ABCMdb

ABCB4 p.Glu558Gln

None has been submitted yet.

Publications

PMID: 21820390 [PubMed] Groen A et al: "Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity."

No. Sentence Comment

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

PMID: 23593265 [PubMed] Ellinger P et al: "Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11) and MDR3 (ABCB4) expressed in the yeast Pichia pastoris."

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). 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

PMID: 25533467 [PubMed] Kluth M et al: "A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3."

No. Sentence Comment

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