ABCMdb

ABCC1 p.Lys513Ala

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Publications

PMID: 21177244 [PubMed] Iram SH et al: "Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2."

No. Sentence Comment

4 Ala substitution of Lys513 , Lys516 , Glu521 , and Glu535 markedly reduced MRP1 levels. Login to 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. Login to comment
110 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). Login to comment
116 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. Login to comment
118 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)). Login to comment
122 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. Login to comment
135 Thus, at 37 °C the MRP1 signals from cells expressing the K513A, E521A, and E535A mutants were very faint; however, the signals were found mostly at the plasma membrane as observed for wild-type MRP1 (Fig. 3B). Login to comment
138 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). Login to comment
140 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). Login to comment
210 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). Login to comment
213 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. Login to comment
223 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. Login to comment

PMID: 22232552 [PubMed] Iram SH et al: "Mutation of Glu521 or Glu535 in cytoplasmic loop 5 causes differential misfolding in multiple domains of multidrug and organic anion transporter MRP1 (ABCC1)."

No. Sentence Comment

6 Thus Ala substitution of Lys513 , Lys516 , Glu521 , and Glu535 all cause misfolding of MRP1 and target the protein for proteasome-mediated degradation. Login to 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. Login to comment
8 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. Login to comment
54 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). Login to comment
92 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. Login to comment
94 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. Login to comment
100 Moreover, under these conditions a significant portion of three of the bortezomib-rescued mutant proteins (K513A, E521A, and E535A) migrated as the fully glycosylated mature form of MRP1. Login to comment
102 Together, these observations indicate that Ala substitution of Lys513 , Lys516 , Glu521 , or Glu535 causes misfolding of MRP1, which in turn targets the transporter for degradation by the Differential Rescue of Misfolded Cytoplasmic Mutants of MRP1 MARCH 2, 2012•VOLUME 287•NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7545 proteasome. Login to comment
106 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. Login to comment
109 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(2.5␮M for16h).Untransfectedcellswereusedasanegativecontrol,andMRP1proteinsweredetectedwith mAb QCRL-1. Equal protein loading was confirmed by blotting for ␣-tubulin. Login to comment
114 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. Login to comment
118 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. Login to comment
122 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. Login to comment
127 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. Login to comment
128 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). Login to comment
138 After 48 h of 4-PBA treatment, levels of K513A, E521A, and E535A were further increased, whereas K516A levels remained unchanged. Login to comment
141 As shown in Fig. 4C, MRP1 signals from three of four 4-PBA-rescued mutants (K513A, E521A, and E535A) were mostly at the plasma membrane as observed for wild-type MRP1. Login to comment
144 Functional Characterization of CL5 Mutants Rescued by 4-PBA-To determine if the 4-PBA-rescued K513A, E521A, and E535A mutants were functional, their vesicular transport activities were measured using the prototypical MRP1 substrates LTC4 and E217betaG. Login to comment
146 Immunoblots showed that, consistent with results in cell lysates (Fig. 4, A and B), levels of the 4-PBA-rescued K513A mutant in membrane vesicles were comparable with wild-type MRP1 levels in vesicles from untreated cells, whereas levels of the rescued E521A and E535A mutants were moderately reduced (ϳ40%) (Fig. 5A). Login to comment
148 Uptake levels by the 4-PBA- rescued K513A mutant vesicles were also comparable with wild-type MRP1. Login to comment
150 These results indicate that although the 4-PBA-rescued K513A mutant appears fully functional, this is not the case for the E521A and E535A mutants. Login to comment
154 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. Login to comment
155 Shown are representativeimmunoblotsofwholecelllysates(10␮gofprotein/lane)preparedfrom HEK293T cells transfected with wild-type (WT-MRP1) and mutant (K513A, K516A, E521A, and E535A) cDNA expression vectors. Login to comment
184 Further evidence that the nucleotide interactions of 4-PBA- rescued E521A differs from E535A and wild-type MRP1 was obtained by determining the relative Km(ATP) values of the two FIGURE 5. Effect of 4-PBA on the levels and vesicular transport activities of the K513A, E521A, and E535A mutant MRP1 proteins. Login to comment
185 A, shown is a representative immunoblot of membrane vesicles (1 ␮g of protein/lane) prepared from HEK293T cells incubated for 48 h in the presence of 5 mM 4-PBA after transfection with wild-type (WT-MRP1) and mutant (K513A, E521A, and E535A) cDNA expression vectors. Login to comment
241 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). Login to comment
243 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. Login to comment
244 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. Login to comment
264 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. Login to comment
265 On the one hand, the K513A mutant was restored to wild-type MRP1 levels (and transport activity) by all four treatments. Login to comment
266 This indicates that if the conformation of the 4-PBA-rescued K513A mutant differs from wild-type MRP1, the differences are subtle and do not constitute a significant kinetic energy barrier to shift the folding equilibrium of the K513A protein toward a more native transport-competent state. Login to comment
277 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). Login to comment
280 In contrast to K513A, the LTC4 and E217betaG transport activities of the 4-PBA-rescued E521A and E535A mutants were significantly reduced to a similar degree. Login to comment
296 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. Login to comment

