ABCMdb

PMID: 18361776

Loo TW, Bartlett MC, Clarke DM
Correctors promote folding of the CFTR in the endoplasmic reticulum.
Biochem J. 2008 Jul 1;413(1):29-36., 2008-07-01 [PubMed]

Sentences

No. Mutations Sentence Comment

40 ABCC7 p.Val510Ala The location of the V510A and cross-linkable cysteine residues [(M348C(TM6)/T1142C(TM12) (dashed line), T351C(TM6)/T1142C(TM12) (solid line) and W356C(TM6)/W1145C(TM12) (dotted line) mutants] are indicated. Login to comment 46 ABCC7 p.Cys590Leu ABCC7 p.Cys592Leu A cysteine-less CFTR was constructed by replacing Cys590 and Cys592 with leucine and by changing all other endogenous cysteine residues to alanine [23]. Login to comment 47 ABCC7 p.Val510Ala ABCC7 p.Val510Ala The cysteine-less CFTR also contained a V510A mutation (cysteine-less/V510A) that promoted maturation at 37◦ C [17]. Login to comment 48 ABCC7 p.Val510Ala The double-cysteine mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12) and W356C(TM6)/W1145C(TM12) that were shown to be cross-linkable with M8M (3,6-dioxaoctane-1,8-bismethanethiosulfonate) cross-linker [3] were introduced into the cysteine-less/V510A CFTR. Login to comment 49 ABCC7 p.Tyr563Asn ABCC7 p.Asp567Ala ABCC7 p.Asp565Ala The COPII exit motif (Y563 KDAD567 ) was disrupted by introducing the Y563N or D565A/D567A changes into wild-type CFTR [21] or into the double-cysteine mutants. Login to comment 50 ABCC7 p.Tyr563Asn The F508 mutation was also introduced into the Y563N double-cysteine mutants. Login to comment 63 ABCC7 p.Tyr563Asn ABCC7 p.Asp567Ala ABCC7 p.Asp565Ala Cell-surface labelling of CFTR Wild-type CFTR or COPII exit motif mutant Y563N or D565A/ D567A was transiently expressed in HEK-293 cells. Login to comment 83 ABCC7 p.Trp1145Cys It was shown that pairs of cysteine residues introduced into TM6 and TM12 [M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C- (TM12)] of CFTR (Figure 1) could be cross-linked with MTS (methanethiosulfonate) cross-linkers when the protein matured and was delivered to the cell surface. Login to comment 89 ABCC7 p.Thr351Cys The aggregates probably formed because of cross-linking between the 18 endogenous cysteine residues as the M348C(TM6)/T1142C(TM12), T351C- (TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutations were introduced into a wild-type CFTR background. Login to comment 92 ABCC7 p.Cys590Leu ABCC7 p.Cys592Leu Cysteine-less CFTR, in which Cys590 and Cys592 were replaced with leucine and the remaining cysteine residues were changed to alanine [23], did not mature at 37◦ C unless Val510 (in NBD1) was changed to alanine [17]. Login to comment 93 ABCC7 p.Val510Ala The cysteine-less/ V510A CFTR mutant exhibited channel activity at the cell surface [17]. Login to comment 95 ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala To test whether the mature and immature forms of cysteine-less CFTR/V510A still exhibited structural differences, the Figure 2 Disulfide cross-linking of cysteine mutants in wild-type or cysteine-less/V510A CFTR background Wild-type CFTR containing the M348C(TM6)/T1142C(TM12) mutations (A) or cysteine-less/V510A CFTR containing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutation (B) were expressed in HEK-293 cells. Login to comment 99 ABCC7 p.Val510Ala M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutations were introduced into cysteine-less/V510A CFTR. Login to comment 103 ABCC7 p.Thr1142Cys ABCC7 p.Val510Ala There was a little aggregation of immature CFTR, however, when cysteine-less/V510A containing the M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/ W1145C(TM12) mutations were treated with M8M cross-linker (Figure 2B). Login to comment 105 ABCC7 p.Val510Ala In contrast, no cross-linked product was detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142(TM12) and W1145C(TM12) in the cysteine-less/V510A background were each treated with M8M cross-linker (results not shown). Login to comment 106 ABCC7 p.Val510Ala These results suggest that the M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutations in the cysteine-less/V510A CFTR background could act as reporters for monitoring folding of the TMDs because only mature CFTR shows cross-linking. Login to comment 112 ABCC7 p.Val510Ala The reactions were Figure 3 Concentration-dependence of M8M cross-linking of cysteine mutants (A) HEK-293 cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background were suspended in PBS. Login to comment 124 ABCC7 p.Thr1142Cys ABCC7 p.Val510Ala Slow-migrating product was not detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142C(TM12) and W1145(TM12) in cysteine-less CFTR/V510A were each treated with M8M (results not shown) We then examined whether correctors, channel blockers or potentiators inhibited cross-linking of M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) mutants. Login to comment 135 ABCC7 p.Trp1145Cys The reactions were stopped by addition of SDS sample buffer containing no reducing agent, and samples were subjected to immunoblot analysis. Figure 4 shows that the channel blockers benzbromarone and glibenclamide (structures shown in Figure 1B) almost completely inhibited cross-linking of M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C- (TM12) mutants. Login to comment 145 ABCC7 p.Tyr563Asn ABCC7 p.Asp567Ala ABCC7 p.Asp565Ala Tyr563 , Asp565 and Asp567 in the exit motif are evolutionarily Figure 5 Effect of COPII mutations on cross-linking of cysteine mutants (A) Whole-cell SDS extracts of HEK-293 cells expressing wild-type CFTR and wild-type CFTR containing Y563N or D565A/D567A mutation were subjected to immunoblot analysis. Login to comment 146 ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Asp567Ala