Localization of Sequences within the C-terminal Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Which Impact Maturation and Stability
2001; Elsevier BV; Volume: 276; Issue: 2 Linguagem: Inglês
10.1074/jbc.m003672200
ISSN1083-351X
AutoresMartina Gentzsch, John R. Riordan,
Tópico(s)Cellular transport and secretion
ResumoSome disease-associated truncations within the 100-residue domain C-terminal of the second nucleotide-binding domain destabilize the mature protein (Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N., and Lukacs, G. L. (1999) J. Biol. Chem. 274, 21873–21877). We now have identified three short oligopeptide regions in the C-terminal domain which impact cystic fibrosis transmembrane conductance regulator (CFTR) maturation and stability in different ways. A highly conserved hydrophobic patch (region I) formed by residues 1413–1416 (FLVI) was found to be crucial for the stability of the mature protein. Nascent chain stability was severely decreased by shortening the protein by 81 amino acids (1400X). This accelerated degradation was sensitive to proteasome inhibitors but not influenced by brefeldin A, indicating that it occurred at the endoplasmic reticulum. The five residues at positions 1400 to 1404 (region II) normally maintain nascent CFTR stability in a positional rather than a sequence-specific manner. A third modulating region (III) constituted by residues 1390 to 1394 destabilizes the protein. Hence the nascent form regains stability on further truncation back to residues 1390 or 1380, permitting some degree of maturation and a low level of cyclic AMP-stimulated chloride channel activity at the cell surface. Thus while not absolutely essential, the C-terminal domain strongly modulates the biogenesis and maturation of CFTR. Some disease-associated truncations within the 100-residue domain C-terminal of the second nucleotide-binding domain destabilize the mature protein (Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N., and Lukacs, G. L. (1999) J. Biol. Chem. 274, 21873–21877). We now have identified three short oligopeptide regions in the C-terminal domain which impact cystic fibrosis transmembrane conductance regulator (CFTR) maturation and stability in different ways. A highly conserved hydrophobic patch (region I) formed by residues 1413–1416 (FLVI) was found to be crucial for the stability of the mature protein. Nascent chain stability was severely decreased by shortening the protein by 81 amino acids (1400X). This accelerated degradation was sensitive to proteasome inhibitors but not influenced by brefeldin A, indicating that it occurred at the endoplasmic reticulum. The five residues at positions 1400 to 1404 (region II) normally maintain nascent CFTR stability in a positional rather than a sequence-specific manner. A third modulating region (III) constituted by residues 1390 to 1394 destabilizes the protein. Hence the nascent form regains stability on further truncation back to residues 1390 or 1380, permitting some degree of maturation and a low level of cyclic AMP-stimulated chloride channel activity at the cell surface. Thus while not absolutely essential, the C-terminal domain strongly modulates the biogenesis and maturation of CFTR. cystic fibrosis transmembrane conductance regulator endoplasmic reticulum nucleotide-binding domain transmembrane domain adenine nucleotide binding cassette PSD-95, disc-large, ZO-1 brefeldin A N-acetyl-leucinyl-leucinyl-norleucinal N-carbobenzoxyl-leucinyl-leucinyl-leucinal 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucinyl-leucinyl-leucinyl-vinylsulfone phosphate-buffered saline The cystic fibrosis transmembrane conductance regulator (CFTR)1 is a large multidomain membrane protein that forms a tightly regulated chloride channel in the apical membrane of many chloride secreting and reabsorbing epithelial cells (1Seibert F.S. Loo T.W. Clarke D.M. Riordan J.R. J. Bioenerg. Biomembr. 