ABCC1 p.Lys516Ala
| Predicted by SNAP2: | A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (91%), H: D (85%), I: D (91%), L: D (91%), M: D (91%), N: D (91%), P: D (95%), Q: D (85%), R: D (85%), S: D (85%), T: D (91%), V: D (91%), W: D (91%), 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, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Expression and function of human MRP1 (ABCC1) is d... J Biol Chem. 2011 Mar 4;286(9):7202-13. Epub 2010 Dec 20. Iram SH, Cole SP
Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2.
J Biol Chem. 2011 Mar 4;286(9):7202-13. Epub 2010 Dec 20., 2011-03-04 [PMID:21177244]
Abstract [show]
Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.
Comments [show]
None has been submitted yet.
No. Sentence Comment
47 To generate point mutations in CL5, plasmid pBluescriptSK(ϩ)BamHI/SpHI-MRP1 containing a 1.9-kb fragment from pcDNA3.1(-)-MRP1k was used as the template with the following mutagenic primers (substituted nucleotides are underlined): R501A, 5Ј-GAG CAA AGA CAA TGC GAT CAA GCT GAT G-3Ј; K503A, 5Ј-GAC AAT CGG ATC GCG CTG ATG AAC G-3Ј; E507A, 5Ј-GCT GAT GAA CGC AAT TCT CAA TGG G-3Ј; G511I, 5Ј-CTC AAT ATT ATC AAA GTG CTA AAG CTT TAT GCC TGG GAG CTG GC-3Ј; K513A, 5Ј-CTC AAT GGG ATC GCA GTG CTA AAG C-3Ј; K513R, 5Ј-CTC AAT GGG ATC AGA GTG CTA AAG C-3Ј; K516A, 5Ј-GAT CAA AGT GCT AGC GCT TTA TGC CTG-3Ј; K516R, 5Ј-GAT CAA AGT GCT AAG ACT TTA TGC CTG-3Ј; E521A, 5Ј-CTT TAT GCC TGG GCG CTG GCA TTC-3Ј; E521D, 5Ј-CTT TAT GCC TGG GAC CTG GCA TTC-3Ј; R532A, 5Ј-GTG CTG GCC ATT GCG CAG GAG GAG CT-3Ј; E535A, 5Ј-CAT CAG GCA GGA GGC CTT GAA GGT GCT GAA G-3Ј; and E535D, 5Ј- CAG GCA GGA GGA TCT GAA GGT GCT GAA G-3Ј.
X
ABCC1 p.Lys516Ala 21177244:47:627
status: NEW110 Densitometric analysis of immunoblots showed that the K516A, E521A, and E535A mutants were consistently expressed very poorly (levels Ͻ10% of wild-type MRP1), whereas the levels of K513A were reduced by Ͼ70% (Fig. 2).
X
ABCC1 p.Lys516Ala 21177244:110:54
status: NEW116 A marked increase in the levels of the K513A, K516A, E521A, and E535A mutants was observed in cells incubated at the lower temperature; however, most of this increase was attributable to the underglycosylated form of the MRP1. Furthermore, despite the increase, the levels of K513A, K516A, E521A, and E535A FIGURE 2.
X
ABCC1 p.Lys516Ala 21177244:116:46
status: NEWX
ABCC1 p.Lys516Ala 21177244:116:283
status: NEW118 Shown is a representative immunoblot of whole cell lysates (WCL) (10 g of protein/lane) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (K513A, K516A, E521A, and E535A) cDNA expression vectors. Untransfected cells were used as a negative control. MRP1 proteins were detected with mAb QCRL-1 (top), and the relative levels, estimated by densitometry, are indicated below the blot (after correction for loading based on ␣-tubulin levels detected using an anti-␣-tubulin mAb (bottom)).
X
ABCC1 p.Lys516Ala 21177244:118:180
status: NEW122 A, immunoblots of whole cell lysates (WCL) (10 g of protein/lane) prepared from HEK293T cells incubated at 28 °C for 60 h after transfection with mutant (K513A, K516A, E521A, and E535A) and wild-type MRP1 cDNA expression vectors.
X
ABCC1 p.Lys516Ala 21177244:122:174
status: NEW137 In contrast, K516A was not detectable at all in the HEK cells at 37 °C, suggesting that the misfolding of this mutant is so severe that none of this mutant protein escapes the ERAD pathways of the cell.
