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• W2129208664 abstract "We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum. We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum. CFTR 2The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; Csp, cysteine string protein; ER, endoplasmic reticulum; GST, glutathione S-transferase; GST-R, CFTR R-domain; HEK, human embryonic kidney; Hsp, heat shock protein; R-domain, regulatory domain; GFP, green fluorescent protein; SPQ, 6-methyoxyl-N-(3-sulfopropyl) quinolinium; WT, wild type; dsRNA, double-stranded RNAs; BSA, bovine serum albumin; IP, immunoprecipitation; CHIP, carboxyl terminus of Hsp70-interacting protein; IBMX, isobutylmethylxanthine. is a phosphorylation-activated Cl- channel expressed at the apical membranes of epithelial cells that are involved in salt secretion or absorption. Mutations in the CFTR gene produce cystic fibrosis by reducing or eliminating this primary anion conductance pathway and the salt and water transport processes it underlies (1Pilewski J.M. Frizzell R.A. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (384) Google Scholar, 2Boucher R.C. Eur. Respir. J. 2004; 23: 146-158Crossref PubMed Scopus (496) Google Scholar, 3Nilius B. Droogmans G. Acta Physiol. Scand. 2003; 177: 119-147Crossref PubMed Scopus (371) Google Scholar). The most common CFTR mutation, ΔF508, results in biosynthetic arrest and ER-associated degradation of the mutant protein so that little, if any, ΔF508 CFTR progresses to the apical plasma membrane. Experimental rescue of ΔF508 CFTR by a variety of methods (4Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 358: 761-764Crossref PubMed Scopus (1061) Google Scholar, 5Sato S. Ward C.L. Krauss M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 6Welch W.J. Semin. Cell Dev. Biol. 2004; 15: 31-38Crossref PubMed Scopus (97) Google Scholar) has demonstrated its partial functional competence, suggesting the potential value of therapies aimed at promoting the trafficking of CFTR processing mutants, like ΔF508, to the cell surface. Achieving this goal will be facilitated by an understanding of the chaperone and other protein interactions that regulate CFTR biogenesis. Early steps in this process are inefficient, leading to degradation of ∼70% of wild type (WT) CFTR in most cell types (7Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar, 8Qu B.H. Thomas P.J. J. Biol. Chem. 1996; 271: 7261-7264Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Several of the protein components involved in the folding and domain assembly of native CFTR have been identified, including the molecular chaperones Hdj-2, Hsp70, Hsp90, and calnexin (9Yang Y. Janich S. Cohn J.A. Wilson J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9480-9484Crossref PubMed Scopus (282) Google Scholar, 10Pind S. Riordan J.R. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar, 11Loo M.A. Jensen T.J. Cui L. Hou Y. Chang X.B. Riordan J.R. EMBO J. 1998; 17: 6879-6887Crossref PubMed Scopus (299) Google Scholar, 12Meacham G.C. Lu Z. King S. Sorscher E. Tousson A. Cyr D.M. EMBO J. 1999; 18: 1492-1505Crossref PubMed Scopus (271) Google Scholar). Their interactions are thought to facilitate CFTR folding; for example, Hsp70 and Hdj-2 interact with the first nucleotide binding domain (NBD1, the site of the ΔF508 mutation) to inhibit its aggregation in vitro (12Meacham G.C. Lu Z. King S. Sorscher E. Tousson A. Cyr D.M. EMBO J. 1999; 18: 1492-1505Crossref PubMed Scopus (271) Google Scholar, 13Strickland E. Qu B.H. Millen L. Thomas P.J. J. Biol. Chem. 1997; 272: 25421-25424Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Both Hsp70 and calnexin exhibit prolonged associations with ΔF508 CFTR relative to the wild type (WT) protein (9Yang Y. Janich S. Cohn J.A. Wilson J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9480-9484Crossref PubMed Scopus (282) Google Scholar, 10Pind S. Riordan J.R. