ABCB1 p.Ile541Phe
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PMID: 19185004
[PubMed]
Delaunay JL et al: "A missense mutation in ABCB4 gene involved in progressive familial intrahepatic cholestasis type 3 leads to a folding defect that can be rescued by low temperature."
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Sentence
Comment
3
ABCB4 is highly homologous to ATP-binding cassette, sub-family B, member 1 (ABCB1) (MDR1), themultidrugtransporterresponsiblefordrugresistanceofcancercells.Wehavestudiedtheeffectof mutation I541F localized to the first nucleotide-binding domain, which is highly conserved between ABCB4andABCB1.Plasmidsencodingthewild-typehumanABCB4orratABCB1-greenfluoresc- ing protein (GFP) construct, and corresponding I541F-mutants, were expressed in hepatocellular carcinoma, human (HepG2) and Madin-Darby canine kidney (MDCK) cells.
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ABCB1 p.Ile541Phe 19185004:3:190
status: NEWX
ABCB1 p.Ile541Phe 19185004:3:405
status: NEW61 For construction of the ABCB4-I541F and ABCB1-I541F-GFP mutants, site-directed mutagenesis was performed using the QuikChange XL mutagenesis kit (Stratagene Europe, Amsterdam Zvidoost, The Netherlands).
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ABCB1 p.Ile541Phe 19185004:61:30
status: NEWX
ABCB1 p.Ile541Phe 19185004:61:46
status: NEW68 Enrichment of the cell population with ABCB1-I541F-GFP-expressing cells was performed by fluorescence-activated cell sorting.
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ABCB1 p.Ile541Phe 19185004:68:45
status: NEW133 These expression experiments show that mutation I541F causes a trafficking defect both in ABCB1 and ABCB4, and that ABCB1-GFP chimera provides an interesting model to further investigate the effect of I541F mutation.
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ABCB1 p.Ile541Phe 19185004:133:48
status: NEW145 The location of mutation I541F is indicated by an arrow.
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ABCB1 p.Ile541Phe 19185004:145:25
status: NEW148 Expression of ABCB1-GFP and ABCB1-I541F-GFP in MDCK cells.
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ABCB1 p.Ile541Phe 19185004:148:34
status: NEW149 (A) Confluent filter-grown MDCK cells stably expressing ABCB1-GFP (A, C) or ABCB1-I541F-GFP (B, D) were fixed with paraformaldehyde and examined for the fluorescence of GFP.
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ABCB1 p.Ile541Phe 19185004:149:82
status: NEW152 (B) Subconfluent MDCK cells stably expressing ABCB1-I541F-GFP (B, D) were fixed with paraformaldehyde and ethanol, and processed for immunofluorescence using the monoclonal C219 antibody and Cy3-conjugated anti-mouse immunoglobulin G.
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ABCB1 p.Ile541Phe 19185004:152:52
status: NEW158 ABCB1-I541F-GFP Is Not Processed Correctly.
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ABCB1 p.Ile541Phe 19185004:158:6
status: NEW161 To assess more precisely the processing defect caused by the mutation, the glycosylation status of ABCB1 and ABCB1-I541F was studied with endoglycosidases.
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ABCB1 p.Ile541Phe 19185004:161:115
status: NEW166 In contrast, ABCB1-I541F-GFP migrated as two bands of roughly equal intensity of 180 and 190 kDa (Fig. 5).
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ABCB1 p.Ile541Phe 19185004:166:19
status: NEW168 Thus, biochemical studies confirmed that the I541F-mutant does not mature as the wild-type protein during the biosynthetic process, and remains largely in a high-mannose endoH-sensitive form.
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ABCB1 p.Ile541Phe 19185004:168:45
status: NEW172 Colocalization of ABCB1 and ABCB1-I541F with markers of specific cellular compartments.
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ABCB1 p.Ile541Phe 19185004:172:34
status: NEW173 Subconfluent MDCK cells stably expressing ABCB1-GFP (A) or ABCB1-I541F-GFP (B) were fixed with 4% paraformaldehyde, permeabilized with 0.075% saponin, and processed for immunofluorescence using antibodies to calnexin, protein disulfide isomerase, giantin, Golgi matrix protein, early endosomal antigen, or lysosomal-associated membrane protein 2, and appropriate Cy3-conjugated secondary antibodies.
