ABCMdb

PMID: 21998193

Patrick AE, Karamyshev AL, Millen L, Thomas PJ
Alteration of CFTR transmembrane span integration by disease-causing mutations.
Mol Biol Cell. 2011 Dec;22(23):4461-71. Epub 2011 Oct 12., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg In this study, the ER luminal integration profiles of TM1 and TM2 were determined using the ER glycosylation machinery, and the effects of the CF-causing mutations G85E and G91R thereon were assessed. Login to comment 5 ABCC7 p.Gly85Glu G85E misfolding is based in TM1 destabilization by glutamic acid and loss of glycine and correlates with the temperature-insensitive ER accumulation of immature full-length CFTR harboring the mutation. Login to comment 6 ABCC7 p.Gly91Arg By contrast, temperature-dependent misfolding owing to the G91R mutation depends on the introduction of the basic side chain rather than the loss of the glycine. Login to comment 36 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg The CF-causing mutants G91R and G85E are in the original predicted TM1 span, residues 81-102 (Riordan et al., 1989). Login to comment 41 ABCC7 p.Gly91Arg Consistent with reduced stability, full-length G91R CFTR accumulates in the ER, and multiple domains exhibit increased proteolytic susceptibility in mammalian cells (Du and Lukacs, 2009). Login to comment 66 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg In this study, the span boundaries or integration profiles of TM1 and TM2 were determined and the effects of the CF-causing mutations G85E and G91R assessed. Login to comment 67 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg The G91R and G85E mutations Figure 1: Predicted TM1 and TM2 spans. Login to comment 69 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg The positions of the CF-causing mutants G85E and G91R are indicated by triangles. Login to comment 71 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg This work further elucidates CFTR membrane-spanning structures and provides mechanistic insight into the molecular pathology of the G85E and G91R CF-causing mutations. Login to comment 76 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Moreover, when the CF-causing mutations G85E and G91R were analyzed using these algorithms, variable effects of mutations were predicted, including shortening, no affect, no TM, or shifting TM1 boundaries (Supplemental Table S1). Login to comment 114 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Core glycosylation analysis of WT, G91R, and G85E CFTR containing the artificial ECL1 site and lacking the ECL4 sites was performed by deletion of residues between the glycosylation site and TM1 (A) or between the glycosylation site and TM2 (B). Login to comment 117 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Schematics of the experimentally identified ER integration profiles in WT (C) or G91R (D) and G85E (E) mutants. Login to comment 130 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Determining the ER integration profiles of CF-causing mutants To assess effects of the CF-causing mutants G91R and G85E on the TM1 ER integration profile, these mutations were analyzed using the ECL1 site core glycosylation assay. Login to comment 131 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg The G85E and G91R mutations introduce an ionizable group into or near the predicted TM1 span and might be reasonably expected to alter its ER integration profile (Xiong et al., 1997). Login to comment 133 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg The G91R and G85E mutations in CFTR containing the natural ECL4 sites or the ECL1 site resulted in misfolding and accumulation in the ER (Supplemental Figure S3). Login to comment 134 ABCC7 p.Gly85Glu G85E reduced CFTR maturation and degradation in constructs containing either the natural ECL4 sites or the artificial ECL1 site (Supplemental Figure S4). Login to comment 136 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Thus, in the same manner as WT ECL1, glycosylation analysis was used to characterize the TM1 integration profiles for the G91R and G85E mutants. Login to comment 137 ABCC7 p.Gly91Arg ABCC7 p.Gly91Arg Cystic fibrosis-causing mutant G91R shifts the ER integrated profile of TM1 The effect of the G91R mutation on the TM1 ER integrated profile was tested by its introduction into the ECL1 core glycosylation assay. Login to comment 139 