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar), suggesting that these chaperones are part of the quality control apparatus that identifies non-native CFTR conformations for degradation. A key component involved in removing CFTR from the productive folding pathway appears to be the ubiquitin ligase, CHIP, which poly-ubiquitinates Hsp70-ladened CFTR, thereby targeting the protein for proteasome-mediated degradation (14Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (705) Google Scholar). Similarly, calnexin undergoes transient cyclic associations with newly synthesized glycoproteins like CFTR to facilitate their folding, but components of this quality control cycle also can contribute to the ER retention and degradation of proteins whose native conformations are not achieved. Calnexin associates with both WT and ΔF508 CFTR during their biogenesis, but only the WT protein escapes the ER for Golgi-mediated glycosylation and apical membrane targeting (10Pind S. Riordan J.R. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar). The biogenesis of a protein that assumes its native conformation with difficulty, like CFTR, represents the net result of the two opposing influences of molecular chaperones: facilitation of folding and targeting for degradation if folding does not succeed (15Younger J.M. Ren H.Y. Chen L. Fan C.Y. Fields A. Patterson C. Cyr D.M. J. Cell Biol. 2004; 167: 1075-1085Crossref PubMed Scopus (145) Google Scholar, 16Fewell S.W. Pipas J.M. Brodsky J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2002-2007Crossref PubMed Scopus (25) Google Scholar, 17Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (916) Google Scholar). The co-translational association with CFTR of a DnaJ family co-chaperone, Hdj-2, represents a case in point. In combination with Hsp70, Hdj-2 is thought to play a significant role in the folding of NBD1 and its appropriate interaction with other CFTR domains, particularly the R-domain (12Meacham G.C. Lu Z. King S. Sorscher E. Tousson A. Cyr D.M. EMBO J. 1999; 18: 1492-1505Crossref PubMed Scopus (271) Google Scholar). In contrast, prolonged or excessive interactions with Hsp70 and Hdj-2 can facilitate the efficient CHIP-mediated ubiquitination and degradation of CFTR (14Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (705) Google Scholar). We have reported the interaction of CFTR with another J-domain protein, the cysteine string protein (Csp), and we found that the coexpression of Csp with CFTR reduced cAMP-stimulated CFTR currents (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Ostensibly, this finding was compatible with the known action of Csp, identified originally as a synaptic vesicle-associated protein that plays an essential role in neurosecretory vesicle insertion into the plasma membrane (19Zinsmaier K.E. Eberle K.K. Buchner E. Walter N. Benzer S. Science. 1994; 263: 977-980Crossref PubMed Scopus (318) Google Scholar). Subsequent studies of Csp demonstrated that it is required for other regulated exocytic processes, including insulin secretion (20Zhang H. Kelley W.L. Chamberlain L.H. Burgoyne R.D. Lang J. J. Cell Sci. 1999; 112: 1345-1351Crossref PubMed Google Scholar). Nevertheless, although Csp localized partially to the apical membrane domain in polarized CFTR expressing epithelia, as expected for its participation in regulated exocytosis, its co-localization with calnexin also suggested the potential for Csp to function in the endoplasmic reticulum (ER). Further studies showed that the Csp-induced inhibition of CFTR currents was explained by a primary effect on the production of mature CFTR protein (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), implying that Csp may also function at early steps in the protein secretory pathway. In this study, we examined the physiological role of Csp in CFTR protein processing by experimentally increasing or decreasing its expression level. The effects of varying Csp expression on CFTR biogenesis were compared