X
ABCC1 p.Lys516Ala 21177244:137:13
status: NEW138 On the other hand, when cells were incubated at 28 °C, the amount of mutant proteins increased (more so for K513A and K516A than for E521A and E535A) (Fig. 3, A and C).
X
ABCC1 p.Lys516Ala 21177244:138:123
status: NEW140 Although the "rescue" of K516A and K513A was greater than for E521A and E535A (Fig. 3A), the retention of K516A and K513A in the endoplasmic reticulum was more pronounced, whereas a small amount of E521A and E535A could be detected at the plasma membrane (Fig. 3C).
X
ABCC1 p.Lys516Ala 21177244:140:25
status: NEWX
ABCC1 p.Lys516Ala 21177244:140:106
status: NEW159 As observed for the CL5 mutant K516A, the NBD2 mutant R1367A was not detectable by immunoblot analysis (Fig. 5C).
X
ABCC1 p.Lys516Ala 21177244:159:31
status: NEW210 Of the charged amino acid mutants, only K503A exhibited properties comparable with those of wild-type MRP1, whereas the seven remaining Ala-substituted mutants were either poorly expressed (K513A, K516A, E521A, and E535A) or exhibited significantly reduced transport activity (R501A, E507A, and R532A).
X
ABCC1 p.Lys516Ala 21177244:210:197
status: NEW213 The substantially reduced levels of the K513A, K516A, E521A, and E535A mutants indicates that introducing a neutral cavity-creating Ala residue at these positions is not compatible with some aspect(s) of MRP1 biosynthesis or assembly, at least in HEK cells. When cells transfected with the FIGURE 7.
X
ABCC1 p.Lys516Ala 21177244:213:47
status: NEW223 Role of Cytoplasmic Loop 5 in MRP1 Expression and Function 7210 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 9•MARCH 4, poorly expressed mutant MRP1 cDNA expression vectors were incubated at a subphysiological temperature, marked increases in the levels of the K513A, K516A, E521A, and E535A mutants were observed, although they remained well below those of wild-type MRP1 and consisted of both mature and underglycosylated forms of the transporter.
X
ABCC1 p.Lys516Ala 21177244:223:288
status: NEW[hide] Mutation of Glu521 or Glu535 in cytoplasmic loop 5... J Biol Chem. 2012 Mar 2;287(10):7543-55. Epub 2012 Jan 9. Iram SH, Cole SP
Mutation of Glu521 or Glu535 in cytoplasmic loop 5 causes differential misfolding in multiple domains of multidrug and organic anion transporter MRP1 (ABCC1).
J Biol Chem. 2012 Mar 2;287(10):7543-55. Epub 2012 Jan 9., [PMID:22232552]
Abstract [show]
The polytopic 5-domain multidrug resistance protein 1 (MRP1/ABCC1) extrudes a variety of drugs and organic anions across the plasma membrane. Four charged residues in the fifth cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 are critical for stable expression of MRP1 at the plasma membrane. Thus Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) all cause misfolding of MRP1 and target the protein for proteasome-mediated degradation. Of four chemical chaperones tested, 4-phenylbutyric acid (4-PBA) was the most effective at restoring expression of MRP1 mutants K513A, K516A, E521A, and E535A. However, although 4-PBA treatment of K513A resulted in wild-type protein levels (and activity), the same treatment had little or no effect on the expression of K516A. On the other hand, 4-PBA treatment allowed both E521A and E535A to exit the endoplasmic reticulum and be stably expressed at the plasma membrane. However, the 4-PBA-rescued E535A mutant exhibited decreased transport activity associated with reduced substrate affinity and conformational changes in both halves of the transporter. By contrast, E521A exhibited reduced transport activity associated with alterations in the mutant interactions with ATP as well as a distinct conformational change in the COOH-proximal half of MRP1. These findings illustrate the critical and complex role of CL5 for stable expression of MRP1 at the plasma membrane and more specifically show the differential importance of Glu(521) and Glu(535) in interdomain interactions required for proper folding and assembly of MRP1 into a fully transport competent native structure.
Comments [show]
None has been submitted yet.
No. Sentence Comment
7 Of four chemical chaperones tested, 4-phenylbutyric acid (4-PBA) was the most effective at restoring expression of MRP1 mutants K513A, K516A, E521A, and E535A.