PMID: 24231430 [PubMed] Iram SH et al: "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."

No. Sentence Comment

2 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. Login to comment
3 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. Login to comment
5 In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. Login to comment
26 Thus, Ala substitution of Arg501 , Glu507 or Arg532 caused a significant reduction in organic anion transport activity while Ala substitution of Lys513 , Lys516 , Glu521 or Glu535 abrogated MRP1 expression by disrupting the folding and assembly of the transporter, targeting it for degradation by the proteasome [8,9]. Login to comment
27 In the case of three of these misfolded CL5 mutants (K513A, E521A and E535A), plasma membrane MRP1 levels could be improved significantly by exposing transfected HEK cells to the widely used chemical chaperone, 4-phenylbutyric acid (4-PBA) [9,10]. Login to comment
29 In contrast to E521A and E535A, the 4-PBA rescued K513A mutant exhibited transport properties comparable to wild-type MRP1. Login to comment
31 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. Login to comment
38 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]. Login to comment
74 Photolabeling of MRP1 with 8-azido-[b1;-32 P]ATP K513A mutant and wild-type MRP1 from transfected HEK cells were photolabeled with 8-azido-[b1;-32 P]ATP essentially as described [22]. Login to comment
76 After 5 min incubation on ice, the samples were cross-linked at 302 nm, washed, and then subjected to SDS-PAGE. After drying, the gel was exposed to film for 2-4 h. K513A mutant and wild-type MRP1 membrane proteins were also incubated in transport buffer containing 5 mM MgCl2, 1 mM freshly prepared sodium orthovanadate, and 5 bc;M 8-azido-[b1;-32 P]ATP for 15 - min at 37 &#b0;C to measure orthovanadate-induced trapping of 8-azido- [b1;-32 P]ADP by MRP1. Login to comment
79 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]. Login to comment
85 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. Login to comment
86 As shown in Fig. 2A, all treatments enhanced levels of the K513A mutant to a similar degree while the same treatments did not significantly alter wild-type MRP1 levels indicating that the rescue is not a result of transcriptional upregulation. Login to comment
87 Confocal microscopy confirmed the plasma membrane localization of the rescued K513A mutants indicating their ability to bypass the quality control mechanisms for protein folding in the endoplasmic reticulum of HEK cells and to traffic to the plasma membrane (Supplemental Fig. S1). Login to comment
88 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). Login to comment
94 Functional characterization of chemically rescued K513A mutant MRP1 proteins To determine whether or not the chemically rescued K513A mutants were transport competent, membrane vesicles were prepared (Fig. 3A) and vesicular transport activities were measured using the prototypical organic anion substrates E217b2;G and LTC4. Login to comment
95 As shown in Fig. 3, [3 H] E217b2;G and [3 H]LTC4 uptake levels by K513A mutant membrane vesicles prepared from cells treated with 4-PBA or DMSO were comparable to wild-type MRP1. Login to comment
98 These results indicate that despite restoring similar levels of the K513A mutant, the chemical chaperones altered MRP1 function to different extents. Login to comment
100 Conformational differences in chemically rescued K513A mutant proteins To determine whether the transport properties of the K513A mutants rescued by the different chemical chaperones were associated with any differences in protein folding, membrane vesicles containing K513A were subjected to limited trypsinolysis and tryptic fragments detected by immunoblotting with mAbs directed against epitopes in the NH2-proximal (mAb MRPr1) and COOH-proximal (mAb MRPm6) halves of MRP1 (Fig. 4A) [6,29]. Login to comment
102 Effect of chemical chaperones on relative levels of K513A and K516A mutant MRP1 proteins in HEK293T cells. Login to comment