with those of Hdj-2, an acknowledged CFTR co-chaperone (12Meacham G.C. Lu Z. King S. Sorscher E. Tousson A. Cyr D.M. EMBO J. 1999; 18: 1492-1505Crossref PubMed Scopus (271) Google Scholar). Our results are consistent with the concept that these J-domain proteins play different roles in the processing of CFTR; Hdj-2 facilitates CFTR biogenesis, whereas Csp regulates the export of CFTR to post-ER compartments. Materials and Reagents—Wild type or mutant Csp constructs were tagged with the Myc epitope as described (20Zhang H. Kelley W.L. Chamberlain L.H. Burgoyne R.D. Lang J. J. Cell Sci. 1999; 112: 1345-1351Crossref PubMed Google Scholar). Anti-CFTR monoclonal antibodies (M3A7) were kindly provided by Dr. John Riordan (Mayo Clinic, Scottsdale, AZ) or purchased from Chemicon (MAB3480). Anti-Csp was also from Chemicon. Anti-CFTR polyclonal antibodies (R3195 and MD1314) were generated and affinity-purified, as described previously (21French P.J. van Doornick J.H. Peters R.H. Verbeek E. Ameen N.A. Marino C.R. deJonge H.R. Bijman J. Scholte B.J. J. Clin. Investig. 1996; 98: 1304-1312Crossref PubMed Scopus (124) Google Scholar), and kindly provided by Dr. Christopher Marino, University of Tennessee, Memphis. The monoclonal antibody to human c-Myc developed by Dr. Michael Bishop was obtained from the Developmental Studies Hybridoma Bank, under the auspices of the NICHD, National Institutes of Health, and maintained by the University of Iowa. Monoclonal anti-Hsp70 and Hsp90 antibodies were purchased from Sigma. Anti-tubulin antibody was a gift from Dr. Linton Traub (University of Pittsburgh). Rabbit anti-green fluorescent protein (GFP) was purchased from Abcam (Ab6556). Mouse anti-Hsp70 (SPA-810) was obtained from Stressgen. Goat antibody to Grp78 was purchased from Santa Cruz Biotechnology (SC-1050). Mouse anti-glyceraldehyde 3-phosphate dehydrogenase was from U. S. Biochemical Corp. Mouse anti-calnexin was purchased from Affinity Bioreagents (MA3-027). Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were obtained from Sigma. Double-stranded RNAs (dsRNA) to interfere with the expression of endogenous mammalian Csp (CCUCGGAUGACAUUAAGAA) or Xenopus Csp (GUAUCACCCCGACAAGAAC) mRNAs were designed by Oligogene software and obtained from Dharmacon Research. Lipofectamine was obtained from Invitrogen. Redivue Pro-mix L-35S was obtained from Amersham Biosciences. Protein A/G-agarose beads and Taq polymerase were purchased from Invitrogen; glutathione-Sepharose® 4B was purchased from Amersham Biosciences. DNA restriction endonucleases were from New England Biolabs. DNA purification kits were obtained from Qiagen. Reagents for analytical gel electrophoresis were from Bio-Rad. Pre-stained protein standard used for immunoblotting was purchased from Invitrogen. BCA protein assay, Supersignal West Dura chemiluminescent substrate, and Restore stripping reagent were from Pierce. Other reagent grade chemicals were purchased from Sigma. Cell Culture and Transient Transfection—HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) and supplemented with 10% fetal bovine serum, 2 mm glutamine, 10 mm HEPES, and antibiotics (Invitrogen). For transient transfection of cDNAs encoding Myc-tagged Csp and CFTR, 106 cells per well in 6-well plates were plated and transfected using Lipofectamine 2000 the following day according to the manufacturer's instructions and the methods described by Zhang et al. (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Calu-3 cells were cultured as described (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 22Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Immunoblots and Pulse-Chase Assays—Transfected cells were lysed in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA supplemented with complete protease inhibitor mixture (Roche Applied Science) by rocking for 30 min at 4 °C. To determine the distribution of CFTR between soluble and insoluble fractions, we used the following