ABCC7 p.Asp567Ala ABCC7 p.Asp565Ala ABCC7 p.Asp565Ala (B) HEK293 cells expressing wild-type CFTR or wild-type CFTR containing Y563N or D565A/D567A mutant were surface-labelled with sulfo-NHS-SS-biotin. Login to comment 148 ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala (C) M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A CFTR background was expressed in the absence (-) or presence (+) of 15 μM corr-4a. Login to comment 151 ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala (D) Membranes were prepared from HEK-293 cells expressing M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A background that were grown in the absence (-) or presence (+) 15 μM corr-4a. Login to comment 152 ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn Samples were then treated with 0.025 mM M8M [M348C(TM6)/T1142C(TM12)/Y563N, W356C(TM6)/W1145C(TM12)/Y563N] or 0.2 mM M8M [T351C(TM6)/T1142C(TM12)/Y563N] for 10 min at 20◦C. The reactions were stopped by addition of SDS sample buffer containing 50 mM EDTA with (+) or without (-) 20 mM dithiothreitol (+DTT). Login to comment 158 ABCC7 p.Tyr563Asn ABCC7 p.Asp567Ala ABCC7 p.Asp565Ala Accordingly, we introduced the Y563N or D565A/D567A mutations in the COPII exit motif of wild-type CFTR. Login to comment 161 ABCC7 p.Tyr563Asn ABCC7 p.Asp567Ala ABCC7 p.Asp565Ala Cells expressing wild-type CFTR or Y563N or D565A/D567A mutant were treated with sulfo-NHS-SS-biotin, and then lysed with detergent. Login to comment 166 ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala The Y563N mutation was then introduced into M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background. Login to comment 167 ABCC7 p.Tyr563Asn The mutants were each expressed in HEK-293 cells to test whether the Y563N mutation would block maturation when they were expressed in the absence or presence of corr-4a. Login to comment 170 ABCC7 p.Val510Ala These results indicate that the COPII exit motif mutations in cysteine-less/V510A CFTR still blocked maturation even when expressed in the presence of a corrector. Login to comment 171 ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala Membranes were then prepared from cells transfected with M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant cDNA in the cysteine-less/V510A/Y563N background and grown in the presence or absence of corr-4a. Login to comment 178 ABCC7 p.Tyr563Asn ABCC7 p.Thr1142Cys ABCC7 p.Val510Ala To confirm that the corrector was modulating folding in the ER, we expressed wild-type CFTR and T351C(TM6)/T1142C- (TM12)/Y563N/cysteine-less/V510A mutant in the absence or presence of brefeldin A before cross-linking with M8M cross-linker. Login to comment 182 ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala Cross-linking of T351C(TM6)/ T1142C(TM12)/Y563N/cysteine-less/V510A mutant expressed in the presence of brefeldin A with or without corr-4a showed that there was more cross-linked product when corr-4a was present (Figure 6B). Login to comment 186 ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala To test whether corr-4a promoted folding of the F508 mutant in the ER, the F508 mutation was introduced into Figure 6 Effect of brefeldin A on maturation of wild-type CFTR and cross-linking analysis of T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant HEK-293 cells were transfected with wild-type or T351C(TM6)/T1142C(TM12)/Y563N/cysteineless/V510A mutant CFTR cDNAs. Login to comment 189 ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala (B) After 16 h, the medium in cells expressing T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant was replaced with fresh medium containing 10 μg/ml brefeldin A with (+) or without (-) 15 μM corr-4a. Login to comment 193 ABCC7 p.Tyr563Asn ABCC7 p.Trp1145Cys ABCC7 p.Thr1142Cys ABCC7 p.Val510Ala the double-cysteine mutants M348C(TM6)/T1142C-(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C- (TM12) in the Y563N/cysteine-less/V510A CFTR background. Login to comment 196 ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala ABCC7 p.Val510Ala Expression in the presence of corr-4a, however, increased the yield of cross-linked product of F508/M348C(TM6)/T1142C(TM12)/Y563N/cysteine-less/ V510A, F508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510Aand F508/W356C(TM6)/W1145C(TM12)/Y563N/ cysteine-less/V510A mutants to 5, 11 and 10% respectively (Figure 7). Login to comment 207 ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Tyr563Asn ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala The presence Figure 7 Effects of F508 mutation on cross-linking of COPII cysteine mutants HEK-293 cells expressing CFTR F508/M348C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A, F508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A or F508/ W356C(TM6)/W1145C(TM12)/Y563N/cysteine-less/V510A mutant were grown in the absence (-) or presence (+) of 15 μM corr-4a. Membranes were prepared, and cross-linking with M8M cross-linker was performed. Login to comment 230 ABCC7 p.Met348Cys An example is the channel blocker, benzbromarone, which also inhibited cross-linking between cysteine residues in TM6 and TM7 [I340C(TM6)/S877C(TM7) mutant] [17], as well as between cysteine residues in TM6 and TM12 [M348C- (TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants] (Figure 4). Login to comment 242 ABCC7 p.Thr1142Cys This delayed interaction between the TMDs might be reflected in the relatively low amount (2-4% of total CFTR) of cross-linked product in M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) mutants when grown in the absence of corr-4a, but is increased to 22-35% when grown in the presence of corr-4a (Figures 5D and 5E). Login to comment