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ABCB1 p.Ile541Phe 19185004:173:65
status: NEW176 Note that the calnexin pattern (B, upper middle picture) is different in ABCB1-I541F-GFP-expressing cells (large asterisks) than in nontransfected cells (small asterisks), and colocalizes with the GFP signal.
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ABCB1 p.Ile541Phe 19185004:176:79
status: NEW178 Expression and glycosylation status of ABCB1-GFP and ABCB1-I541F-GFP in MDCK cells.
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ABCB1 p.Ile541Phe 19185004:178:59
status: NEW179 MDCK/ABCB1-GFP and MDCK/ ABCB1-I541F-GFP cells were lysed and immunoprecipitation was performed using a polyclonal anti-GFP antibody absorbed onto protein A-sepharose.
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ABCB1 p.Ile541Phe 19185004:179:31
status: NEW183 defects that can be overcome by lowering the temperature.25 MDCK-ABCB1-I541F-GFP cells were filter grown to confluence at 37°C, then the cells were switched or not to 27°C for 24 hours.
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ABCB1 p.Ile541Phe 19185004:183:71
status: NEW184 Figure 6A shows that in control cells grown at 37°C, ABCB1-I541F-GFP was intracellular, whereas in cells grown at 27°C, ABCB1-I541F-GFP was expressed at the apical surface.
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ABCB1 p.Ile541Phe 19185004:184:64
status: NEWX
ABCB1 p.Ile541Phe 19185004:184:136
status: NEW187 These results show that I541F is a temperature-sensitive mutation and that processing and trafficking of the mutant reverts toward that of wild-type on lowering temperature.
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ABCB1 p.Ile541Phe 19185004:187:24
status: NEW188 ABCB1-I541F-GFP Is Functional When Expressed at the Apical Membrane.
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ABCB1 p.Ile541Phe 19185004:188:6
status: NEW194 Only very weak activity was measured in MDCK/ ABCB1-I541F-GFP cells grown at 37°C; however, when cells were grown at 27°C for 24 hours before the assay, they accumulated significantly less fluorescent calcein (Fig. 6C).
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ABCB1 p.Ile541Phe 19185004:194:52
status: NEW195 These results show that mutation I541F does not impair activity, provided that the transporter can reach the plasma membrane.
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ABCB1 p.Ile541Phe 19185004:195:33
status: NEW231 Here, we also used ABCB1 and showed that the I541F mutation caused similar intracellular retention in both ABCB1 and ABCB4.
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ABCB1 p.Ile541Phe 19185004:231:45
status: NEW
PMID: 22184139
[PubMed]
Gautherot J et al: "Effects of cellular, chemical, and pharmacological chaperones on the rescue of a trafficking-defective mutant of the ATP-binding cassette transporter proteins ABCB1/ABCB4."
No.
Sentence
Comment
1
Results: The I541F mutation induces misfolding and intracellular retention that is rescued by the ABCB1-competitive substrate cyclosporin A but not by modulating the chaperones calnexin or Hsp/Hsc70.
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ABCB1 p.Ile541Phe 22184139:1:13
status: NEW50 ABCB1-I541F has the advantage that it can be expressed at detectable level as a GFP fusion protein and ABCB1 activity is easy to measure.
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ABCB1 p.Ile541Phe 22184139:50:6
status: NEW52 We found that cyclosporin A, an ABCB1 substrate, induced the best rescue of the ABCB1-I541F mutant and also provides rescue of the ABCB4-I541F mutant.
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ABCB1 p.Ile541Phe 22184139:52:86
status: NEWX
ABCB1 p.Ile541Phe 22184139:52:137
status: NEW110 An ϳ40-kDa fragment was resistant to kallikrein both in ABCB1 and ABCB1-I541F, again indicating that the C terminus of the molecule was less affected by the mutation.
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ABCB1 p.Ile541Phe 22184139:110:78
status: NEW111 These proteolysis experiments provided evidence for a conformational defect of the I541F mutant.
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ABCB1 p.Ile541Phe 22184139:111:83
status: NEW112 The I541F Mutant Colocalizes and Coprecipitates with Calnexin-Calnexin is a chaperone of the ER that is involved in the folding of ABCB1 (19).
