient was layered above the 4-ml plasma membrane preparations. Gradients were then centrifuged at 52,000 × g, for 90 min, in a Beckman SW41 rotor and Beckman Coulter Optima L-90K Ultracentrifuge. 10-ml Optiprep gradients were then collected in 1-ml fractions, and protein was trichloroacetic acid-precipitated. The pellets from all fractions were dissolved in sample buffer and loading buffer (60 μl of total volume) and analyzed by SDS-PAGE. All fractions were probed for caveolin-1 and clathrin by Western blot. Subcellular [3H]Cholesterol Distribution—Mock, ABCA1, and A937V mutant cells were grown to 80% confluence in 20-cm plates (two for each cell line). Radiolabeling of the cellular cholesterol pool was performed by incubation with 0.5 μCi/ml [3H]cholesterol for 24 h. Cells were then incubated in DMEM with 1 mg/ml BSA and 10 nm mifepristone for 18 h. All of the following steps were carried out at 4 °C. Plates were washed twice with 3 ml of ice-cold buffer A (250 mm sucrose, 2.0 mm EDTA, 40 mm Tricine, pH 7.8). Cells were then scraped and collected in 3 ml of buffer A. Pelletting of cells was performed by centrifugation at 1,000 × g, for 5 min, in a Beckman Coulter Allegra 6R centrifuge. The pellets were resuspended in 1 ml of buffer A, and then homogenized with 20 strokes of a Dounce homogenizer. Homogenates were transferred to 1.5-ml Eppendorf tubes and centrifuged at 1000 × g, for 10 min, in a Fischer Scientific accuSpin MicroR centrifuge, to remove nuclei. Postnuclear supernatant fractions were collected and completed to 2 ml with buffer A. 45-10% continuous sucrose gradients were formed in SW41 tubes, and the 2-ml postnuclear supernatant fractions were layered on top of the gradients. The gradients were subjected to centrifugation at 137,000 × g for 20 h at 4 °C. Each gradient was collected into 12 fractions (1 ml each). Equal volumes of each fraction were analyzed by SDS-PAGE, followed by Western blotting. Fractions were probed for the following cellular markers: caveolin-1 (plasma membrane), hsp-70 (cytosol), and calnexin (endoplasmic reticulum). [3H]Cholesterol levels in each fraction were analyzed by mixing 300 μl of sample with 2 ml of liquid scintillation mixture, and radioactivity was detected using a Beckman Coulter LS 6500 multipurpose scintillation counter. Determination of Akt Activation—The procedure used was based on a protocol developed by Pike et al. (20Pike L.J. Casey L. Biochemistry. 2002; 41: 10315-10322Crossref PubMed Scopus (166) Google Scholar). Briefly, mock, ABCA1, and A937V mutant cells were grown to 80% confluence in 20-cm plates. Cells were incubated in DMEM with 1 mg/ml BSA and 10 nm mifepristone for 18 h. The use of serum-free DMEM effectively starved the cells during the 18-h incubation. Cells were then stimulated with 50 ng/ml EGF. The cells were washed twice with 5 ml of ice-cold PBS. Once again, all of the following steps were carried out at 4 °C. Cells were washed twice with 3 ml of ice-cold radioimmune precipitation assay buffer (150 mm NaCl, 10 mm Tris, pH 7.2, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mm EDTA), scraped, and collected in 3 ml of radioimmune precipitation assay buffer, supplemented with 100 μm orthovanadate, 20 nm p-nitrophenylmethylsulfonyl fluoride, 1 μg/ml leupeptin. Pelletting of cells was performed by centrifugation at 1000 × g, for 5 min, in a Beckman Coulter Allegra 6R centrifuge. The pellets were resuspended in 1 ml of supplemented radioimmune precipitation assay buffer, and then homogenized with 20 strokes of a Dounce homogenizer. Homogenates were transferred to 1.5-ml Eppendorf tubes and centrifuged at 1000 × g for 10 min. The supernatants were collected, and aliquots were assayed for protein. Equivalent amounts of protein were mixed with SDS loading buffer and analyzed by SDS-PAGE and Western blotting. Phospho-Akt (activated) was probed using a goat polyclonal antibody recognizing phosphorylated Akt1 (Ser-473). Total Akt was probed using a goat polyclonal antibody against Akt1 (C-20). Akt activation was expressed as a ratio of phospho-Akt1/total Akt1. 