ABCMdb

ABCC1 p.Lys516Ala

None has been submitted yet.

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

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
137 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. 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
159 As observed for the CL5 mutant K516A, the NBD2 mutant R1367A was not detectable by immunoblot analysis (Fig. 5C). 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

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
101 In contrast, only the underglycosylated form of the fourth mutant, K516A, was detected (Fig. 2B). Login to comment
103 In addition, the properties of the misfolded mutants, particularly in the case of K516A, appear distinct from one another. Login to comment
105 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. 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
112 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. 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
119 Furthermore, the K516A protein detected after the chemical treatments was again predominantly the underglycosylated form (170 kDa) of the transporter. 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
142 In marked contrast, the 4-PBA-rescued K516A mutant co-localized primarily with the ER marker calnexin. 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
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
267 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. Login to comment
268 Rescue of the E521A and E535A mutants was achieved only with 4-PBA, but unlike K516A, these mutants were fully glycosylated. 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
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

5 In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. Login to comment
6 In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Login to comment
7 Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by N80% due to reduced substrate Km. Login to comment
30 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]. 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
46 Wild-type and K516A mutant MRP1 were also cloned into pFastBac vectors (Life Technologies Inc.) for expression in Sf9 insect cells. Login to comment
50 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. 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
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
90 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. Login to comment
92 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. 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
154 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. Login to comment
155 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. Login to comment
156 However, the LTC4 transport activity of the heterologously expressed K516A was reduced by 80% relative to wild-type MRP1 (Fig. 7B). Login to comment
157 [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. Login to comment
171 Expression and function of K516A mutant MRP1 expressed in insect cells. Login to comment
172 (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. Login to comment
175 (B) [3 H]LTC4 transport activity of insect cell membrane vesicles enriched for K516A mutant and wild-type MRP1. Login to comment
179 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. Login to comment
183 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). Login to comment
184 Differences in the wild-type and K516A tryptic fragmentation patterns were detected with both antibodies. Login to comment
185 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. Login to comment
186 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. Login to comment
187 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. Login to comment
188 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. Login to comment
199 Fig. 8. Limited trypsin digests of transport-deficient K516A mutant MRP1 in insect cell membranes. Login to comment
201 (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. Login to comment
203 The boxes highlight significant differences between the patterns of the tryptic fragments of the wild-type and transport-deficient K516A mutant proteins. 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
225 In contrast to K513A, the K516A processing mutant was remarkably resistant to rescue by the chemical chaperones tested. Login to comment
231 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]. Login to comment
232 However, despite the robust levels of K516A mutant achieved in this system, the transport activity of this mutant was severely diminished. Login to comment
233 Thus, LTC4 transport levels were just 10% those of wild-type MRP1, and the K516A mutant could no longer be photolabeled with this substrate. Login to comment
234 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. Login to comment
235 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. Login to comment
236 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. 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