X
ABCC1 p.Lys516Ala 22232552:7:135
status: NEW8 However, although 4-PBA treatment of K513A resulted in wild-type protein levels (and activity), the same treatment had little or no effect on the expression of K516A.
X
ABCC1 p.Lys516Ala 22232552:8:160
status: NEW54 Transfections, Preparation of Membrane Vesicles, and Measurements of MRP1 Protein Levels in Transfected HEK Cells-The generation of wild-type, K513A, K516A, E521A, and E535A mutant MRP1 pcDNA3.1 expression vectors has been described previously (19, 21).
X
ABCC1 p.Lys516Ala 22232552:54:150
status: NEW92 RESULTS Proteasome Inhibition Restores Expression Levels of K513A, K516A, E521A, and E535A Mutants-To better understand the molecular mechanisms responsible for the poor expression of CL5 mutants K513A, K516A, E521A, and E535A in HEK cells, we first investigated whether proteasome-mediated degradation of the mutants might be involved.
X
ABCC1 p.Lys516Ala 22232552:92:67
status: NEWX
ABCC1 p.Lys516Ala 22232552:92:203
status: NEW94 As shown in Fig. 1C, mutants K513A, K516A, E521A, and E535A were present at very low or nondetectable levels in HEK cells in the absence of MG-132, but after exposure to this proteasome inhibitor (2.5 M for 16 h), levels of the mutants were substantially increased, in some cases to levels comparable with wild-type MRP1.
X
ABCC1 p.Lys516Ala 22232552:94:36
status: NEW101 In contrast, only the underglycosylated form of the fourth mutant, K516A, was detected (Fig. 2B).
X
ABCC1 p.Lys516Ala 22232552:101:67
status: NEW103 In addition, the properties of the misfolded mutants, particularly in the case of K516A, appear distinct from one another.
X
ABCC1 p.Lys516Ala 22232552:103:82
status: NEW105 As shown in Fig. 2C, signals corresponding to the K516A, E521A, and E535A mutants at the plasma membrane remained substantially lower than for wild-type MRP1.
X
ABCC1 p.Lys516Ala 22232552:105:50
status: NEW106 The four mutants also showed some (but not exclusive) co-localization with calnexin, indicating that a significant fraction of the FIGURE 1. Effect of proteasome inhibition by MG-132 on levels of MRP1 CL5 mutant proteins K513A, K516A, E521A, and E535A.
X
ABCC1 p.Lys516Ala 22232552:106:228
status: NEW109 C, representative immunoblots of whole cell lysates (WCL) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (K513A, K516A, E521A, and E535A) MRP1 cDNAexpressionvectorsafterexposuretoMG-132(2.5M for16h).Untransfectedcellswereusedasanegativecontrol,andMRP1proteinsweredetectedwith mAb QCRL-1. Equal protein loading was confirmed by blotting for ␣-tubulin.
X
ABCC1 p.Lys516Ala 22232552:109:142
status: NEW112 This was particularly evident for K516A where co-localization with calnexin was the most pronounced and is consistent with the absence of any detectable glycosylated form of this mutant.
X
ABCC1 p.Lys516Ala 22232552:112:34
status: NEW114 We have now investigated the effect of several commonly used low molecular weight chemical chaperones on the levels and trafficking of K513A, K516A, E521A, and E535A.
X
ABCC1 p.Lys516Ala 22232552:114:142
status: NEW118 Thus, K513A levels were increased by all four chemicals to levels comparable with untreated wild-type MRP1, whereas the same chemical treatments only slightly increased K516A levels.
X
ABCC1 p.Lys516Ala 22232552:118:169
status: NEW119 Furthermore, the K516A protein detected after the chemical treatments was again predominantly the underglycosylated form (170 kDa) of the transporter.
X
ABCC1 p.Lys516Ala 22232552:119:17
status: NEW122 Effect of 4-PBA on Levels and Localization of K513A, K516A, E521A, and E535A-The effect of 4-PBA was further explored by determining the concentration and time dependence of its ability to increase levels of the four CL5 mutants.
X
ABCC1 p.Lys516Ala 22232552:122:53
status: NEW127 As observed with other chemical treatments, the mutant K516A protein was mostly FIGURE 2. Effect of bortezomib on the levels and localization of MRP1 CL5-processing mutants K513A, K516A, E521A, and E535A.
