Mutations in the Nucleotide Binding Domain 1 Signature Motif Region Rescue Processing and Functional Defects of Cystic Fibrosis Transmembrane Conductance Regulator ΔF508
2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês
10.1074/jbc.m205644200
ISSN1083-351X
AutoresAna C. deCarvalho, Lisa J. Gansheroff, John L. Teem,
Tópico(s)Neonatal Respiratory Health Research
ResumoThe gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (ΔF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRΔF508 chimera system in yeast, we isolated two novel ΔF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRΔF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRΔF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRΔF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRΔF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine. The data show that the ΔF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif. The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (ΔF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRΔF508 chimera system in yeast, we isolated two novel ΔF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRΔF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRΔF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRΔF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRΔF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine. The data show that the ΔF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif. cystic fibrosis CF transmembrane conductance regulator ATP binding cassette nucleotide binding domain CAMP-dependent protein kinase wild type Fischer rat thyroid 3-isobutyl-1-methylxanthine analysis of variance multidrug resistance-related protein Cystic fibrosis (CF)1 is the most frequent lethal genetic disease associated with a single gene in Caucasians (1Collins F.S. Science. 1992; 256: 774-779Crossref PubMed Scopus (735) Google Scholar). CF results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an ATP binding cassette (ABC) transporter that functions as a phosphorylation and nucleotide-regulated chloride channel located in the apical membrane of epithelial cells (2Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (6181) Google Scholar, 3Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (824) Google Scholar). The ABC transporters constitute a large family of ubiquitously expressed proteins, mostly involved in ATP-driven translocation of diverse substrates across biological membranes (4Young J. Holland I.B. Biochim. Biophys. Acta. 1999; 1461: 177-200Crossref PubMed Scopus (136) Google Scholar, 5Higgins C.F. Annu. Rev. 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Hwang T.C. J. Physiol. (Lond.). 2000; 524: 637-648Crossref Scopus (89) Google Scholar). The defective activity of CFTRΔF508 channel can be ameliorated pharmacologically (23Haws C.M. Nepomuceno I.B. Krouse M.E. Wakelee H. Law T. Xia Y. Nguyen H. Wine J.J. Am. J. Physiol. 1996; 270: C1544-C1555Crossref PubMed Google Scholar, 24Al-Nakkash L. Hwang T.C. Pfluegers Arch. 1999; 437: 553-561Crossref PubMed Scopus (62) Google Scholar, 25Hwang T.C. Wang F. Yang I.C. Reenstra W.W. Am. J. Physiol. 1997; 273: C988-C998Crossref PubMed Google Scholar). To identify regions in the NBD1 that are affected by ΔF508, we isolated point mutations that rescued the functional and processing defects of CFTRΔF508. Making use of the sequence homology of NBDs of CFTR and the Saccharomyces cerevisiae-mating peptide pheromone transporter, Ste6p (26McGrath J.P. Varshavsky A. Nature. 1989; 340: 400-404Crossref PubMed Scopus (493) Google Scholar, 27Kuchler K. Sterne R.E. Thorner J. EMBO J. 1989; 8: 3973-3984Crossref PubMed Scopus (352) Google Scholar), a STE6/CFTR chimera was previously developed to study the ΔF508 mutation in yeast (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 29Teem J.L. Carson M.R. Welsh M.J. Receptors Channels. 1996; 4: 63-72PubMed Google Scholar). Mutations analogous to ΔF508 disrupt the function of other ABC transporters (30Goldman B.S. Sherman D.A. Kranz R.G. J. Bacteriol. 1997; 179: 7869-7871Crossref PubMed Google Scholar, 31Gibson A.L. Wagner L.M. Collins F.S. Oxender D.L. Science. 1991; 254: 109-111Crossref PubMed Scopus (15) Google Scholar, 32Katzmann D.J. Epping E.A. Moye-Rowley W.S. Mol. Cell. Biol. 1999; 19: 2998-3009Crossref PubMed Google Scholar, 33Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 34Wemmie J.A. Moye-Rowley W.S. Mol. Microbiol. 