125I-ApoA-I Association—Purified plasma apoA-I (Biodesign) solubilized in 4 m guanidine-HCl was dialyzed extensively against PBS buffer. ApoA-I was iodinated with 125-iodine by IODO-GEN® (Pierce) to a specific activity of 2800 cpm/ng of apoA-I, dialyzed, and used within 48 h. Mifepristone-induced BHK cells were washed two times with DMEM and incubated for 30 min at 37 °C in DMEM containing 10 μg/ml 125I-apoA-I. The medium was removed, cells were washed three times with ice-cold PBS, chilled on ice for 30 min in DMEM plus 10 mm Hepes, pH 7.4, and cold Triton X-100 extraction was performed. Cells were lysed with 0.5 n NaOH, and the protein concentration was determined by using the Lowry method. Radioactivity found in the medium and in the cells was determined by gamma counting. We hypothesized that ABCA1 modifies the plasma membrane raft/non-raft microdomains to facilitate apoA-I mediated efflux. To examine this, we used a BHK cell model stably transfected with a mifepristone-inducible plasmid containing an ABCA1 insert. A cell line containing identical mifepristone-inducible plasmid but without insert serves as control (Mock) (14Vaughan A.M. Oram J.F. J. Lipid Res. 2003; 44: 1373-1380Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). These BHK cells do not normally express ABCA1. An 18- to 20-h induction led to ABCA1 expression (Fig. 1, A). The expression level of ABCA1 in these cells is comparable to that of cAMP-induced RAW264 macrophages and ∼5-fold higher than that in human THP-1 macrophages induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid, as determined by immunoblot (data not shown). Accordingly, ABCA1-expressing BHK cells had a high efficiency to efflux cholesterol to apoA-I (Fig. 1B), comparable to RAW264 cells, which typically efflux ∼15% cholesterol in 4 h (21Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar) or THP cells (1.7% cholesterol in 2 h (22Kiss R.S. Maric J. Marcel Y.L. J. Lipid Res. 2005; 46: 1877-1887Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar)). These results indicate that wt ABCA1 expressed in BHK cells is fully functional. We then examined ABCA1 localization in BHK cells by immunofluorescent staining. We found that ABCA1 was mainly on the plasma membrane. Using confocal microscopy we took either a single slice (Fig. 2A, first and third columns) or serial images along the Z-axis that cover the whole cell volume and then project onto a single image (second and fourth columns). We observed that ABCA1 predominantly decorated the plasma membrane and projections (Fig. 2A), in agreement with previous reports using GFP-ABCA1 (10Hamon Y. Broccardo C. Chambenoit O. Luciani M.F. Toti F. Chaslin S. Freyssinet J.M. Devaux P.F. McNeish J. Marguet D. Chimini G. Nat. Cell Biol. 2000; 2: 399-406Crossref PubMed Scopus (467) Google Scholar, 23Wang N. Silver D.L. Thiele C. Tall A.R. J. Biol. Chem. 2001; 276: 23742-23747Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Details of Z-stacks are shown in the supplement data. At steady state, ABCA1 can also be found intracellularly (first and second columns). These intracellular structures have the characteristics of the endoplasmic reticulum and the Golgi. To examine whether these ABCA1-containing intracellular structures represent the biosynthesis pathway that delivers ABCA1 to the plasma membrane, we treated cells with a general protein synthesis inhibitor, cycloheximide. We found that, when the biosynthesis pathway is blocked, ABCA1 can only be found on the plasma membrane (Fig. 2A, third and fourth columns, also see supplement data). This indicates that the intracellular portion at steady state most likely represents newly synthesized ABCA1 that transiently passes through these organelles. Furthermore, we examined free cholesterol in these cells and found that total free cholesterol mass was identical between Mock- and ABCA1-expressing cells (4.87 ± 0.88 μg/mg of protein versus 4.56 ± 0.77 μg/mg of protein, respectively). To rule out the possibility that cholesterol might be abnormally sequestered to some intracellular compartments in ABCA1 cells, which could significantly diminish cholesterol in the plasma membrane without apparent alteration in total cholesterol, we stained cells with filipin. We found that filipin staining patterns were comparable in all cell types (Fig. 2B), ensuring that ABCA1 expression did not alter either cholesterol content or subcellular distribution. Furthermore, we preformed cell fractionation in these cells to characterize the cholesterol distribution biochemically. Results, shown in Fig. 2C, demonstrate that the plasma membrane is peaked at fraction 7 as marked by caveolin and so is the cellular cho" @default.
• W1993012149 created "2016-06-24" @default.
• W1993012149 creator A5011375774 @default.
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• W1993012149 creator A5068787947 @default.
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• W1993012149 date "2006-11-01" @default.
• W1993012149 modified "2023-09-28" @default.
• W1993012149 title "ATP-binding Cassette Transporter A1 Expression Disrupts Raft Membrane Microdomains through Its ATPase-related Functions" @default.
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