X
ABCC1 p.Lys516Ala 22232552:127:55
status: NEWX
ABCC1 p.Lys516Ala 22232552:127:180
status: NEW128 A and B, shown are representativeimmunoblotsofwholecelllysates(WCL)preparedfromHEK293Tcellstransfectedwithwild-type(WT-MRP1)andmutant(K513A,K516A,E521A, and E535A) MRP1 cDNA expression vectors and exposed to bortezomib (100 nM for 24 h).
X
ABCC1 p.Lys516Ala 22232552:128:140
status: NEW138 After 48 h of 4-PBA treatment, levels of K513A, E521A, and E535A were further increased, whereas K516A levels remained unchanged.
X
ABCC1 p.Lys516Ala 22232552:138:97
status: NEW142 In marked contrast, the 4-PBA-rescued K516A mutant co-localized primarily with the ER marker calnexin.
X
ABCC1 p.Lys516Ala 22232552:142:38
status: NEW154 In contrast, the E535A mutant exhibited a 3-6-fold increase in its Km values (22 M for E217betaG and 478 nM for LTC4) relative to wild-type MRP1, whereas the Vmax values FIGURE 3. Effect of chemical chaperones on levels of wild-type and K513A, K516A, E521A, and E535A mutant proteins.
X
ABCC1 p.Lys516Ala 22232552:154:252
status: NEW155 Shown are representativeimmunoblotsofwholecelllysates(10gofprotein/lane)preparedfrom HEK293T cells transfected with wild-type (WT-MRP1) and mutant (K513A, K516A, E521A, and E535A) cDNA expression vectors.
X
ABCC1 p.Lys516Ala 22232552:155:163
status: NEW241 The levels of these CL5 processing mutants K513A, K516A, E521A, and E535A could be increased by incubating cells at subphysiologi- cal temperatures, but these increases were relatively modest (particularly for E521A and E535A) and could be largely attributed to increased amounts of the underglycosylated form(s) of MRP1, which was retained in the ER (19).
X
ABCC1 p.Lys516Ala 22232552:241:50
status: NEW243 In this study the involvement of proteasome-mediated degradation of the K513A, K516A, E521A, and E535A mutants was confirmed by demonstrating that exposure of HEK cells expressing these mutants to proteasome inhibitors markedly increased the levels of all four mutant proteins.
X
ABCC1 p.Lys516Ala 22232552:243:79
status: NEW244 However, except for K513A, trafficking of the mutants to the plasma membrane remained impaired, and substantial retention in the ER, particularly for the underglycosylated K516A, was observed.
X
ABCC1 p.Lys516Ala 22232552:244:172
status: NEW264 Somewhat surprisingly, the MRP1 processing mutants K513A, K516A, E521A, and E535A varied substantially in their response to the different chemicals despite the relatively close proximity of the affected amino acids in the linear MRP1 sequence.
X
ABCC1 p.Lys516Ala 22232552:264:58
status: NEW267 On the other hand, the K516A mutant was very resistant to rescue by the chemical chaperones and was the only one among the rescued mutants where mature glycosylated MRP1 was not detected.
X
ABCC1 p.Lys516Ala 22232552:267:23
status: NEW268 Rescue of the E521A and E535A mutants was achieved only with 4-PBA, but unlike K516A, these mutants were fully glycosylated.
X
ABCC1 p.Lys516Ala 22232552:268:79
status: NEW277 However, treatment with sodium butyrate, in contrast to 4-PBA, caused little or no increase in levels of K513A, K516A, E521A, and E535A (results not shown).
X
ABCC1 p.Lys516Ala 22232552:277:112
status: NEW296 Thus, although exposure to 4-PBA was able to fully overcome the folding defect of the K513A mutant, it had little or no effect on the K516A mutant.
X
ABCC1 p.Lys516Ala 22232552:296:134
status: NEW[hide] Differential functional rescue of Lys(513) and Lys... Biochim Biophys Acta. 2014 Mar;1838(3):756-65. doi: 10.1016/j.bbamem.2013.11.002. Epub 2013 Nov 11. Iram SH, Cole SP
Differential functional rescue of Lys(513) and Lys(516) processing mutants of MRP1 (ABCC1) by chemical chaperones reveals different domain-domain interactions of the transporter.