1997; 29: 429-442Crossref PubMed Scopus (38) Google Scholar, 2Akabas M.H. J. Biol. Chem. 2000; 275: 3729-3732Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). As an adenine nucleotide binding cassette (ABC) protein it contains two nucleotide-binding domains (NBD1 and NBD2) and two transmembrane domains (TMD1 and TMD2), each spanning the membrane several times. CFTR also has a large R-domain between NBD1 and TMD2 which is not common to other ABC proteins and is the site of regulatory phosphorylation and dephosphorylation (3Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (555) Google Scholar, 4Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (475) Google Scholar, 5Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-1410Crossref PubMed Google Scholar, 6Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (375) Google Scholar). Additionally there are N- and C-terminal cytoplasmic domains of less than 100 amino acids each that precede TMD1 and follow NBD2, respectively. The former has recently been shown to participate in channel regulation by interacting with the R-domain (7Naren A.P. Cormet-Boyaka E. Fu J. Villain M. Blalock J.E. Quick M.W. Kirk K.L. Science. 1999; 286: 544-548Crossref PubMed Scopus (117) Google Scholar). The C-terminal domain, however, is apparently not essential to channel function (8Rich D.P. Gregory R.J. Cheng S.H. Smith A.E. Welsh M.J. Receptors Channels. 1993; 1: 221-232PubMed Google Scholar, 9Zhang L. Wang D. Fischer H. Fan P.D. Widdicombe J.H. Kan Y.W. Dong J.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10158-10163Crossref PubMed Scopus (54) Google Scholar, 10Haardt M. Benharouga M. Lechardeur D. Kartner N. Lukacs G.L. J. Biol. Chem. 1999; 274: 21873-21877Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) but the very C terminus can tether the protein to PDZ domain containing proteins (11Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (250) Google Scholar, 12Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar), possibly to localize CFTR within regulatory complexes. Wild-type CFTR matures very inefficiently following synthesis on membrane-bound ribosomes (13Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar) and many disease-associated mutations in different domains preclude the formation of any mature protein that can be transported to the cell surface. Hence, it is important to understand the role of each domain of the molecule in achieving and maintaining a mature and stable conformation. Frameshift or premature stop mutations found in patients with cystic fibrosis that cause truncations at several locations in the C-terminal domain were shown to destabilize the mature CFTR protein so that its lifetime was greatly shortened (10Haardt M. Benharouga M. Lechardeur D. Kartner N. Lukacs G.L. J. Biol. Chem. 1999; 274: 21873-21877Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We have now made systematic stepwise truncations as well as deletions and substitutions across the entire C-terminal domain and identified different short sequences which strongly influence not only the stability of the mature protein but also the maturation and stability by the nascent chain. The major determinant of the steady state amount of mature protein is a hydrophobic "patch" formed by residues 1413 to 1416 whereas there are strong "positional effects" on the nascent chain of sequences closer to NBD2. Truncation back to residue 1400 resulted in an extremely unstable nascent chain, degraded at the endoplasmic reticulum (ER) by the proteasome. Strikingly, on further truncation back to residues 1390 or 1380 the nascent protein regains stability such that some mature protein is again formed that mediates a low but detectable level of cAMP-stimulated chloride efflux. These findings reveal that C-terminal motifs and their positioning have a major impact on the assembly and stability of the CFTR ion channel. All C-terminal truncations were constructed by polymerase chain reaction introducing a TAG stop codon flanked by an ApaI restriction site. The polymerase chain reactions were performed on pBQ4.7 CFTR plasmid DNA. The antisense primers introducing the stop codon were the following: A1440X, 5′-TGATATCGGGCCCCTATTGCCGGAAGAGGCTCCTCTCGTT-3′; S1435X, 5′-TGATATCGGGCCCCTACCTCTCGTTCAGCAGTTTCTGGATGG-3′; L1430X, 