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• W2012602248 abstract "The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-ΔF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000) Traffic 1, 852–870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNApREceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells. The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-ΔF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000) Traffic 1, 852–870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNApREceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells. The cystic fibrosis transmembrane conductance regulator (CFTR) 1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorArfADP-ribosylation factorBFAbrefeldin ACFcystic fibrosisCOPIIcoat protein complex IICOPIcoat protein complex IERendoplasmic reticulumVSV-Gvesicular stomatitis virus G proteinSynsyntaxinendo Hendoglycosidase HDMEMDulbecco's modified Eagle's mediumBFAbrefeldin AVTCsvesicular tubular elementsTGNtrans-Golgi networkCHOChinese hamster ovarySNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptors 1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorArfADP-ribosylation factorBFAbrefeldin ACFcystic fibrosisCOPIIcoat protein complex IICOPIcoat protein complex IERendoplasmic reticulumVSV-Gvesicular stomatitis virus G proteinSynsyntaxinendo Hendoglycosidase HDMEMDulbecco's modified Eagle's mediumBFAbrefeldin AVTCsvesicular tubular elementsTGNtrans-Golgi networkCHOChinese hamster ovarySNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptors is a cAMP-regulated chloride channel polarized to the apical membrane in numerous epithelia including those found in lung, pancreas, intestine, and kidney (1.Stanton B.A. Wien. Klin. Wochenschr. 1997; 109: 457-464PubMed Google Scholar, 2.Jilling T. Kirk K.L. Int. Rev. Cytol. 1997; 172: 193-241Crossref PubMed Google Scholar). Although nearly 1000 mutations have been identified in the CFTR gene, ∼70% of CF chromosomes contain the ΔF508 mutation, which leads to severe forms of the genetic disease cystic fibrosis (CF) (3.Mickle J.E. Cutting G.R. Clin. Chest Med. 1998; 19: 443-458Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4.Davis P.B. Drumm M. Konstan M.W. Am. J. Respir. Crit. Care Med. 1996; 154: 1229-1256Crossref PubMed Scopus (826) Google Scholar, 5.Riordan 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 (5858) Google Scholar). Deletion of F508 prevents proper folding and trafficking of CFTR from the endoplasmic reticulum (ER) to the plasma membrane (6.Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1407) Google Scholar, 7.Kopito R.R. Physiol. Rev. 1999; 79: 167-173Crossref PubMed Scopus (373) Google Scholar). Both wild-type CFTR and CFTR-ΔF508 can be detected in ER and ER-Golgi transport intermediates, but only wild-type CFTR is readily expressed at the cell surface (2.Jilling T. Kirk K.L. Int. Rev. Cytol. 1997; 172: 193-241Crossref PubMed Google Scholar, 8.Gilbert A. Jadot M. Leontieva E. Wattiaux-De Coninck S. Wattiaux R. Exp. Cell Res. 1998; 242: 144-152Crossref PubMed Scopus (65) Google Scholar, 9.Bradbury N.A. Physiol. Rev. 1999; 79: 175-191Crossref PubMed Scopus (140) Google Scholar). Both newly synthesized wild-type CFTR and CFTR-ΔF508 molecules that neither fold productively nor achieve an ER export-competent conformation are eliminated by covalent addition of ubiquitin followed by degradation via the proteosome (10.Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1127) Google Scholar, 11.Jensen 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 (767) Google Scholar). Whereas ∼20–40% of wild-type CFTR nascent chains become properly folded, are exported from the ER, and acquire complex carbohydrates characteristic of passage through the Golgi apparatus, negligible levels of CFTR-ΔF508 follow this pathway, and CFTR-Δ508 is quantitatively degraded (12.Lukacs G.L. Mohamed A. Kartner N. Chang X.B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (341) Google Scholar, 13.Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar).To rationally develop therapeutic means to stimulate CFTR-ΔF508 trafficking from the ER to the cell surface, it is first necessary to understand the mechanisms and pathways directing wild-type CFTR trafficking. By using morphological analyses, we recently reported that although wild-type CFTR was readily detectable in ER membranes, ER-Golgi transport intermediates, endosomes, and the plasma membrane, it was largely absent from the Golgi stack in vivo and in situ (14.Bannykh S.I. Bannykh G.I. Fish K.N. Moyer B.D. Riordan J.R. Balch W.E. Traffic. 2000; 1: 852-870Crossref PubMed Scopus (56) Google Scholar). However, the molecular mechanism of CFTR export from the ER as well as the biochemical foundation for its absence from Golgi cisternae were not addressed. We have therefore examined the biosynthetic pathway of CFTR trafficking through the early secretory pathway at the molecular level. We report that not only is the trafficking pathway by which CFTR acquires complex carbohydrates cell type-specific but that CFTR maturation can utilize a non-conventional pathway whereby the protein is first likely to be transported to distal Golgi compartments and/or the endosomal system prior to retrieval to earlier Golgi compartments for oligosaccharide processing to the complex form. Pharmacological modulation of this non-conventional pathway may be useful to selectively stimulate CFTR-ΔF508 transport to the plasma membrane.EXPERIMENTAL PROCEDURESMaterialsThe following antibodies were used in this study: a monoclonal antibody (M3A7) against the second nucleotide-binding domain of CFTR (15.Kartner N. Riordan J.R. Methods Enzymol. 1998; 292: 629-652Crossref PubMed Scopus (24) Google Scholar), a monoclonal antibody (p5D4) against the C-terminal cytoplasmic tail of the vesicular stomatitis virus G protein (VSV-G) (16.Kreis T.E. EMBO J. 1986; 5: 931-941Crossref PubMed Scopus (282) Google Scholar), and a polyclonal antibody against Rab6 (B. Goud, Institut Curie, Paris, France) (17.Goud B. Zahraoui A. Tavitian A. Saraste J. Nature. 1990; 345: 553-556Crossref PubMed Scopus (251) Google Scholar). Protein G-Sepharose 4 Fast Flow beads were from Amersham Biosciences; BFA was from Calbiochem; Complete Protease Inhibitor Cocktail Tablets were from Roche Molecular Biochemicals; LipofectAMINE Plus Reagent, Opti-MEM I reduced serum medium, DMEM, and α-minimum Eagle's medium were from Invitrogen; and Easy Tag Express Protein Labeling 35S-Met Mix was from PerkinElmer Life Sciences.Vaccinia Virus Infection and TransfectionBHK, HeLa, and HEK- 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum and 100 units/ml each of penicillin and streptomycin in a 5% humidified CO2 incubator at 37 °C. CHO and CHO-15B cells were maintained α-minimum Eagle's medium as above. Infection with T7 RNA polymerase-recombinant vaccinia virus and transfection were performed as described (18.Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (417) Google Scholar) with minor modifications. Briefly, BHK cells (1.5 × 106) were plated in 60-mm cell culture dishes 1 day prior to experimentation. Before infection, cells were washed twice with Opti-MEM I and then infected with recombinant vaccinia virus expressing the T7 RNA polymerase (19.Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (1864) Google Scholar, 20.Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5987) Google Scholar) at a multiplicity of 10 plaque-forming units/cell in 1 ml of Opti-MEM I for 30 min with rocking in a 5% humidified CO2 incubator at 37 °C. Infection media were removed; cells were washed twice with Opti-MEM I, and cells were co-transfected with 7 μg of pcDNA3.1 wild-type CFTR and 7 μg of pcDNA3.1/pET vector (21.Dascher C. Tisdale E.J. Balch W.E. Methods Enzymol. 1995; 257: 165-173Crossref PubMed Scopus (5) Google Scholar) containing trafficking machinery using LipofectAMINE Plus Reagent, following the manufacturer's instructions. Transfection medium was aspirated after 3 h and replaced with DMEM containing 10% fetal bovine serum for an additional 5–7 h. Expression of recombinant GTPases and SNARE components were verified using immunoblotting with specific antibodies (data not shown) to ensure typical expression levels of 5–10-fold over endogenous protein levels (21.Dascher C. Tisdale E.J. Balch W.E. Methods Enzymol. 