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• W2070602012 abstract "The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that 125I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I Δ(187–243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of 125I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of 125I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 °C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL. The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that 125I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I Δ(187–243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of 125I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of 125I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 °C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL. The molecular interaction of apoA-I with the cell membrane ABCA1 transporter has important implications in reverse cholesterol transport. It provides a mechanism whereby excess cholesterol is removed from peripheral cells that are unable to catabolize cholesterol, including macrophages in the vessel wall. This process plays a crucial role in both the formation and maintenance of HDL 3The abbreviations used are: HDL, high density lipoprotein; 22OH, 22(R)-hydroxycholesterol; 9CRA, 9-cis-retinoic acid; PAGGE, polyacrylamide nondenaturing gradient gel electrophoresis; ABCA1, ATP-binding cassette AI; apo, apolipoprotein; HCBS, high capacity binding site; ICCs, intracellular compartments; LpA-I, nascent apoA-I-containing particle; rLpA-I, recombinant LpA-I; PLC, phospholipase C; PC, phosphatidylcholine; PM, plasma membrane; TD, Tangier disease; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; Tf, transferrin; WT, wild type; SMase, sphingomyelinase; BHK, baby hamster kidney; CsA, cyclosporin A. levels in plasma and is believed to be one of the major mechanisms by which HDL protects against atherosclerotic cardiovascular disease (1Brewer Jr., H.B. Remaley A.T. Neufeld E.B. Basso F. Joyce C. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1755-1760Crossref PubMed Scopus (151) Google Scholar, 2Linsel-Nitschke P. Tall A.R. Nat. Rev. Drug Discov. 2005; 4: 193-205Crossref PubMed Scopus (417) Google Scholar). Despite a large body of information identifying HDL as a potent physiological protector against atherosclerotic cardiovascular disease, the fundamental mechanisms underlying the genesis of HDL at the cellular level remain complex and poorly understood. It is generally thought that the lipidation of apoA-I occurs inside the cell as a part of a retroendocytosis pathway. Of particular interest is the concept of Smith and co-workers (3Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar) that cellular cholesterol efflux involves endocytosis and resecretion of apoA-I. This concept falls in line with the idea that the ABCA1 transporter may play an important role in the apoA-I retroendocytosis pathway and is supported by previous studies documenting the following: 1) apoA-I colocalizes with ABCA1-containing endosomes (4Smith J.D. Waelde C. Horwitz A. Zheng P. J. Biol. Chem. 2002; 277: 17797-17803Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar); and 2) apoA-I-mediated lipid efflux is defective in the lysosomal storage diseases Niemann-Pick type C disease (5Chen W. Sun Y. Welch C. Gorelik A. Leventhal A.R. Tabas I. Tall A.R. J. Biol. Chem. 2001; 276: 43564-43569Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and Niemann-Pick type B disease (6Leventhal A.R. Chen W. Tall A.R. Tabas I. J. Biol. Chem. 2001; 276: 44976-44983Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The pioneering biophysical and biochemical studies by Neufeld et al. (7Neufeld 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. Brewer Jr., H.B. J. Biol. Chem. 2001; 276: 27584-27590Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 8Neufeld E.B. Stonik J.A. Demosky Jr., S.J. Knapper C.L. Combs C.A. Cooney A. Comly M. Dwyer N. Blanchette-Mackie J. Remaley A.T. Santamarina-Fojo S. Brewer Jr., H.B. J. Biol. Chem. 2004; 279: 15571-15578Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) carried out with a functional fluorescent chimeric human ABCA1-GFP protein expressed in living cells have led to important new insights into the structural mechanisms involved in the lipidation of apoA-I. Indeed, ABCA1 was found to be present in late endosomes and lysosomes and to traffic between late endosomes and the plasma membrane (PM). The presence of both internalized apoA-I and ABCA1 in late endosomes is thought to be functionally important in mediating lipid efflux from this intracellular location. It is not clear, however, whether apoA-I solubilizes a membrane domain that is created by ABCA1 (9Chroni A. Liu T. Fitzgerald M.L. Freeman M.W. Zannis V.I. Biochemistry. 