5′-TGATATCGGGCCCCTATTTCTGGATGGAATCGTACTGCCGCAC-3′; D1425X, 5′-TGATATCGGGCCCCTAGTACTGCCGCACTTTGTTCTCTTCTATG-3′; K1420X, 5′-TGATATCGGGCCCCTAGTTCTCTTCTATGACCAAAAATTGTTGGC-3′; V1415X, 5′-TGATATCGGGCCCCTACAAAAATTGTTGGCATTCCAGCATTGC-3′; C1410X, 5′-TGATATCGGGCCCCTATTCCAGCATTGCTTCTATCCTGTGTTC-3′; E1405X, 5′-TGATATCGGGCCCCTATATCCTGTGTTCACAGAGAATTACTGTGC-3′; C1400X, 5′-TGATATCGGGCCCCTAGAGAATTACTGTGCAATCAGCAAATGC-3′; E1405X, 5′-TGATATCGGGCCCCTATATCCTGTGTTCACAGAGAATTACTGTGC-3′; C1395X, 5′-TGATATCGGGCCCCTAATCAGCAAATGCTTGTTTTAGAGTTCTTC-3′; Q1390X, 5′-TGATATCGGGCCCCTATTTTAGAGTTCTTCTAATTATTTGGTATGTTAC-3′; T1380X, 5′-TGATATCGGGCCCCTATACTGGATCCAAATGAGCACTGGGTTC-3′. The sense primer was derived from nucleotides 3551–3579 (5′-TGAGTACATTGCAGTGGGCTGTAAACTCC-3′). The fragments generated by polymerase chain reaction were subcloned into pBluescript SK+. The BprPI-ApaI fragment of the created plasmids was then used to replace the corresponding fragment of pNUT-D1270V CFTR. This plasmid, derived from pNUT-CFTR, contains an engineered ApaI site at the 3′ end of CFTR. 2X.-B. Chang and J. R. Riordan, unpublished data. Replacements of various amino acids with alanine and the deletion of amino acids 1400 to 1404 (Δ1400–1404) were constructed using the QuickChange Site-directed Mutagenesis kit from Stratagene. A modified pBQ4.7 (M1475V CFTR, with CFTR coding sequence identical to the one in pBQ6.2 (14Rommens J.M. Dho S. Bear C.E. Kartner N. Kennedy D. Riordan J.R. Tsui L.-C. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7500-7504Crossref PubMed Scopus (89) Google Scholar)) was utilized as a template for the polymerase chain reaction mutagenesis. The following oligonucleotides were used to introduce the indicated mutations into wild-type CFTR cDNA. F1413A/L1414A/V1415A/I1416A/E1417A, 5′-GCTGGAATGCCAACAAGCTGCGGCCGCAGCAGAGAACAAAGTGCGG-3′ and 5′-CCGCACTTTGTTCTCTGCTGCGGCCGCAGCTTGTTGGCATTCCAGC-3′; F1413A/L1414A/V1415A/I1416A, 5′-GCTGGAATGCCAACAAGCTGCGGCCGCAGAAGAGAACAAAGTGCGG-3′ and 5′-CCGCACTTTGTTCTCTTCTGCGGCCGCAGCTTGTTGGCATTCCAGC-3′; Q1411A/Q1412A, 5′-GGATAGAAGCAATGCTGGAATGCGCAGCATTTTTGGTCATAGAAG-3 and 5′-CTTCTATGACCAAAAATGCTGCGCATTCCAGCATTGCTTCTATCC-3; F1413A/L1414A, 5′-GCTGGAATGCCAACAAGCTGCGGTCATAGAAGAGAACAAAGTGCG-3′ and 5′-CGCACTTTGTTCTCTTCTATGACCGCAGCTTGTTGGCATTCCAGC-3′; L1414A/V1415A, 5′-GCTGGAATGCCAACAATTTGCGGCCATAGAAGAGAACAAAGTGCGG-3′ and 5′-CCGCACTTTGTTCTCTTCTATGGCCGCAAATTGTTGGCATTCCAGC-3′; V1415A/I1416A, 5′-GGAATGCCAACAATTTTTGGCCGCAGAAGAGAACAAAGTGCGGCAG-3′ and 5′-CTGCCGCACTTTGTTCTCTTCTGCGGCCAAAAATTGTTGGCATTCC-3′; E1417A/E1418A, 5′-GCCAACAATTTTTGGTCATAGCAGCGAACAAAGTGCGGCAGTACG-3′ and 5′-CGTACTGCCGCACTTTGTTCGCTGCTATGACCAAAAATTGTTGGC-3′; F1413A, 5′-GCAATGCTGGAATGCCAACAAGCTTTGGTCATAGAAGAGAAC-3′ and 5′-GTTCTCTTCTATGACCAAAGCTTGTTGGCATTCCAGCATTGC-3′; L1414A, 5′-GCTGGAATGCCAACAATTTGCGGTCATAGAAGAGAACAAAGTGCG-3′ and 5′-CGCACTTTGTTCTCTTCTATGACCGCAAATTGTTGGCATTCCAGC-3′; V1415A, 5′-GGAATGCCAACAATTTTTGGCCATAGAAGAGAACAAAGTGCGGCAG-3′ and 5′-CTGCCGCACTTTGTTCTCTTCTATGGCCAAAAATTGTTGGCATTCC-3′; I1416A, 5′-GGAATGCCAACAATTTTTGGTCGCAGAAGAGAACAAAGTGCGGCAG-3′ and 5′-CTGCCGCACTTTGTTCTCTTCTGCGACCAAAAATTGTTGGCATTCC-3′; E1417A, 5′-GCCAACAATTTTTGGTCATAGCAGAGAACAAAGTGCGGCAGTACG-3′ and 5′-CGTACTGCCGCACTTTGTTCTCTGCTATGACCAAAAATTGTTGGC-3′; C1400A/E1401A/H1402A/R1403A/I1404A, 5′-GCACAGTAATTCTCGCTGCAGCCGCGGCAGAAGCAATGCTGGAATGCC-3′ and 5′-GGCATTCCAGCATTGCTTCTGCCGCGGCTGCAGCGAGAATTACTGTGC-3′; Δ1400–1404: 5′-GCATTTGCTGATTGCACAGTAATTCTCGAAGCAATGCTGGAATGCC-3′ and 5′-GGCATTCCAGCATTGCTTCGAGAATTACTGTGCAATCAGCAAATGC-3′; C1400A/E1401A, 5′-GATTGCACAGTAATTCTCGCTGCACACAGGATAGAAGCAATGC-3′ and 5′-GCATTGCTTCTATCCTGTGTGCAGCGAGAATTACTGTGCAATC-3′; H1402A/R1403A, 5′-CAGTAATTCTCTGTGAAGCCGCGATAGAAGCAATGCTGGAATGCC-3′ and 5′-GGCATTCCAGCATTGCTTCTATCGCGGCTTCACAGAGAATTAC TG-3′; I1404A/E1405A, 5′-CTCTGTGAACACAGGGCAGCAGCAATGCTGGAATGCCAAC-3′ and 5′-GTTGGCATTCCAGCATTGCTGCTGCCCTGTGTTCACAGAG-3′, Q1390A/A1391A/F1392A/A1393A/D1394A, 5′-GAAGAACTCTAAAAGCAGCAGCTGCTGCTTGCACAGTAATTCTC-3′ and 5′-GAGAATTACTGTGCAAGCAGCAGCTGCTGCTTTTAGAGTTCTTC-3′. The BprPI-ApaI fragment of pBQ 4.7 M1475VCFTR and the derived mutated plasmids were used to replace the corresponding fragment of pNUT-D1270V CFTR. The sequences of all inserted fragments were confirmed by DNA sequencing. Baby hamster kidney cells grown at 37 °C in 5% CO2 were stably transfected and analyzed as described earlier (15Loo M.A. Jensen T.J. Cui L. Hou Y.