ABCC1 p.Asp1454Leu
Predicted by SNAP2: | A: D (95%), C: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
Predicted by PROVEAN: | A: D, C: D, E: 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] Allosteric interactions between the two non-equiva... J Biol Chem. 2000 Jul 7;275(27):20280-7. Hou Y, Cui L, Riordan JR, Chang X
Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1.
J Biol Chem. 2000 Jul 7;275(27):20280-7., 2000-07-07 [PMID:10781583]
Abstract [show]
Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.
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No. Sentence Comment
33 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L, were generated by using procedures described previously (11).
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ABCC1 p.Asp1454Leu 10781583:33:89
status: NEW205 Fig. 5C shows that although labeling by N3[␣-32 P]ATP was not abolished by the mutations, it was greatly reduced: K684L was ϳ10% of wild-type; K1333L, ϳ5%; D1454L/D1455L, ϳ15%.
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ABCC1 p.Asp1454Leu 10781583:205:175
status: NEW207 Fig. 5D demonstrates that labeling of K684L by N3[␥-32 P]ATP was almost eliminated and labeling of K1333L and D1454L/E1455L were decreased to ϳ10% and ϳ15% of the wild-type levels, respectively.
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ABCC1 p.Asp1454Leu 10781583:207:117
status: NEW232 Lane 1, 10 g of wild-type MRP1; lane 2, 20 g of K684L; lane 3, 10 g of K1333L; lane 4, 10 g of D1454L/ E1455L.
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ABCC1 p.Asp1454Leu 10781583:232:127
status: NEW234 Lane 1, 10 g of wild-type MRP1; lane 2, 20 g of K684L; lane 3, 10 g of K1333L; lane 4, 10 g of D1454L/ E1455L.
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ABCC1 p.Asp1454Leu 10781583:234:127
status: NEW235 E, ATP-dependent LTC4 uptake by membrane vesicles containing wild-type (closed diamonds) and mutant MRPs: NBD1 Walker A lysine mutant K684L (open circles), NBD2 Walker A lysine mutant K1333L (open square), NBD2 Walker B aspartate mutant D1454L/ E1455L (closed circles).
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ABCC1 p.Asp1454Leu 10781583:235:237
status: NEW[hide] ATP binding to the first nucleotide-binding domain... J Biol Chem. 2002 Feb 15;277(7):5110-9. Epub 2001 Dec 7. Hou YX, Cui L, Riordan JR, Chang XB
ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain.
J Biol Chem. 2002 Feb 15;277(7):5110-9. Epub 2001 Dec 7., 2002-02-15 [PMID:11741902]
Abstract [show]
Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.
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No. Sentence Comment
50 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792A, K1333L, and D1454L/E1455L were established previously (2, 31).
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ABCC1 p.Asp1454Leu 11741902:50:83
status: NEW117 Essentially the stimulation was much reduced in the NBD1 mutants, K684L and D792A (Fig. 4, C and D), and in the NBD2 mutants, K1333L and D1454L/E1455L (Fig. 4, E and F), and the stimulation effects were shifted to higher ATP concentrations (Fig. 4, C-F).
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ABCC1 p.Asp1454Leu 11741902:117:137
status: NEW118 Trypsin digestion of either [␣-32 P]8-N3ATP or [␣-32 P]8N3ADP-labeled K1333L and D1454L/E1455L proved that the mutated NBD2 fragment can still be labeled (data not shown).
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ABCC1 p.Asp1454Leu 11741902:118:95
status: NEW173 Lane 1, 10 g of wild-type MRP1; lane 2, 15 g of K684L; lane 3, 20 g of D792A; lane 4, 10 g of K1333L; lane 5, 10 g of D1454L/E1455L.
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ABCC1 p.Asp1454Leu 11741902:173:158
status: NEW175 The results for K684L and D792A are the average of three independent experiments and for K1333L and D1454L/E1455L are the average of two independent experiments. C, influence of ATP on the [␣-32 P]8-N3ADP labeling of K684L.
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ABCC1 p.Asp1454Leu 11741902:175:100
status: NEW180 F, influence of ATP on the [␣-32 P]8-N3ADP labeling of D1454L/E1455L.
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ABCC1 p.Asp1454Leu 11741902:180:62
status: NEW181 10 g of D1454L/E1455L was labeled in the presence of varying amounts of ATP indicated above each lane.
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ABCC1 p.Asp1454Leu 11741902:181:16
status: NEW[hide] Nucleotide dissociation from NBD1 promotes solute ... Biochim Biophys Acta. 2005 Mar 1;1668(2):248-61. Yang R, McBride A, Hou YX, Goldberg A, Chang XB
Nucleotide dissociation from NBD1 promotes solute transport by MRP1.