2004; 43: 2126-2139Crossref PubMed Scopus (96) Google Scholar, 10Fitzgerald M.L. Morris A.L. Chroni A. Mendez A.J. Zannis V.I. Freeman M.W. J. Lipid Res. 2004; 45: 287-294Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Vedhachalam C. Liu L. Nickel M. Dhanasekaran P. Anantharamaiah G.M. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Biol. Chem. 2004; 279: 49931-49939Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) or if apoA-I/ABCA1 must interact directly and be internalized together for subsequent nascent HDL formation. This information is critical to the ongoing assessment of whether PM or intracellular compartments (ICCs) represent active cellular sites for the lipidation of apoA-I. Although it is accepted that a retroendocytosis pathway plays an important role in the formation of nascent HDL particles, the structural determinants governing the dynamics of apoA-I lipidation at different cellular sites have not yet been elucidated. In this study, we have used a quantitative assay based on cell surface biotinylation to investigate the cellular compartmentalization and trafficking of apoA-I/ABCA1 in relation to the biogenesis of nascent HDL particles. Patient Selection—For this study, we selected fibroblasts from three normal control subjects and two patients with TD (homozygous for Q597R at the ABCA1 gene and compound heterozygous for C1477R as described previously (12Brooks-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. Genest Jr., J. Hayden M.R. Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1509) Google Scholar)). The protocol for the study was reviewed and accepted by the Research Ethics Board of the McGill University Health Centre. Separate consent forms for blood sampling, DNA isolation, and skin biopsy were provided. Cell Culture—BHK cells stably transfected with an ABCA1 expression vector that is inducible by treating the cells with mifepristone and cells transfected with the same vector lacking the ABCA1 cDNA insert (mock-transfected) were generously provided by Dr. John F. Oram from the Department of Medicine, University of Washington, and were characterized and cultured as described previously (13Oram J.F. Vaughan A.M. Stocker R. J. Biol. Chem. 2001; 276: 39898-39902Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 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. Human skin fibroblasts were obtained from 3.0-mm punch biopsies of the forearm of patients and healthy control subjects and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.1% nonessential amino acids, penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum. Human Plasma ApoA-I—Purified plasma apoA-I (Biodesign) was resolubilized in 4 m guanidine-HCl and dialyzed extensively against PBS buffer. Freshly resolubilized apoA-I was iodinated with 125I by IODO-GEN® (Pierce) to a specific activity of 3000–3500 cpm/ng apoA-I and used within 48 h. Expression of wild type (WT) apoA-I and its mutants in a bacterial system and the isolation and purification of these proteins have been described previously by Marcel et al. (15Frank P.G. Bergeron J. Emmanuel F. Lavigne J.P. Sparks D.L. Denefle P. Rassart E. Marcel Y.L. Biochemistry. 1997; 36: 1798-1806Crossref PubMed Scopus (35) Google Scholar). Deletion mutant apoA-I Δ(187–234) was provided by Dr. Yves L. Marcel. Cell Surface Biotinylation Assay—BHK cells stably expressing ABCA1 or mock were treated or not with mifepristone as described by Oram and co-workers (13Oram J.F. Vaughan A.M. Stocker R. J. Biol. Chem. 2001; 276: 39898-39902Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 14Vaughan A.M. Oram J.F. J. Lipid Res. 2003; 44: 1373-1380Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Confluent fibroblasts were stimulated or not with 2.5 μg/ml 22(R)-hydroxycholesterol and 10 μm 9-cis-retinoic acid for 20 h. Cells were incubated in the presence of 10 μg/ml of apoA-I for 45 min at 37 °C or with increasing concentrations of apoA-I. Cells were washed three times with PBS, and surface proteins were biotinylated with 500 μg/ml sulfosuccinimidobiotin (sulfo-NHS-biotin; Pierce) for 30 min at 4 °C. The biotinylation reaction was quenched for 10 min at 4 °C by removal of the biotin solution and addition of 20 mm Tris-HCl (pH 7.5). Cells were washed twice with ice-cold PBS, lysed, and homogenized. 