ABCC7 p.Gly91Arg By contrast, G91R N-terminal deletion constructs were completely core glycosylated until a 14-residue deletion resulted in partial core glycosylation, and a 16-residue deletion was completely nonglycosylated (Figure 4A), indicating the edge of the mutant TM integration profile shifted by two or three residues. Login to comment 140 ABCC7 p.Gly91Arg Thus, instead of I86 as in the wild type, L88 is 12 residues from the ECL1 site in the mutant, positioning it at the G91R TM1 ER luminal integration profile edge (Figure 4D). Login to comment 141 ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu Cystic fibrosis-causing mutant G85E dramatically alters the TM1 integration profile in the ER membrane The effect of the G85E mutation on the TM1 ER integration profile was examined by introducing it into the ECL1 core glycosylation assay. Login to comment 143 ABCC7 p.Gly85Glu In the presence of G85E, the unmodified ELC1 site is not glycosylated (Figure 4A). Login to comment 146 ABCC7 p.Gly85Glu This aberrant pattern is in stark contrast to WT TM1 and is consistent with multiple conformations and/or profiles of G85E TM1 in the ER membrane, with the two extreme positions defined by the addition of two residues and the deletion of 14 residues. Login to comment 148 ABCC7 p.Gly91Arg As is the case for G91R, the L88 TM1 integration profile edge is slightly shifted from WT. Login to comment 149 ABCC7 p.Gly85Glu However, the R104 TM1 integration profile edge is dramatically shifted from WT, indicating a significant disruption in the G85E TM1. Login to comment 150 ABCC7 p.Gly85Glu Comparison of the experimental integration profile edge to predicted TM1 boundaries reveals that it is surprisingly close to several of the original predicted WT TM1 boundaries (Figure 1) and the Kyte and Doolittle scale-predicted G85E TM1 (Supplemental Table S1). Login to comment 151 ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu G85E alters the position of TM1 in the membrane The positioning of G85E TM1 in the membrane as monitored by OST accessibility is altered as compared with WT. Login to comment 154 ABCC7 p.Gly85Glu CFTR has a cysteine at position 76, which is within the experimentally derived WT, but not G85E, TM1 integration profile. Login to comment 163 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg The reaction of C76 with AMS in the presence of G91R and G85E was also tested. Login to comment 164 ABCC7 p.Gly91Arg In WT and G91R, no shift is observed, indicating that position 76 is protected by the membrane (Figure 5, B and C). Login to comment 165 ABCC7 p.Gly85Glu In contrast, G85E C76 reacts with AMS, indicating that this position is no longer protected by the membrane (Figure 5, B and C). Login to comment 166 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg This result is consistent with glycosylation scanning results and an initial positioning of TM1 in the presence of G85E that is more C-terminal than for either WT or G91R. Login to comment 168 ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Arg Role of the ionizable side chain in altered G91R and G85E TM1 ER integration profiles In the G91R and G85E mutants, an ionizable side chain replaces the glycine Cα hydrogen. Login to comment 170 ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala To investigate whether the mutant effects on folding and TM boundaries are caused by the loss of glycine or by the introduction of the ionizable group, the 85 and 91 positions were mutated to the neutral residue alanine (G91A and G85A). Login to comment 172 ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala In the ECL-site assay, G91A and G85A N-terminal deletion constructs were glycosylated until a 14-residue deletion was partially glycosylated and a 16-residue deletion was not glycosylated (Figure 6A). Login to comment 173 ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala L88 is at the edge of the TM1 ER integration profile edges for both G91A and G85A (Figure 6B), which are shifted by two or three residues from the WT TM1 ER integration profile edge. Login to comment 174 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg This integration profile is the same as the G91R TM1integration profile and one of the two extreme G85E TM1 integration profiles. Login to comment 175 ABCC7 p.Gly85Glu ABCC7 p.Gly85Ala The two-residue boundary shifts are consistent with