ABCMdb

ABCA1 p.Lys1952Met

Predicted by SNAP2:	A: D (91%), C: D (75%), D: D (95%), E: D (95%), F: D (91%), G: D (95%), H: D (91%), I: D (91%), L: D (85%), M: D (85%), N: D (91%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] The mammalian ABC transporter ABCA1 induces lipid-... Biochim Biophys Acta. 2012 Mar;1821(3):373-80. doi: 10.1016/j.bbalip.2011.07.005. Epub 2011 Jul 20. Bocer T, Zarubica A, Roussel A, Flis K, Trombik T, Goffeau A, Ulaszewski S, Chimini G
The mammalian ABC transporter ABCA1 induces lipid-dependent drug sensitivity in yeast.
Biochim Biophys Acta. 2012 Mar;1821(3):373-80. doi: 10.1016/j.bbalip.2011.07.005. Epub 2011 Jul 20., [PMID:21787882]

Abstract [show]
ABCA1 belongs to the A class of ABC transporter, which is absent in yeast. ABCA1 elicits lipid translocation at the plasma membrane through yet elusive processes. We successfully expressed the mouse Abca1 gene in Saccharomyces cerevisiae. The cloned ABCA1 distributed at the yeast plasma membrane in stable discrete domains that we name MCA (membrane cluster containing ABCA1) and that do not overlap with the previously identified punctate structures MCC (membrane cluster containing Can1p) and MCP (membrane cluster containing Pma1p). By comparison with a nonfunctional mutant, we demonstrated that ABCA1 elicits specific phenotypes in response to compounds known to interact with membrane lipids, such as papuamide B, amphotericin B and pimaricin. The sensitivity of these novel phenotypes to the genetic modification of the membrane lipid composition was studied by the introduction of the cho1 and lcb1-100 mutations involved respectively in phosphatidylserine or sphingolipid biosynthesis in yeast cells. The results, corroborated by the analysis of equivalent mammalian mutant cell lines, demonstrate that membrane composition, in particular its phosphatidylserine content, influences the function of the transporter. We thus have reconstituted in yeast the essential functions associated to the expression of ABCA1 in mammals and characterized new physiological phenotypes prone to genetic analysis. This article is a part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

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39 Two additional vectors, bearing the nonfunctional mutant, pTB1A1MM and pTB2A1MM were generated by replacing the central fragment of ABCA1 gene by a fragment containing K939M and K1952M mutations in the Walker A motif in both nucleotide binding domains [2,4]. Login to comment

[hide] ATP hydrolysis-dependent conformational changes in... J Lipid Res. 2012 Jan;53(1):126-36. Epub 2011 Oct 25. Nagao K, Takahashi K, Azuma Y, Takada M, Kimura Y, Matsuo M, Kioka N, Ueda K
ATP hydrolysis-dependent conformational changes in the extracellular domain of ABCA1 are associated with apoA-I binding.
J Lipid Res. 2012 Jan;53(1):126-36. Epub 2011 Oct 25., [PMID:22028339]

Abstract [show]
ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high-density lipoprotein (HDL) metabolism. Although it is predicted that apolipoprotein A-I (apoA-I) directly binds to ABCA1, the physiological importance of this interaction is still controversial and the conformation required for apoA-I binding is unclear. In this study, the role of the two nucleotide-binding domains (NBD) of ABCA1 in apoA-I binding was determined by inserting a TEV protease recognition sequence in the linker region of ABCA1. Analyses of ATP binding and occlusion to wild-type ABCA1 and various NBD mutants revealed that ATP binds equally to both NBDs and is hydrolyzed at both NBDs. The interaction with apoA-I and the apoA-I-dependent cholesterol efflux required not only ATP binding but also hydrolysis in both NBDs. NBD mutations and cellular ATP depletion decreased the accessibility of antibodies to a hemagglutinin (HA) epitope that was inserted at position 443 in the extracellular domain (ECD), suggesting that the conformation of ECDs is altered by ATP hydrolysis at both NBDs. These results suggest that ATP hydrolysis at both NBDs induces conformational changes in the ECDs, which are associated with apoA-I binding and cholesterol efflux.

