ABCMdb

PMID: 7682896

Teem JL, Berger HA, Ostedgaard LS, Rich DP, Tsui LC, Welsh MJ
Identification of revertants for the cystic fibrosis delta F508 mutation using STE6-CFTR chimeras in yeast.
Cell. 1993 Apr 23;73(2):335-46., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met We isolated two AF508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBDl sequence. Login to comment 91 ABCC7 p.Arg553Met These yeast transformants each contained an H5-AF508 plasmid with a mutation at amino acid R553 of CFTR; in one case, R553 was replaced by methionine (H5-AF508/R553M), and in the other plasmid, R553 was replaced by glutamine (H5-AF508IR553Q). Login to comment 92 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met It is possible that other mutations within the R553-L558 region of the H5-AF508 plasmid could also result in increased mating efficiency; however, we proceeded to analyze the R553Q and R553M mutants in greater detail without further mutagenesis of the R553-L558 region. Login to comment 94 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met Whereas the mating efficiency of the H5-AF508 yeast strain is approximately lo/a of the H5 strain, yeast containing the H5-AF508/R553Q and H5-AF508/R553M plasmids mated at 3% and 3204 respec- 330 tively. Login to comment 96 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met However, when the mutations R553Q and R553M press the AF508 mating defect. Login to comment 97 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met ABCC7 p.Arg553Met The R553M mutation were introduced into CFTRAF508 (CFTRAF508/R553Q alone had little effect on H5 (H5R553M); when this mutant and CFTRAF508/R553M, respectively), CAMP-dependent was transformed into yeast, no further increase in mating anion permeability was restored. Login to comment 101 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met The R553Q and R553M mutations partially correct the defect in the H5-AF508 chimera and should also correct the defect in CFTRAF508 if a similar structure exists for NBDl in both proteins. Login to comment 102 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met As a test of this hypothesis, we introduced the R553Q and R553M mutations into CFTRAF508 cDNA and transfected mammalian cells with these constructs to determine whether the revertant mutations would correct the defect in CAMP-regulated Cl- transport of CFTRAF508. Login to comment 105 ABCC7 p.Arg553Gln First, the initial rate of halide efflux after CAMP stimulation was less for CFTRAF508IR553M and CFTRAF508/R553Q than for wild-type CFTR (Figure 3). Login to comment 108 ABCC7 p.Arg553Gln Thus, CAMP-stimulated halide efflux from the CFTRAF5081 R553Q- and CFTRAF508/R553Mtransfected cells was less efficient than that in cells transfected with wild-type CFTR.ThisobservationsuggeststhattheAF508CI-channel defect was only partially reversed by the reversion mutations. Login to comment 109 ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Arg553Met ABCC7 p.Arg553Met R553Q and R553M Suppress the CFTRAF508 Anion Transport Defect We assessed the effect of the R553Q and R553M mutations on CFTR function by assaying for CAMP-stimulated halide efflux using the halide-sensitive fluorophore 8-methoxy-N-(3sulfopropyl)-quinolinium (SPQ) (Illsley and Verkman, 1987). Login to comment 110 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met Expression of CFTR cDNA containing either the R553Q or the R553M mutation alone (without the AF508 mutation) in HeLa cells generated CAMP-stimulated halide efflux like wild-type CFTR (Figure 3). Login to comment 112 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met Relative Mating Efficiency of AF5OS Revertants Genotype H5R553M H5-AF508/R553M H&AF508/R553Q HSAF508 Mating Efficiency Relative to H5 (%) 77.4 f 3.7 34.2 f 7.0 3.2 i 0.8 1.1 f 0.5 Mating efficiencies were determined by quantitative mating assays and are expressed as a percentage relative to H5 (100%). Login to comment 118 ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Arg553Met ABCC7 p.Arg553Met Correction of CFTRAF508 Processing and Localization Cl- transport by CFTRAF508 containing the R553Q and R553M mutations would be detected only if the processing defect of CFTRAF508 was suppressed. Login to comment 122 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met ABCC7 p.Arg553Met