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• W2159836929 abstract "ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis. ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis. high density lipoprotein ATP-binding cassette protein-A1 brefeldin A Chinese hamster ovary 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate low density lipoprotein Dulbecco's modified Eagle's medium endoplasmic reticulum green fluorescent protein lysosomal-associated membrane protein 2 Cholesterol is a membrane constituent that maintains structural domains that are important in the regulation of vesicular trafficking and signal transduction (1Simons K. Ikonen E. Science. 2000; 290: 1721-1726Crossref PubMed Scopus (1078) Google Scholar). In most cells, cholesterol is not catabolized. Thus, the regulation of cellular sterol efflux plays a crucial role in cellular sterol homeostasis. Cellular sterol can efflux to extracellular sterol acceptors by both nonregulated, passive diffusion mechanisms (2Johnson W.J. Mahlberg F.H. Rothblat G.H. Phillips M.C. Biochim. Biophys. Acta. 1991; 1085: 273-298Crossref PubMed Scopus (389) Google Scholar) as well as by an active, regulated, energy-dependent process (3Oram J.F. Yokoyama S. J. Lipid Res. 1996; 37: 2473-2491Abstract Full Text PDF PubMed Google Scholar, 4Mendez A.J. J. Lipid Res. 1997; 38: 1807-1821Abstract Full Text PDF PubMed Google Scholar) mediated by the ABCA1 transporter (5Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (658) Google Scholar). The human ABCA1 transporter is a polytopic membrane-spanning ATP-binding cassette protein (6Fitzgerald M.L. Mendez A.J. Moore K.J. Andersson L.P. Panjeton H.A. Freeman M.W. J. Biol. Chem. 2001; 276: 15137-15145Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) that is essential for the formation of HDL1 via apoA-I-mediated efflux of cholesterol and phospholipids from peripheral cells (3Oram J.F. Yokoyama S. J. Lipid Res. 1996; 37: 2473-2491Abstract Full Text PDF PubMed Google Scholar, 4Mendez A.J. J. Lipid Res. 1997; 38: 1807-1821Abstract Full Text PDF PubMed Google Scholar, 5Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (658) Google Scholar, 7Francis G.A. Knopp R.H. Oram J.F. J. Clin. Invest. 1995; 96: 78-87Crossref PubMed Scopus (373) Google Scholar,8Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H. Brewer Jr., H.B. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1813-1821Crossref PubMed Scopus (191) Google Scholar). Recent studies suggest that the ABCA1 transporter may function to regulate cellular sterol efflux by modifying adjacent membrane lipid domains, thereby allowing apoA-I and other apolipoproteins to bind to the cell membrane and remove cholesterol and phospholipids from cells (9Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (279) Google Scholar, 10Chambenoit O. Hamon Y. Marguet D. Rigneault H. Rosseneu M. Chimini G. J. Biol. Chem. 2001; 276: 9955-9960Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 11Wang 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, 12Orso E. Broccardo C. Kaminski W.E. Bottcher A. Liebisch G. Drobnik W. Gotz A. Chambenoit O. Diederich W. Langmann T. Spruss T. Luciani M.-F. Rothe G. Lackner K.J. Chimini G. Schmitz Nat. Genet. 2000; 24: 192-196Crossref PubMed Scopus (431) Google Scholar). ABCA1-mediated cellular sterol efflux constitutes the initial step in the pathway of reverse cholesterol transport that ultimately leads to elimination of cholesterol from the body. Macrophages, which scavenge serum lipoproteins in an unregulated manner, are particularly dependent on ABCA1-mediated sterol efflux to prevent pathogenic sterol accumulation. Mutations in the human ABCA1 transporter cause Tangier disease (13Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer Jr., H.B. Duverger N. Denefle P. Assmann G. Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1269) Google Scholar, 14Remaley A.T. Rosier M. Knapper C. Peterson K.M. Koch C. Dinger M. Duverger N. Assmann G. Dean M. Santamarina-Fojo S. Fredrickson D.S. Denefle P. Brewer Jr., H.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12685-12690Crossref PubMed Scopus (230) Google Scholar, 15Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez M. Kaminski W.E. Hahmann H.W. Oette K. Rothe G. Aslanidis C. Lackner K.J. Schmitz G. Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1349) Google Scholar, 16Brooks-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, 17Assmann G. von Eckardstein A. Brewer Jr., H.