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ABCB1 p.Ile541Phe 22184139:112:4
status: NEW113 To study whether the mutant interacts with calnexin, we performed colocalization and coimmunoprecipitation experiments using MDCK cells stably transfected with ABCB1-WT or ABCB1-I541F.
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ABCB1 p.Ile541Phe 22184139:113:178
status: NEW115 By contrast, the ABCB1-I541F mutant colocalized strongly with calnexin, which appeared to be relocalized near the nucleus, together with the mutant (Fig. 2A).
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ABCB1 p.Ile541Phe 22184139:115:23
status: NEW117 In situ protease susceptibility of ABCB1-WT and ABCB1-I541F.
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ABCB1 p.Ile541Phe 22184139:117:54
status: NEW118 Microsomes obtained from MDCK cells expressing GFP-tagged ABCB1-WT or ABCB1-I541F were submitted to limited proteolysis at the indicated concentrations of thermolysin or kallikrein for 30 min at 4 °C.
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ABCB1 p.Ile541Phe 22184139:118:76
status: NEW119 Samples (ABCB1, 30 g and ABCB1-I541F, 50 g protein per lane) were immunoblotted with the anti-GFP monoclonal antibody.
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ABCB1 p.Ile541Phe 22184139:119:39
status: NEW123 ABCB1 migrated as two bands of ϳ190 kDa and 175 kDa (bands A and B), whereas ABCB1-I541F presented only the 175-kDa band.
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ABCB1 p.Ile541Phe 22184139:123:89
status: NEW125 Immunoprecipitation of calnexin also pulled down ABCB1-WT and ABCB1-I541F.
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ABCB1 p.Ile541Phe 22184139:125:68
status: NEW127 Effect of Calnexin Down-expression-Interaction between ABCB1-I541F and calnexin suggested that the chaperone might retain the I541F mutant in the ER.
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ABCB1 p.Ile541Phe 22184139:127:61
status: NEWX
ABCB1 p.Ile541Phe 22184139:127:126
status: NEW131 However, it had little or no effect on the expression of ABCB1-I541F (Fig. 3) and did not induce the appearance of the mature form, which would have indicated a rescue effect.
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ABCB1 p.Ile541Phe 22184139:131:63
status: NEW132 The I541F Mutant Coprecipitation with Hsc70 and Effect of Hsp70 Overexpression-The cognate Hsp70 family member Hsc70 is another ubiquitous chaperone that is involved in ABCB1 folding (20).
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ABCB1 p.Ile541Phe 22184139:132:4
status: NEW135 However, Hsc70 coprecipitated significantly with the mutant ABCB1-I541F, and in higher amounts than with FIGURE 2.
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ABCB1 p.Ile541Phe 22184139:135:66
status: NEW136 ABCB1-I541F colocalizes and coprecipitates with calnexin.
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ABCB1 p.Ile541Phe 22184139:136:6
status: NEW137 A, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were fixed, and immunolocalization of calnexin was performed using a Cy3-conjugated secondary antibody.
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ABCB1 p.Ile541Phe 22184139:137:67
status: NEW139 Note that the staining pattern of calnexin in ABCB1-I541F cells is different from that in ABCB1-WT cells because of ER accumulation of the mutant.
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ABCB1 p.Ile541Phe 22184139:139:52
status: NEW140 Scale bars ϭ 10 m. B, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F and control MDCK cells were lysed, and immunoprecipitation (IP) was performed with the anti-GFP antibody. Samples were analyzed by immunoblotting using anti-GFP or anti-calnexin monoclonal antibodies.
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ABCB1 p.Ile541Phe 22184139:140:100
status: NEW143 D, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F and control MDCK cells were lysed, and immunoprecipitation was performed with the anti-calnexin antibody. Samples were analyzed by immunoblotting using the anti-GFP antibody.
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ABCB1 p.Ile541Phe 22184139:143:67
status: NEW145 Effect of calnexin silencing on ABCB1-I541F expression.
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ABCB1 p.Ile541Phe 22184139:145:38
status: NEW146 MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were transfected with control or calnexin (CLNX) siRNA.