Biochim Biophys Acta. 2014 Mar;1838(3):756-65. doi: 10.1016/j.bbamem.2013.11.002. Epub 2013 Nov 11., [PMID:24231430]
Abstract [show]
Multidrug resistance protein 1 (MRP1) extrudes drugs as well as pharmacologically and physiologically important organic anions across the plasma membrane in an ATP-dependent manner. We previously showed that Ala substitutions of Lys(513) and Lys(516) in the cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 cause misfolding of MRP1, abrogating its expression at the plasma membrane in transfected human embryonic kidney (HEK) cells. Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. This structural change was accompanied by differences in ATP binding and hydrolysis but no changes in substrate Km. In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by >80% due to reduced substrate Km. Tryptic fragmentation patterns indicated a more open conformation of the third membrane spanning domain of MRP1. Thus, despite their close proximity to one another in CL5, Lys(513) and Lys(516) participate in different interdomain interactions crucial for the proper folding and assembly of MRP1.
Comments [show]
None has been submitted yet.
No. Sentence Comment
5 In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones.
X
ABCC1 p.Lys516Ala 24231430:5:22
status: NEW6 In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1.
X
ABCC1 p.Lys516Ala 24231430:6:42
status: NEW7 Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by N80% due to reduced substrate Km.
X
ABCC1 p.Lys516Ala 24231430:7:41
status: NEW30 Consequently, the failure of 4-PBA to rescue expression of the fourth processing mutant, K516A, was unexpected, particularly since Lys516 is within one turn of Lys513 in the same b1;-helix (the so-called coupling b1;-helix) that atomic homology models predict runs roughly parallel to the plasma membrane and interfaces with NBD1 and NBD2 [7,8].
X
ABCC1 p.Lys516Ala 24231430:30:89
status: NEW31 In the present study, we have further investigated the relative abilities of several chemical chaperones to rescue the expression and/or function of K513A and K516A to better understand the distinct roles that Lys513 and Lys516 play in promoting the proper folding and assembly of MRP1, and its trafficking to the plasma membrane.
X
ABCC1 p.Lys516Ala 24231430:31:159
status: NEW38 Preparation of MRP1-enriched membrane vesicles and measurement of MRP1 levels in HEK293T and Sf9 insect cells The generation of wild-type, K513A and K516A mutant MRP1 pcDNA3.1 expression vectors has been described previously [8,16].
X
ABCC1 p.Lys516Ala 24231430:38:149
status: NEW46 Wild-type and K516A mutant MRP1 were also cloned into pFastBac vectors (Life Technologies Inc.) for expression in Sf9 insect cells.
X
ABCC1 p.Lys516Ala 24231430:46:14
status: NEW50 Sf9 cells grown as monolayers were infected at 80% confluency with 1.25 ml of wild-type or K516A MRP1 baculoviruses (8 &#d7; 107 IFU ml-1 ) per 150 mm plate.
X
ABCC1 p.Lys516Ala 24231430:50:91
status: NEW79 2.7. Limited trypsin digestions HEK or Sf9 insect cell membrane vesicles (2.0 bc;g per reaction) enriched for K513A or K516A mutant or wild-type MRP1 were digested with trypsin over a range of trypsin:protein ratios for 15 min essentially as described [9,23].
X
ABCC1 p.Lys516Ala 24231430:79:122
status: NEW85 K513A and K516A mutants differ in their ability to be rescued by chemical chaperones The relative effectiveness of glycerol, DMSO, PEG and 4-PBA to rescue the processing mutants K513A and K516A was evaluated by comparing MRP1 protein levels in lysates prepared from transfected HEK293T cells treated with or without these chemical chaperones.
X
ABCC1 p.Lys516Ala 24231430:85:10
status: NEWX
ABCC1 p.Lys516Ala 24231430:85:188
status: NEW88 In contrast to K513A, and consistent with our earlier observations [9], exposure of K516A transfected cells to glycerol, DMSO, PEG and 4-PBA only slightly increased MRP1 levels (Fig. 2B).
X
ABCC1 p.Lys516Ala 24231430:88:84
status: NEW90 In addition, given the multiple intra-and inter-domain interactions that appear to be involved in the optimal expression of MRP1 at the plasma membrane, it may not be reasonable to expect a single small molecule to 'correct` the conformation and transport activity of K516A.