1997; 25: 683-694Crossref PubMed Scopus (45) Google Scholar). The mutation equivalent to ΔF508 in the STE6 gene, ΔL455, did not result in a defective phenotype (35Berkower C. Michaelis S. EMBO J. 1991; 10: 3777-3785Crossref PubMed Scopus (166) Google Scholar), but in the context of the CFTR sequences, ΔF508 disrupts the a-factor transport function of STE6/CFTR chimera (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 36Paddon C. Loayza D. Vangelista L. Solari R. Michaelis S. Mol. Microbiol. 1996; 19: 1007-1017Crossref PubMed Scopus (21) Google Scholar), providing a yeast system for identification of ΔF508 revertant mutations within CFTR sequences. Here we used the STE/CFTRΔF508 chimera system to identify novel amino acid substitutions just upstream (I539T) and within (G550E) the ABC signature motif of CFTR NBD1 that partially suppressed the CFTRΔF508 defect in HeLa cells and in Fischer rat thyroid (FRT) cells. The G550E mutation introduces a negatively charged amino acid in the highly conserved LSGGQ core signature motif of CFTR NBD1. Interestingly, the site of this mutation is flanked by two residues where CF-causing mutations have been identified that impair folding/trafficking of CFTR (S549R) or ATP-dependent channel gating (G551D) (37Frossard P.M. Hertecant J. Bossaert Y. Dawson K.P. Eur. Respir. J. 1999; 13: 100-102Crossref PubMed Scopus (24) Google Scholar, 38Frossard P.M. Bakalinova D. Hertecant J. Bossaert Y. Dawson K.P. J. Trop. Pediatr. 1999; 45: 158-160Crossref PubMed Scopus (4) Google Scholar, 39Van Oene M. Lukacs G.L. Rommens J.M. J. Biol. Chem. 2000; 275: 19577-19584Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 40Qu B.H. Strickland E.H. Thomas P.J. J. Biol. Chem. 1997; 272: 15739-15744Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 41Logan J. Hiestand D. Daram P. Huang Z. Muccio D.D. Hartman J. Haley B. Cook W.J. Sorscher E.J. J. Clin. Invest. 1994; 94: 228-236Crossref PubMed Scopus (77) Google Scholar). We assessed the effect of the G550E mutation on the PKA-dependent activation of wild type and mutant CFTR chloride channel. We also investigated the effect of two compounds known to optimize PKA-dependent CFTRΔF508 activity, genistein and 3-isobutyl-1-methylxanthine (IBMX) (23Haws C.M. Nepomuceno I.B. Krouse M.E. Wakelee H. Law T. Xia Y. Nguyen H. Wine J.J. Am. J. Physiol. 1996; 270: C1544-C1555Crossref PubMed Google Scholar, 24Al-Nakkash L. Hwang T.C. Pfluegers Arch. 1999; 437: 553-561Crossref PubMed Scopus (62) Google Scholar, 25Hwang T.C. Wang F. Yang I.C. Reenstra W.W. Am. J. Physiol. 1997; 273: C988-C998Crossref PubMed Google Scholar), on the ΔF508 revertant channels. The construction of the plasmids for expression of the STE6/CFTR and STE6/CFTRΔF508 chimeras in yeast has been previously described (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar). Derived from JTS6 plasmid carrying the STE6 gene, JTS6-H5 is a single copy CEN plasmid that carries the selectable marker URA3 and the STE6/CFTR hybrid gene H5, where sequences corresponding to Ste6p amino acid residues Arg-441–Ile-516 were replaced by the corresponding region from CFTR (F494-L558). Similarly, the CFTRΔF508 sequence was used to make plasmid H5-ΔF508. A fragment containing 193 bp of CFTRΔF508 DNA flanked by STE6 sequences, 101 bp on the amino terminus and 100 bp on the carboxyl terminus, was generated by PCR amplification of plasmid H5-ΔF508 using the following primers: forward (5′-GTTCTACGATAGCTATAATGGAT-3′), and reverse (5′-GCCTAATTGCCTTCATCAACAG-3′). To generate random point mutations within this fragment, PCR reactions were performed under mutagenic conditions (42Tindall K.R. Kunkel T.A. Biochemistry. 1988; 27: 6008-6013Crossref PubMed Scopus (671) Google Scholar, 43Leung D.W. Chen E. Goeddel D.V. Technique (Phila.). 1989; 1: 11-15Google Scholar) using Taq DNA polymerase (Promega). For site-directed mutagenesis at the position corresponding to CFTR G550, degenerate DNA oligos were used in the PCR reactions to generate multiple codons. Yeast strain JPY201 (MAT a,STE6Δ::HIS3, gal2,ura5-52, lys2-801, trp1,leu2-3,112,his3Δ200) (26McGrath J.P. Varshavsky A. Nature. 1989; 340: 400-404Crossref PubMed Scopus (493) Google Scholar) was co-transformed with the linearized H5-ΔF508 plasmid and the mutagenized 394-bp DNA fragments using the lithium acetate method. Mutations were inserted into H5-ΔF508 as a result of homologous recombination (44Orr-Weaver T.L. Szostack J.W. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6354-6358Crossref PubMed Scopus (973) Google Scholar). The transformed JPY201 cells were plated in Sc-Ura medium, selective for transformants expressing the URA3 gene, and then mated with the yeast strain 22–2D4 (MATα, ura3-52,leu2-3,112, trp1). The yeast mating assays were performed as previously described (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 29Teem J.L. Carson M.R. Welsh M.J. Receptors Channels. 1996; 4: 63-72PubMed Google Scholar). Briefly, a lawn of 22-2D4 cells and transformed JPY201 colonies were replica-printed to a non-selective medium (yeast extract/peptone/dextrose) and incubated at 30 °C to allow mating. After 8 h, cells were replica-printed to a medium selective for diploids (yeast nitrogen base supplemented with leucine). After a retest, plasmid DNA was isolated from single haploid JPY201 colonies that gave rise to diploid colonies and sequenced. For a quantitative mating assay, JPY201 cells transformed with each H5 variant were grown to log phase in 0.1% glucose Sc-URA medium. From each culture, 3 × 106cells were mixed with an equal number of 22-2D4 cells and collected by filtration onto a filter that was then placed on a yeast extract/peptone/dextrose plate for 4 h at 30 °C. Cells were resuspended, sonicated briefly, and plated from serial dilutions onto selective plates. Plates were incubated for 3 days at 30 °C, and diploid colonies were counted (45Trueheart J. Boeke J.D. Fink G.R. Mol. Cell. Biol. 1987; 7: 2316-2328Crossref PubMed Scopus (358) Google Scholar). For the expression of CFTR variants in HeLa cells, pTM CFTR (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar) digested at unique SmaI and XhoI sites flanking the Phe-508 region was used to clone the sequences from the H5-ΔF508 variants amplified by PCR. For expression in FRT cells, the pSwick (pMT3-Swick) vector (46Swick A.G. Janicot M. Cheneval-Kastelic T. McLenithan J.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1812-1816Crossref PubMed Scopus (113) Google Scholar) was similarly constructed. The CFTR region of the H5-ΔF508 hybrid gene containing second site mutations was amplified by PCR using the oligos Sma-L (forward, 5′-GGATTATGCCCGGGACCATTAAAG-3′) and Xho-R (reverse, 5′-GATGAATGCTCGAGCTAAAGAAA-3′). The PCR product was digested withSmaI and XhoI, resulting in a 178-bp fragment corresponding to CFTR cDNA nucleotide residues 1630–1808 and introduced in-frame into pTM-CFTR and pSwick CFTR. Constructs were verified by DNA sequencing. HeLa cells were maintained at 37 °C in a humidified, 5% carbon dioxide atmosphere. Growth medium was minimum essential medium with Earle's salts (Sigma) supplemented with 10% fetal bovine serum (Summit Biotechnology) and 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B (Invitrogen). The vaccinia virus/bacteriophage T7 hybrid expression system (47Elroy-Stein O. Fuerst T.R. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6126-6130Crossref PubMed Scopus (385) Google Scholar) was used for transient expression of the CFTR variants in HeLa cells, as described previously (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar). Briefly, sub-confluent 100-mm plates of HeLa cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase (10 MOI) and transfected with pTM plasmids carrying each CFTR variant cDNA under the control of the T7 promoter. Cells were incubated for 18 h and lysed. FRT epitheloid cells and FRT cell lines stably expressing CFTR wt and CFTR ΔF508 (48Sheppard D.N. Carson M.R. Ostedgaard L.S. Denning G.M. Welsh M.J. Am. J. Physiol. 1994; 266: L405-L413PubMed Google Scholar) were gifts from Michael Welsh, University of Iowa. FRT cells were maintained at 37 °C in a humidified, 5% carbon dioxide atmosphere. Growth medium was Coon's modification of Ham's F-12 (Sigma) supplemented with 5% fetal bovine serum (Summit Biotechnology) and 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B (Invitrogen). Dividing FRT cells were trypsinized in a solution containing 0.25% trypsin and 0.1% EDTA, pelleted, and resuspended in serum-free F-12 Coon's medium. Aliquots of cell suspension equivalent to 5 × 105 cells were transferred to microcentrifuge tubes, pelleted, and resuspended in F-12 Coon's medium containing DMRIE-cholesterol reagent (Invitrogen) complexed with pSwick plasmid, carrying each CFTR variant cDNA. Cells were transfected at 37 °C for 2 h with slow rotation and plated on Millicell hemagglutinin-permeable cell culture inserts (pore size, 0.45 μm, Millipore Co.). Transepithelial chloride currents were recorded 4 days after transfection. FRT cells were co-transfected