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7 Chambenoit et al. reported that substituting the Walker A lysine residue in either NBD with methionine (K939M or K1952M) abolishes the ability of apoA-I to bind to ABCA1-expressing cells and inhibits cholesterol secretion to apoA-I (12). Login to comment
26 Plasmids The expression vectors for wild-type ABCA1, ABCA1-K939M, ABCA1-K1952M, and ABCA1-K939M,K1952M that were tagged with GFP at the C terminus were generated as previously described (7, 13). Login to comment
88 HEK293 cells stably expressing wild-type (wt) ABCA1 or the Walker A lysine mutants ABCA1-K939M, ABCA1-K1952M, or ABCA1-K939M,K1952M were incubated for 24 h in DMEM containing 0.02% BSA with or without 5 ␮g/ml apoA-I. Login to comment
119 However, cholesterol efflux was impaired in cells expressing ABCA1-K939M,K1952M, a mutant in which the Walker A lysines in both NBDs are replaced with methionines, or the single Walker A lysine mutants ABCA1-K939M or ABCA1-K1952M, in which the Walker A lysine in NBD1 or NBD2 is replaced with methionine (Fig. 3B). Login to comment
135 We consistently observed stronger ATP binding to NBD2 in ABCA1-K939M,K1952M-TEV-GFP, although the reason for this higher-affinity interaction is unknown. Login to comment
143 On the other hand, the K1952M mutation drastically decreased the photoaffinity labeling by approximately 95%. Login to comment
144 Interestingly, the addition of vanadate significantly (1.7-fold) enhanced the photoaffinity labeling of ABCA1-K1952M, suggesting that vanadate stabilizes the nucleotide at NBD1 after ATP hydrolysis. Login to comment
145 There was no photoaffinity-labeled band with ABCA1-K939M,K1952M. Login to comment
147 These results suggest that the occluded nucleotide in the NBD of wild-type ABCA1, ABCA1-K939M, and ABCA1-K1952M is not ATP but ADP, that both NBDs occlude ADP after ATP hydrolysis, and that the features of the two NBDs of ABCA1 are quite different. Login to comment
162 In addition, there were no detectable nucleotide occlusions at either NBD in the K1952M mutant. Login to comment
27 Plasmids The expression vectors for wild-type ABCA1, ABCA1-K939M, ABCA1-K1952M, and ABCA1-K939M,K1952M that were tagged with GFP at the C terminus were generated as previously described (7, 13). Login to comment
89 HEK293 cells stably expressing wild-type (wt) ABCA1 or the Walker A lysine mutants ABCA1-K939M, ABCA1-K1952M, or ABCA1-K939M,K1952M were incubated for 24 h in DMEM containing 0.02% BSA with or without 5 òe;g/ml apoA-I. Login to comment
120 However, cholesterol efflux was impaired in cells expressing ABCA1-K939M,K1952M, a mutant in which the Walker A lysines in both NBDs are replaced with methionines, or the single Walker A lysine mutants ABCA1-K939M or ABCA1-K1952M, in which the Walker A lysine in NBD1 or NBD2 is replaced with methionine (Fig. 3B). Login to comment
137 We consistently observed stronger ATP binding to NBD2 in ABCA1-K939M,K1952M-TEV-GFP, although the reason for this higher-affinity interaction is unknown. Login to comment
146 Interestingly, the addition of vanadate significantly (1.7-fold) enhanced the photoaffinity labeling of ABCA1-K1952M, suggesting that vanadate stabilizes the nucleotide at NBD1 after ATP hydrolysis. Login to comment
149 These results suggest that the occluded nucleotide in the NBD of wild-type ABCA1, ABCA1-K939M, and ABCA1-K1952M is not ATP but ADP, that both NBDs occlude ADP after ATP hydrolysis, and that the features of the two NBDs of ABCA1 are quite different. Login to comment
165 In addition, there were no detectable nucleotide occlusions at either NBD in the K1952M mutant. Login to comment