200 μg of protein was added to 50 μl of streptavidin-Sepharose beads and incubated overnight on a platform mixer at 4 °C. The pellet (PM) or supernatant (ICCs) was washed with lysis buffer and counted for radioactivity. To test whether the biotinylation of cell surface proteins was complete, BHK cells induced with mifepristone and incubated with 10 μg/ml of 125I-apoA-I for 45 min at 4 °C were washed, and biotinylation was performed as described above. After streptavidin pulldown, the recovery of 125I-apoA-I associated with PM was estimated from total 125I-apoA-I (counts/min) added to the cells. We found that more than 85% of apoA-I at the cell surface was accessible to biotin. Preparation of Reconstituted HDL Particles—Complexes comprising apoA-I and palmitoyloleoylphosphatidylcholine were prepared using the sodium cholate dialysis method as described by Jonas et al. (16Jonas A. Steinmetz A. Churgay L. J. Biol. Chem. 1993; 268: 1596-1602Abstract Full Text PDF PubMed Google Scholar). An apoA-I/palmitoyloleoylphosphatidylcholine molar ratio of 1:100 was used. rHDL particles were further concentrated by ultrafiltration (spiral ultrafiltration cartridge, molecular weight cutoff 50,000, Amicon) to discard any lipid-free apoA-I or proteolytic peptides. ApoA-I-lipid complex formation was verified by analysis with two-dimensional PAGGE as we have described previously (17Krimbou L. Hajj H.H. Blain S. Rashid S. Denis M. Marcil M. Genest J. J. Lipid Res. 2005; 46: 1668-1677Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). ABCA1 Endocytosis Assay—Endocytosis assays were performed as described previously (18Le T.L. Yap A.S. Stow J.L. J. Cell Biol. 1999; 146: 219-232Crossref PubMed Scopus (484) Google Scholar). Briefly, mifepristone-induced BHK-ABCA1 or 22OH/9CRA-stimulated normal fibroblasts or ABCA1 mutant fibroblasts were biotinylated with a cleavable form of biotin (sulfosuccinimidyl 2-(biotinamido)-ethyl-1, 3-dithiopropionate (sulfo-NHS-SS-biotin); Pierce) at 4 °C for 30 min. Excess biotin was removed, and cells were incubated with warm DMEM containing 10 μg/ml apoA-I or not. Cells were maintained at 37 °C for various times to permit cellular trafficking. Subsequently, cells were washed with ice-cold PBS, and biotin cleavage was performed using a non-cell-permeable reducing reagent (50 mm glutathione in 75 mm NaCl, 10 mm EDTA containing 1% bovine serum albumin (pH 7.4)), which was applied to the cells twice for 15 min. Reducing reagent was quenched by two 5-min incubations with iodoacetamide (5 mg/ml) at 4 °C. Biotinylated cells incubated with or without apoA-I at 4 °C throughout each assay and subjected to reducing agent were used as controls for the efficacy of biotin cleavage. Under these conditions, more than 95% of biotinylated ABCA1 was cleaved by glutathione. Integrin α4 and activin receptor type II, which localize to the plasma membrane in BHK cells and human fibroblasts, respectively, were used as controls for protein loading. The ratios of endocytosed ABCA1 over integrin α4 and ABCA1 over activin receptor type II were determined. To ensure that application of cleavable biotin did not alter the function of ABCA1, cholesterol efflux assays were carried out on biotinylated cells. We found that labeling cell surface protein with cleavable biotin did not affect significantly apoA-I-mediated cholesterol efflux via ABCA1 pathway. Dissociation of 125I-ApoA-I from Intact Cells—BHK-ABCA1, normal human fibroblasts, or fibroblasts with ABCA1 mutations (Q597R and C1477R) from Tangier disease subjects were used. Fibroblasts were grown to confluence in 100-mm diameter dishes and stimulated with 2.5 μg/ml 22(R)-hydroxycholesterol and 10 μm 9-cis-retinoic acid for 20 h in DMEM/bovine serum albumin. BHK cells stably expressing ABCA1 or mock were treated or not with mifepristone as