ABCMdb

ABCB4 p.Glu558Gln

Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (59%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: N (57%), N: D (91%), P: D (95%), Q: D (63%), R: D (91%), S: D (91%), T: D (91%), V: D (91%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, F: D, G: D, H: D, I: D, K: 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] Complementary functions of the flippase ATP8B1 and... Gastroenterology. 2011 Nov;141(5):1927-37.e1-4. Epub 2011 Aug 4. Groen A, Romero MR, Kunne C, Hoosdally SJ, Dixon PH, Wooding C, Williamson C, Seppen J, Van den Oever K, Mok KS, Paulusma CC, Linton KJ, Oude Elferink RP
Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.
Gastroenterology. 2011 Nov;141(5):1927-37.e1-4. Epub 2011 Aug 4., [PMID:21820390]

Abstract [show]
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS: To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS: Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS: ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

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23 Materials and Methods Plasmids Generation of the pcDNA3.1ϩ vector (Invitrogen, Carlsbad, CA) expressing human ABCB4 and its K435M derivative (replacing the conserved lysine within the Walker A motif of the first nucleotide binding domain to inhibit adenosine triphosphate binding) was described previously.11 The E558Q derivative (replacing a conserved glutamate in the Walker B motif with a glutamine to prevent adenosine triphosphate hydrolysis) was derived from the wild-type ABCB4 by site-directed mutagenesis using the mutagenic oligonucleotide 5=- GATCCTTCTGCTG- GATCAGGCGACGTCAGCATTGGAC-3= and its reverse complement to prime synthesis of the mutant vector using Quikchange II (Agilent Technologies, Santa Clara, CA). Login to comment
65 We also noted that inactive derivatives of ABCB4 carrying a point mutation in either the Walker A (K435M) or Walker B (E558Q) motifs of the first nucleotide binding domain, expressed to much higher levels than wild-type protein, suggesting a negative selective pressure against ABCB4 function (Figure 1A). Login to comment
88 (A) Western analysis of mutant and wild-type ABCB4 in HEK293T whole cell lysates. Lane 1, mock-transfected cells; lane 2, wild-type ABCB4 alone; lane 3, ABCB4 mutant K435M alone; lane 4, ABCB4 mutant E558Q alone. Login to comment
66 We also noted that inactive derivatives of ABCB4 carrying a point mutation in either the Walker A (K435M) or Walker B (E558Q) motifs of the first nucleotide binding domain, expressed to much higher levels than wild-type protein, suggesting a negative selective pressure against ABCB4 function (Figure 1A). Login to comment
89 (A) Western analysis of mutant and wild-type ABCB4 in HEK293T whole cell lysates. Lane 1, mock-transfected cells; lane 2, wild-type ABCB4 alone; lane 3, ABCB4 mutant K435M alone; lane 4, ABCB4 mutant E558Q alone. Login to comment

[hide] Detergent screening and purification of the human ... PLoS One. 2013 Apr 4;8(4):e60620. doi: 10.1371/journal.pone.0060620. Print 2013. Ellinger P, Kluth M, Stindt J, Smits SH, Schmitt L
Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11) and MDR3 (ABCB4) expressed in the yeast Pichia pastoris.
PLoS One. 2013 Apr 4;8(4):e60620. doi: 10.1371/journal.pone.0060620. Print 2013., [PMID:23593265]

Abstract [show]
The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding approximately 1 mg and approximately 6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-beta-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.