[hide] Sodium taurocholate-dependent lipid efflux by ABCA... J Lipid Res. 2009 Jun;50(6):1165-72. Epub 2009 Feb 8. Nagao K, Zhao Y, Takahashi K, Kimura Y, Ueda K
Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation.
J Lipid Res. 2009 Jun;50(6):1165-72. Epub 2009 Feb 8., [PMID:19202195]

Abstract [show]
ABCA1 plays a major role in HDL metabolism. Cholesterol secretion by ABCA1 is dependent on the presence of extracellular acceptors, such as lipid-free apolipoprotein A-I (apoA-I). However, the importance of the direct interaction between apoA-I and ABCA1 in HDL formation remains unclear. In contrast, ABCB4 mediates the secretion of phospholipids and cholesterol in the presence of sodium taurocholate (NaTC) but not in the presence of apoA-I. In this study, we analyzed apoA-I binding and NaTC-dependent lipid efflux by ABCA1. ABCA1 mediated the efflux of cholesterol and phospholipids in the presence of NaTC as well as in the presence of apoA-I in an ATP-dependent manner. The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. However, the W590S mutation did not impair apoA-I binding and, conversely, retarded the dissociation of apoA-I from ABCA1. These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading, facilitates apoA-I dissociation from ABCA1. NaTC is a good tool for analyzing ABCA1-mediated lipid efflux and allows dissection of the steps of HDL formation by ABCA1.

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45 Cell culture Human embryonic kidney (HEK293) cells and WI-38 fibroblasts were grown in a humidified incubator (5% CO2) at 37°C in DMEM supplemented with 10% heat-inactivated FBS. Plasmids The expression vectors for wild-type ABCA1, ABCA1-W590S, and ABCA1-K939M,K1952M (MM), fused to GFP at their C termini, were generated as described previously (3, 18). Login to comment
43 Plasmids The expression vectors for wild-type ABCA1, ABCA1-W590S, and ABCA1-K939M,K1952M (MM), fused to GFP at their C termini, were generated as described previously (3, 18). Login to comment

[hide] Formation of two intramolecular disulfide bonds is... J Biol Chem. 2009 Apr 24;284(17):11293-300. Epub 2009 Mar 3. Hozoji M, Kimura Y, Kioka N, Ueda K
Formation of two intramolecular disulfide bonds is necessary for ApoA-I-dependent cholesterol efflux mediated by ABCA1.
J Biol Chem. 2009 Apr 24;284(17):11293-300. Epub 2009 Mar 3., [PMID:19258317]

Abstract [show]
ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. ABCA1 contains disulfide bond(s) between its N- and C-terminal halves, but it remains unclear whether disulfide bond formation is important for the functions of ABCA1 and which cysteines are involved in disulfide bond formation. To answer these questions, we constructed >30 ABCA1 mutants in which 16 extracellular domain (ECD) cysteines were replaced with serines and examined disulfide bond formation, apoA-I binding, and HDL formation in these mutants. From the single cysteine replacements, two cysteines (Cys(75) and Cys(309)) in ECD1 were found to be essential for apoA-I binding. In contrast, in ECD2, only Cys(1477) was found to be essential for HDL formation, and no single cysteine replacement impaired apoA-I binding. The concurrent replacement of two cysteines, Cys(1463) and Cys(1465), impaired apoA-I binding and HDL formation, suggesting that four of five extracellular cysteines (Cys(75), Cys(309), Cys(1463), Cys(1465), and Cys(1477)) are involved in these functions of ABCA1. Trypsin digestion experiments suggested that one disulfide bond is not sufficient and that two intramolecular disulfide bonds (between Cys(75) and Cys(309) in ECD1 and either Cys(1463) or Cys(1465) and Cys(1477) in ECD2) are required for ABCA1 to be fully functional.