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw Hill, New York2001: 2937-2960Google Scholar), which is characterized by the accumulation of cholesterol ester in reticuloendothelial cells, hypercatabolism of poorly lipidated serum apoA-I, exceedingly low serum HDL, and increased risk of coronary heart disease (17Assmann G. von Eckardstein A. Brewer Jr., H.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw Hill, New York2001: 2937-2960Google Scholar). To date, the cellular distribution of ABCA1 and its possible site(s) of function are not fully understood. Immunocytochemical studies have suggested that endogenously expressed human ABCA1 resides solely on the plasma membrane (5Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (658) Google Scholar, 11Wang 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, 12Orso E. Broccardo C. Kaminski W.E. Bottcher A. Liebisch G. Drobnik W. Gotz A. Chambenoit O. Diederich W. Langmann T. Spruss T. Luciani M.-F. Rothe G. Lackner K.J. Chimini G. Schmitz Nat. Genet. 2000; 24: 192-196Crossref PubMed Scopus (431) Google Scholar). Stably and transiently expressed chimeric ABCA1-GFP has been reported to reside in intracellular compartments as well as on the plasma membrane (6Fitzgerald M.L. Mendez A.J. Moore K.J. Andersson L.P. Panjeton H.A. Freeman M.W. J. Biol. Chem. 2001; 276: 15137-15145Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 9Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (279) Google Scholar, 18Hamon 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). It is currently thought that ABCA1 at the plasma membrane functions in cellular lipid efflux. Several investigators have provided evidence suggesting that ABCA1-mediated lipid efflux involves intracellular trafficking of substrate lipids (19Oram J.F. Mendez A.J. Slotte J.P. Johnson T.F. Arterioscler. Thromb. 1991; 11: 403-414Crossref PubMed Scopus (140) Google Scholar, 20Bielicki J.K. Johnson W.J. Weinberg R.B. Glick J.M. Rothblat G.H. J. Lipid Res. 1992; 33: 1699-1709Abstract Full Text PDF PubMed Google Scholar) or the apoA-I acceptor (21Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar). In the present study, a functional fluorescent chimeric human ABCA1-GFP protein expressed in living cells has revealed that early endosomes containing the ABCA1 transporter shuttle between the plasma membrane and other endocytic compartments. These studies suggest that the trafficking of ABCA1 in endocytic compartments may play important roles in apoA-I-mediated efflux of cellular lipids. HeLa and Chinese hamster vary (CHO) cells were grown in DMEM (Life Technologies, Inc.), supplemented with 10% fetal calf serum, 2 mm glutamine, 100 IU/ml of penicillin, 100 µg/ml streptomycin, and 100 µg/ml G418. CHO cells were transiently transfected with Fugene-6 (Roche Molecular Biochemicals, Indianapolis, IN), using the expression plasmid pTRE2 (CLONTECH, Palo Alto, CA), encoding a chimeric ABCA1-GFP protein (pTRE2-ABCA1-GFP). Enhanced GFP along with a 5 amino acid glycine linker (Quantum Biologics, Vancouver, Canada) was fused in frame to the carboxyl terminus of human ABCA1, after first deleting the stop codon from the full-length ABCA1 cDNA (9Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (279) Google Scholar). HeLa cells were also transiently and stably transfected with a chimeric ABCA1-GFP protein, as previously described (9Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (279) Google Scholar). Briefly, HeLa (Tet-off) cells (CLONTECH, PaloAlto, CA) were co-transfected with pTRE2-ABCA1-GFP and pTK-Hyg (CLONTECH, PaloAlto, CA) at a ratio of 1:20 and selected with 500 µg/ml of hygromycin. Hygromycin-resistant cells were screened for expression of the fusion protein by fluorescence microscopy and positive clones were further purified by limiting dilution. Control cells were co-transfected with pTRE2 and pTK-Hyg (CLONTECH, PaloAlto, CA) at a ratio of 1:20 and selected with 500 µg/ml of hygromycin. ApoA-I purified from human plasma (22Brewer H.B. Ronan R. Meng M. Bishop C. Methods Enzymol. 1986; 128: 223-246Crossref PubMed Scopus (130) Google Scholar) was over 99% pure, as determined by SDS-PAGE and amino-terminal sequence analysis. Cholesterol and phospholipid efflux were performed as previously described (8Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H. Brewer Jr., H.B. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1813-1821Crossref PubMed Scopus (191) Google Scholar). For cholesterol efflux, nearly confluent cells were labeled with 3H-cholesterol for 48 h, washed, and then incubated for the indicated times with in DMEM containing 1 mg/ml of bovine serum albumin in the presence or absence of 30 µg/ml apoA-I. Percentage efflux was calculated by subtracting the radioactive counts of blank media (DMEM/bovine serum albumin) from the radioactive counts in the presence of apoA-I, and then dividing the result by the sum of the radioactive counts in the medium plus the cell fraction. For phospholipid efflux, cells were labeled with methyl-3Hcholine chloride for 24 h, washed, and incubated for 24 h in DMEM/bovine serum albumin in the presence or absence of 30 µg/ml apoA-I. Cells in glass chamber slides were washed in phosphate-buffered saline and fixed in 3% paraformaldehyde for 30 min. Cells were immunolabeled using an indirect procedure in which all incubations were performed either in blocker solution containing filipin (0.05%) and goat IgG (2.5 mg/ml) or 10% fetal bovine serum in phosphate-buffered saline containing saponin (0.2%). Primary antibodies used were raised against human LAMP2, transferrin receptor, and p58K protein. Secondary Alexa-568-labeled antibodies were used at a 1:100 dilution. Fluorescence was viewed with a Zeiss 410 or 510 laser scanning confocal microscope, using a krypton-argon-Omnichrome laser with excitation wavelengths of 488 and 568 nm for enhanced green fluorescent protein and Alexa-568, respectively. Time-lapse images were taken with a Zeiss Axiovert 35 microscope equipped with a charged-coupled device camera (TEA/CCD-1317K/1, Princeton Instruments, Trenton, NJ). For live cell imaging, cells were prepared on 40-mm coverslips, and temperature was maintained at 37 °C in Focht Chamber System 2 with an Objective Heater System (Bioptechs, Butler, PA). A total of 40 GFP images were acquired at the rate of 1/s (0.3-s exposure). Capture, animation, and export to QuickTime movie were performed using the IPLab software system (Scanalytics, Fairfax, VA). Structures in QuickTime movies were pseudocolored using Adobe AfterEffects 4.1 and Adobe Photoshop 5.0 software. ApoA-I-mediated efflux of cellular cholesterol and choline-containing phospholipids was enhanced 5-fold or more by ABCA1-GFP expression in stably transfected HeLa cells compared with control cells (Fig. 1). Thus, as previously shown, the fusion of enhanced green fluorescent protein to the C terminus of ABCA1 does not interfere with its function (9Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (279) Google Scholar, 18Hamon 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). ABCA1-GFP expressed either transiently or stably in HeLa cells resides at the cell surface and in intracellular compartments (Fig. 2,A and B). Similar patterns of distribution were observed in both cell types (HeLa, CHO) and expression systems examined. To identify the endocytic compartments containing ABCA1, HeLa cells transiently expressing ABCA1-GFP were immunostained with antibodies to the human transferrin receptor (early endocytic compartments (23Klausner R.D. Ashwell G. Renswoude J.V. Harford J.B. Bridges K.R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2263-2266Crossref PubMed Scopus (476) Google Scholar)) or LAMP2 protein (late endocytic compartments (24Chen J.W. Murphy T.L. Willingham M.C. Pastan I. August J.T. J. Cell Biol. 1985; 101: 85-95Crossref PubMed Scopus (387) Google Scholar)). As shown in Fig. 2 C, ABCA1-GFP does not appreciably localize in the punctate intracellular structures containing the transferrin receptor. As shown in Fig. 2 D, ABCA1-GFP does co-localize with LAMP2, a marker for late endocytic compartments, which include late endosomes and lysosomes (24Chen J.W. Murphy T.L. Willingham M.C. Pastan I. August J.T. J. Cell Biol. 1985; 101: 85-95Crossref PubMed Scopus (387) Google Scholar). Endocytosed DiI-LDL as well as tetramethylrhodamine-dextran when used as vital markers for late endocytic compartments (25Ghosh R.N. Gelman D.L. Maxfield F.R. J. Cell Sci. 1994; 107: 2177-2189Crossref PubMed Google Scholar, 26Chen C.S. Bach G Pagano R.