1995; 257: 165-173Crossref PubMed Scopus (5) Google Scholar). Experiments comparing effects of trafficking machinery components on transport of VSV-G and CFTR were performed in parallel.Transport AnalysisTransport analysis of VSV-G was performed as described previously (18.Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (417) Google Scholar). For transport analysis of CFTR, medium was removed after 8–10 h of transfection, and cells were washed two times with phosphate-buffered saline (pre-warmed to 37 °C) and incubated for 30 min in methionine-deficient minimum Eagle's medium. Cells were radiolabeled for 20–30 min with 500 μCi of Trans35S-label and then chased in DMEM containing 10% fetal calf serum and 10 mm cold methionine. For experiments utilizing BFA, cells were chased in the above medium containing 5 μg/ml brefeldin A (BFA) or equivalent levels of ethanol as control. After the indicated chase periods, cells were washed twice with ice-cold phosphate-buffered saline and lysed for 30 min on ice with RIPA buffer (50 mm Tris-HCl (pH 7.4), 150 mmNaCl, 0.1% SDS, 1% deoxycholate, and 1% Triton X-100) supplemented with a Complete Protease Inhibitor Cocktail Tablet and 1 mmphenylmethylsulfonyl fluoride. Lysates were spun at 14,000 ×g for 10 min at 4 °C to pellet insoluble material, and supernatants were transferred to fresh tubes and incubated with M3A7 monoclonal antibody (5 μg/ml) overnight at 4 °C with rotation. Immunoprecipitates were isolated with 50 μl of 50% protein G-Sepharose beads at 4 °C for 3 h. Beads were washed 4 times with ice-cold RIPA buffer and eluted with 1× Laemmli sample buffer (22.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206057) Google Scholar) for 1 h at room temperature. Beads were pelleted by brief centrifugation, and supernatants, containing CFTR protein, were separated using 7.5% SDS-PAGE gels. Following electrophoresis, gels were treated with enhancing solution (125 mm sodium salicylate, 35% methanol) for 1 h and dried. Signals were developed by autoradiography and quantitated using a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). Graphical results represent the mean ± S.E. of at least three independent experiments for all conditions tested.RESULTSCFTR Maturation Is Blocked by Dominant Negative Sar1-GTPTo investigate the mechanism(s) of wild-type CFTR trafficking in the early secretory pathway, we used a recombinant T7-vaccinia virus expression system (19.Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (1864) Google Scholar, 20.Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5987) Google Scholar) to transiently co-express wild-type CFTR and key components regulating the formation, targeting, and fusion of endoplasmic reticulum (ER)-derived transport intermediates with Golgi membranes. This approach has been used previously (23.Ostedgaard L.S. Zeiher B. Welsh M.J. J. Cell Sci. 1999; 112: 2091-2098Crossref PubMed Google Scholar) to examine transport of CFTR through the secretory pathway, the functional roles of the coat protein complex II (COPII) and coat protein complex I (COPI) involved in transport vesicle assembly, and the role of Rab GTPases and SNAp REceptor (SNARE) proteins involved in membrane fusion in the trafficking of cargo through the early secretory pathway (18.Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (417) Google Scholar, 23.Ostedgaard L.S. Zeiher B. Welsh M.J. J. Cell Sci. 1999; 112: 2091-2098Crossref PubMed Google Scholar, 24.Dascher C. Balch W.E. J. Biol. Chem. 1994; 269: 1437-1448Abstract Full Text PDF PubMed Google Scholar, 25.Dascher C. Matteson J. Balch W.E. J. Biol. Chem. 1994; 269: 29363-29366Abstract Full Text PDF PubMed Google Scholar, 26.Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. Cell Biol. 1994; 125: 51-65Crossref PubMed Scopus (253) Google Scholar).We first examined the role of the COPII complex, composed of the small molecular weight GTPase Sar1 and the heterodimeric Sec23–24 and Sec13–31 complexes in CFTR export from the ER (26.Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. Cell Biol. 1994; 125: 51-65Crossref PubMed Scopus (253) Google Scholar, 27.Scales S.J. Gomez M. Kreis T.E. Int. Rev. Cytol. 