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90 ApoA-I bound to cells expressing wild-type ABCA1 but not to cells expressing ABCA1-MM (ABCA1(K939M/K1952M)), a mutant in which the Walker A lysines in both nucleotide-binding domains are replaced by methionines, although the expression level (data not shown) and surface expression (Fig. 4A) of the mutant were comparable with those of the wild-type protein, as reported (24). Login to comment
88 ApoA-I bound to cells expressing wild-type ABCA1 but not to cells expressing ABCA1-MM (ABCA1(K939M/K1952M)), a mutant in which the Walker A lysines in both nucleotide-binding domains are replaced by methionines, although the expression level (data not shown) and surface expression (Fig. 4A) of the mutant were comparable with those of the wild-type protein, as reported (24). Login to comment

[hide] Retroendocytosis pathway of ABCA1/apoA-I contribut... Genes Cells. 2009 Feb;14(2):191-204. Epub 2008 Jan 6. Azuma Y, Takada M, Shin HW, Kioka N, Nakayama K, Ueda K
Retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation.
Genes Cells. 2009 Feb;14(2):191-204. Epub 2008 Jan 6., [PMID:19170766]

Abstract [show]
ATP-binding cassette protein A1 (ABCA1) mediates transfer of cellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor in plasma, to form high-density lipoprotein (HDL). It is currently unknown to what extent ABCA1 endocytosis and recycling contribute to the HDL formation. To address this issue, we expressed human ABCA1 constructs with either an extracellular HA tag or an intracellular GFP tag in cells, and used this system to characterize endocytosis and recycling of ABCA1 and apoA-I. Under basal conditions, ABCA1 and apoA-I are endocytosed via a clathrin- and Rab5-mediated pathway and recycled rapidly back to the cell surface, at least in part via a Rab4-mediated route; approximately 30% of the endocytosed ABCA1 is recycled back to the cell surface. When receptor-mediated endocytosis is inhibited, the level of ABCA1 at the cell surface increases and apoA-I internalization is blocked. Under these conditions, apoA-I mediated cholesterol efflux from cells that have accumulated lipoprotein-derived cholesterol is decreased, whereas efflux from cells without excess cholesterol is increased. These results suggest that the retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation when excess lipoprotein-derived cholesterol has accumulated in cells.

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197 DNA construction We constructed plasmids for expressing human wild-type ABCA1, ABCA1(W590S) and ABCA1(K939M,K1952M)(MM) bearing an insertion of the influenza virus hemagglutinin (HA) epitope sequence between residues 207 and 208 (within the first extracellular loop), using the bicistronic expression vector pHaMAIRESneo. Login to comment

[hide] ABCA1 mediates high-affinity uptake of 25-hydroxyc... Am J Physiol Cell Physiol. 2006 Sep;291(3):C490-502. Epub 2006 Apr 12. Tam SP, Mok L, Chimini G, Vasa M, Deeley RG
ABCA1 mediates high-affinity uptake of 25-hydroxycholesterol by membrane vesicles and rapid efflux of oxysterol by intact cells.
Am J Physiol Cell Physiol. 2006 Sep;291(3):C490-502. Epub 2006 Apr 12., [PMID:16611739]

Abstract [show]
ATP Binding Cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport by mediating the cellular efflux of phospholipid and cholesterol. Studies using intact cells strongly suggest that ABCA1 acts as a phospholipid floppase, but there has been no direct demonstration that the protein is a primary active sterol transporter. Using membrane vesicles from insect Sf21 cells, we found that ABCA1 mediated ATP-dependent uptake of [(3)H]25-hydroxycholesterol with an apparent K(m) of 0.7 muM. Consistent with this high apparent affinity, expression of ABCA1 in human embryonic kidney cells both increased rapid efflux of 25-hydroxcholesterol and prevented oxysterol-mediated repression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase mRNAs. Comparison of wild-type and ABCA1(-/-) murine fibroblasts indicates that 25-hydroxycholesterol is effluxed approximately 5-fold more rapidly by wild-type cells. In addition, the rate of efflux from the wild-type but not the ABCA1(-/-) fibroblasts is increased a further twofold by inducers of ABCA1 expression. Thus under the experimental conditions employed, endogenous ABCA1 is a major contributor to 25-hydroxycholesterol efflux from wild-type fibroblasts. Evidence from in vitro studies indicates that oxysterols are potent inducers of genes involved in cellular cholesterol efflux and metabolism, including the ABCA1 gene, and repressors of genes involved in cholesterol synthesis or uptake. Our observations raise the possibility that efflux of oxysterols by ABCA1 could contribute to a homeostatic mechanism, which both attenuates oxysterol-induced expression of its cognate gene and alleviates repression of genes encoding proteins, such as HMG-CoA reductase and LDL receptor.