described above. Cells were incubated in the presence of 10 μg/ml 125I-apoA-I for 45 min at 37 °C. After washing to remove unbound 125I-apoA-I, DMEM was added, and the plates were immediately incubated at 37 °C for increasing times. Biotinylation was performed, and 125I-apoA-I dissociation from both plasma membrane and intracellular compartments was quantitated as described above. Analysis of Nascent ApoA-I-containing Particles—125I-ApoA-I released to the medium at the time point (6 h) from mifepristone-induced BHK-ABCA1 cells was analyzed by two-dimensional PAGGE, and the number of apoA-I molecules per particle was assessed by cross-linking with dithiobis(succinimidyl propionate), as described previously (20Denis M. Haidar B. Marcil M. Bouvier M. Krimbou L. Genest Jr., J. J. Biol. Chem. 2004; 279: 7384-7394Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Treatment with Phospholipases—Confluent BHK-ABCA1 cells were incubated for 45 min in DMEM containing 10 μg/ml 125I-apoA-I. After washing to remove unbound 125I-apoA-I, cells were subjected to treatment with 2.5 units/ml phosphatidylcholine-specific phospholipase C (PC-PLC) or 0.4 units/ml sphingomyelinase (SMase) (Sigma) for 30 min at 37 °C. Following washes, cell surface biotinylation was performed as described above. Statistical Analysis—Results were compared statistically by Student's t test. Two-tailed p values <0.05 were considered as significantly different. Development of a Quantitative Biotinylation Assay—To investigate the cellular compartmentalization and trafficking pattern of apoA-I in a cell culture model, we developed a quantitative assay based on cell surface biotinylation. This permitted us to quantify the amount of apoA-I associated with the PM and ICCs as described under “Experimental Procedures.” However, both the incomplete biotinylation of cell surface proteins or the possible contamination of PM with ICCs fractions could result in an inaccurate quantification. To test whether the biotinylation of cell surface proteins was complete, 22OH/9CRA-stimulated fibroblasts were incubated with 10 μg/ml 125I-apoA-I for 45 min at 37 °C, washed, and incubated with increasing amounts of biotin (0.25–3 mg/ml). After streptavidin pulldown, 125I-apoA-I associated with PM (pellets) or ICCs (supernatants) was determined by γ-counting. Increasing the amount of biotin beyond 0.5 mg/ml did not result in any significant increase in 125I-apoA-I association with either the PM or ICCs (Fig. 1A). To examine the specificity of the biotinylation reaction, a 30-fold excess of unlabeled apoA-I, absence of ABCA1 stimulation with 22OH/9CRA, and ABCA1 mutant fibroblasts (Q597R) were used as controls. As shown in Fig. 1B, the presence of excess unlabeled apoA-I, the absence of stimulation with 22OH/9CRA, or utilization of an ABCA1 mutant (Q597R) drastically reduced the association of 125I-apoA-I with both the PM and ICCs. We have previously reported that the Q597R-ABCA1 mutant does not bind apoA-I but is expressed normally and localizes to the cell surface (19Haidar B. Denis M. Marcil M. Krimbou L. Genest Jr., J. J. Biol. Chem. 2004; 279: 9963-9969Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). To ascertain the integrity of the PM and ICCs fractions, both fractions were separated by SDS-PAGE and monitored by appropriate antibodies for the colocalization of specific cellular markers. No significant contamination of the PM with ICCs was observed as assessed by the absence of VAL-α2 from ICCs, which is considered a marker for the PM. Similarly, Hsp-70 and tubulin were found exclusively associated with ICCs (Fig. 1C). These results indicate that the biotinylation is complete and limited to the PM. In light of these findings, a concentration of 0.5 mg/ml biotin and an incubation period of 30 min at 4 °C were used for biotinylation throughout this study. Importantly, a period of 45 min of incubation of 125I-apoA-I with cells at 37 °C was chosen to permit sufficient time for equilibration of apoA-I with different cellular compartments. Although several groups used binding at 4 °C to determine the association of apoA-I with the PM, we obtained evidence that incubation at 4 °C alters the association of apoA-I with ABCA1 at the PM (data not shown), in agreement with previous studies (20Denis M. Haidar B. Marcil