CFTRAF508 is only present as the unglycosylated band A and the core glycosyl- 6000 -CFTR - AF508/R553M -c- R553M + AF508lR553Q I R553Q ,+ AF508 0 0 2 4 6 6 10 12 TIME (MIN) ated band B protein, consistent with its failure to traverse the Golgi complex and reach the plasma membrane (Cheng et al., 1990) (Figure 4). Login to comment 124 ABCC7 p.Arg553Gln Analysis of Processed Forms of CFTR (A) Expression of wild-type CFTR (lane I), CFlWR553M (lane 2) CFTR/R553Q (lane 3) and CFTRAF508 (lane 4) in HeLa cells. Login to comment 130 ABCC7 p.Arg553Met (B) Expression of wild-type CFTR (lane 1) or CFTR mutants CFTRAF508/R553M (lane 2) CFTRAF508IR553Q (lane 3) and CFTRAF508 (lane 4) in HeLa cells. Login to comment 140 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met R553Q and R553M mutations alone (without the AF508 mutation). Login to comment 141 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met As shown in Figure 4A, band C is present in cells expressing wild-type CFTR (lane 1) and also mutant CFTR containing either the R553Q or R553M mutation (lanes 2 and 3). Login to comment 143 ABCC7 p.Arg553Met Thus, the R5530 and R553M mutationsalone do not affect the glycosylation of CFTR. Login to comment 144 ABCC7 p.Arg553Met In cells transfected with the CFTRAF508/R553M (Figure 48, lane 2) a small increase in band C is detectable as compared with CFTRAF508 (lane 4). Login to comment 146 ABCC7 p.Arg553Gln We were not able to consistently detect band C CFTR in cells expressing CFTRAF508/R553Q, possibly owing to limitation8 in the sensitivity of the assay. Login to comment 147 ABCC7 p.Arg553Met Thus, a detectable increase in band C occurs only with CFTRAF508/R553M as compared with CFTRAF508. Login to comment 148 ABCC7 p.Arg553Met However, the total amount of fully glycosylated CFTRAF508/R553M is still small relative to wild-type CFfR. Login to comment 153 ABCC7 p.Arg553Met However, when cells expressed CFTRAF508/ R553M, CFTR was detected at the plasma membrane (Figure 5C). Login to comment 154 ABCC7 p.Arg553Gln For CFTRAF508/R553Q, plasma membrane staining of CFTR was weak and variable and could not be demonstrated consistently (Figure 5D). Login to comment 156 ABCC7 p.Arg553Met ABCC7 p.Arg553Met These results are consistent with the observation that more CFTRAF5081 R553M than CFTRAF508Kt553Q is found in the band C Figure 5. lmmunolocalization of Wild-Type and Mutant CFTR HeLa cells were transfected with wild-type CFTR (A) and CFTR mutants CFTRAF508 (B), CFTRAF508/R553M (C), and CFTRAF5081R553Q (D). Login to comment 160 ABCC7 p.Arg553Gln form and suggest that the amount of CFTRAF508/R553Q Single-Channel Analysis of at the plasma membrane is exceedingly low. Login to comment 161 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met Thus, these CFTRAF508/R553Q CFTR data indicate that only the CFTRAF508/R553M mutant is To determine whether the suppressor mutations altered detectable in the plasma membrane at levels greater than the single-channel properties of CFTRAF508, the re- that observed for CFTRAF508. Login to comment 162 ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln vertant mutant CFTRAF508/R553Q was expressed in A R553Q c AF508/R553Q Y---M W---I w---m m--k w----m m-----v w---w 60mV 4OmV 20mV OmV -20 mV -40 mV -60 mV -60 mV -100 mV -120 mV 1P d 1 .4ms Figure 6. Login to comment 174 ABCC7 p.Arg553Gln We chose the R553Q mutant because, as discussed below, it has been observed in a CF patient. Login to comment 185 ABCC7 p.Arg553Gln The results also indicate that, as predicted by the functional analysis of CFTRAF508/R553Q by the SPQ halide efflux assay above, at least some CFTRAF508/R5530 is localized in the plasma membrane. Login to comment 186 ABCC7 p.Arg553Gln Following activation with PKA and ATP, we measured the P, for CFfRIR553Q and CFTRAF508/R553Q at different MgATP concentrations. Login to comment 187 ABCC7 p.Arg553Gln Figures 7A and 78 show examples of the activity of single channels at different concentrations of MgATP; as the concentration of MgATP increased, both CFTRIR553Q and CFTRAF508/R553Q spent more time in the open state. Login to comment 188 ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln Figure 7C shows that the P, of CFTRIR553Q Cl- channels was similar to that previously reported for wild-type CFTR Cl- channels (An- Cdl A R553Q B