ABCMdb

ABCG1 p.Lys124Met

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
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] ATP binding cassette transporter G1 (ABCG1) is an ... Proc Natl Acad Sci U S A. 2011 Dec 6;108(49):19719-24. Epub 2011 Nov 17. Tarling EJ, Edwards PA
ATP binding cassette transporter G1 (ABCG1) is an intracellular sterol transporter.
Proc Natl Acad Sci U S A. 2011 Dec 6;108(49):19719-24. Epub 2011 Nov 17., [PMID:22095132]

Abstract [show]
Four members of the mammalian ATP binding cassette (ABC) transporter G subfamily are thought to be involved in transmembrane (TM) transport of sterols. The residues responsible for this transport are unknown. The mechanism of action of ABCG1 is controversial and it has been proposed to act at the plasma membrane to facilitate the efflux of cellular sterols to exogenous high-density lipoprotein (HDL). Here we show that ABCG1 function is dependent on localization to intracellular endosomes. Importantly, localization to the endosome pathway distinguishes ABCG1 and/or ABCG4 from all other mammalian members of this superfamily, including other sterol transporters. We have identified critical residues within the TM domains of ABCG1 that are both essential for sterol transport and conserved in some other members of the ABCG subfamily and/or the insulin-induced gene 2 (INSIG-2). Our conclusions are based on studies in which (i) biotinylation of peritoneal macrophages showed that endogenous ABCG1 is intracellular and undetectable at the cell surface, (ii) a chimeric protein containing the TM of ABCG1 and the cytoplasmic domains of the nonsterol transporter ABCG2 is both targeted to endosomes and functional, and (iii) ABCG1 colocalizes with multiple proteins that mark late endosomes and recycling endosomes. Mutagenesis studies identify critical residues in the TM domains that are important for ABCG1 to alter sterol efflux, induce sterol regulatory element binding protein-2 (SREBP-2) processing, and selectively attenuate the oxysterol-mediated repression of SREBP-2 processing. Our data demonstrate that ABCG1 is an intracellular sterol transporter that localizes to endocytic vesicles to facilitate the redistribution of specific intracellular sterols away from the endoplasmic reticulum (ER).

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No. Sentence Comment
101 In contrast, luciferase activity did not increase significantly following overexpression of ABCG1-K124M that contains a mutation in a critical lysine in the Walker A motif (Fig. 5). Login to comment
112 Interestingly, overexpression of the remaining eight ABCG1 constructs failed to induce luciferase activity above the level seen with the ABCG1-K124M mutant (Fig. 5). Login to comment
128 Importantly, this cholesterol-dependent repression of luciferase activity was attenuated in cells overexpressing wild-type ABCG1, but not the inactive mutant ABCG1-K124M (Fig. 6A). Login to comment
148 Further, heterodimeric ABCG5:ABCG8 promotes the efflux of cholesterol ABCG1 -Actin Empty Wildtype K124M G422A F455A Y479A Q498A W511A T513A L534A L541A G569A S573A 8 - 6 - 4 - 2 - 0 - Luciferase(FoldChange) *** *** *** Fig. 5. Login to comment
175 A B C D Luciferase(FoldChange) 1.0 - 0.0 - 0.4 - 0.20.6 - 0.8 - 1.2 - CD CD:25-OHC CD:27-OHCCD:7- OHC CD:7-KetoC NO ABCG1 ABCG1 K124M *** *** Luciferase(FoldChange) 1.0 - 0.0 - 0.4 - 0.2 - 0.6 - 0.8 - 1.2 - 0 0.075 0.25 0.5 1 NO ABCG1 ABCG1 K124M Concentration of 25-OHC ( M) ** * Luciferase(FoldChange) 1.0 - 0.0 - 0.4 - 0.20.6 - 0.8 - NO ABCG1 ABCG1 K124M CD CD:CHOL 1.2 - 1.4 - *** *** * Luciferase(FoldChange) 1.0 - 0.0 - 0.4 - 0.2 - 0.6 - 0.8 - 1.2 - 0 0.075 0.25 0.5 1 NO ABCG1 ABCG1 K124M Concentration of 27-OHC ( M) *** *** *** * Fig. 6. Login to comment
177 (A) CHO-K1 cells were cotransfected with pSynSRE and either wild-type or mutant ABCG1 (ABCG1-K124M). Login to comment