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158 A: immunoblots of total membrane proteins (15 ␮g) from vesicles containing wild-type ABCA1 (KK) and mutant proteins containing Met substitutions of the highly conserved Walker A Lys of nucleotide binding domain (NBD1; K939M, MK), NBD2 (K1952M, KM), or both NBDs (K939M/K1952M, MM), as well as control vesicles of beta-glucuronidase (beta-gus). Login to comment

[hide] Purification and ATPase activity of human ABCA1. J Biol Chem. 2006 Apr 21;281(16):10760-8. Epub 2006 Feb 24. Takahashi K, Kimura Y, Kioka N, Matsuo M, Ueda K
Purification and ATPase activity of human ABCA1.
J Biol Chem. 2006 Apr 21;281(16):10760-8. Epub 2006 Feb 24., [PMID:16500904]

Abstract [show]
ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.

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33 2 The abbreviations used are: ABCA1, ATP-binding cassette protein A1; ABCA1 MM, ABCA1 K939M-K1952M; MDR, multidrug resistance; MRP, multidrug resistance-related protein; NBF, nucleotide-binding fold; apo, apolipoprotein; HDL, high density lipoprotein; FC, free cholesterol; PL, phospholipid; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SM, sphingomyelin; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; DPPC, 1,2-dipalmitoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; DPPE, 1,2- dipalmitoylphosphatidylethanolamine; DPPG, 1,2-dipalmitoylphosphatidylglycerol; Sf9, Spodoptera frugiperda 9; HEK, human embryo kidney; NGF, N-glycosidase F; DDM, n-dodecyl-beta-D-maltoside; NTA, nitrilotriacetic acid. Login to comment
117 ABCA1 K939M-K1952M protein, expressed and purified in the same procedure, was as pure as the wild type (Figs. Login to comment
149 To confirm that ATPase activity is derived from purified ABCA1, a baculovirus for ABCA1 K939M-K1952M mutant, in which lysine 939 and lysine 1952 in the Walker A motif of nucleotide binding folds were replaced by methionines, was constructed. Login to comment
151 Purified ABCA1 K939M-K1952M protein showed little ATPase activity, less than 10 nmol/min/mg of protein (Fig. 5). Login to comment
166 E, purified ABCA1 K939M-K1952M mutant, 0.5 ␮g (Coomassie Brilliant Blue R-250 staining). Login to comment
214 A, ATPase activity was measured in the presence of various concen- trationsofMgATP.F,wildtype;E,K939M-K1952M mutant. Login to comment
32 2 The abbreviations used are: ABCA1, ATP-binding cassette protein A1; ABCA1 MM, ABCA1 K939M-K1952M; MDR, multidrug resistance; MRP, multidrug resistance-related protein; NBF, nucleotide-binding fold; apo, apolipoprotein; HDL, high density lipoprotein; FC, free cholesterol; PL, phospholipid; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SM, sphingomyelin; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; DPPC, 1,2-dipalmitoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; DPPE, 1,2- dipalmitoylphosphatidylethanolamine; DPPG, 1,2-dipalmitoylphosphatidylglycerol; Sf9, Spodoptera frugiperda 9; HEK, human embryo kidney; NGF, N-glycosidase F; DDM, n-dodecyl-beta-D-maltoside; NTA, nitrilotriacetic acid. Login to comment
114 ABCA1 K939M-K1952M protein, expressed and purified in the same procedure, was as pure as the wild type (Figs. 1B and 2E). Login to comment
145 To confirm that ATPase activity is derived from purified ABCA1, a baculovirus for ABCA1 K939M-K1952M mutant, in which lysine 939 and lysine 1952 in the Walker A motif of nucleotide binding folds were replaced by methionines, was constructed. Login to comment
147 Purified ABCA1 K939M-K1952M protein showed little ATPase activity, less than 10 nmol/min/mg of protein (Fig. 5). Login to comment
162 E, purified ABCA1 K939M-K1952M mutant, 0.5 òe;g (Coomassie Brilliant Blue R-250 staining). Login to comment
208 A, ATPase activity was measured in the presence of various concen- trationsofMgATP.F,wildtype;E,K939M-K1952M mutant. Login to comment