M. Bouvier M. Krimbou L. Genest Jr., J. J. Biol. Chem. 2004; 279: 7384-7394Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 21Fitzgerald 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). These results underscore the importance of using physiological temperatures to study apoA-I/ABCA1 interactions. Association of ApoA-I with the PM and ICCs—To determine the association of apoA-I with the PM and ICCs, either BHK cells treated or not with mifepristone or fibroblasts treated or not with 22OH/9CRA were incubated with increasing concentrations of 125I-apoA-I for 45 min at 37 °C. After washing to remove unbound 125I-apoA-I, biotinylation was performed and 125I-apoA-I associated with the PM and ICCs was determined by γ-counting. As shown in Fig. 2A, 125I-apoA-I exhibited saturable, concentration-dependent association with the PM and ICCs in BHK cells induced with mifepristone. Conversely, 125I-apoA-I showed no significant association with either fraction in noninduced BHK cells (Fig. 2B). Similarly, 125I-apoA-I exhibited saturable association with both PM and ICCs in 22OH/9CRA-stimulated fibroblasts (data not shown). Importantly, analysis of affinity parameters for the association of apoA-I with different cellular compartments revealed that the PM possessed nearly a 2-fold higher capacity (Bmax) to accommodate apoA-I as compared with ICCs in BHK expressing ABCA1 and stimulated fibroblasts. In contrast, apoA-I exhibited similar affinity for both the PM and ICCs (Table 1). This result indicates that under conditions of continuous exposure to an excess of apoA-I, two-thirds of apoA-I is found associated with the PM and the remaining one-third with the ICCs.TABLE 1Kinetics of apoA-I association and dissociation from the PM and ICCs in BHK cells and fibroblastsKinetic parametersAssociationDissociationApparent BmaxApparent KdKofft½ng/ μg cell proteinng/ μg cell proteinmin-1minBHK-ABCA1 PM0.58 ± 0.05aFor apparent Bmax, p < 0.001 for PM is compared with ICCs in BHK cells.2.52 ± 0.2030 ± 582 ± 7cFor t½, p < 0.001 for PM is compared with ICCs in BHK cells. ICCs0.31 ± 0.042.20 ± 0.15127 ± 1120 ± 3Fibroblasts + 22OH/9CRA PM0.21 ± 0.01bFor apparent Bmax, p < 0.001 for PM is compared with ICCs in fibroblasts.1.52 ± 0.1418 ± 6126 ± 24dFor t½, p < 0.001 for PM is compared with ICCs in fibroblasts. ICCs0.10 ± 0.021.68 ± 0.2584 ± 1829 ± 2a For apparent Bmax, p < 0.001 for PM is compared with ICCs in BHK cells.b For apparent Bmax, p < 0.001 for PM is compared with ICCs in fibroblasts.c For t½, p < 0.001 for PM is compared with ICCs in BHK cells.d For t½, p < 0.001 for PM is compared with ICCs in fibroblasts. Open table in a new tab Transferrin (Tf), a ligand that is known to specifically interact with the Tf receptor and recycle to the cell surface (22Raub T.J. Newton C.R. J. Cell. Physiol. 1991; 149: 141-151Crossref PubMed Scopus (68) Google Scholar) was used as a control. We found that 77% of 125I-Tf was found associated with the PM and 23% with ICCs in HepG2 (0.76 ± 0.02 versus 0.22 ± 0.01 ng of Tf/μg of cell protein). Effect of the C-terminal Deletion of ApoA-I and the Lipidation of WT ApoA-I on the Association with the PM and ICCs—It is well documented that the C-terminal region of apoA-I is important in the ABCA1-mediated lipid efflux pathway. Indeed, previous studies have documented that apoA-I deletion mutants lacking residues 187–243 of the C-terminal domain (Δ(187–243)) exhibit both reduced cell surface binding and the ability to promote lipid efflux (9Chroni A. Liu T. Fitzgerald M.L. Freeman M.W. Zannis V.I. Biochemistry. 2004; 43: 2126-2139Crossref PubMed Scopus (96) Google Scholar, 11Vedhachalam C. Liu L. Nickel M. Dhanasekaran P. Anantharamaiah G.M. Lund-Katz S. Rothblat G.H. Phillips M.C. J. Biol. Chem. 2004; 279: 49931-49939Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 15Frank P.G. Bergeron J. Emmanuel F. Lavigne J.P. Sparks D.L. Denefle P. Rassart E. Marcel Y.L. Biochemistry. 