[hide] Expression and functional characterization of ABCG... FEBS Lett. 2006 Aug 7;580(18):4551-9. Epub 2006 Jul 13. Engel T, Bode G, Lueken A, Knop M, Kannenberg F, Nofer JR, Assmann G, Seedorf U
Expression and functional characterization of ABCG1 splice variant ABCG1(666).
FEBS Lett. 2006 Aug 7;580(18):4551-9. Epub 2006 Jul 13., [PMID:16870176]

Abstract [show]
The ATP-binding cassette transporter ABCG1 mediates the transport of excess cholesterol from macrophages and other cell types to high density lipoprotein (HDL) but not to lipid-depleted apolipoprotein AI. Several splice variants which may have different functions have been identified in mammals. In the current study, we characterized the human splice variant ABCG1(666), which differs from full-length ABCG1(678) by absence of an internal segment of 12 amino acids (VKQTKRLKGLRK). Accordingly spliced ABCG1 transcripts were detected in macrophages and liver in approximately twofold higher amounts than the alternatively spliced ABCG1 form encoding full-length ABCG1. We used transient and stable expression of ABCG1(666) fusion proteins to characterize glycosylation, subcellular localization, molecular interaction and functions of this ABCG1 variant. It could be demonstrated that ABCG1(666) is located at the cell surface and has the ability to form cholesterol transport competent homodimers which affect cellular cholesterol export in a similar manner as previously characterized forms of ABCG1. Our results support that ABCG1(666) may in fact be the most prominent form of functional ABCG1 expressed in the human.

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138 Both, lipoprotein particles as well as serum albumin take up cholesterol twice as efficiently from wt ABCG1 expressing cells as compared to the same cell line where ABCG1 expression had been turned off by application of doxycycline, the parental TetOff HeLa cells or a cell line expressing inactive mutant ABCG1(666; K124M). Login to comment
170 ABCG1(666; K124M) mutant localizes at the cell surface and expression can be regulated by doxycycline. Login to comment
171 TetOff HeLa cells were stable transfected with plasmids coding for wt (HA)3-ABCG1(666), mutant (HA)3-ABCG1(666; K124M) or (HA)3-ABCG2, respectively. Login to comment
174 (B) Lanes 5 and 6: (HA)3-ABCG1(666; K124M) clone 47. Login to comment
184 [3 H]cholesterol efflux to various acceptors was measured for 1.5 h in TetOff HeLa cells either stably transfected with (HA)3-ABCG1(666) (G1-94; white, black), (HA)3-ABCG1(666; K124M) (G1(KM)-47; light/dark grey), or non-transfected (TetOff-HeLa; cross-hatched) in the presence (white, light gray, hatched) or absence (black, dark gray, hatched gray) of 1 lg/ml doxycycline, as described in Section 2. differed from previously characterized ABCG1 variants by absence of an internal segment of 12 amino acids (Fig. 8). Login to comment
204 (A) Samples from TetOff HeLa cell lines expressing wt or mutant (K124M) (HA)3- ABCG transporter, as indicated, were subjected to reducing (right panel) or non-reducing (left panel) SDS-PAGE and subsequent Western blot analysis with HA.11 antibody followed by goat anti-mouse HRP-labeled secondary antibody. Login to comment
206 G1-94, (HA)3-ABCG1(666) clone 94; G1KM-47, (HA)3-ABCG1(666, K124M) clone 47; G2-63, (HA)3-ABCG2 clone 63. Login to comment
235 We thank Dr. Helen Hobbs (UTSW, Dallas, TX, USA) for providing us with an ABCG5-(FLAG)3 plasmid used as a template for cloning ABCG5 employed in control 1_HSG1A 1 SDHKRDLGGDAEVNPFLWHRPSEE------------DSSSMEGCHSFSASCLTQFCILFKRTFL 2_HSG1B 1 SDHKRDLGGDAEVNPFLWHRPSEEVKQTKRLKGLRKDSSSMEGCHSFSASCLTQFCILFKRTFL 3_MMG1_ 1 ADYKRDLGGDTDVNPFLWHRPAEE------------DSASMEGCHSFSASCLTQFCILFKRTFL 4_RNG1_ 1 SDYKRELGGDGDVNPFLWHRPAEE------------DSASMEGCHSFSASCLTQFCILFKRTFL 6_CFG1_ 1 SEHRREPGGDAEVNPFLWHRPSEE------------DSTSMEGCHSFSASCLTQFCILFKRTFL 5_BTG1_ 1 SDCRREPGGDAEVNPFIWHRPSEE------------DSTSMEGCHSFSASCLTQFCILFKRTFL 7_DRG1_ 1 KDYKTEMNGNGVHNPFLWHRPSDE------------DSSSSEGCHSFSASCLTQFCILFKRTFL 8_HSG4_ 1 MAEKKSSPEKNEVPAPCPPCPPEV------------DPIES---HTFATSTLTQFCILFKRTFL hotloop hotloop ordered dis- HSG1A HSG1B A B COOH NH2 Walker B Signature, motif C Walker A outside cytoplasma K124M potential NGS * # 379-VKQTKRLKGLRK-390 Fig. 8. Login to comment
243 The mutated site in the Walker-A motif of the transporter, used for generating ABCG1(666; K124M) mutant, is indicated as well (#). Login to comment