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106 Oligonucleotide Sequence 59R 39 pSGP18-2m-ori-S1 TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTAAATATTGCGAATACCGCTTCCACAAACATTG pSGP18-2m-ori-S2 AACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTATTTCACACCGCATATATCGGATCGTACT BSEP-HR-PP-S1 ATCAAAAAACAACTAATTATTCGAACGAGGTAAAAGAATGTCTGACTCAGTAATTCTTCGAAGT ATA BSEP-HR-PP-S2 ACGTTTGGACCTTGGAAAAGACTTCTAAGGAGTTGGAGGCACTGATGGGGGATCCAGTGGTGACTAGTTT MDR3-HR-PP-S1 ATCAAAAAACAACTAATTATTCGAACGAGGTAAAAGAATGGATCTTGAGGCGGCAAAGAACGGAACA MDR3-HR-PP-S2 ACGTTTGGACCTTGGAATAAGACTTCTAAGGAGTTGGAGGCTAAGTTCTGTGTTCCAGCCTGGACACTGACCATTGAAAAATAG YEpN14HIS-BSEP-S2 GAATAAGGTAAACATGGTAGCGATGTCGACCTCGAGACGCGTCTAACTGATGGGGGATCCAGTGGTGACT YEpN14HIS-MDR3-S2 GAATAAGGTAAACATGGTAGCGATGTCGACCTCGAGACGCGTCTATAAGTTCTGTGTCCCAGCCTGGACACTGACCATT GFP-BSEP-HR-S1 AGCCTACTACAAACTAGTCACCACTGGATCCCCCATCAGTGGTGGTGGTCGACGGATCCCCGGGTTA GFP-PP-HR-S2 ACGTTTGGACCTTGGAATAAGACTTCTAAGGAGTTGGAGGCTATTATTTGTATAGTTCATCCATGCCATGT GFP-MDR3-HR-S1 TTTCAATGGTCAGTGTCCAGGCTGGAACAAAGAGACAAGGTGGTGGTCGACGGATCCCCGGGTTA MDR3-E558Q S1 GATCCTTCTGCTGGATCAAGCCACGTCAGCATTGGACAC MDR3-E558Q S2 GTGTCCAATGCTGACGTGGCTTGATCCAGCAGAAGGATC MDR3-E1207Q S1 CAAATCCTCCTGTTGGATCAAGCTACATCAGCTCTGGATAC MDR3-E1207Q S2 GTATCCAGAGCTGATGTAGCTTGATCCAACAGGAGGATTTG doi:10.1371/journal.pone.0060620.t001 Preparation of crude membrane vesicles for protein purification 100 g batches of P. pastoris cells expressing BSEP or MDR3 were thawed on ice, washed with ddH2O and re-suspended at a concentration of 0.5 g cells/ml in homogenization buffer containing protease inhibitor cocktail (Roche). Login to comment
248 The ATPase inactive mutant (E558Q, E1207Q, further called EQ/EQ mutant) exhibited basal ATPase activity comparable to the wild-type protein. Login to comment
280 Molecular weight markers are shown on the left. B Normalized ATPase activity of MDR3 wild-type (black) and of an ATPase deficient mutant (E558Q E1207Q, white) in FC-16 without and with different phospholipids. Login to comment
322 This observation was sustained by analysis of an ATP hydrolysis deficient EQ double mutant (E558Q, E1207Q). Login to comment
105 Oligonucleotide Sequence 59R 39 pSGP18-2m-ori-S1 TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTAAATATTGCGAATACCGCTTCCACAAACATTG pSGP18-2m-ori-S2 AACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTATTTCACACCGCATATATCGGATCGTACT BSEP-HR-PP-S1 ATCAAAAAACAACTAATTATTCGAACGAGGTAAAAGAATGTCTGACTCAGTAATTCTTCGAAGT ATA BSEP-HR-PP-S2 ACGTTTGGACCTTGGAAAAGACTTCTAAGGAGTTGGAGGCACTGATGGGGGATCCAGTGGTGACTAGTTT MDR3-HR-PP-S1 ATCAAAAAACAACTAATTATTCGAACGAGGTAAAAGAATGGATCTTGAGGCGGCAAAGAACGGAACA MDR3-HR-PP-S2 ACGTTTGGACCTTGGAATAAGACTTCTAAGGAGTTGGAGGCTAAGTTCTGTGTTCCAGCCTGGACACTGACCATTGAAAAATAG YEpN14HIS-BSEP-S2 GAATAAGGTAAACATGGTAGCGATGTCGACCTCGAGACGCGTCTAACTGATGGGGGATCCAGTGGTGACT YEpN14HIS-MDR3-S2 GAATAAGGTAAACATGGTAGCGATGTCGACCTCGAGACGCGTCTATAAGTTCTGTGTCCCAGCCTGGACACTGACCATT GFP-BSEP-HR-S1 AGCCTACTACAAACTAGTCACCACTGGATCCCCCATCAGTGGTGGTGGTCGACGGATCCCCGGGTTA GFP-PP-HR-S2 ACGTTTGGACCTTGGAATAAGACTTCTAAGGAGTTGGAGGCTATTATTTGTATAGTTCATCCATGCCATGT GFP-MDR3-HR-S1 TTTCAATGGTCAGTGTCCAGGCTGGAACAAAGAGACAAGGTGGTGGTCGACGGATCCCCGGGTTA MDR3-E558Q S1 GATCCTTCTGCTGGATCAAGCCACGTCAGCATTGGACAC MDR3-E558Q S2 GTGTCCAATGCTGACGTGGCTTGATCCAGCAGAAGGATC MDR3-E1207Q S1 CAAATCCTCCTGTTGGATCAAGCTACATCAGCTCTGGATAC MDR3-E1207Q S2 GTATCCAGAGCTGATGTAGCTTGATCCAACAGGAGGATTTG doi:10.1371/journal.pone.0060620.t001 Preparation of crude membrane vesicles for protein purification 100 g batches of P. pastoris cells expressing BSEP or MDR3 were thawed on ice, washed with ddH2O and re-suspended at a concentration of 0.5 g cells/ml in homogenization buffer containing protease inhibitor cocktail (Roche). Login to comment
247 The ATPase inactive mutant (E558Q, E1207Q, further called EQ/EQ mutant) exhibited basal ATPase activity comparable to the wild-type protein. Login to comment
279 Molecular weight markers are shown on the left. B Normalized ATPase activity of MDR3 wild-type (black) and of an ATPase deficient mutant (E558Q E1207Q, white) in FC-16 without and with different phospholipids. Login to comment
321 This observation was sustained by analysis of an ATP hydrolysis deficient EQ double mutant (E558Q, E1207Q). Login to comment