-X. Chang X.-B. Riordan J.R. EMBO J. 1998; 17: 6879-6887Crossref PubMed Scopus (303) Google Scholar). Cells were washed twice with PBS containing 0.1 m CaCl2 and 1 mmMgCl2 (PBS++) and incubated for 2 min with 10 mm sodium periodate in PBS++ in the dark. Cells were washed twice with 0.1 m sodium acetate, pH 5.5, with 0.1 mm CaCl2 and 1 mm MgCl2and incubated for 1 min with 1 mm biotin-LC-hydrazide (Pierce), in the same buffer, in the dark. The labeling buffer was aspirated and cells were incubated for 2 min in 0.1 mTris-HCl, pH 7.5, with 1% bovine serum albumin, 0.1 mCaCl2, and 1 mm MgCl2 to stop the reaction. After washing 5 times with ice-cold PBS++, cells were lysed and biotinylated proteins were isolated with immobilized streptavidin (Pierce) as we have described recently (17Chang X. Cui L. Hou Y. Jensen T.J. Aleksandrov A.A. Mengos A. Riordan J.R. Mol. Cell. 1999; 4: 137-142Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Stably transfected BHK-21 cells were starved in methionine-free medium for 30 min, labeled for 20 min with 0.1 mCi/ml [35S]methionine, and chased with Dulbecco's modified Eagle's medium/F-12 supplemented with 1 mm methionine and 5% fetal bovine serum. Cell lysis and immunoprecipitation were performed as described before (15Loo M.A. Jensen T.J. Cui L. Hou Y.-X. Chang X.-B. Riordan J.R. EMBO J. 1998; 17: 6879-6887Crossref PubMed Scopus (303) Google Scholar). With the exception of the 1380X CFTR mutant, all immunoprecipitations were performed using the monoclonal antibody M3A7 (16Kartner N. Augustinas O. Jensen T.J. Naismith A.L. Riordan J.R. Nat. Genet.. 1992; 1: 321-327Crossref PubMed Scopus (330) Google Scholar). Since the molecule 1380X does not contain the complete M3A7 epitope, it was immunoprecipitated with the L12B4 antibody (16Kartner N. Augustinas O. Jensen T.J. Naismith A.L. Riordan J.R. Nat. Genet.. 1992; 1: 321-327Crossref PubMed Scopus (330) Google Scholar). The amount of35S-radioactivity in each band was quantified by electronic autoradiography using a Packard Instant Imager. Brefeldin A (BFA), lactacystin,N-acetyl-leucinyl-leucinyl-norleucinal (ALLN),N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucinyl-leucinyl-leucinyl-vinylsulfone (NLVS) were added to cells 90 min prior to methionine starvation to a final concentration of 10 μg/ml (BFA) and 10 μm (NLVS) or 50 μm (lactacystin, MG132, and ALLN) and were present during the pulse labeling and chase period. Cells were grown on coverslips and fixed in 100% methanol at −20 °C for 10 min. CFTR was detected by indirect immunofluorescence as described previously (17Chang X. Cui L. Hou Y. Jensen T.J. Aleksandrov A.A. Mengos A. Riordan J.R. Mol. Cell. 1999; 4: 137-142Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) using the mouse monoclonal antibody M3A7 (16Kartner N. Augustinas O. Jensen T.J. Naismith A.L. Riordan J.R. Nat. Genet.. 1992; 1: 321-327Crossref PubMed Scopus (330) Google Scholar) or BJ570 which recognizes the R-domain 3T. J. Jensen, B. G. Bone, and J. R. Riordan, unpublished data. and visualized with Alexa Fluor 488 goat anti-mouse IgG conjugate (dilution 1:250, Molecular Probes). The plasma membrane Ca2+-ATPase was detected by the mouse monoclonal antibody 5F10 (Affinity Bioreagents, Inc.) and visualized with Alexa Fluor 594 goat anti-mouse IgG conjugate (dilution 1:250, Molecular Probes). To visualize CFTR and the plasma membrane ATPase simultaneously, the mouse monoclonal anti-CFTR antibody BJ570 was biotinylated with NHS-biotin (Pierce) as described by Harlow and Lane (18Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) and detected with streptavidin Alexa Fluor 488 conjugate (dilution 1:1500, Molecular Probes). The chloride efflux assay was performed exactly as we have described recently (17Chang X. Cui L. Hou Y. Jensen T.J. Aleksandrov A.A. Mengos A. Riordan J.R. Mol. Cell. 1999; 4: 137-142Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Cells