with pSwick CFTR and pcDNA plasmid (Invitrogen) using the DMRIE-cholesterol reagent. Clonal cell lines resistant to zeocin (Invitrogen) were expanded and screened for CFTR expression using short circuit chloride current measurements in Ussing chambers. For analysis of CFTR processing, transiently transfected HeLa cells were washed 3 times with phosphate-buffered saline and lysed with radioimmune precipitation assay buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mm Tris-HCl, pH 8.0) containing a protease inhibitor mixture (1 mm benzamidine, 5 μg/ml pepstatin, 5 μg/ml leupeptin, 1 μg/ml aprotinin, and 17.4 μg/ml phenylmethylsulfonyl fluoride). After unbroken cells and nuclei were discarded by low speed centrifugation, proteins in the lysates were denatured in SDS-gel loading buffer and stored at −20 °C. Cells growing on 100-mm plates were washed 3 times with phosphate-buffered saline, collected in 2 ml of phosphate-buffered saline, pelleted, and resuspended in ice-cold 1 ml TEAA buffer (20 mm Tris/HCl, pH 8.0, 1 mmEDTA, 3 mm EGTA) supplemented with protease inhibitor mixture (1 mm benzamidine, 5 μg/ml pepstatin, 5 μg/ml leupeptin, 1 μg/ml aprotinin, and 17.4 μg/ml phenylmethylsulfonyl fluoride). After a 5-min incubation in TEAA, the cell suspension was passed 10 times through a 27-gauge needle. Nuclei and cell debris were pelleted (5 min/4,000 × g) and discarded, and the cleared supernatant was submitted to high speed centrifugation (100,000 × g/30 min). The pelleted membranes were resuspended in SDS-gel loading buffer and stored at −20 °C. Fifty micrograms of total protein were separated by SDS-PAGE on 6% gels. Proteins were electrophoretically transferred to nitrocellulose membranes (Micron Separations Inc.). CFTR variants were probed with the monoclonal antibody M3A7, which recognizes an epitope within the CFTR NBD2 (9Kartner N. Augustinas O. Jensen T.J. Naismith A.L. Riordan J.R. Nat. Genet. 1992; 1: 321-327Crossref PubMed Scopus (334) Google Scholar). A secondary anti-mouse IgG antibody peroxidase conjugate (Jackson ImmunoResearch Laboratories Inc.) was added, and protein bands were visualized using ECL (AmershamBiosciences). FRT cell lines stably expressing CFTR variants or transiently transfected were plated at 2.5 × 105cells/cm2 on Millicell-hemagglutinin cell culture inserts. Transepithelial resistance was monitored daily (Millicell Electrical Resistance System, Millipore Co.) and was typically greater than 3000 ohms/cm2 after 4 days. FRT monolayers were mounted in modified Ussing chambers (Jim's Instruments, Iowa City, IA) and continually gassed with O2. Temperature was maintained at 37 °C. Transepithelial chloride gradient was imposed by bathing the basolateral surface with a recording solution containing 135 mm NaCl, 1.2 mm CaCl2, 1.2 mm MgCl2, 2.4 mmK2HPO4, 0.6 mmKH2PO4, 10 mm HEPES, and 10 mm dextrose, pH 7.4, and the apical surface with a similar recording solution, except that 135 mm sodium gluconate replaced the 135 mm NaCl, bringing the chloride concentration to 4.8 mm. The potential difference between the potential sensing electrodes was compensated. The transepithelial voltage was clamped to zero (voltage clamp channel module, model 558C-5, Dept. of Bioengineering, University of Iowa), and transepithelial resistance was monitored by recording current deflections in response to 2-s pulses of 1 or 5 mV every 50 s. The short circuit currents were recorded continuously on a charter recorder (model SR6335, Western Graphtec, Inc.). After a stable base-line current was observed (usually within less than 10 min), IBMX, forskolin, and genistein (Sigma) were added to the apical chamber, as described for each experiment, and the current, reflecting the flow of chloride promoted by its concentration gradient (I sc), was recorded as a downward deflection. The I sc was calculated as the difference between the base-line and the sustained phase of the response (plateau), or the peak current. Values were normalized by the area of the insert (0.6 cm2), and results were then expressed in μA/cm2. Data are expressed as the mean ± S.E. Current values were compared before normalization by the Student's t test or by one-way ANOVA with a Student-Newman-Keuls follow-up test at a 95% (p < 0.05) or greater confidence level. A STE6/CFTR hybrid gene (H5-wt), in which a region coding for 74 amino acid residues within NBD1 of STE6 (R441 to I516) was replaced by the corresponding region of the CFTR NBD1 (F494 to L558), has been