1997; 36: 1798-1806Crossref PubMed Scopus (35) Google Scholar). Furthermore, we and others have previously shown that lipid association with apoA-I or apoE3 reduced their ability to interact with ABCA1 (20Denis M. Haidar B. Marcil M. Bouvier M. Krimbou L. Genest Jr., J. J. Biol. Chem. 2004; 279: 7384-7394Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 25Hajj H.H. Blain S. Boucher B. Denis M. Krimbou L. Genest J. J. Lipid Res. 2005; 46: 1457-1465Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 26Krimbou L. Denis M. Haidar B. Carrier M. Marcil M. Genest Jr., J. J. Lipid Res. 2004; 45: 839-848Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 27Wang N. Silver D.L. Tall A.R. J. Biol. Chem. 2001; 276: 23742-23747Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). We further examined the role of the C-terminal domain of apoA-I and the lipidation of WT apoA-I on the association with different cellular compartments. As shown in Fig. 3, A and B, apoA-I Δ(187–243) and rLpA-I exhibited drastically reduced cell association and association with both the PM and ICCs as compared with lipid-free WT apoA-I. These results indicate that the C-terminal domain of apoA-I is important for the compartmentalization of apoA-I between the PM and ICCs. Similarly, they indicate a requirement of lipid-free apoA-I for efficient compartmentalization. Overexpressing ABCA1 Promotes the Compartmentalization of ApoA-I between the PM and ICCs—It is well documented that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux (4Smith J.D. Waelde C. Horwitz A. Zheng P. J. Biol. Chem. 2002; 277: 17797-17803Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 8Neufeld E.B. Stonik J.A. Demosky Jr., S.J. Knapper C.L. Combs C.A. Cooney A. Comly M. Dwyer N. Blanchette-Mackie J. Remaley A.T. Santamarina-Fojo S. Brewer Jr., H.B. J. Biol. Chem. 2004; 279: 15571-15578Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 23Cavelier C. Rohrer L. von Eckardstein A. Circ. Res. 2006; 99: 1060-1066Crossref PubMed Scopus (83) Google Scholar, 24Chen W. Wang N. Tall A.R. J. Biol. Chem. 2005; 280: 29277-29281Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To further examine the role of ABCA1 in the cellular compartmentalization of apoA-I, different levels of ABCA1 were expressed in BHK cells under a mifepristone-inducible ABCA1 gene. As shown in Fig. 2C (inset), without induction, BHK-ABCA1 cells had no detectable ABCA1 protein, whereas cells induced with 0.1 and 10 nm mifepristone for 20 h expressed increasing levels of ABCA1. Concomitantly, cellular cholesterol efflux was significantly increased (2 ± 0.35, 8 ± 0.22, and 13 ± 0.68%; 0, 0.1, and 10 nm mifepristone; respectively). Furthermore, ABCA1 induction by mifepristone was paralleled by elevated 125I-apoA-I association to both the PM and ICCs (Fig. 2D). Similarly, stimulation of normal fibroblasts with 22OH/9CRA increased the association of 125I-apoA-I with both the PM and ICCs (Fig. 1B). These results indicate that expression of ABCA1 is required for the association of apoA-I with both the PM and ICCs. The Endocytotic Pathway Is Required for the Association of ApoA-I with ICCs—Having demonstrated that ABCA1 expression is required for the compartmentalization of apoA-I, we examined the impact of ABCA1 trafficking on apoA-I association with the PM and ICCs. Cells were treated with either cyclosporin A (CsA) or probucol, which are known to specifically inhibit ABCA1-mediated cholesterol efflux to apoA-I (28Le Goff W. Peng D.Q. Settle M. Brubaker G. Morton R.E. Smith J.D. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2155-2161Crossref PubMed Scopus (74) Google Scholar, 29Wu C.A. Tsujita M. Hayashi M. Yokoyama S. J. Biol. Chem. 2004; 279: 30168-30174Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). CsA is a potent inhibitor of ABCA1 shown to block recycling of ABCA1 from the PM to endosomes, resulting in the inhibition of HDL biogenesis and of ABCA1 degradation by calpain proteases (28Le Goff W. Peng D.Q. Settle M. Brubaker G. Morton R.E. Smith J.D. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2155-2161Crossref PubMed Scopus (74) Google Scholar). Alternatively, Rothblat and co-workers (42Favari E. Zanotti I. Zimetti F. Ronda N. Bernini F. Rothblat G.H. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2345-2350Crossref PubMed Scopus (117) Google Scholar) suggest that probucol inhibits cholesterol efflux by preventing traffickin" @default.
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• W2070602012 title "Quantitative Analysis of ABCA1-dependent Compartmentalization and Trafficking of Apolipoprotein A-I" @default.
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