AF508/R553Q Figure 7. Login to comment 193 ABCC7 p.Arg553Gln A single CFTWR553Q Cl-channel (A) and a single CFTFtAF506/R553Q Cl-channel (6) are shown at different MgATP concentrations. Login to comment 198 ABCC7 p.Arg553Gln Thus, the R553Q mutation corrected the functional defect in gating of the CFTRAF508 Cl- channels. Login to comment 208 ABCC7 p.Arg553Gln Although the R553Q revertant was less eff icient at correcting processing of CFTRAF508 as measured by the amount of fully processed CFTR present and by immunocytochemistry, it produced functional channels as measured by the SPQ halide efflux assay. Login to comment 209 ABCC7 p.Arg553Gln In addition, R553Q corrected the altered gating of CFTRAF508. Login to comment 213 ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln We speculate that the R553Q mutation must change the conformation of the CFTRAF5081 R553Q protein sufficiently to revert gating to normal and that both CFTRAF508lR553Q and CFTRAF508lR553M must adopt a conformation that allows at least some of the mutant protein to escape the cellular quality control mechanisms. Login to comment 215 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met The CFTRAF508 revertant mutations R553Q and R553M were initially identified as revertants of the mating defect in yeast containing the H5-AF508 chimera, suggesting that the structure of NBDl in the chimera (and the effect of CF mutations on NBDl structure) resembles that of CFTR. Login to comment 223 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met Because wild-type phenotypes were observed with R553Q and R553M mutations alone, these mutations may be compensatory mutations that cause no detectable phenotype themselves. Login to comment 235 ABCC7 p.Arg553Gln Interestingly, a CF patient with both the AF508 and R553Q mutations on the same chromosome has been described (Dork et al., 1991). Login to comment 236 ABCC7 p.Arg553* On the non-AF508 chromosome, the nonsense mutation R553X was found. Login to comment 238 ABCC7 p.Arg553* In contrast, the mean value for patients (n = 80) homozygous for the AF508 mutation in that clinic was 109 mmolll and was 86 mmolll for patients (n = 9) heterozygous for R553X and AF508; these values are characteristic of CF patients and significantly higher than the normal range. Login to comment 239 ABCC7 p.Arg553Gln It is interesting to speculate that the R553Q mutation may partially suppress the AF508 Cl- transport defect in the sweat gland in this patient, but is unable to suppress the defect in other affected tissues, such as the lung and pancreas, sufficiently to prevent clinical disease. Login to comment 240 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met Although the R553Q mutation corrected the defect in the P, of CFTRAF508, it was less effective than R553M in correcting the processing defect of CFTRAF508. Login to comment 241 ABCC7 p.Arg553Gln Thus, the in vivo effect of the R553Q mutation on processing and function of CFTRAF508 might be quite small. Login to comment 242 ABCC7 p.Arg553Gln However, a combination of restored CFTRAF508 channel function and improved processing might be sufficient to allow some of the CFTRAF508/R553Q mutant protein to reach the plasma membrane, where it could mediate Cl- transport in the duct of the sweat gland. Login to comment 243 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met In this regard, one might predict that the R553M mutation (or the AF508/R553Q on both chromosomes) might have a greater effect in suppressing the AF508 Cl- transport defect in the sweat gland and other organs. Login to comment 275 ABCC7 p.Arg553Gln ABCC7 p.Arg553Met Two such colonies were identified (which contained the R553Q and R553M mutations), plasmid DNA was isolated from each, and the DNA sequence of the NED1 region was determined. Login to comment 284 ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Arg553Met To express CFTR in HeLa cells transiently, cells were infected with recombinant vaccinia virus (vTF7-3) to express the T7 bacteriophage RNA polymerase and then transfected with plasmid DNA containing either wild-type CFTR (pTM-CFTR-4) or CFTR mutants (pTMCFTRAF508, pTMCFTRAF508/R553Q, pTMCFTRAF508/R553M, pTMCFTR/R553Q, and pTMCFTWR553M) under the control of the T7 promoter, essentially as described in Rich et al. (1990). Login to comment