ABCMdb

ABCC1 p.Glu535Ala

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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

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
233 If such interdomain interactions exist and are important for the proper folding and assembly of MRP1, it was reasonable to expect that, as we observed for the Ala substitution of Glu535 , replacing His1049 with Ala would also affect the levels of MRP1. Login to comment
234 Indeed, this was the case, although the reduction in E535A protein levels was significantly greater (Ͼ90%) than the reduction observed for H1049A (60%). 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
9 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. Login to comment
10 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. 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
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
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
120 Levels of E521A and E535A were most effectively restored by 4-PBA treatment with only modest increases observed after treatment with glycerol, DMSO, or PEG. 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
149 By contrast, the E521A and E535A mutants showed a 45-55% decrease in [3 H]E217betaG and [3 H]LTC4 uptake (even after differences in MRP1 levels were taken into account). 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
151 The reduced transport activity of the 4-PBA-rescued E521A and E535A mutants was further investigated by determining the kinetic parameters of E217betaG and LTC4 vesicular uptake. 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
174 These differences in kinetic parameters indicate that the mechanisms responsible for the reduced transport activities of the 4-PBA-rescued E521A and E535A mutants differ. Login to comment
175 Decreased Transport of E535A Mutant Is Associated with Diminished Photolabeling by [3 H]LTC4-To further examine the interaction of the 4-PBA-rescued E521A and E535A mutants with LTC4, membrane vesicles enriched for the wild-type or mutant MRP1 proteins were photolabeled with [3 H]LTC4. Login to comment
176 As shown in Fig. 6A, [3 H]LTC4 labeling of the E521A mutant was comparable with labeling of wild-type MRP1, whereas labeling of the E535A mutant was decreased by 40% (after correcting for differences in protein levels). Login to comment
178 Nucleotide Interactions of Mutants E521A and E535A-Because its apparent affinity (Km) for LTC4 and E217betaG and its photolabeling by [3 H]LTC4 were not significantly different from wild-type MRP1, the possibility that the transport defect of the 4-PBA-rescued E521A mutant could involve altered nucleotide interactions was explored. Login to comment
179 In these experiments, in addition to wild-type MRP1, the Glu535 mutant was included as a "control" because (a) Glu535 is predicted to lie outside the CL5/NBD2 interface region and thus is unlikely to be involved in the catalytic activity of the transporter (11, 19), and (b) the reduced transport activity of the E535A mutant appears to be accounted for by changes in Km rather than Vmax (Table 1). Login to comment
180 Thus, the ability of E521A, E535A, and wild-type MRP1 to bind ATP was first assessed by photolabeling the proteins with 8-azido-[␣-32 P]ATP under conditions of minimal hydrolysis (4 °C). Login to comment
181 As shown in Fig. 6B, photolabeling of the rescued E535A mutant and wild-type MRP1 were comparable, whereas photolabeling of the rescued E521A mutant was ϳ30% less. Login to comment
183 As shown in Fig. 6C, [32 P]ADP trapping by the 4-PBA- rescued E521A mutant was reduced by 40%, whereas trapping by the E535A mutant was comparable with wild-type MRP1. 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
193 TABLE 1 Kinetic parameters of E217betaG and LTC4 uptake by 4-PBA-rescued MRP1 mutants E521A and E535A Km and Vmax values for E217betaG and LTC4 uptake by membrane vesicles prepared from HEK293T cells transfected with wild-type or mutant proteins were determined by measuring ATP-dependent uptake of the indicated 3 H-labeled organic anion at eight different concentrations as described under "Experimental Procedures." Login to comment
195 The values shown are the means of results from two independent experiments. Transfectant Km Vmax a E217betaG LTC4 E217betaG LTC4 ␮M nM pmol-1 mg-1 min-1 WT-MRP1 4.0 134 791 302 E521A 5.5 131 251 122 E535A 22.0 483 920 289 a Vmax values have been corrected to take into account differences in levels of the mutant proteins relative to wild-type MRP1 (WT-MRP1). Login to comment
197 As summarized in Table 2, the Km(ATP) values of E521A for both E217betaG and LTC4 uptake were ϳ2.5-fold lower than those for both wild-type MRP1 and E535A. Login to comment
199 Mutations E521A and E535A Cause Distinct Conformational Changes in MRP1-Because the mechanisms responsible for the reduced transport activities of the 4-PBA-rescued E521A and E535A mutants differed, it was of interest to determine whether differences in the conformation of the two mutant proteins were involved. Login to comment