2000; 195: 67-144Crossref PubMed Google Scholar, 28.Rowe T. Aridor M. McCaffery J.M. Plutner H. Nuoffer C. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (147) Google Scholar, 29.Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar). To block COPII vesicle function in BHK cells, we co-expressed wild-type CFTR with Sar1-GTP(H79G), a constitutively active mutant locked in the GTP-bound form that inhibits vesicle uncoating leading to the accumulation of ER to Golgi transport intermediates (28.Rowe T. Aridor M. McCaffery J.M. Plutner H. Nuoffer C. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (147) Google Scholar, 30.Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (340) Google Scholar). Co-expression of Sar1 or other components of the vesicular trafficking pathway with CFTR (see below) generally leads to a 5–10-fold elevated level of the component over the endogenous pool, consistent with previous results (18.Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (417) Google Scholar, 23.Ostedgaard L.S. Zeiher B. Welsh M.J. J. Cell Sci. 1999; 112: 2091-2098Crossref PubMed Google Scholar, 24.Dascher C. Balch W.E. J. Biol. Chem. 1994; 269: 1437-1448Abstract Full Text PDF PubMed Google Scholar, 25.Dascher C. Matteson J. Balch W.E. J. Biol. Chem. 1994; 269: 29363-29366Abstract Full Text PDF PubMed Google Scholar, 26.Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. Cell Biol. 1994; 125: 51-65Crossref PubMed Scopus (253) Google Scholar) (data not shown). Following labeling of cells with a pulse of [35S]methionine, delivery of CFTR to the Golgi was measured by the conversion of the ∼140–150-kDa core-glycosylated band B pre-Golgi form to ∼170–190-kDa complex glycosylated band C isoform diagnostic of processing by Golgi-associated mannosidases and glycosyltransferases leading to the addition of polylactosamine (31.O'Riordan C.R. Lachapelle A.L. Marshall J. Higgins E.A. Cheng S.H. Glycobiology. 2000; 10: 1225-1233Crossref PubMed Scopus (26) Google Scholar) (Fig. 1, A–C).In control cells, ∼30% of CFTR was processed to the mature band C glycoisoform over 3 h, consistent with the efficiency and rate of CFTR maturation reported in other stably transfected cell lines (12.Lukacs G.L. Mohamed A. Kartner N. Chang X.B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (341) Google Scholar,13.Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar). By contrast, no detectable levels of CFTR in the band C glycoisoform were observed following overexpression of Sar1-GTP(H79G) (Fig. 1B). As a control, under identical conditions we also tested the effect of Sar1-GTP(H79G) on the processing of the reporter cargo type 1 transmembrane protein vesicular stomatitis virus glycoprotein (VSV-G) from the endoglycosidase H (endo H)-sensitive pre-Golgi form to the endo H-resistant, Golgi-processed glycoisoform as described previously (28.Rowe T. Aridor M. McCaffery J.M. Plutner H. Nuoffer C. Balch W.E. J. Cell Biol. 1996; 135: 895-911Crossref PubMed Scopus (147) Google Scholar, 30.Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (340) Google Scholar). Consistent with previous findings (26.Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. Cell Biol. 1994; 125: 51-65Crossref PubMed Scopus (253) Google Scholar,30.Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (340) Google Scholar, 32.Aridor M. Balch W.E. J. Biol. Chem. 2000; 275: 35673-35676Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33.Aridor M. Fish K.N. Bannykh S. Weissman J. Roberts T.H. Lippincott-Schwartz J. Balch W.E. J. Cell Biol. 2001; 152: 213-229Crossref PubMed Scopus (203) Google Scholar, 34.Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (244) Google Scholar), transport of VSV-G was potently inhibited by the Sar1-GTP(H79G) mutant (Fig. 1D). Thus, export of CFTR from the ER, like VSV-G, is dependent on a COPII-mediated mechanism. Consistent with this interpretation, morphological analyses of cells expressing wild-type CFTR using high resolution deconvolution microscopy showed a significant co-localization of wild-type CFTR with the Sar1 GTPase (∼70% of Sar1 containing punctate structures) and the Sec23 component of the COPII coat complex (∼40% of punctate Sec23 containing structures) (Fig. 1E) that was significantly reduced in cells expressing the ΔF508 mutant that cannot exit the ER (∼15% of Sar1 containing punctate structures; <10% of Sec23 containing structures) (Fig. 1E) (35.Riordan J.R. Am. J. Hum. Genet. 