[hide] Expression of ATP binding cassette-transporter ABC... FEBS Lett. 2007 Apr 17;581(8):1673-80. Epub 2007 Mar 28. Engel T, Kannenberg F, Fobker M, Nofer JR, Bode G, Lueken A, Assmann G, Seedorf U
Expression of ATP binding cassette-transporter ABCG1 prevents cell death by transporting cytotoxic 7beta-hydroxycholesterol.
FEBS Lett. 2007 Apr 17;581(8):1673-80. Epub 2007 Mar 28., [PMID:17408620]

Abstract [show]
Oxysterols result from cholesterol by enzymatic or oxidative processes. Some exert cytotoxic effects leading to necrosis or apoptosis. Detoxification of these compounds mainly occurs in the liver and requires transport from peripheral tissues towards it. Some ATP-binding cassette transporters are involved in export of cytotoxic compounds. In the current study, we investigated whether ABC transporter family member G1 (ABCG1) may be involved in oxysterol transport, since its gene expression is highly responsive to oxysterol loading. TetOff HeLa cells stably expressing ABCG1 showed decreased mass uptake of 7beta-hydroxycholesterol (7beta-HC) whereas that of other physiologically relevant oxysterols was unaffected. Application of 7beta-HC to ABCG1 expressing cells induced hyperpolarization of mitochondrial membrane potential and production of reactive oxygen species, indicating energy consumption by the ATP-binding cassette transporter when it is activated by its correct substrate. Our study points to detoxification as one of potential cellular functions of ABCG1. We assume that ABCG1 protects against 7beta-HC-induced cell death, an important role in prevention of neurodegenerative and cardiovascular disease.

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No. Sentence Comment
94 Results In order to identify whether ABCG1 is able to transport oxysterols we used our previously characterized TetOff HeLa cell lines which allow regulatable expression of ABCG1(666) or the ABCG1(666,K124M) defective mutant [16] over four orders of magnitude. Login to comment