previously described (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar). H5-wt complements the yeast ste6Δ mutation (JPY201 strain), restoring a-factor transport and consequent mating. However, when the ΔF508 mutation was introduced in the CFTR portion of H5 (H5-ΔF508), the mating efficiency, assessed by a quantitative mating assay, was reduced to 0.28% of H5-wt (Table I) (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar). Because the ΔF508 mutation can be modeled in yeast, this system can be used for the identification of second site mutations within the CFTR NBD1 region that restore a-factor transport function to the H5-ΔF508 chimera. To generate random point mutations, the entire CFTR portion of H5-ΔF508 was subjected to in vitro PCR mutagenesis and introduced into JPY201 yeast. Transformants were screened using a qualitative mating assay to identify colonies with an increased mating efficiency relative to H5-ΔF508. Plasmids yielding a revertant phenotype were rescued and reintroduced into JPY201 to confirm the phenotype by a quantitative mating assay. Plasmids associated with improved mating efficiencies were sequenced. Two novel point mutations were isolated in the CFTR sequence that substantially rescued the H5-ΔF508 mating defect (Table I), resulting in the change of Ile-539 of the CFTR sequence to a Thr residue (I539T) and of Gly to Glu at the 550 position (G550E).Table IYeast mating efficiency mediated by the STE6/CFTR chimerasSTE6/CFTR (H5) variantMating efficiency% of H5ΔF5080.28 ± 0.04ΔF508/I539T42.70 ± 0.40ΔF508/G550E79.90 ± 4.50Results of quantitative mating are expressed as percentage of H5-wt and represent the mean ± S.E. for n = 3 experiments. Open table in a new tab Results of quantitative mating are expressed as percentage of H5-wt and represent the mean ± S.E. for n = 3 experiments. The two novel ΔF508 revertant mutations isolated in yeast were located either just upstream (I539T) or within (G550E) the CFTR NBD1 signature motif (Fig. 1). Interestingly, the three ΔF508 revertant mutations previously isolated using the STE6/CFTR system, R553Q, R553M, and R555K, are also located within the NBD1 signature motif (28Teem J.L. Berger H.A. Ostedgaard L.S. Rich D.P. Tsui L.C. Welsh M.J. Cell. 1993; 73: 335-346Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 29Teem J.L. Carson M.R. Welsh M.J. Receptors Channels. 1996; 4: 63-72PubMed Google Scholar). To evaluate the effect of the novel revertant mutations on CFTRΔF508 processing, I539T and G550E mutations were introduced into the full-length CFTRΔF508 cDNA (ΔF/I539T and ΔF/G550E) for expression in mammalian cells. To test whether the combination of the I539T and G550E mutations would result in an additive or synergistic effect in correcting the ΔF508 phenotype, we also constructed a CFTRΔF508 allele containing both revertant mutations (ΔF/DB). The CFTR variants were expressed in HeLa cells using a vaccinia virus hybrid expression system, and the steady-state level of CFTR protein in the cell lysates was analyzed by SDS-PAGE followed by Western analysis (Fig.2 A). Only the core-glycosylated endoplasmic reticulum form of CFTR, referred to as "band B," was observed for CFTRΔF508. CFTR wt was present as both band B and "band C" forms. The latter represents mature CFTR that trafficked to the Golgi, where complex oligosaccharide processing takes place. We observed that I539T and, to a lesser extent, G550E partially rescued the CFTRΔF508-processing defect. The steady-state level of mature CFTRΔF/DB protein was higher compared with each revertant alone (Fig. 2 A).Figure 2The I539T and G550E mutations partially rescue CFTRΔF508-processing and functional defects. A, the steady-state level of CFTR protein resulting from transient expression in HeLa cells. HeLa cells were infected with vTF7–3 and transfected with pTM plasmid carrying each CFTR variant cDNA. Cell lysates were obtained 18 h after transfection, and 50-μg protein samples were separated by SDS-PAGE. Western blots were probed with anti-CFTR monoclonal antibody M3A7. HeLa cells infected with vTF7–3 served as the control. Positions of the core glycosylated (band B) and complex glycosylated CFTR (band C) are indicated by the arrows. B, FRT monolayers transiently expressing the CFTRΔF508 variants were incubated in permeable supports for 4 days at 37 °C. Monolayers were mounted in Ussing chambers, and transepithelial chloride currents were recorded after
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