grown in 6-well culture dishes were loaded with loading buffer containing 0.5 μCi of Na36Cl (Amersham Pharmacia Biotech), washed with efflux buffer, and stimulated with forskolin-containing buffer. Haardt et al. (10Haardt M. Benharouga M. Lechardeur D. Kartner N. Lukacs G.L. J. Biol. Chem. 1999; 274: 21873-21877Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) had previously shown that truncations at several positions within the C-terminal domain destabilized mature CFTR. We decided to further elucidate sites within the C-terminal domain that impact maturation and stability of both nascent and mature CFTR. As a first step, systematic incremental truncations terminating at the points indicated in Fig.1 A were constructed and expressed. As shown in Fig. 1 B the most C-terminal 40 residues of the CFTR sequence distinguish it entirely from other related human ABC proteins. The final four residues of this CFTR-specific segment form a PDZ domain binding motif (11Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (250) Google Scholar, 19Saras J. Heldin C.H. Trends Biochem. Sci. 1996; 21: 455-458Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 20Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1235) Google Scholar), which may contribute to apical localization (21Moyer B.D. Denton J. Karlson K.H. Reynolds D. Wang S. Mickle J.E. Milewski M. Cutting G.R. Guggino W.B. Li M. Stanton B.A. J. Clin. Invest. 1999; 104: 1353-1361Crossref PubMed Scopus (248) Google Scholar) and be involved in the correct placement of CFTR within multimolecular regulatory complexes (12Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 22Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar, 23Fanning A.S. Anderson J.M. J. Clin. Invest. 1999; 103: 767-772Crossref PubMed Scopus (406) Google Scholar). No function has yet been attributed to the remainder of this 40 residue block (residues 1440–1476). Fig. 1 C shows that truncation at this point to form a 1440X variant had no apparent effect on the steady state amounts of the immature, core-glycosylated form of CFTR (lower band) or the mature form with complex oligosaccharide chains (upper band). Apparently no major changes in these amounts occur when truncation of as many as 61 residues occurs, i.e. to form 1420X. However, truncation of a further five residues from this point resulted in a major reduction in the amount of the mature band (1415X). Shortening the C-terminal region by an additional five residues (1410X) nearly eliminated the mature protein band. Hence a portion of the polypeptide between residues 1410 and 1419 appears to play an important role in the formation or maintenance of mature CFTR. The intensity of the immature band also appeared to be significantly reduced on shortening from 1415X to 1410X (Fig. 1 C). The next three incremental shortenings to 1405X, 1400X, and 1395X were without apparent additional effect; the mature band was barely detectable and the immature band remained fainter when compared with 1415X and was weakest at 1400X. Strikingly, deeper truncation to 1390X resulted in a significant increase in the amounts of both bands, the immature band appearing nearly as strong as in the much longer constructs and the mature band as strong as at 1415X. Further shortening to 1380X resulted in a still larger amount of the mature band. To ensure that the mature form of CFTR reach the plasma membrane surface, labeling was carried out with the membrane-impermeable reagent biotin-LC-hydrazide (Fig.1 D). Mature forms of 1390X CFTR and 1380X CFTR were found to be biotinylated. These observations suggest that some sequence C-terminal of residue 1390 is actually destabilizing and emphasizes the fact that CFTR biogenesis and maturation can occur reasonably effectively in the complete absence of the C-terminal domain beyond NBD2. However, certain short regions within the domain have strong modulating effects, both positive and negative. At least three different short segments of the C-terminal domain have significant impact on the steady state amounts of immature and mature CFTR observed. The influence of these