201 Consequently, membrane vesicles containing wild-type MRP1 and 4-PBA-rescued E521A and E535A were subjected to limited trypsinolysis, and tryptic fragments were detected by serial immunoblotting with MRP1-specific antibodies directed against epitopes in both the NH2-proximal half (mAbs MRPr1 and 42.4) (MSD0-MSD1-NBD1) (Fig. 7A) and the COOH-proximal half (mAbs MRPm6, 897.2 and MRPm5) (MSD2-NBD2) (Fig. 8A) of the transporter. Login to comment
208 When immunoblots of the E521A and E535A mutants were probed with mAb 42.4, reduced levels of the N1 fragment were detected relative to wild-type MRP1, as observed with mAb MRPr1. Login to comment
212 [3 H]LTC4 and azido-[␣32 P]ATP photolabeling of wild-type and E521A and E535A mutant MRP1 proteins. Login to comment
221 TABLE 2 ATP dependence of [3 H]E217betaG and [3 H]LTC4 uptake by 4-PBA-rescued MRP1 mutants E521A and E535A Initial rates of [3 H]E217betaG and [3 H]LTC4 uptake by wild-type and 4-PBA-rescued K521A and K535A mutant MRP1 were measured for 1 min in the presence of eight different concentrations of ATP (2.5-2000 ␮M). Login to comment
223 The values shown are means of two independent experiments. Transfectant [3 H]E217betaG uptake [3 H]LTC4 uptake Km(ATP) Vmax a Km(ATP) Vmax a ␮M pmol mg-1 min-1 ␮M pmol mg-1 min-1 WT-MRP1 350 48 71 53 E521A 823 20 185 25 E535A 350 22 76 26 a Vmax values have been corrected for differences in protein levels of the 4-PBA- rescued mutants relative to WT-MRP1. Login to comment
224 Differential Rescue of Misfolded Cytoplasmic Mutants of MRP1 7550 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 10•MARCH the 4-PBA-rescued E521A and E535A mutants differs from that of the same region of the wild-type protein. Login to comment
225 Trypsin digests of the E521A and E535A mutants and wild-type MRP1 were then probed with mAbs that detect epitopes in the COOH-proximal half of MRP1. Login to comment
227 Immunoblots of the 4-PBA-rescued E521A and E535A mutants with the same mAb showed a very similar pattern of disappearance of full-length protein, indicating that accessibility of the cleavage site in the linker region between N1 and C1 of wild-type MRP1 and the 4-PBA-rescued E521A and E535A mutants is the same (Fig. 8B). Login to comment
229 In contrast, levels of C1 fragment (containing MSD2-NBD2) were substantially lower for both the E521A and E535A mutants when probed with mAbs MRPm6 (Fig. 8B), 897.2 (Fig. 8C), or MRPm5 (Fig. 8D), indicating conformational differences in this protein fragment. Login to comment
230 These observations provide strong evidence that the E521A and E535A mutations in the NH2-proximal MSD1 cause conformational changes in the COOH-proximal half of MRP1. Login to comment
237 Differential Rescue of Misfolded Cytoplasmic Mutants of MRP1 MARCH 2, 2012•VOLUME 287•NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7551 the E535A mutant but not for the E521A mutant. Login to comment
238 The C2 fragment of the E535A mutant appeared at lower trypsin:protein ratios than did the corresponding fragment of wild-type MRP1 (Fig. 8, B and C, lanes 6-9 for E535A versus lanes 8-9 for wild-type), suggesting an altered conformation of the C1 fragment of E535A. 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
251 Detection of COOH-proximal tryptic fragments of E521A and E535A mutant and wild-type MRP1. 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
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
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
282 Thus, the decreased transport activity of the E535A mutant could be largely, if not completely, attributed to a reduced affinity for its organic anion substrates. Login to comment
284 In seeking an explanation for the mechanistic differences underlying the transport deficiencies of E521A and E535A, we explored the possibility that differences in protein conformation might be involved by using limited proteolysis with a panel of mAbs able to detect tryptic fragments both close and distal to CL5. Login to comment
287 Thus, the MSD1-NBD1 region (N1 fragment) of the E521A and E535A mutants was relatively more sensitive to trypsin than wild-type MRP1 (Fig. 7), but given that N1 contains CL5 where the mutations reside, a conformational change in this region is not surprising. Login to comment
290 Differences between the two mutants were also observed in the C2 fragment (containing NBD2), with the E521A fragment being more sensitive to trypsin than the same fragment in E535A and wild-type MRP1. Login to comment
297 Between these two extremes was the partial and differential rescue of the E521A and E535A mutants. 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

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
28 Although the 4-PBA- rescued E521A and E535A mutants were stably expressed, they exhibited mechanistically distinct transport deficiencies that were associated with different structural conformations of the transporter that in turn indicated that the folding and assembly processes for MRP1 are distinct from those of its homolog CFTR [9]. 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