ABCB4 p.Glu1207Gln
| Predicted by SNAP2: | A: D (91%), C: D (91%), D: D (91%), F: D (95%), G: D (95%), H: D (91%), I: D (95%), K: D (95%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), R: D (95%), S: D (95%), T: D (95%), V: D (91%), W: D (95%), Y: D (95%), |
| 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] 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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No. Sentence Comment
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).
X
ABCB4 p.Glu1207Gln 23593265:106:1128
status: NEWX
ABCB4 p.Glu1207Gln 23593265:106:1185
status: NEW248 The ATPase inactive mutant (E558Q, E1207Q, further called EQ/EQ mutant) exhibited basal ATPase activity comparable to the wild-type protein.
X
ABCB4 p.Glu1207Gln 23593265:248:35
status: NEW280 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.
X
ABCB4 p.Glu1207Gln 23593265:280:144
status: NEW322 This observation was sustained by analysis of an ATP hydrolysis deficient EQ double mutant (E558Q, E1207Q).
X
ABCB4 p.Glu1207Gln 23593265:322:99
status: NEW105 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).
X
ABCB4 p.Glu1207Gln 23593265:105:1128
status: NEWX
ABCB4 p.Glu1207Gln 23593265:105:1185
status: NEW247 The ATPase inactive mutant (E558Q, E1207Q, further called EQ/EQ mutant) exhibited basal ATPase activity comparable to the wild-type protein.
X
ABCB4 p.Glu1207Gln 23593265:247:35
status: NEW279 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.
X
ABCB4 p.Glu1207Gln 23593265:279:144
status: NEW321 This observation was sustained by analysis of an ATP hydrolysis deficient EQ double mutant (E558Q, E1207Q).
X
ABCB4 p.Glu1207Gln 23593265:321:99
status: NEW[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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No. Sentence 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.
X
ABCB4 p.Glu1207Gln 25533467:118:190
status: NEW156 A, human wild type MDR3, the E558Q/E1207Q double mutant, and the Q1174E mutant purified from P. pastoris.
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ABCB4 p.Glu1207Gln 25533467:156:35
status: NEW182 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.
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ABCB4 p.Glu1207Gln 25533467:182:364
status: NEW270 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).
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ABCB4 p.Glu1207Gln 25533467:270:356
status: NEW269 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).
X
ABCB4 p.Glu1207Gln 25533467:269:356
status: NEW