protocol. The Triton X-100-insoluble fraction was separated by centrifugation at 16,000 × g for 15 min at 4 °C. The soluble fraction was decanted, and the insoluble pellets were washed twice with 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, and re-solubilized in SDS-PAGE loading buffer by sonication. Crude membranes and membrane extracts from cell lines were prepared as described (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, 50 μg of membrane extract per lane was separated on 12% SDS-polyacrylamide minigels and transferred to polyvinylidene difluoride membranes. Blots were probed with the indicated antibodies and diluted as follows: anti-Csp, 1:2000; anti-Hsp70, 1:3000; anti-Hsp90, 1:2000; and anti-CFTR, 1:2000. Immunoblots were scanned and quantitated using ImageJ, a public domain Java image-processing program (downloaded from the National Institutes of Health, Bethesda). Statistics and t test calculations were done with Excel 2000 (Microsoft, Redmond, WA). Metabolic labeling and immunoprecipitation (IP) for detection of protein binding to CFTR were performed using methods described previously (9Yang Y. Janich S. Cohn J.A. Wilson J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9480-9484Crossref PubMed Scopus (282) Google Scholar, 18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Immunofluorescence—Cells were replated on 12-mm poly-l-lysinecoated coverslips (BD Biosciences) in 24-well plates 1 day after transfection and then fixed and permeabilized on day 2. Antibody incubations were done in phosphate-buffered saline containing 2% BSA, and all washes were done with phosphate-buffered saline containing 0.5% BSA. Cy3- and Cy5-labeled secondary antibodies were from Jackson ImmunoResearch (West Grove, PA), and nuclei were stained with the DNA dye Hoechst 33342 (bisbenzamide) purchased from Sigma. Images were obtained with an Olympus Fluoview 1000 confocal microscope (see also Refs. 22Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar and 23Butteworth M.B. Frizzell R.A. Johnson J.P. Peters K.W. Edinger R.S. Am. J. Physiol. 2005; 289: F969-F977Google Scholar). In Vitro Binding Assays and Co-immunoprecipitation—In vitro binding assays were performed as described by Sun et al. (24Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Briefly, 10 μg of GST or GST-R domain fusion protein (GST-CFTR-R) were incubated with 20 μl of pre-equilibrated glutathione-Sepharose 4B beads in 200 μl of DIGNAM-D buffer containing 0.1% BSA at 4 °C for 1 h (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 25Condliffe S.B. Carattino M.D. Frizzell R.A. Zhang H. J. Biol. Chem. 2003; 278: 12796-12804Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In vitro competitive binding assays were performed as described (24Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) with modifications. Glutathione beads were incubated with 35S-labeled Csp or Hdj-2 at the indicated concentrations. After five washes with binding buffer, samples were resuspended in SDS sample buffer, and the proteins were resolved by SDS-PAGE; the signals were visualized by autoradiography. Co-IPs were performed using either 3 μl of anti-Csp or anti-CFTR (M3A7) antibody as described (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 22Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). As a control for efficiency in the Csp/CFTR IPs, supernatant fractions obtained after IP of myc-Csp were incubated overnight with 3 μl of anti-CFTR (M3A7) antibody in the presence of protein G-agarose at 4 °C. The next day, samples were washed four times with DIGNAM-D buffer and separated by 5% SDS-PAGE. Blots were probed for CFTR (re-IP) using polyclonal anti-CFTR (R3195; 1:2000). RNA Interference—Six μg of dsRNA was transfected using 20 μl of Lipofectamine 2000 into 50% confluent 3T3 cells stably expressing CFTR (kindly provided by Dr. Michael Welsh, University of Iowa), which had been seeded onto 35-mm dishes 1 day earlier. Two and 4 