the substitution for glycine causing a small but consistent decreased distance between the ECL1 site and the ER membrane. It is striking that the G85A TM1 glycosylation pattern does not indicate multiple profiles for TM1, indicating that the aberrant pattern of G85E TM1 results from introduction of the glutamate side chain (Figure 6A). Login to comment 176 ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Arg ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala Role of the ionizable side chain in trafficking of G91R and G85E The data from the glycosylation assay demonstrate that the G85E mutant splits the integration profile of TM1, whereas the G91R, G85A, and G91A mutants do not. Login to comment 179 ABCC7 p.Gly91Arg ABCC7 p.Gly91Ala G91A is both core and complex glycosylated and traffics to the cell surface, indicating that introduction of arginine rather than loss of glycine causes G91R ER accumulation. Login to comment 180 ABCC7 p.Gly91Ala Consistent with this, the G91A mutant has unaltered topology and WT-like degradation in X. laevis oocytes (Xiong et al., 1997). Login to comment 181 ABCC7 p.Gly85Ala However, G85A was not assessed in the Xenopus system. Login to comment 182 ABCC7 p.Gly85Glu ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala In stark contrast to G91A, G85A accumulates in the ER, suggesting that both introduction of charge and loss of glycine at position 85 contribute to G85E ER accumulation. Login to comment 185 ABCC7 p.Gly91Ala At both temperatures, G91A traffics like WT CFTR. Login to comment 186 ABCC7 p.Gly91Arg ABCC7 p.Gly85Ala At the lower temperature, both G91R and G85A mutants partially traffic from the ER and are thus temperature sensitive, similar to ΔF508. Login to comment 187 ABCC7 p.Gly85Glu It is striking that the G85E mutant exhibited temperature-insensitive ER accumulation. Login to comment 188 ABCC7 p.Gly85Glu This observation cannot be accounted for by lower protein expression of G85E, as band B is not measurably altered. Login to comment 189 ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu Of importance, G85E temperature-insensitive ER accumulation correlates with the G85E- perturbed TM1 integration profile with an edge at R104. Login to comment 194 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg This study determined the TM1 and TM2 ER luminal integration profile edges and CF-causing mutant G91R and G85E effects on TM1, using the mammalian ER luminal core glycosylation machinery. Login to comment 204 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg (B) A CFTR construct containing C76 with G91R or G85E was exposed to AMS and examined for the presence of an interaction by gel shift. Login to comment 205 ABCC7 p.Gly85Glu G85E has a gel shift, indicating that C76 is exposed to cytosol. Login to comment 206 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg (C) Schematics of G91R and G85E mutant CFTR three-TM constructs with the relative positions of C76 and the mutants labeled. Login to comment 235 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala HeLa cell trafficking of WT, ΔF508, G85E, G85A, G91R, and G91A mutant CFTR at 37°C was analyzed by Western blot analysis (A). Login to comment 240 ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala Figure 6: Experimental TM1 ER luminal integration profile edges for G91A and G85A mutant CFTR. Login to comment 241 ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala (A) Core glycosylation analysis of G91A and G85A mutant CFTR containing the artificial ECL1 site by deletion of residues between the glycosylation site and TM1. Login to comment 244 ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala (B) A combined schematic of the experimentally identified TM1 ER integration profiles for the G91A and G85A mutants. Login to comment 249 ABCC7 p.Gly91Arg Thus CF mutations, such as G91R, in the C-terminal region may be within or exposed to the ER lumen during translation and integration. Login to comment 250 ABCC7 p.Gly91Arg Consistent with this is the modest shift in the G91R-CFTR TM1 ER integration profile. Login to comment 251 ABCC7 p.Gly91Arg ABCC7 p.Gly91Ala The data here and in previous reports indicate that G91R, but not G91A, disrupts CFTR trafficking in the cell and has significant effects on the stability and assembly of full-length CFTR (Xiong et al., 1997; Younger et al., 2006; Rosser et