X
ABCC1 p.Lys516Ala 24231430:90:268
status: NEW92 However, none of the chemicals, alone or in combination, enhanced K516A levels substantially, leading to the conclusion that the folding defects introduced by mutation of Lys516 must differ from the defects introduced by mutation of Lys513 , despite the close proximity of the two basic residues in the coupling helix in CL5.
X
ABCC1 p.Lys516Ala 24231430:92:66
status: NEW102 Effect of chemical chaperones on relative levels of K513A and K516A mutant MRP1 proteins in HEK293T cells.
X
ABCC1 p.Lys516Ala 24231430:102:62
status: NEW103 Shown are representative immunoblots of whole cell lysates prepared from HEK293T cells transfected with K513A or K516A mutant or wild-type (WT) MRP1 cDNA expression vectors, with or without exposure to the chemicals indicated at the top of the blots.
X
ABCC1 p.Lys516Ala 24231430:103:113
status: NEW106 (A) WT-MRP1 (left panel), K513A (right panel); (B) K516A.
X
ABCC1 p.Lys516Ala 24231430:106:51
status: NEW154 Expression and functional characterization of K516A in Sf9 insect cells Since treatment of HEK293T cells expressing MRP1 K516A with chemical chaperones alone, and in combination, failed to significantly increase levels of this mutant protein (Fig. 2C), K516A was cloned into a baculovirus expression vector and expressed in Sf9 insect cells which exert less stringent quality control mechanisms during biosynthesis of human proteins.
X
ABCC1 p.Lys516Ala 24231430:154:46
status: NEWX
ABCC1 p.Lys516Ala 24231430:154:121
status: NEWX
ABCC1 p.Lys516Ala 24231430:154:253
status: NEW155 As shown in Fig. 7A, this approach was successful since K516A was expressed at levels comparable to those of wild-type MRP1 in insect cells.
X
ABCC1 p.Lys516Ala 24231430:155:56
status: NEW156 However, the LTC4 transport activity of the heterologously expressed K516A was reduced by 80% relative to wild-type MRP1 (Fig. 7B).
X
ABCC1 p.Lys516Ala 24231430:156:69
status: NEW157 [3 H]LTC4 labeling of the heterologously expressed K516A mutant was also diminished by 90% (Fig. 7C), indicating the LTC4 substrate binding site of MRP1 had been compromised.
X
ABCC1 p.Lys516Ala 24231430:157:51
status: NEW171 Expression and function of K516A mutant MRP1 expressed in insect cells.
X
ABCC1 p.Lys516Ala 24231430:171:27
status: NEW172 (A) Representative immunoblot of membrane vesicles (1 bc;g protein per lane) prepared from Sf9 insect cells infected with K516A mutant and wild-type MRP1 recombinant baculoviruses.
X
ABCC1 p.Lys516Ala 24231430:172:125
status: NEW175 (B) [3 H]LTC4 transport activity of insect cell membrane vesicles enriched for K516A mutant and wild-type MRP1.
X
ABCC1 p.Lys516Ala 24231430:175:79
status: NEW179 Control insect cell membrane vesicles (b2;-gus) and vesicles enriched for K516A mutant and wild-type MRP1 (50 bc;g protein) were photolabeled with [3 H]LTC4 as described in the legend to Fig. 6.
X
ABCC1 p.Lys516Ala 24231430:179:77
status: NEW183 K516A mutation causes 'long range` conformational changes in MSD2 In seeking a structural explanation for the reduced substrate binding of the heterologously expressed K516A mutant, the conformation of the mutant protein was probed by limited trypsinolysis followed by immunoblotting with mAbs directed against epitopes in the NH2- and COOH-proximal halves of MRP1 as before (Fig. 8A).
X
ABCC1 p.Lys516Ala 24231430:183:0
status: NEWX
ABCC1 p.Lys516Ala 24231430:183:168
status: NEW184 Differences in the wild-type and K516A tryptic fragmentation patterns were detected with both antibodies.
X
ABCC1 p.Lys516Ala 24231430:184:33
status: NEW185 Thus, when probed with mAb MRPr1 (Fig. 8B, left panel), fragments of unknown identity (50-65 kDa) accumulated in the case of the K516A mutant but not wild-type MRP1, whereas the trypsin sensitivity of the full-length, as well as the expected N1 and N3 (MSD0) fragments, were quite similar.
X
ABCC1 p.Lys516Ala 24231430:185:129
status: NEW186 In contrast, when probed with mAb 897.2 (Fig. 8B, right panel) the tryptic fragmentation of the K516A mutant was strikingly different from wild-type MRP1.