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6373-6378Crossref PubMed Scopus (167) Google Scholar) also co-localized with a subset of intracellular vesicles containing ABCA1-GFP (data not shown). To further elucidate the intracellular trafficking pathways used by ABCA1, HeLa cells transiently expressing ABCA1-GFP were treated with cycloheximide, to prevent further delivery of newly synthesized protein to the cell surface, as well as with brefeldin A or monensin, agents that block both intracellular vesicular trafficking (27Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1548) Google Scholar, 28Mollenhauer H.H. Morre D.J. Rowe L.D. Biochim. Biophys. Acta. 1990; 1031: 225-246Crossref PubMed Scopus (533) Google Scholar) and apoA-I-mediated cellular lipid efflux (8Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H. Brewer Jr., H.B. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1813-1821Crossref PubMed Scopus (191) Google Scholar, 29Mendez A.J. J. Biol. Chem. 1995; 270: 5891-5900Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The effect of these agents on the cellular distribution of ABCA1-GFP and their effects on cellular lipid efflux by ABCA1-GFP overexpression were monitored. Confocal microscopy reveals that blocking cellular protein synthesis with cycloheximide substantially reduced the total cellular levels of ABCA1-GFP including the amount residing at the cell surface (Fig.3 B). Cycloheximide treatment reduced ABCA1-GFP-mediated cellular sterol efflux to 51.9 ± 7.0% of controls (Fig. 5). These results are readily explained if in the absence of delivery of newly synthesized ABCA1-GFP to the cell surface the amount of ABCA1-GFP at the cell surface is reduced with time, as the existing cellular pool of protein is degraded, presumably in lysosomes.Figure 5Inhibition of protein synthesis or transport inhibits apoA-I-mediated efflux in HeLa cells expressing ABCA1-GFP. The effect of treatment with 5 µg/ml BFA or 10 µm monensin in the absence or presence of 100 µg/ml cycloheximide (CHX) for 6 h on the ability of apoA-I to mediate cholesterol efflux (as described under “Experimental Procedures”) in stably transfected HeLa cells expressing the human ABCA1-GFP fusion protein was examined.View Large Image Figure ViewerDownload Hi-res image Download (PPT) BFA causes the Golgi to fuse with the endoplasmic reticulum and blocks vesicular transport to the cell surface along the secretory pathway (27Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1548) Google Scholar). Thus, BFA would be expected to cause newly synthesized ABCA1-GFP to accumulate in the fused Golgi-ER. In addition, BFA would be expected to reduce the amount of ABCA1-GFP at the cell surface and in endocytic compartments as ABCA1-GFP at the cell surface traffics along the endocytic pathway. As expected, ABCA1-GFP is considerably reduced at the surface of BFA-treated HeLa cells expressing ABCA1-GFP (Fig.3 C). Instead, ABCA1-GFP is seen to distribute in a cytosolic reticular pattern (Fig. 3 C), consistent with the trapping of newly synthesized ABCA1-GFP in the fused Golgi-ER. Treatment with BFA in the presence of cycloheximide resulted in a similar pattern of distribution of ABCA1-GFP in the Golgi-ER (Fig. 3 D). However, blocking protein synthesis greatly reduced the amount of ABCA1-GFP trapped by BFA in the hybrid organelle (Fig.3 D). BFA reduced ABCA1-GFP-induced cellular efflux of cellular sterol to 60.3 ± 6.5% of control values (Fig. 5). Thus, together with the progressive loss of ABCA1-GFP at the cell surface, there is a concomitant loss of ABCA1-stimulated cellular sterol efflux. Co-treatment with cycloheximide did not further reduce cellular sterol efflux (49.7 ± 8.4% of control values; Fig. 5). This latter finding suggests that the ABCA1-GFP trapped in the hybrid Golgi-ER organelle by the action of BFA does not promote efflux of cellular sterol. Monensin (28Mollenhauer H.H. Morre D.J. Rowe L.D. Biochim. Biophys. Acta. 1990; 1031: 225-246Crossref PubMed Scopus (533) Google Scholar), like brefeldin A (27Klausner R.D. Donaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1548) Google Scholar), blocks delivery of newly synthesized protein to the cell surface and thus is expected to reduce surface expression of ABCA1-GFP as well as apoA-I-mediated efflux. In addition, because monensin blocks protein degradation and trafficking out of late endosomes and lysosomes (28Mollenhauer H.H. Morre D.J. Rowe L.D. Biochim. Biophys. Acta. 1990; 1031: 225-246Crossref PubMed Scopus (533) Google Scholar), ABCA1-GFP would be expected to accumulate in LAMP2(+) late endosomes and lysosomes. Monensin treatment reduced the level of ABCA1-GFP residing at the cell surface, as revealed by confocal microscopy (Figs. 3 E and4). Monensin treatment also induced the localization of ABCA1-GFP in intracellular vesicles containing LAMP2 (Fig.4) as well as in late endocytic vesicles vitally labeled with endocytosed DiI-LDL (25Ghosh R.N. Gelman D.L. Maxfield F.R. J. Cell Sci. 1994; 107: 2177-2189Crossref PubMed Google Scholar) or endocytosed tetramethylrhodamine-dextran (26Chen C.S. Bach G Pagano R.