[hide] A mutation within the extended X loop abolished su... J Biol Chem. 2015 Feb 20;290(8):4896-907. doi: 10.1074/jbc.M114.588566. Epub 2014 Dec 22. Kluth M, Stindt J, Droge C, Linnemann D, Kubitz R, Schmitt L
A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.
J Biol Chem. 2015 Feb 20;290(8):4896-907. doi: 10.1074/jbc.M114.588566. Epub 2014 Dec 22., [PMID:25533467]

Abstract [show]
The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.

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64 Fermentation of MDR3 Transformed P. pastoris Cells-For large-scale expression, P. pastoris cells containing the chromosomally integrated wild type MDR3, the E558Q/E1702Q double mutant, or the Q1174E mutant gene were fermented in a 15-liter table-top glass fermenter (Applikon Biotechnology) according to the P. pastoris fermentation guidelines from Invitrogen. Login to comment
118 RESULTS Expression and Purification of the Human ABC Transporter MDR3 in P. pastoris-Previously, we described the expression of wild type MDR3 and the ATP hydrolysis-deficient mutant (E558Q/E1207Q, later called the EQ/EQ mutant) in the methylotrophic yeast P. pastoris. Login to comment
136 Groen et al. (7) reported an important cytotoxicity caused by expression of wild type MDR3 in HEK293T cells, which was counteracted by the single mutation E558Q of the Walker B motif of the first NBD resulting in an inactive floppase. Login to comment
156 A, human wild type MDR3, the E558Q/E1207Q double mutant, and the Q1174E mutant purified from P. pastoris. Login to comment
182 of purifications Km (MgATP) vmax kcat mg/100 g wet cell weight mM nmol minafa;1 mgafa;1 safa;1 Wild type 6.3 afe; 1.2 8 2.17 afe; 0.20 354 afe; 13 0.83 afe; 0.03 1.78 afe; 0.10a 536 afe; 11a 1.26 afe; 0.03a Wild type-BODIPY 1.26 afe; 0.10 186 afe; 6 0.44 afe; 0.01 1.43 afe; 0.19a 175 afe; 10a 0.41 afe; 0.02a E558Q/E1207Q 3.4 afe; 0.6 5 b b b Q1174E 2.0 afe; 0.2 5 1.04 afe; 0.15 286 afe; 16 0.64 afe; 0.04 Q1174E-BODIPY 0.74 afe; 0.10 198 afe; 5 0.46 afe; 0.01 a ATPase activity was in the presence of 300 òe;M DOPC lipids. Login to comment
270 ATPase Activity of Human MDR3 4904 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 8ߦFEBRUARY 20, 2015 at SEMMELWEIS UNIV OF MEDICINE on December , Cross-linking of wild type MDR3 with maleimide-BODIPY blocks basal and PC-induced ATPase activity as demonstrated for MDR1 (33-35), whereas the ATP hydrolysis-deficient EQ double mutant (E558Q/E1207Q) showed no PC stimulation, and ATPase activity of the labeled EQ/EQ mutant was only marginally reduced (Table 1 and Fig. 1). Login to comment
65 Fermentation of MDR3 Transformed P. pastoris Cells-For large-scale expression, P. pastoris cells containing the chromosomally integrated wild type MDR3, the E558Q/E1702Q double mutant, or the Q1174E mutant gene were fermented in a 15-liter table-top glass fermenter (Applikon Biotechnology) according to the P. pastoris fermentation guidelines from Invitrogen. Login to comment
269 ATPase Activity of Human MDR3 4904 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 8ߦFEBRUARY 20, 2015 at SEMMELWEIS UNIV OF MEDICINE on December , Cross-linking of wild type MDR3 with maleimide-BODIPY blocks basal and PC-induced ATPase activity as demonstrated for MDR1 (33-35), whereas the ATP hydrolysis-deficient EQ double mutant (E558Q/E1207Q) showed no PC stimulation, and ATPase activity of the labeled EQ/EQ mutant was only marginally reduced (Table 1 and Fig. 1). Login to comment