X
ABCC1 p.Lys516Ala 24231430:186:96
status: NEW187 Thus, the levels of the C1 fragment (MSD2-NBD2) for K516A were substantially reduced relative to wild-type MRP1, despite very similar patterns of disappearance for the two full-length proteins.
X
ABCC1 p.Lys516Ala 24231430:187:52
status: NEW188 Furthermore, the levels of C2 fragment (NBD2) were also comparable between the wild-type and K516A mutant suggesting that the trypsin hypersensitivity of the C1 fragment (MSD2-NBD2) of K516A is mainly due to conformational changes in MSD2.
X
ABCC1 p.Lys516Ala 24231430:188:93
status: NEWX
ABCC1 p.Lys516Ala 24231430:188:185
status: NEW199 Fig. 8. Limited trypsin digests of transport-deficient K516A mutant MRP1 in insect cell membranes.
X
ABCC1 p.Lys516Ala 24231430:199:55
status: NEW201 (B) Membrane vesicles (2 bc;g protein) prepared from insect cells expressing transport-deficient K516A mutant and wild-type MRP1 were incubated at 37 &#b0;C for 15 min with trypsin at different trypsin:protein ratios (range 1:500 to 1:12.5) and then immunoblotted with (left panel) mAb MRPr1 and (right panel) mAb 897.2.
X
ABCC1 p.Lys516Ala 24231430:201:100
status: NEW203 The boxes highlight significant differences between the patterns of the tryptic fragments of the wild-type and transport-deficient K516A mutant proteins.
X
ABCC1 p.Lys516Ala 24231430:203:131
status: NEW210 In the present study, we have shown that despite the proximity of the affected amino acids to one another in CL5, the MRP1 processing mutants K513A and K516A display remarkable differences in their ability to be functionally rescued by four commonly used chemical chaperones.
X
ABCC1 p.Lys516Ala 24231430:210:152
status: NEW225 In contrast to K513A, the K516A processing mutant was remarkably resistant to rescue by the chemical chaperones tested.
X
ABCC1 p.Lys516Ala 24231430:225:26
status: NEW231 Despite the inability of chemical chaperones to rescue K516A expression in HEK cells, heterologous expression in Sf9 insect cells yielded levels of the mutant transporter comparable to wild-type MRP1 which is likely attributable to the lower stringency of the protein quality control processes in these cells [44-47].
X
ABCC1 p.Lys516Ala 24231430:231:55
status: NEW232 However, despite the robust levels of K516A mutant achieved in this system, the transport activity of this mutant was severely diminished.
X
ABCC1 p.Lys516Ala 24231430:232:38
status: NEW233 Thus, LTC4 transport levels were just 10% those of wild-type MRP1, and the K516A mutant could no longer be photolabeled with this substrate.
X
ABCC1 p.Lys516Ala 24231430:233:75
status: NEW234 These observations indicated that the substrate binding pocket of MRP1 had been compromised which in turn suggested structural differences between the K516A mutant and wild-type MRP1 proteins, despite the fact that both were expressed equally well.
X
ABCC1 p.Lys516Ala 24231430:234:151
status: NEW235 Support for this conclusion was obtained by comparing the tryptic digest patterns of the two proteins which indicate that the CL5 K516A mutation in MSD1 introduces a change in the COOH-proximal half of MRP1 (MSD2-NBD2) to a more open, trypsin-sensitive conformation.
X
ABCC1 p.Lys516Ala 24231430:235:130
status: NEW236 Since the tryptic fragments of NBD2 (C2) in both wild-type and K516A were similar, it may be inferred that this conformational change lies predominantly in MSD2.
X
ABCC1 p.Lys516Ala 24231430:236:63
status: NEW238 According to ab initio analyses, both K513A and K516A mutations are predicted to introduce significant rigidity into the tertiary structure of the coupling helix in CL5 where these Lys residues are found.
X
ABCC1 p.Lys516Ala 24231430:238:48
status: NEW241 This difference would provide a plausible explanation for why mutation of Lys516 might be more disruptive to normal domain-domain interactions than mutation of Lys513 , which in turn, could explain why the K516A mutant is more resistant to rescue by chemical chaperones than K513A.
X
ABCC1 p.Lys516Ala 24231430:241:206
status: NEW