days later, the transfections were repeated with the same procedure. Three days thereafter, cell lysates or membrane extracts were prepared and analyzed for CFTR, chaperone protein, and control protein expression, as described previously (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Western blots were probed simultaneously with anti-Csp and anti-Hsp70 antibodies or with anti-CFTR and anti-Hsp90 antibodies. The blots were stripped and re-probed for tubulin as control. SPQ Assays of Regulated Anion Transport—Experimental procedures for monitoring forskolin-stimulated halide transport across the plasma membranes of HEK293 cells using SPQ fluorescence were performed as described (26Strong T.V. Wilkinson D.J. Mansoura M.K. Devor D.C. Henze K. Yang Y. Wilson J.M. Cohn J.A. Dawson D.C. Frizzell R.A. Collns F.S. Hum. Mol. Genet. 1993; 2: 225-230Crossref PubMed Scopus (94) Google Scholar) with modifications. HEK293 cells were seeded onto coverslips pre-coated with poly-l-lysine and cultured for 24-36 h. When cells reached ∼60% confluence, 1.5 μg of cDNA encoding human CFTR and/or wild type or mutant Csp was co-transfected using Lipofectamine, as described (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). After 1 day, the cells were loaded with SPQ using hypotonic loading conditions as follows. Cells were incubated at room temperature for 5 min in a 1:1 mixture of Cl- buffer (126 mm NaCl, 5 mm KCl, 1.5 mm CaCl2, 1.0 mm MgCl2, 20 mm HEPES, 0.1% bovine serum albumin, 0.1% d-glucose, pH 7.2) and distilled H2O, containing 5 mm SPQ. After a 30-min recovery period in culture medium, the coverslips were then mounted in a chamber heated to 37 °C on the stage of a fluorescence microscope. The chamber was supplied continually with warmed buffer solution, and individual cells (regions of interest) were selected for study. Fluorescence intensity was collected as the perfused solutions were changed from sodium iodide to sodium nitrate to dequench SPQ fluorescence and expressed relative to intensity at time 0. Further fluorescence dequenching elicited by forskolin (10 μm) plus IBMX (100 μm) was indicative of cAMP-stimulated iodide efflux from preloaded cells and provides an assay for CFTR channel activity. Re-addition of iodide buffer then quenches SPQ fluorescence to base-line levels. Data were collected from at least 20 cells per coverslip, with images acquired at 0.2 Hz. The blank field obtained by closing the camera shutter was used as background and subtracted prior to image acquisition. Oocyte Expression—Oocyte preparation and cRNA injections were carried out essentially as described by Takahashi et al. (27Takahashi A. Watkins S.C. Howard M. Frizzell R.A. Am. J. Physiol. 1996; 271: C1887-C1894Crossref PubMed Google Scholar). A total of 1 ng of cRNA encoding CFTR with or without 100 ng of dsRNA targeting endogenous Xenopus Csp were injected, and expression was allowed to proceed for 4-5 days before current recordings. All data are provided as the mean ± S.E. Hsp70 ATPase Assays—The ability of WT or H43Q Csp to stimulate Hsp70 ATPase activity was determined as described previously (28Chamberlain L.H. Burgoyne R.D. Biochem. J. 1997; 322: 853-858Crossref PubMed Scopus (106) Google Scholar, 29Fewell S.W. Smith C.M. Lyon M.A. Dumitrescu T.P. Wipf P. Day B.W. Brodsky J.L. J. Biol. Chem. 2004; 279: 51131-51140Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) using the yeast homologue, Ssa1p. Briefly, Ssa1p-ATP complexes were formed as described (30Sullivan C.S. Tremblay J.D. Fewell S.W. Lewis J.A. Brodsky J.L. Pipas J.M. Mol. Cell. Biol. 2000; 20: 5749-5757Crossref PubMed Scopus (74) Google Scholar) by incubating Ssa1p (25 μg) with 100 μCi of [α-32P]ATP (PerkinElmer Life Sciences) and 25 μm ATP in complex buffer (25 mm HEPES-KOH, pH 7.5, 100 mm KCl, 11 mm magnesium acetate) for 30 min on ice. Free ATP was removed on a NICK spin column (Amersham Biosciences) pre-equilibrated with complex buffer. Using a