1999; 64: 1499-1504Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar).To quantitatively express the efficiency of maturation of CFTR from band B to band C glycisoforms, we compared the ratio of CFTR band C to CFTR band B as a function of time. If the band C/band B (C/B) ratio remains unaltered relative to the control by co-expression with dominant negative mutants, CFTR processing to the mature glycoisoform is not affected. If the C/B ratio is reduced, CFTR processing to the mature glycoisoform is inhibited. By using this method, we found that although expression of CFTR alone exhibited a C/B ratio of ∼6 after a 3-h chase period, co-transfection with Sar1-GTP(H79G) yielded a C/B ratio of ∼0.1 (Fig. 1C). Interestingly, not only was CFTR processing blocked by the Sar1-GTP(H79G) mutant but Sar1-GTP(H79G) stabilized immature CFTR band B and reduced its rate of degradation ∼3–6-fold (Fig. 1A).CFTR Maturation Is Not Blocked by Dominant Negative Arf1-GTPWhereas the above results are consistent with the generally accepted view that the COPII machinery is involved in the export of cargo proteins from the ER (26.Kuge O. Dascher C. Orci L. Rowe T. Amherdt M. Plutner H. Ravazzola M. Tanigawa G. Rothman J.E. Balch W.E. J. Cell Biol. 1994; 125: 51-65Crossref PubMed Scopus (253) Google Scholar, 29.Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar, 32.Aridor M. Balch W.E. J. Biol. Chem. 2000; 275: 35673-35676Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33.Aridor M. Fish K.N. Bannykh S. Weissman J. Roberts T.H. Lippincott-Schwartz J. Balch W.E. J. Cell Biol. 2001; 152: 213-229Crossref PubMed Scopus (203) Google Scholar, 34.Aridor M. Weissman J. Bannykh S. Nouffer C. Balch W.E. J. Cell Biol. 1998; 141: 61-70Crossref PubMed Scopus (244) Google Scholar, 36.Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Biol. Chem. 1999; 274: 4389-4399Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), the step following export from the ER generally requires the function(s) of the coat complex I (COPI) machinery that is recruited to pre-Golgi intermediates consisting of vesicular tubular clusters (VTCs) (14.Bannykh S.I. Bannykh G.I. Fish K.N. Moyer B.D. Riordan J.R. Balch W.E. Traffic. 2000; 1: 852-870Crossref PubMed Scopus (56) Google Scholar) that are potentially formed from the fusion of COPII vesicles (37.Rowe T. Dascher C. Bannykh S. Plutner H. Balch W.E. Science. 1998; 279: 696-700Crossref PubMed Scopus (97) Google Scholar). COPI components include the small molecular weight GTPase Arf1 and the coatomer coat complex. The function of the COPI coat complex remains controversial. COPI may function to regulate anterograde transport of VTCs from the ER to the Golgi stack, transport within the Golgi stack, and/or the retrograde transport of recycling components from pre-Golgi and Golgi membranes to the ER (27.Scales S.J. Gomez M. Kreis T.E. Int. Rev. Cytol. 2000; 195: 67-144Crossref PubMed Google Scholar, 38.Pelham H.R. Rothman J.E. Cell. 2000; 102: 713-719Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 39.Storrie B. Pepperkok R. Nilsson T. Trends Cell Biol. 2000; 10: 385-391Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).To investigate the requirement for COPI in the processing of CFTR, we co-expressed CFTR with Arf1-GTP(Q71L), a dominant negative mutant locked in the GTP-bound form that inhibits ER to Golgi transport of VSV-G by blocking COPI coat disassembly (24.Dascher C. Balch W.E. J. Biol. Chem. 1994; 269: 1437-1448Abstract Full Text PDF PubMed Google Scholar). Control experiments demonstrated that the Arf1-GTP(Q71L) mutant was functional under these conditions as it stimulated coatomer (β-COP) recruitment to Golgi membranes (data not shown) and completely blocked the processing of VSV-G to the endo H-resistant form (Fig. 2D) as reported previously (24.Dascher C. Balch