103 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. Login to comment
106 (A) WT-MRP1 (left panel), K513A (right panel); (B) K516A. Login to comment
109 Effect of chemical chaperones on the levels and vesicular transport activity of K513A mutant MRP1 expressed in HEK cells. Login to comment
110 (A) Shown is a representative immunoblot of membrane vesicles (1 bc;g protein per lane) prepared from transfected HEK293T cells, untreated (WT-MRP1) or treated with the indicated chemicals (K513A) for 48 h. Untransfected cells were used as a negative control (ctrl). Login to comment
117 The four chemically rescued K513A mutants had similar tryptic profiles, indicating that the conformation of the NH2-proximal halves of these mutant proteins were comparable to wild-type MRP1. Login to comment
118 To probe for conformational changes in the COOH-proximal halves of the rescued K513A mutants, the trypsin digests were then immunoblotted with mAb MRPm6 (Fig. 4B, right panel). Login to comment
119 Under these conditions, the wild-type MRP1 digests yielded first a larger 75-kDa fragment (C1, corresponding to MSD2-NBD2) which was then cleaved Fig. 4. Limited trypsin digests of chemically rescued K513A mutants and wild-type MRP1 expressed in HEK293T cells. Login to comment
121 (B) Membrane vesicle proteins (2 bc;g per lane) from cells expressing wild-type MRP1 or K513A mutant MRP1 rescued with the indicated chemical chaperones were incubated at 37 &#b0;C for 15 min with trypsin at different trypsin:protein ratios (range 1:5000 to 2.5:1), and then immunoblotted with (left panel) mAb MRPr1 and (right panel) mAb MRPm6. Login to comment
125 The tryptic profiles for the K513A mutants rescued by DMSO or 4-PBA were similar to wild-type MRP1, suggesting that no significant conformational changes had occurred in the COOH-proximal halves of these mutants. Login to comment
126 In contrast, the tryptic profiles of the glycerol and PEG rescued K513A mutants were substantially different. Login to comment
127 Most notably, the NBD2 (C2) fragments were not detectable in the glycerol or PEG rescued K513A proteins. Login to comment
128 These observations indicate that NBD2 in the glycerol and PEG-rescued K513A mutants is hypersensitive to trypsin suggesting that this region has adopted a more open (less compact) conformation than is the case in wild-type MRP1. Login to comment
129 Conformational changes in NBD2 in the glycerol and PEG rescued K513A proteins were further corroborated by taking advantage of two MRP1-specific conformation-dependent antibodies, mAb QCRL-2 (which recognizes a discontinuous epitope within NBD1) and mAb QCRL-4 (which recognizes a discontinuous epitope within NBD2) [15]. Login to comment
131 As shown in Fig. 5A, the immunoreactivity of the glycerol and PEG rescued K513A proteins detected by mAb QCRL-4 was diminished by N50% compared to wild-type MRP1, consistent with substantial changes in the conformation of NBD2. Login to comment
133 Together with the tryptic fragment analyses (Fig. 4), these results indicate that the transport deficiencies of K513A rescued by glycerol or PEG are associated with a change to a less compact conformation of NBD2 in MRP1. Login to comment
135 E217b2;G uptake and [3 H]LTC4 photolabeling of glycerol and PEG rescued K513A MRP1 To further explore the consequences of the conformational changes detected in the glycerol or PEG rescued K513A proteins, kinetic parameters of E217b2;G uptake were determined. Login to comment
137 The glycerol and PEG rescued K513A proteins exhibited comparable Km (E217b2;G) values of 3.8 and 5.6 bc;M, respectively, but their Vmax (E217b2;G) values were decreased 3 to 4-fold relative to wild-type MRP1 (230 and 317 pmol mg-1 min-1 , respectively). Login to comment
138 [3 H]LTC4 photolabeling of these two rescued K513A proteins and wild-type MRP1 were also comparable (Fig. 6A). Login to comment
139 Together, these results indicated that the reduced transport levels of the PEG and glycerol rescued K513A mutant were not caused by decreased Km (E217b2;G) or reduced binding of LTC4 (at 200 nM). Login to comment