Biochim Biophys Acta. 2005 Mar 1;1668(2):248-61., 2005-03-01 [PMID:15737336]
Abstract [show]
MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear. We now report that ATP hydrolysis at NBD1 may not be essential for transport, but that the dissociation of the NBD1-bound nucleotide facilitates ATP-dependent LTC4 transport. These conclusions are supported by the following results. The substitution of the putative catalytic E1455 with a non-acidic residue in NBD2 greatly decreases the ATPase activity of NBD2 and the ATP-dependent LTC4 transport, indicating that E1455 participates in ATP hydrolysis. The mutation of the corresponding D793 residue in NBD1 to a different acidic residue has little effect on ATP-dependent LTC4 transport. The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Along with their higher transport activities, their Michaelis constant Kms (ATP) are also higher than that of wild-type. Coincident with their higher Kms (ATP), their Kds derived from ATP binding are also higher than that of wild-type, implying that the rate of dissociation of the bound nucleotide from the mutated NBD1 is faster than that of wild-type. Therefore, regardless of whether the bound ATP at NBD1 is hydrolyzed or not, the release of the bound nucleotide from NBD1 may bring the molecule back to its original conformation and facilitate the protein to start a new cycle of ATP-dependent solute transport.
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No. Sentence Comment
159 Interestingly, this result is also similar to that of the double mutant D1454L/E1455L [28] including the mutations of the D1454 residue in the Walker B motif and the putative catalytic base E1455 residue directly adjacent to the D1454, implying that the mutation of the putative catalytic base E1455 to a non-acidic amino acid affecting ATP hydrolysis [43] has the same effects as the D1454L/E1455L double mutant affecting ATP binding [28] and hydrolysis.
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ABCC1 p.Asp1454Leu 15737336:159:72
status: NEWX
ABCC1 p.Asp1454Leu 15737336:159:385
status: NEW[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]
Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.
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No. Sentence Comment
160 D792A (NBD1) and D1454L/E1455L (NBD2) diminished nucleotide-binding abilities rather than completely abolishing binding [100].
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ABCC1 p.Asp1454Leu 16442101:160:17
status: NEW[hide] The hydroxyl group of S685 in Walker A motif and t... Biochim Biophys Acta. 2008 Feb;1778(2):454-65. Epub 2007 Nov 29. Yang R, Scavetta R, Chang XB
The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function.
Biochim Biophys Acta. 2008 Feb;1778(2):454-65. Epub 2007 Nov 29., [PMID:18088596]
Abstract [show]
Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the beta-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding. Indeed, substitution of the S685 with residues potentially preventing formation of this hydrogen-bond resulted in misfolding of the protein. In addition, some substitutions that might form hydrogen-bond with D792 also yielded immature protein. All these mutants are temperature-sensitive variants. However, these complex-glycosylated mature mutants prepared from the cells grown at 27 degrees C still significantly affect ATP binding and ATP-dependent solute transport. In contrast, substitution of the S685 with threonine yielded complex-glycosylated mature protein that is more active than the wild-type MRP1, indicating that the interaction between the hydroxyl group of 685 residue and the carboxyl group of D792 plays a crucial role for the protein folding and the interactions of the hydroxyl group at 685 with magnesium and the beta-phosphate of the bound ATP play an important role for ATP-binding and ATP-dependent solute transport.
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No. Sentence Comment
253 In addition, substitutions of the Walker B motif D1454 and E1455 in NBD2 of MRP1 with a leucine residue (D1454L/E1455L) also did not cause misfolding of the protein [20].
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ABCC1 p.Asp1454Leu 18088596:253:105
status: NEW254 In order to rule out the possibility that the double mutant D1454L/E1455L might rescue the misfolding caused by D14 54L mutation, we have made single mutants including D1454L, D1454N, S1334A, S1334T, S1334C, S1334H, S1334D and S1334N.
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ABCC1 p.Asp1454Leu 18088596:254:60
status: NEWX
ABCC1 p.Asp1454Leu 18088596:254:168
status: NEW261 It is also true for the corresponding mutations in Walker B motif of NBD2 in MRP1, such as D1454L (manuscript in preparation) and D1454N [46], have a significant effect on the ATP-dependent LTC4 transport.
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ABCC1 p.Asp1454Leu 18088596:261:91
status: NEW[hide] Interaction between the bound Mg.ATP and the Walke... Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18. Yang R, Scavetta R, Chang XB
Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport.
Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18., 2008-08-12 [PMID:18636743]
Abstract [show]
Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport. In this report we tested whether the corresponding substitution in NBD2 would also result in misfolding of the protein. In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Due to this low efficient ATP binding at the mutated NBD2, the inhibitory effect of ATP on the LTC4 binding is significantly decreased. Furthermore, ATP bound to the mutated NBD2 cannot be efficiently hydrolyzed, leading to almost completely abolishing the ATP-dependent LTC4 transport. In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport.
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12 In contrast, however, substitution of the corresponding Walker B D1454 in NBD2 with the hydrophobic residue leucine (D1454L/E1455L) did not cause misfolding of the mutated MRP1 protein (8), suggesting distinct structures in NBD1 and NBD2 of MRP1.
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ABCC1 p.Asp1454Leu 18636743:12:117
status: NEW166 Indeed, elimination of the carboxyl group at D1454, such as D1454L/E1455L (8), had no effect on the protein folding and processing.
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ABCC1 p.Asp1454Leu 18636743:166:60
status: NEW[hide] Characterization of the ATPase activity of a novel... Arch Biochem Biophys. 2009 May 15;485(2):102-8. Epub 2009 Mar 11. Wan L, Liang X, Huang Y
Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1.
Arch Biochem Biophys. 2009 May 15;485(2):102-8. Epub 2009 Mar 11., 2009-05-15 [PMID:19285030]
Abstract [show]
Nucleotide Binding Domains (NBDs) are responsible for the ATPase activity of the multidrug resistance protein 1 (MRP1). A series of NBD1-linker-NBD2 chimeric fusion proteins were constructed, expressed and purified, and their ATPase activities were analyzed. We report here that a GST linked NBD1(642-890)-GST-NBD2(1286-1531) was able to hydrolyze ATP at a rate of about 4.6 nmol/mg/min (K(m)=2.17 mM, V(max)=12.36 nmol/mg/min), which was comparable to the purified and reconstituted MRP1. In contrast, neither a mixture of NBD1 and GST-NBD2 nor the NBD1-GST-NBD1 fusion protein showed detectable ATPase activity. Additionally, the E1455Q mutant was found to be nonfunctional. Measurements by both MIANS labeling and circular dichroism spectroscopy revealed significant conformational differences in the NBD1-GST-NBD2 chimeric fusion protein compared to the mixture of NBD1 and GST-NBD2. The results suggest a direct interaction mediated by GST between the two NBDs of MRP1 leading to conformational changes which would enhance its ATPase activity.
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163 Among the NBD1-GST-NBD2 mutants, K684L in Walker A of NBD1, K1333L in Walker A of NBD2, and D1454L/E1455L in Walker B of NBD2 were expressed mainly as inclusion bodies in E. coli, and only the E1455Q mutant was expressed in a sufficient quantity of soluble protein to allow activity analysis.
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ABCC1 p.Asp1454Leu 19285030:163:92
status: NEW[hide] Mutations of the Walker B motif in the first nucle... Arch Biochem Biophys. 2001 Aug 1;392(1):153-61. Cui L, Hou YX, Riordan JR, Chang XB
Mutations of the Walker B motif in the first nucleotide binding domain of multidrug resistance protein MRP1 prevent conformational maturation.
Arch Biochem Biophys. 2001 Aug 1;392(1):153-61., [PMID:11469806]
Abstract [show]
ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes. The so-called "Walker motifs" in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may interfere with function. This is the case with the MRP1 multidrug transporter. However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. We have determined now that this latter effect is entirely due to the D792L substitution. This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein. In vivo, the core-glycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome. A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function.
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No. Sentence Comment
42 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L were established previously (8).
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ABCC1 p.Asp1454Leu 11469806:42:89
status: NEW147 This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L).
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ABCC1 p.Asp1454Leu 11469806:147:262
status: NEW174 Since hydrolysis is believed to drive MRP1 transport it would be expected that the mature D792A protein would not be capable of active transport.
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ABCC1 p.Asp1454Leu 11469806:174:286
status: NEW175 The data in Fig. 7 confirm this expectation, i.e., there is not significantly more ATP-dependent LTC4 uptake by vesicles containing D792A protein that does mature than by the other variants that do not mature (Fig. 7, D792L and D792L/D793L), nor by the NBD2 mutants (Fig. 7, K1333L and D1454L/E1455L) that do mature but have difficulties to hydrolyze ATP and to trap the hydrolysis product, ADP (8).
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ABCC1 p.Asp1454Leu 11469806:175:286
status: NEW208 (G) K1333L, 0.3 g protein in each lane.
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ABCC1 p.Asp1454Leu 11469806:208:4
status: NEW209 (H) D1454L/E1455L, 0.3 g protein in each lane.
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ABCC1 p.Asp1454Leu 11469806:209:4
status: NEW146 This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L).
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ABCC1 p.Asp1454Leu 11469806:146:262
status: NEW