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6373-6378Crossref PubMed Scopus (167) Google Scholar) (data not shown). As seen in Fig.3 F, co-treatment with monensin and cycloheximide resulted in the accumulation of ABCA1-GFP in punctate cytoplasmic structures identified to be late endosomes/lysosomes by their co-localization with endocytosed DiI-LDL or tetramethylrhodamine-dextran (data not shown). The Golgi-specific marker p58 protein (30Bloom G.S. Brashear T.A J. Biol. Chem. 1989; 264: 16083Abstract Full Text PDF PubMed Google Scholar) did not appreciably colocalize with LAMP2 (data not shown). Taken together, these results indicate that monensin causes ABCA1-GFP derived from the cell surface to become trapped in late endocytic compartments. As expected, based on the results of BFA treatment (Figs. 3 and5), associated with the monensin-induced loss of surface expression a considerable decrease (47.3 ± 3.8%) in apoA-I-mediated sterol efflux in HeLa cells stably transfected with ABCA1-GFP was observed (Fig. 5). Blocking protein synthesis with cycloheximide did not further reduce the monensin-induced block in ABCA1-GFP-stimulated sterol efflux (43.3 ± 6.4%) (Fig. 5). Taken together, these results suggest that monensin blocks the movement of newly synthesized ABCA1-GFP to the cell surface and traps plasma membrane-derived ABCA1-GFP in late endocytic compartments. Time-lapse digital video fluorescence microscopy of ABCA1-GFP expressed in living cells revealed a complex intracellular trafficking pathway for the ABCA1-GFP transporter (Figs. 6 and 7; Movies 1–3 in the Supplemental Material). The fluorescent chimeric protein was seen to traffic in at least two types of intracellular vesicles: (i) small rapidly moving vesicles and (ii) large, static perinuclear vesicles (Figs. 6 and 7 and Movie 1 in Supplemental Material). Treatment of cells expressing ABCA1-GFP with U18666A (31Liscum L. Faust J.R. J. Biol. Chem. 1989; 264: 11796-11806Abstract Full Text PDF PubMed Google Scholar) selectively enlarged the static, large ABCA1-GFP-containing perinuclear vesicles (data not shown). U18666A enlarges late endosomes/lysosomes as a result of the accumulation of cholesterol in these compartments (Refs. 32Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A.M. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar and33Zhang M. Dwyer N.K. Neufeld E.B. Love D.C. Cooney A.D. Comly M. Patel S. Watari H. Strauss III, J.F. Pentchev P.G. Hanover J.A. Blanchette-Mackie E.J. J. Biol. Chem. 2001; 276: 3417-3425Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar; data not shown). Thus, the large, static ABCA1-GFP-containing perinuclear vesicles are likely to represent late endocytic compartments, whereas the small, fast-moving ABCA1-GFP-containing vesicles are likely to represent, for the most part, early endosomes.Figure 7ABCA1-GFP-containing endosomes interact with the cell surface in living cells. CHO cells were transiently transfected with ABCA1-GFP, and time-lapse image acquisition of GFP fluorescence was performed at 37 °C as described under “Experimental Procedures.” The large top panel shows a cell imaged in the first frame. The white arrowheads indicate the site where an ABCA1-GFP-containing endosome contacts the cell surface. Small, mobile ABCA1-GFP-containing endosomes are pseudocoloredmagenta, green, and blue. High magnification time-lapse images of a selected region of the cell (yellow box) are indicated by the image number. The dynamic interaction of a small, rapidly moving ABCA1-GFP vesicle (green) with the plasma membrane (arrowhead) can be seen. Note that the ABCA1-GFP-containing endosomes pseudocolored magenta and blue are identical to the corresponding vesicles seen in Fig. 6, whereas the ABCA1-GFP-containing endosome pseudocolored green represents a different vesicle than that in Fig. 6.