al., 2008; Du and Lukacs, 2009). Login to comment 253 ABCC7 p.Gly85Glu The core glycosylation experiments demonstrate that G85E is within TM1 and causes at least two TM1 positions with distinct ER integration profiles. Login to comment 254 ABCC7 p.Gly85Glu There are several alternate integration profiles within the monitored G85E constructs, for which multiple potential explanations exist. Login to comment 255 ABCC7 p.Gly85Glu One is that G85E samples distinct conformations during integration, and specific conformational distributions between the different constructs are being monitored. Login to comment 256 ABCC7 p.Gly85Glu The mutation G85E might also lead to different interactions between the TM span and the translocation machinery, similar to interactions mediated by an acidic residue within TM8 of CFTR (Pitonzo et al., 2009). Login to comment 257 ABCC7 p.Gly85Glu Polar residues have also been shown to drive associations between TM spans (Choma et al., 2000; Zhou et al., 2000, 2001); therefore G85E could result in altered interactions between TM1 and other TM spans that are reflected in the glycosylation pattern. Login to comment 258 ABCC7 p.Gly85Glu This would indicate that the perturbed pattern for G85E could be due to more than a simple alteration of the 12-residue rule and that perturbations associated with introduction of an acidic residue may be occurring. Login to comment 259 ABCC7 p.Gly85Glu However, positioning of the G85E TM1 in the membrane monitored by cysteine exposure was found to be more C-terminal in a three-TM span construct that lacked the other nine CFTR TM spans. Login to comment 260 ABCC7 p.Gly85Glu Thus the simplest interpretation is that the integration profile defect is consistent with a distinct placement or destabilization of G85E TM1 in the membrane. Login to comment 261 ABCC7 p.Gly85Glu Of interest, the most C-terminal extreme G85E TM1 boundary is within several residues of the original predicted WT TM1 span boundary (Figure 1) and a boundary predicted by TopPred KD (Supplemental Table S1). Login to comment 263 ABCC7 p.Gly85Glu Thus G85E destabilization is an early-folding defect potentially recognizable in the ER before translation is complete. Login to comment 264 ABCC7 p.Gly85Ala Furthermore, the G85A nonpolar mutation does not perturb the TM1 integration profile and continues to cause CFTR ER accumulation. Login to comment 265 ABCC7 p.Gly85Glu Consequently, G85E misfolding results from both introduction of an ionizable group and glycine loss, which respectively correlate with temperature-insensitive and temperature-sensitive accumulation in the ER. Login to comment 268 ABCC7 p.Gly85Glu This complex has been implicated in the recognition and removal of improperly folded CFTR, including G85E mutant CFTR (Sun et al., 2006; Younger et al., 2006; Wang et al., 2008). Login to comment 269 ABCC7 p.Gly85Ala Experiments designed to test the role of Derlin-1 in the recognition of the G85A mutant would be a reasonable future step toward distinguishing between these two models. Login to comment 270 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Previous work demonstrated that the G85E and G91R mutations also disrupt later steps in CFTR folding, particularly interdomain interactions, which were proposed to underlie mutant recognition by ER quality control machinery (Xiong et al., 1997). Login to comment 271 ABCC7 p.Gly85Glu The results presented here demonstrate that G85E dramatically alters the integration profile of TM1. Login to comment 273 ABCC7 p.Gly91Arg The G91R mutant was predicted to have a similar effect on CFTR (Xiong et al., 1997). Login to comment 274 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg Yet the experimental evidence presented here distinguishes G91R from G85E with respect to perturbations from the ionizable side chain, the role of glycine, and temperature sensitivity. Login to comment 277 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg It is striking that the corrector compound 4 exhibited mutant-specific effects, partially rescuing the G91R but not G85E CFTR (Grove et al., 2009). Login to comment 286 ABCC7 p.Gly85Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Ala ABCC7 p.Gly85Ala The mutations G91R, G91A, G85E, and G85A were introduced into CFTR constructs containing the natural, artificial, and deletion mutants on the artificial site. Login to comment