141 Photolabeling of glycerol and PEG rescued K513A MRP1 with 8-azido- [b1;-32 P]ATP and orthovanadate-induced trapping of 8-azido-[b1;-32 P]ADP Since the transport deficiencies of the glycerol- and PEG-rescued K513A proteins were not associated with differences in Km (E217b2;G) or reduced binding of LTC4 (at 200 nM), the nucleotide interactions of the mutants were examined. Login to comment
143 As shown in Fig. 6B, photolabeling of the glycerol- and PEG-rescued K513A proteins at this single concentration of 8-azido-ATP was decreased by 50% and 30%, respectively, compared to wild-type MRP1 (after differences in MRP1 levels were taken into account). Login to comment
145 As shown in Fig. 6C, [32 P]ADP trapping by glycerol-rescued K513A was 60% lower than trapping by wild-type MRP1 while trapping by the PEG-rescued K513A was 40% lower. Login to comment
148 Immunodot blot analyses of the transport-deficient PEG and glycerol rescued K513A mutant MRP1 with conformation-dependent mAbs against NBD1 and NBD2. Login to comment
149 Serial dilutions of non-denatured membrane vesicles (corresponding to 0.5, 1 and 2 bc;g protein) prepared from transfected HEK293T cells expressing K513A mutant (treated with glycerol or PEG for 48 h) or wild-type MRP1 (untreated) were spotted on a membrane as described [18] and then probed with conformation-dependent mAbs (A) QCRL-4 (NBD2); and (B) QCRL-2 (NBD1). Login to comment
152 WT-MRP1 (a0;); glycerol-rescued K513A (cf;); PEG-rescued K513A (੪). Login to comment
159 [3 H]LTC4 photolabeling and azido-[b1;32 P]ATP interactions of transport-deficient PEG and glycerol rescued K513A mutant proteins expressed in HEK293T cells. Login to comment
210 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. Login to comment
212 In the case of K513A, levels of the mutant protein could be improved to wild-type MRP1 levels by treatment of cells with all four chemical chaperones tested (Fig. 2A) [9]. Login to comment
213 However, only the 4-PBA and DMSO-rescued K513A mutants displayed organic anion transport activity comparable to wild-type MRP1. Login to comment
214 The reduced transport activity of the glycerol- and PEG-rescued K513A mutants indicated that the structural defects introduced by the Lys513 substitution were only partially restored by these two chemical chaperones. Login to comment
215 Differences in trypsin fragmentation patterns and immunoreactivity with conformation-dependent antibodies revealed that the NBD2 region of the glycerol and PEG-rescued K513A transport-deficient MRP1 mutants had adopted a more open, trypsin sensitive conformation than the same region of wild-type MRP1 (or the functionally intact DMSO or 4-PBA rescued K513A mutants). Login to comment
216 The kinetic analyses of E217b2;G transport and the nucleotide labeling results further indicated that the transport deficiencies of the DMSO and PEG-rescued MRP1 K513A proteins were caused by changes in the binding and hydrolysis of ATP. Login to comment
220 These observations support the suggestion that the K513A mutation in CL5 of MSD1 interferes with the closure of the NBD2 core subdomain making it less able to form a compact interface with CL5. Login to comment
221 The biochemical properties and mode of action of 4-PBA, glycerol, DMSO, and PEG are known to vary and it is not clear here why all 4 were equally able to improve K513A levels but glycerol and PEG were unable to functionally rescue the mutant. Login to comment
223 Thus, in contrast to MRP1-K513A, glycerol effectively restored the function of CFTR-ƊF508 while DMSO did not [37,39]. Login to comment
225 In contrast to K513A, the K516A processing mutant was remarkably resistant to rescue by the chemical chaperones tested. Login to comment
237 This is in contrast to the K513A mutant where NBD2 folding was mainly affected. Login to comment
238 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. Login to comment
241 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. Login to comment