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of these images revealed the dynamic interactions of ABCA1-GFP-containing early endosomes with one another as well as with ABCA1-GFP-containing late endocytic vesicles and the ABCA1-GFP-containing plasma membrane. As described below, ABCA1-GFP-containing early endosomes appear to shuttle between late endosomes/lysosomes and the plasma membrane in living cells. As seen in Fig. 6 (see also Movie 2 in Supplemental Material), a small ABCA1-GFP endosome (pseudocolored green) interacts with two other small ABCA1-GFP endosomes in frames 1–9 and28–31 (the endosome pseudocolored magenta) and in frames 2 and 3 (the endosome pseudocoloredblue). The small ABCA1-GFP endosome (magenta) remains relatively stationary during frames 1–37, moves rapidly in frame 38, and then appears to move out of the plane of focus in frames 39 and 40. As shown in Fig. 6, the small green ABCA1-GFP endosome appears to shuttle between the small magenta ABCA1-GFP endosome and the ABCA1-GFP-containing late endosomes (shown inwhite). The green ABCA1-GFP endosome remains associated with the magenta ABCA1-GFP endosome duringframes 1–8, then rapidly descends vertically while tubulating (frames 9–11), fuses with the ABCA1-GFP-containing late endosomes (shown in white) duringframes 11–12, and then remains fused to the ABCA1-GFP-containing late endosomes during frames 13–25. The green ABCA1-GFP endosome subsequently re-emerges from the ABCA1-GFP-containing late endosome compartment in frame 26, rapidly ascends vertically during frames 27–28, interacts with the magenta ABCA1-GFP endosome again duringframes 28–31, rapidly descends to the vicinity of the ABCA1-GFP-containing late endosomes during frames 31–34, and then appears to interact with the ABCA1-GFP-containing late endocytic vesicles (white) again during frames 34–40. An example of a small mobile ABCA1-GFP endosome that interacts with other small mobile ABCA1-GFP endosomes, as well as with the cell surface, can be seen in Fig. 7 and Movie 3 (Supplemental Material). The small ABCA1-GFP endosome (pseudocolored green) can be seen to interact intimately with the cell surface during frames 36–40. Note that prior to docking at the cell surface, the green ABCA1-GFP endosome interacts first with another small ABCA1-GFP endosome (shown inblue, during frames 16–18) and then fuses with very small ABCA1-GFP endosomes (shown in white, frames 28–31, 34, and 35). Image analysis further reveals the complex trafficking itinerary that a single ABCA1-GFP endosome can take. As seen in Fig. 6 (see also Movie 2 in Supplemental Material), the small mobile blue ABCA1-GFP endosome moves in a linear fashion across the field in frames 1–8, interacting with the small green ABCA1-GFP endosome in frames 2 and 3, then descends in a linear manner in frames 7–10, briefly interacts (frame 10) with late endosomes (shown in white, highlighted by the yellow arrow in frame 1), and then ascends vertically in a linear manner (frames 11–17) until it goes out of frame (frame 18). Note that the same small mobile blue ABCA1-GFP endosome seen in Fig. 6 can also be seen in Fig. 7 (frames 2–19) interacting subsequently with yet another small mobile ABCA1-GFP endosome (pseudocolored green in Fig. 7) during frames 16–18 (Fig. 7). The blue ABCA1-GFP endosome, which goes out of frame in Fig. 7 (frame 19), can be seen in Movie 1 (Supplemental Material) to ascend vertically and dock at the plasma membrane (during frames 19–28). Thus, the small mobileblue ABCA1-GFP endosome first interacts briefly with another small mobile ABCA1-GFP endosome (green, Fig. 6, frames 2 and 3), then interacts briefly with a large, immobile perinuclear late endosome (Fig. 6, frame 10), and finally interacts with another small, mobile ABCA1-GFP endosome for a longer period of time (pseudocolored green in Fig. 7,frames 16–18) prior to docking at the cell surface. Consistent with the perturbations observed by confocal microscopy (Fig.3), time-lapse video microscopy of living cells expressing ABCA1-GFP (Movie 4, Supplemental Material) revealed that BFA treatment resulted in a loss of ABCA1-GFP fluorescence at the cell surface and trapping of ABCA1-GFP in the cytosolic Golgi-ER compartment. Monensin treatment (Movie 4) resulted in a loss of ABCA1-GFP fluorescence at the cell surface and the trapping of ABCA1-GFP in large, static, intracellular vesicles. The human ABCA1 transporter is a polytopic membrane spanning protein that plays an essential role in apolipoprotein-mediated efflux of cellular cholesterol and phospholipids. We have expressed a chimeric ABCA1-GFP protein to gain insight into the cellular localization and trafficking of the human ABCA1 transporter and