W.E. J. Biol. Chem. 1994; 269: 1437-1448Abstract Full Text PDF PubMed Google Scholar, 40.Zhang C.J. Rosenwald A.G. Willingham M.C. Skuntz S. Clark J. Kahn R.A. J. Cell Biol. 1994; 124: 289-300Crossref PubMed Scopus (150) Google Scholar). Remarkably, overexpression of Arf1-GTP(Q71L) in BHK cells had no effect on the rate of CFTR band B degradation, the rate of CFTR band C formation, or the CFTR C/B ratio (Fig. 2, A–C). Thus, CFTR maturation is Arf1-independent, suggesting that neither the potential role of Arf1 in anterograde transport nor retrograde recycling of transport components perturbs egress or degradation of wild-type CFTR from the ER in BHK cells.Figure 2CFTR processing is independent of Arf1 function in BHK cells. A–C, BHK cells were co-transfected with pcDNA3.1-CFTR and empty vector or with pcDNA3.1-CFTR and pET-Arf1-GTP(Q71L). A, quantitation of CFTR band B. Inset, representative autoradiogram is shown. B, quantitation of the CFTR band C glycoisoform. C, ratio of CFTR band C to band B plotted as a function of time. D, inhibitory effect of Arf1-GTP(Q71L) on processing of VSV-G from the endo H-sensitive (endo Hs) ER form to the endo H-resistant (endo Hr) Golgi form.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CFTR Maturation Is Not Blocked by Dominant Negative Rab1a or Rab2Rab1 and Rab2 are members of the ubiquitous Rab family of small molecular weight GTPases that are required for vesicle targeting and fusion in both the exocytic and endocytic pathways (41.Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2683) Google Scholar, 42.Martinez O. Goud B. Biochim. Biophys. Acta. 1998; 1404: 101-112Crossref PubMed Scopus (229) Google Scholar). Rab1 regulates both anterograde trafficking from the ER to the cis-Golgi network and intra-Golgi transport, whereas Rab2 has been proposed to control retrograde transport from post-ER, pre-Golgi intermediates to the ER (18.Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (417) Google Scholar,43.Plutner H. Cox A.D. Pind S. Khosravi-Far R. Bourne J.R. Schwaninger R. Der C.J. Balch W.E. J. Cell Biol. 1991; 115: 31-43Crossref PubMed Scopus (289) Google Scholar, 44.Tisdale E.J. Mol. Biol. Cell. 1999; 10: 1837-1849Crossref PubMed Scopus (64) Google Scholar, 45.Tisdale E.J. Balch W.E. J. Biol. Chem. 1996; 271: 29372-29379Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 46.Tisdale E.J. Jackson M.R. J. Biol. Chem. 1998; 273: 17269-17277Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar).To examine the requirement for Rab1 and Rab2 function in CFTR maturation, we co-expressed CFTR with Rab1a(N124I) or Rab2(N119I), dominant negative mutants that are defective in guanine nucleotide binding and interfere with ER to Golgi transport in BHK cells (18.Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (417) Google Scholar, 47.Pind S.N. Nuoffer C. McCaffery J.M. Plutner H. Davidson H.W. Farquhar M.G. Balch W.E. J. Cell Biol. 1994; 125: 239-252Crossref PubMed Scopus (130) Google Scholar). Control experiments demonstrated that the Rab mutants were functionally expressed and blocked VSV-G processing to the endo H-resistant complex Golgi glycoisoform as reported previously (18.Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (417) Google Scholar, 47.Pind S.N. Nuoffer C. McCaffery J.M. Plutner H. Davidson H.W. Farquhar M.G. Balch W.E. J. Cell Biol. 1994; 125: 239-252Crossref PubMed Scopus (130) Google Scholar) (Fig. 3D, upper endo Hr bands). In contrast, neither Rab1(N124I) nor Rab2(N119) affected CFTR processing from the band B to the band C glycoisoform or the CFTR C/B ratio (Fig. 3, A–C). Thus, dominant negative Rab GTPases controlling the function of pre-Golgi transport intermediates required for anterograde or retrograde trafficking between the ER and Golgi (37.Rowe T. Dascher C. Bannykh S. Plutner H. Balch W.E. Science. 1998; 279: 696-700Crossref PubMed Scopus (97) Google Scholar, 48.Allan B.B. Moyer B.D. Balch W.E. Science. 2000; 289: 444-448Crossref PubMed Scopus (382) Google Scholar, 49.Moyer B.D. Allan B.B. Balch W.E. Traffic. 2001; 2: 268-276Crossref PubMed Scopus (208) Google S" @default.
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