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ABCB1 p.Ile541Phe 22184139:146:64
status: NEW169 ABCB1-I541F Is Functionally Rescued by Cyclosporin A-Cyclosporin A is an immunosuppressant known to be a substrate for and a competitive inhibitor of ABCB1 (28).
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ABCB1 p.Ile541Phe 22184139:169:6
status: NEW171 We therefore tested the effect of cyclosporin A on the I541F mutant at concentrations ranging from 0.
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ABCB1 p.Ile541Phe 22184139:171:55
status: NEW172 1 to 10 M. Cyclosporin A given for 24 h caused a dose-dependent increase in the mature form of ABCB1-I541F (Fig. 7A and B).
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ABCB1 p.Ile541Phe 22184139:172:109
status: NEW174 Study of the localization of the rescued mutant by confocal microscopy showed that after cyclosporin A treatment, the I541F mutant was expressed at the apical membrane like the wild-type protein (Fig. 7C).
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ABCB1 p.Ile541Phe 22184139:174:118
status: NEW176 ABCB1-I541F coprecipitates with Hsc70.
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ABCB1 p.Ile541Phe 22184139:176:6
status: NEW177 A, MDCK cells stably transfected with GFP-tagged ABCB1-I541F were fixed, and Hsc70 was detected by immunofluorescence using a Cy3-conjugated secondary antibody.
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ABCB1 p.Ile541Phe 22184139:177:55
status: NEW179 Scale bar ϭ 10 m. B, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F and control MDCK cells were lysed, and immunoprecipitation (IP) was performed using the monoclonal anti-GFP antibody. Samples were analyzed by immunoblotting using anti-GFP or anti-Hsc70 monoclonal antibodies. Shown is one representative of three experiments.
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ABCB1 p.Ile541Phe 22184139:179:99
status: NEW181 Effect of Hsp70 overexpression on ABCB1-I541F expression.
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ABCB1 p.Ile541Phe 22184139:181:40
status: NEW182 A, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were maintained at 37 °C or exposed at 42 °C for 2 h and then returned to 37 °C for 24 h.
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ABCB1 p.Ile541Phe 22184139:182:67
status: NEW184 B, MDCK cells stably transfected with GFP-tagged ABCB1-WT or ABCB1-I541F were transfected with the plasmid encoding Hsp70.
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ABCB1 p.Ile541Phe 22184139:184:67
status: NEW190 Fig. 7D shows that ABCB1-I541F MDCK cells treated with cyclosporin A extruded more fluorescent calcein than control cells in a dose-dependent manner.
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ABCB1 p.Ile541Phe 22184139:190:25
status: NEW192 ABCB4-I541F Is Also Rescued by Cyclosporin A-Because cyclosporin A was very efficient at rescuing ABCB1-I541F, we also tested its potential effect on ABCB4-I541F.
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ABCB1 p.Ile541Phe 22184139:192:6
status: NEWX
ABCB1 p.Ile541Phe 22184139:192:104
status: NEWX
ABCB1 p.Ile541Phe 22184139:192:156
status: NEW193 ABCB4-I541F was expressed at a low level in stably transfected MDCK cells and was not reproducibly detected by Western blotting.
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ABCB1 p.Ile541Phe 22184139:193:6
status: NEW194 We therefore studied the effect of cyclosporin A in MDCK cells transiently transfected with the ABCB4-I541F plasmid.
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ABCB1 p.Ile541Phe 22184139:194:102
status: NEW231 Similarly, we did not observe that calnexin silencing induced significant change in the expression of the ABCB1-I541F mutant and that it did not rescue ER retention.
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ABCB1 p.Ile541Phe 22184139:231:112
status: NEW234 We found an increase in the ABCB1-I541F mutant when Hsp70 was overexpressed by transfection of the Hsp70 cDNA but not after heat shock treatment.
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ABCB1 p.Ile541Phe 22184139:234:34
status: NEW266 Here, we found that the ABCB1 inhibitor cyclosporin A was remarkably effective at restoring the traffic of ABCB1 bearing the I541F mutation.
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ABCB1 p.Ile541Phe 22184139:266:125
status: NEW273 However, the fact that the ABCB1 mutant was able to extrude calcein after cyclosporin A treatment suggests that rescued ABCB4-I541F may be functional as well.
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ABCB1 p.Ile541Phe 22184139:273:126
status: NEW