[hide] ABCA1 dimer-monomer interconversion during HDL gen... Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5034-9. doi: 10.1073/pnas.1220703110. Epub 2013 Mar 11. Nagata KO, Nakada C, Kasai RS, Kusumi A, Ueda K
ABCA1 dimer-monomer interconversion during HDL generation revealed by single-molecule imaging.
Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5034-9. doi: 10.1073/pnas.1220703110. Epub 2013 Mar 11., [PMID:23479619]

Abstract [show]
The generation of high-density lipoprotein (HDL), one of the most critical events for preventing atherosclerosis, is mediated by ATP-binding cassette protein A1 (ABCA1). ABCA1 is known to transfer cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I) for generating discoidal HDL (dHDL) particles, composed of 100-200 lipid molecules surrounded by two apoA-I molecules; however, the regulatory mechanisms are still poorly understood. Here we observed ABCA1-GFP and apoA-I at the level of single molecules on the plasma membrane via a total internal reflection fluorescence microscope. We found that about 70% of total ABCA1-GFP spots are immobilized on the plasma membrane and estimated that about 89% of immobile ABCA1 molecules are in dimers. Furthermore, an ATPase-deficient ABCA1 mutant failed to be immobilized or form a dimer. We found that the lipid acceptor apoA-I interacts with the ABCA1 dimer to generate dHDL and is followed by ABCA1 dimer-monomer interconversion. This indicates that the formation of the ABCA1 dimer is the key for apoA-I binding and nascent HDL generation. Our findings suggest the physiological significance of conversion of the ABCA1 monomer to a dimer: The dimer serves as a receptor for two apoA-I molecules for dHDL particle generation.

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131 Human ABCA1 and ABCA1-MM (with two point mutations of K939M and K1952M) were fused with EGFP or monomeric (m)EGFP at their carboxyl termini, where an mEGFP mutant was modified from EGFP by introducing a mutation corresponding to an A206K monomeric mutant (33). Login to comment

[hide] Differential phospholipid substrates and direction... J Biol Chem. 2013 Nov 29;288(48):34414-26. doi: 10.1074/jbc.M113.508812. Epub 2013 Oct 4. Quazi F, Molday RS
Differential phospholipid substrates and directional transport by ATP-binding cassette proteins ABCA1, ABCA7, and ABCA4 and disease-causing mutants.
J Biol Chem. 2013 Nov 29;288(48):34414-26. doi: 10.1074/jbc.M113.508812. Epub 2013 Oct 4., [PMID:24097981]

Abstract [show]
ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation.

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66 Corresponding ABCA4 mutations determined by amino acid alignment with ABCA1 included S100P, W605S, F608L, T959I, N965S, C1502R, T1537M, R2107P, and P2180L. Login to comment
67 ABCA1-MM was constructed to harbor the Walker A-motif lysine-to-methionine mutations K939M/K1952M by the nested PCR method; ABCA4-MM had the corresponding K969M/ K1969M Walker A mutations (37), and ABCA7-MM had the Lipid Transport Activity of ABCA Transporters NOVEMBER 29, 2013ߦVOLUME 288ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 34415 at SEMMELWEIS UNIV OF MEDICINE on December 3, K847M/K1833M Walker A mutations. Login to comment
69 ABCA1-MM was constructed to harbor the Walker A-motif lysine-to-methionine mutations K939M/K1952M by the nested PCR method; ABCA4-MM had the corresponding K969M/ K1969M Walker A mutations (37), and ABCA7-MM had the Lipid Transport Activity of ABCA Transporters NOVEMBER 29, 2013ߦVOLUME 288ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 34415 at SEMMELWEIS UNIV OF MEDICINE on December 3, K847M/K1833M Walker A mutations. Login to comment