Geiger counter, 15 fractions were collected (2 drops per fraction), and peak complex fractions were pooled, adjusted to 10% glycerol, and frozen in liquid nitrogen. Single turnover ATPase assays were performed at 30 °C by mixing 25 μl of thawed Ssa1p-ATP complex and 25 μl of complex buffer with or without Csp or Csp H43Q. At specified time points, a 6-μl aliquot of the reaction was removed and added to 2 μl of stop solution (36 mm ATP, 2 m LiCl, 4 m formic acid) on ice. Triplicate 2-μl aliquots of this mixture were spotted onto a TLC plate, developed, and dried for PhosphorImager analysis. NBD1 Aggregation Assays—The chaperone function of Csp was assayed by its ability to prevent the aggregation of NBD1 (amino acids 404-644), as described previously for other CFTR chaperones (31Youker R.T. Walsh P. Beilharz T. Lithgow T. Brodsky J.L. Mol. Biol. Cell. 2004; 15: 47878-47897Crossref Google Scholar). For the assay, purified hexahistidine-tagged NBD1 was diluted from a denaturing buffer (6 m guanidine-HCl, 20 mm HEPES, pH 7.5) into a refolding buffer (50 mm KCl, 2 mm MgCl2, 20 mm HEPES, pH 7.2) to a final concentration of 2 μm. Protein aggregation was measured by light scattering as a function of time at a wavelength of 320 nm at 30 °C in a 14DS UV-visible IR spectrophotometer (Aviv Associates, Inc.) with and without Csp or Csp H43Q. The results were plotted as relative aggregation normalized to values at 10 min for NBD1 alone (13Strickland E. Qu B.H. Millen L. Thomas P.J. J. Biol. Chem. 1997; 272: 25421-25424Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Csp Block of CFTR Maturation Requires Hsp70—The influence of Csp expression on steady-state levels of CFTR was determined by cotransfection of HEK293 cells with plasmids encoding both proteins (Fig. 1A). As observed previously (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), overexpression of Csp reduced the expression of mature, glycosylated (band C) CFTR and increased the expression of nascent, core glycosylated (band B) CFTR. This finding suggests that excess Csp interferes with the ability of CFTR to progress to Golgi-localized glycosylation sites. In our prior studies (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), a block of CFTR maturation was observed with the co-expression of either Csp isoform, Csp1 or Csp2. The latter variant represents a C-terminal truncation of 31 amino acids from Csp1. It is expressed at lower levels than Csp1 (32Chamberlain L.H. Borgoyne R.D. J. Biol. Chem. 1996; 271: 7320-7323Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 33Cuppola T. Gunderson C. FEBS Lett. 1996; 391: 269-272Crossref PubMed Scopus (30) Google Scholar) and is functionally indistinguishable from it (34Eberle K.K. Zinsmaier K.E. Buchner S. Gruhn M. Jenni M. Arnold C. Leibold C. Reisch D. Walter N. Hafen E. Hofbauer A. Pfugfelder G.O. Buchner E. Cell Tissue Res. 1998; 294: 203-217Crossref PubMed Scopus (32) Google Scholar), as shown in our prior work (18Zhang H. Peters K.W. Sun F. Marino C.R. Lang J. Burgoyne R.D. Frizzell R.A. J. Biol. Chem. 2002; 277: 28948-28958Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Therefore, we elected to focus the present studies on the predominantly expressed form, Csp1. We first examined several Csp mutations shown previously to interfere with regulated exocytic events (20Zhang H. Kelley W.L. Chamberlain L.H. Burgoyne R.D. Lang J. J. Cell Sci. 1999; 112: 1345-1351Crossref PubMed Google Scholar). As illustrated in Fig. 1A, the Csp HPD mutant, H43Q, restored the ability of CFTR to progress to Golgi glycosylation sites, as evidenced by the reappearance of CFTR band C. Mutation of the conserved HPD motif within the J-domain interferes with the ability of" @default.
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• W2129208664 title "Cysteine String Protein Monitors Late Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis" @default.
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