truncations on the intracellular localization of CFTR in stably transfected BHK-21 cells was evaluated by immunofluorescence microscopy. Simultaneous staining with an antibody to the plasma membrane Ca2+-ATPase showed that wild-type CFTR is detectable as a uniform staining over the entire cell surface, some of which is punctate in nature (Fig.2 A). Clearly visible beneath the surface staining was a more intense perinuclear pattern corresponding well with ER localization in these cells. Overall this distribution was similar to that observed with CFTR expression in other nonpolar mammalian cells and the same as that observed in BHK cells expressing a CFTR-green fluorescent protein fusion (15Loo M.A. Jensen T.J. Cui L. Hou Y.-X. Chang X.-B. Riordan J.R. EMBO J. 1998; 17: 6879-6887Crossref PubMed Scopus (303) Google Scholar). Truncation to remove the last 41 C-terminal amino acids (1440X) did not change the wild-type picture (Fig. 2 B). However, shortening by 20 more residues to 1420X resulted in some decrease in cell surface staining while perinuclear staining remained intense. With the 1400X truncation only a very circumscribed perinuclear pattern was seen, consistent with the presence of primarily the core-glycosylated immature band seen in immunoblots (Fig. 1) and immunoprecipitates (Fig.3). Shortening by a further 10 or 20 residues to 1390 or 1380 caused the reappearance of weak staining over the cell surface. Hence there was good correspondence between the relative amount of the mature CFTR band detected in Western blots of whole cell lysates (Fig. 1 C) and cell surface staining by immunofluorescence among the different truncations (Fig. 2).Figure 3Pulse-chase experiments showing the turnover of the immature and mature forms of wild-type and C-terminal truncations of CFTR. Cells were starved of methionine for 30 min, pulse-labeled with [35S]methionine (100 μCi/ml) for 15 min, and chased with methionine replete medium for the times indicated in hours. Immunoprecipitations were preformed as described (15Loo M.A. Jensen T.J. Cui L. Hou Y.-X. Chang X.-B. Riordan J.R. EMBO J. 1998; 17: 6879-6887Crossref PubMed Scopus (303) Google Scholar). Following electrophoresis, gels were dried, exposed to x-ray films, and then separately subjected to quantitative electronic autoradiography to enable estimation of the proportion of the initial 35S radioactivity in the immature band after the pulse, which is present in either band at subsequent chase times.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pulse-chase experiments were performed to determine the kinetic effects of each of these truncations on nascent chain maturation or degradation and on the lifetime of the mature molecule once it was formed (Fig. 3). As has been consistently observed in a variety of different mammalian cell types, wild-type CFTR matures inefficiently with 40% or less of the pulse-labeled immature band converted to the mature band during the chase. This maximal conversion occurs in ∼2 h after which little or no immature band is detectable and the amount of radioactively labeled mature band remains nearly constant until 4 h since it has a half-time of ∼16 h in these cells (13Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar, 24Lukacs G.L. Mohamed A. Kartner N. Chang X.-B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (346) Google Scholar). Other observations showed that the rate of disappearance of the immature band is slowed by proteasome inhibitors without augmenting the amount that is converted to the mature product (25Jensen T.J. Loo M.A. Pind S. Williams D.B. Goldberg A.L. Riordan J.R. Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (777) Google Scholar, 26Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1144) Google Scholar) suggesting that a large proportion (in this case ∼60%) of nascent chain was degraded by the proteasome. Truncations to produce 1440X, 1435X, 1430X, and 1425X did not drastically change this precursor-product relationship of nascent and mature CFTR. Notably the maximal proportion of the pulse-labeled nascent 1420X that matured was closer to 30% than 40% and the mature band decayed somewhat more rapidly than wild-type. As was already apparent from the Western blots (Fig. 1) truncation to 1415X had a more major impact. In the pulse-chase experiment the immature band disappeared somewhat more slowly, less mature band appeared and it then turned over more rapidly. The rates of nascent chain disappearance and mature form appearance and disappearance were similar to that of 1410X and 1405X, although the maximum proportion of mature protein formed decreased progressively with shortening. By 1400X this proportion was minimal (∼7% at 1 h of chase). Strikingly, despite the fact that conversion to the mature form was barely detectable, the immature 1400X band disappeared extremely rapidly, i.e. shortening from 1405X to 1400X greatly accelerated the rate of disappearance of the nascent chain. Unexpectedly, removal of a further 5 residues to produce 1395X seemed to cause a reversion to a situation more similar to 1405X than 1400X. A more pronounced restabilization and maturation was exhibited by 1390X. In fact the curve showing the rates of disappearance of the immature band were again quite similar to much longer variants including the wild-type as if residues C-terminal of 1390 destabilized the nascent chain. Formation and maintenance of mature 1390X, however, remained considerably depressed. Further shortening to 1380X, essentially to the C-terminal end of NBD2, had little additional effect. Overall these kinetic data confirm and extend the detection by the immunoblots of three different short segments, within the N-terminal portion of the C-terminal 100 residue domain of CFTR, which have major influences on the stability of the nascent and mature forms of the molecule. These segments lay within a 40-residue stretch immediately C-terminal of NBD2; the most C-terminal 60 residues have little influence on the turnover of immature and mature CFTR and presumably play other roles. The three short segments delineated by the deletions are highlighted in Fig. 4 A and are seen to be reasonably well conserved in CFTRs from different species. Numbering from the C-terminal end, these segments include region I, which when removed greatly reduced mature CFTR, consisting of amino acids 1413 to 1417, all of which except 1417E are hydrophobic, region II (1400–1404) which seems necessary for nascent chain stability, and region III (1390–1394) which has the opposite effect, i.e.destabilizes the nascent molecule. Haardt et al. (10Haardt M. Benharouga M. Lechardeur D. Kartner N. Lukacs G.L. J. Biol. Chem. 1999; 274: 21873-21877Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) reported that naturally occurring C-terminal truncations increased the rate of turnover of the mature form. We observed that C-terminal truncation of 66 amino acids or more decreased the amount of mature protein drastically. Our truncations in steps of five residues suggested the involvement of region I. When all five residues from 1413 to 1417 (FLVIE) were replaced by alanine, the effect was essentially the same as truncation after position 1410,i.e. no mature CFTR band was detected by Western blotting (Fig. 4 B). To determine the crucial residues within region I, alanine substitutions were also made in pairs and individually. Each of the four contiguous hydrophobic residues appeared to contribute and substitution of the Phe1413-Leu1414 pair by alanines was nearly as detrimental as removing all four. Hence a short hydrophobic patch of at least two residues seems to be required for the appearance of the mature protein. Substitution of the glutamic acid at position 1417 was entirely without effect as was replacement of the nonconserved glutamate at 1418 and substitution of the two glutamines flanking this patch on the N-terminal side (Fig. 4 A). In pulse-chase experiments just traces of the large band with complex oligosaccharides could be detected (less than 5%) which turned over rapidly (Fig. 4 D). The nascent chain, however, persisted with a half-life longer than the wild-type protein. The absence of mature CFTR on subs
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