[hide] 3beta,5alpha,6beta-Cholestanetriol and 25-hydroxyc... Atherosclerosis. 2014 Jul;235(1):122-9. doi: 10.1016/j.atherosclerosis.2014.04.023. Epub 2014 May 1. Engel T, Fobker M, Buchmann J, Kannenberg F, Rust S, Nofer JR, Schurmann A, Seedorf U
3beta,5alpha,6beta-Cholestanetriol and 25-hydroxycholesterol accumulate in ATP-binding cassette transporter G1 (ABCG1)-deficiency.
Atherosclerosis. 2014 Jul;235(1):122-9. doi: 10.1016/j.atherosclerosis.2014.04.023. Epub 2014 May 1., [PMID:24833118]

Abstract [show]
OBJECTIVE: Oxysterols are oxidized derivatives of sterols that have cytotoxic effects and are potent regulators of diverse cellular functions. Efficient oxysterol removal by the sub-family G member 1 of the ATP-binding cassette transporters (ABCG1) is essential for cell survival and control of cellular processes. However, the specific role of ABCG1 in the transport of various oxysterol species has been not systematically investigated to date. Here, we examined the involvement of ABCG1 in the oxysterol metabolism by studying oxysterol tissue levels in a mouse model of Abcg1-deficiency. METHODS AND RESULTS: Analysis of lung tissue of Abcg1(-/-) mice on a standard diet revealed that 3beta,5alpha,6beta-cholestanetriol (CT) and 25-hydroxycholesterol (HC) accumulated at more than 100-fold higher levels in comparison to wild-type mice. 24S-HC and 27-HC levels were also elevated, but were minor constituents. A radiolabeled assay employing regulable ABCG1-expressing HeLa cell lines revealed that 25-HC export to albumin was dependent on functional ABCG1 expression and could be blocked by an excess of unlabeled 25-HC or 27-HC. In this cell line, 25-HC at low doses triggered mitochondrial membrane potential, and reactive oxygen species production, which are both indirect indicators of cellular energy expenditure. CONCLUSION: Our results suggest that 25-HC and CT are physiologic substrates for ABCG1. Excessive accumulation of these oxysterols may explain the increased rate of cell death and the inflammatory phenotype observed in Abcg1-deficient animals and cells.

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37 Cells TetOff-HeLa cell lines TG1-94 and TG1KM-47, stably transfected with pTRE expression plasmids (Clontech) containing wild-type (wt) or K124M-mutant (HA)3-ABCG1(666), respectively [13], were maintained in complete medium (Dulbecco`s modification of Eagle`s medium (DMEM) with 10% fetal calf serum (FCS), antibiotic-antimycotic solution, and glutamine), as previously described [13,14]. Login to comment
106 Analysis of ATPase activation by various oxysterols To address the question whether oxysterol species accumulating in Abcg1 deficiency are direct substrates for this transporter, individual oxysterols were applied to tetracycline-inducible HeLa cell lines expressing wt ABCG1 or mutant ABCG1-K124M variant, which was previously shown to be metabolically inactive [13,25]. Login to comment
112 As shown in Fig. 2, 25-HC (Fig. 2B, upper right panel), CT (Fig. 2B, lower left panel), and 7b-HC (Fig. 2B, lower right panel) induced MMP in wt ABCG1-expressing cells only (Fig. 2B bold lines), whereas no such effect could be noted in the cells expressing mutant ABCG1-K124M variant (Fig. 2B dashed lines). Login to comment
123 As shown in Fig. 4A, export of 25-HC was enhanced significantly in wt ABCG1-expressing cells, whereas cells expressing mutant ABCG1-K124M were ineffective in this respect. Login to comment
163 Expression of exogenous wt and mutant (K124M) HA3-ABCG1, as well as endogenous ABCG1 in a membrane fraction of cells grown as in (Fig. 4A and B) is shown in (A). Login to comment
167 (B) TetOff HeLa cell lines expressing (bold/dashed line) or not expressing (regular/dotted line) wt ABCG1 (bold/regular line) or mutant ABCG1-K124M (dashed/dotted line), were incubated in the presence of vehicle only (top left panel) or either 5 mM 25-HC (top right panel), 14.2 mM CT (bottom left panel), or 10 mM 7b-HC (bottom right panel), for 24 h. TMRE fluorescence was recorded (see Material and Methods). Login to comment