ABCG2 p.Ile573Ala
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PMID: 26294421
[PubMed]
Haider AJ et al: "Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences."
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Sentence
Comment
6
One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics.
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ABCG2 p.Ile573Ala 26294421:6:12
status: NEW7 In contrast with many ABC transporter folding mutations which appear to be 'rescued` by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER).
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ABCG2 p.Ile573Ala 26294421:7:143
status: NEW84 For immunofluorescence determination of the localization of the I573A isoform, cells were fixed on cover slips with 4% PFA in PBS for 5 min at room temperature before treatment with 50 mM NH4Cl for 10 min to quench the free aldehyde groups of the fixative preventing autofluorescence [21].
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ABCG2 p.Ile573Ala 26294421:84:64
status: NEW109 We made individual substitutions of each amino acid to alanine: M131A, S195A, K453A, K473A, P485A, M549A, W564A and I573A.
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ABCG2 p.Ile573Ala 26294421:109:116
status: NEW114 Analysis of the expression level (given by the ratio 5D3-PE fluorescence labelling compared with IgG-PE fluorescence labelling) of all mutants suggested that, of the novel mutants examined in the present study, only I573A had reduced cell surface expression (Figures 2E and 2F).
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ABCG2 p.Ile573Ala 26294421:114:216
status: NEW123 variability in expression of I573A was not a reflection of the conformational sensitivity of the 5D3 antibody [31], we performed western blotting of whole cell lysates with the BXP-21 antibody which recognizes an intracellular epitope.
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ABCG2 p.Ile573Ala 26294421:123:29
status: NEW124 This confirmed that I573A was expressed at reduced levels compared with other isoforms (Figure 2G; I573A expression is essentially undetectable in this exposure).
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ABCG2 p.Ile573Ala 26294421:124:20
status: NEWX
ABCG2 p.Ile573Ala 26294421:124:99
status: NEW125 The failure of one of the isoforms (I573A) to effectively reach the cell surface piqued our interest as membrane trafficking is clearly of cellular and clinical relevance, both for ABCG2 and ...................................................................... .......................................................... .................................................................... ........................................................... ........................................................... ................................................ c 2015 Authors.
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ABCG2 p.Ile573Ala 26294421:125:36
status: NEW127 5 Figure His12-I573A shows impaired glycosylation (A) Following transient transfection cell lysates (30 bc;g) were resolved on 10 % w/v acrylamide gels and blotted with BXP-21 antibody.
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ABCG2 p.Ile573Ala 26294421:127:18
status: NEW130 His12-I573A is exclusively present at an intermediate molecular mass at 24 h and even at 48 h the majority of His12-I573A isoform is not the mature molecular mass.
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ABCG2 p.Ile573Ala 26294421:130:6
status: NEWX
ABCG2 p.Ile573Ala 26294421:130:116
status: NEW134 By contrast, the lower molecular mass band following transfection with the His12-I573A isoform shows a small (estimated < 5 kDa) change in molecular mass upon PNGaseF treatment indicative of the removal of core glycosylation.
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ABCG2 p.Ile573Ala 26294421:134:81
status: NEW142 To enable live cell imaging of this isoform and to rule out the possibility that these results are a consequence of a transient transfection system, we generated stable cell lines expressing WT and I573A isoforms of ABCG2 with an N-terminal sfGFP extension.
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ABCG2 p.Ile573Ala 26294421:142:198
status: NEW145 For sfGFP-I573A, the cellular fluorescence level (i.e. a measure of protein expression) is reduced compared with WT sfGFP-ABCG2 (compare intensity of Figure 4A with Figure 4B obtained using identical image acquisition and processing conditions), in agreement with data from transient expression of untagged isoforms (Figure 2).
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ABCG2 p.Ile573Ala 26294421:145:10
status: NEW146 Moreover, sfGFP-I573A is retained in perinuclear and reticular intracellular compartments with very little protein at the cell surface.
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ABCG2 p.Ile573Ala 26294421:146:16
status: NEW147 Co-localization of the sfGFP-I573A isoform was demonstrated with anti-calnexin antibodies confirming the ER retention (Figure 4C).
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ABCG2 p.Ile573Ala 26294421:147:29
status: NEW149 We investigated both of these possibilities with the sfGFP-I573A isoform and demonstrated that neither incubation at 30e6; C (Figure 4D, right hand panel), nor incubation with the chaperone 4-phenylbutyrate rescued the cell surface expression of I573A (Figure 4D, left hand panel), although the latter did result in a qualitatively higher expression of sfGFP-I573A (compare Figure 4B right hand and 4D left hand panel which are obtained under identical image capture and processing settings on the same day).
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ABCG2 p.Ile573Ala 26294421:149:59
status: NEWX
ABCG2 p.Ile573Ala 26294421:149:250
status: NEWX
ABCG2 p.Ile573Ala 26294421:149:363
status: NEW157 sfGFP- ................................................................... ............................................................ .................................................................. ............................................................. ........................................................ .................................................... 6 Figure sfGFP-I573A is retained in the endoplasmic reticulum (ER) and cell surface expression is not rescued by low temperature incubation To visualize trafficking of I573 in live cells, stable cell lines expressing WT and I573A isoforms of ABCG2 with an N-terminal sfGFP tag were generated and examined by confocal imaging.
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ABCG2 p.Ile573Ala 26294421:157:393
status: NEWX
ABCG2 p.Ile573Ala 26294421:157:602
status: NEW158 The majority of WT ABCG2 is present at the cell surface (A, phase and merged image), whereas the majority of I573A is retained intracellularly (B).
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ABCG2 p.Ile573Ala 26294421:158:109
status: NEW159 (C) Immunostaining of cells with anti-calnexin antibodies (red, left hand panel) shows significant co-localization of the sfGFP-I573A fluorescence signal (green, middle panel and yellow in the merged image, right hand panel) indicative of the retention of the majority of sfGFP-I573A in the ER.
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ABCG2 p.Ile573Ala 26294421:159:128
status: NEWX
ABCG2 p.Ile573Ala 26294421:159:278
status: NEW161 (D) Incubation of sfGFP-I573A expressing cells at 30e6; C had little effect on the localization of the I573A to the cell surface (right hand panel).
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ABCG2 p.Ile573Ala 26294421:161:24
status: NEWX
ABCG2 p.Ile573Ala 26294421:161:107
status: NEW179 Of the eight residues we mutated, two resulted in altered drug export function (P485A and M549A) and a further residue (I573A) perturbs the trafficking and maturation of ABCG2 glycosylation.
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ABCG2 p.Ile573Ala 26294421:179:120
status: NEW188 One of the eight residues we mutated in the present study (I573A) proved to have an effect on protein trafficking and maturation, an effect previously seen with a mutation of a residue located close to the intracellular side of TM1 [39].
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ABCG2 p.Ile573Ala 26294421:188:59
status: NEW190 I573A is located within TM5 in the model of Mao and colleagues [30], although sequence based topological models place it within the extracellular loop between TM5 and TM6.
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ABCG2 p.Ile573Ala 26294421:190:0
status: NEW191 Irrespective of its exact location, the effect of mutating I573A on targeting and glycosylation is consistent with a critical role for the TM5 and 6 region in the integrity of ABCG2 [40,41].
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ABCG2 p.Ile573Ala 26294421:191:59
status: NEW192 In contrast with some recent reports on the rescue of folding mutants by chemical 'correctors` or by low incubation temperatures [6,34,35], we could find not positive effect on I573A.
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ABCG2 p.Ile573Ala 26294421:192:177
status: NEW