its possible sites of function. We have identified at least three major cellular sites of ABCA1 localization in living cells, including the plasma membrane, as well as in early and late endocytic compartments (Fig.8). The ABCA1-GFP chimeric protein functions to efflux both cholesterol and choline-containing phospholipids. The present studies suggest that ABCA1 at the cell surface functions in cellular lipid efflux. This is supported by the observation that a reduction in the amount of ABCA1-GFP residing at the cell surface correlates with a reduction in apoA-I-mediated cellular lipid efflux. The present confocal microscopic immunolocalization studies have revealed that ABCA1-GFP is expressed not only on the plasma membrane but also on the surface of LAMP2(+)/DiI-LDL(+) late endocytic vesicles. ABCA1-GFP residing at the plasma membrane appears to traffic along the endocytic pathway to late endocytic compartments, because monensin causes ABCA1-GFP to accumulate in late endosomes/lysosomes while reducing the amount of ABCA1-GFP at the plasma membrane. Time-lapse microscopy revealed that ABCA1-GFP resides on the surface of both small, mobile vesicles as well as on large, relatively immobile perinuclear vesicles. The small, fast-moving ABCA1-GFP-containing vesicles are likely to represent early endosomes, whereas the ABCA1-GFP-containing large, static, perinuclear vesicles are likely to represent late endocytic compartments. The ABCA1-GFP-containing early endosomes undergo fusion and fission events. The ABCA1-GFP-containing early endosomes interact transiently with one another, with the ABCA1-GFP-containing late endocytic vesicles, and with the cell surface. As illustrated in Fig. 8, the ABCA1-GFP early endosomes appear to shuttle between the ABCA1-GFP late endocytic vesicles and the cell surface. The ABCA1-GFP-containing early endosomes tubulate while moving vectorially, consistent with directed movement along cytoskeletal elements such as microtubules or actin filaments. In addition to ABCA1, the membrane components that may be transferred from the ABCA1-GFP early endosomes to other cellular compartments remain to be determined but may include substrate lipids for ABCA1 and/or acceptor apolipoproteins. Indeed, recent studies have provided evidence that extracellular apolipoproteins are endocytosed and trafficked via a recycling compartment back to the cell surface prior to their release from the cell (21Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar, 34Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar). Interestingly, the compartment that appears to mediate the recycling of apolipoproteins is distinctive from the transferrin receptor recycling compartment (34Heeren J. Weber W. Beisiegel U. J. Cell Sci. 1999; 112: 349-359Crossref PubMed Google Scholar). Thus, the possibility exists that apoA-I and other apolipoproteins that are lipidated in an ABCA1-dependent manner may share an endocytic trafficking itinerary with ABCA1 in a novel endocytic recycling compartment. We have shown that delivery of the ABCA1 transporter to the cell surface is necessary for cellular lipid efflux. It remains to be determined, however, whether residence of the ABCA1 transporter at the surface is sufficient for its function or whether trafficking along the endocytic pathway plays a critical role in ABCA1 transporter function. Support for a potential functional role for ABCA1 in endocytic compartments is provided by the recent reports that the ABCA2 (35Zhou C.-J. Zhao L.-X. Inagaki N. Guan J.-L. Nakajo S. Hirabayashi T. Kikuyama S. Shioda S. J. Neurosci. 2001; 21: 849-857Crossref PubMed Google Scholar) and ABCB9 (36Zhang F. Zhang W. Liu L. Fisher C. Hui D. Childs S. Dorovini-Zis K. Ling V. J. Biol. Chem. 2000; 275: 23287-23294Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) transporters predominantly reside in and may function in endocytic compartments. Finally, delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate the surface expression of ABCA1 and hence cellular lipid efflux. 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• W2159836929 date "2001-07-01" @default.
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• W2159836929 title "Cellular Localization and Trafficking of the Human ABCA1 Transporter" @default.
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• W2159836929 doi "https://doi.org/10.1074/jbc.m103264200" @default.
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