Cystic Fibrosis Transmembrane Conductance Regulator Folding Mutations Reveal Differences in Corrector Efficacy Linked to Increases in Immature Cystic Fibrosis Transmembrane Conductance Regulator Expression.

Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin-proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 µM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain.

ABC transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography. Regulation is driven by the cytosolic domains and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the ABC transporter defective in the most abundant rare inherited disease cystic fibrosis. We have combined biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: (i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and (ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies different modes of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.

From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations.

More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.

Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier-dependent pathway.

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27's ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4's impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin-proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein.

Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform.

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.

Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3ß, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3ß and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3ß increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3ß knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3ß interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3ß interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

CFTR Folding Consortium: methods available for studies of CFTR folding and correction.

The CFTR Folding Consortium (CFC) was formed in 2004 under the auspices of the Cystic Fibrosis Foundation and its drug discovery and development affiliate, CFF Therapeutics. A primary goal of the CFC is the development and distribution of reagents and assay methods designed to better understand the mechanistic basis of mutant CFTR misfolding and to identify targets whose manipulation may correct CFTR folding defects. As such, reagents available from the CFC primarily target wild-type CFTR NBD1 and its common variant, F508del, and they include antibodies, cell lines, constructs, and proteins. These reagents are summarized here, and two protocols are described for the detection of cell surface CFTR: (a) an assay of the density of expressed HA-tagged CFTR by ELISA and (b) the generation and use of an antibody to CFTR's first extracellular loop for the detection of endogenous CFTR. Finally, we highlight a systematic collection of assays, the CFC Roadmap, which is being used to assess the cellular locus and mechanism of mutant CFTR correction. The Roadmap queries CFTR structure-function relations at levels ranging from purified protein to well-differentiated human airway primary cultures.

AS160 modulates aldosterone-stimulated epithelial sodium channel forward trafficking.

Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (I(sc)). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and I(sc) 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and I(sc) under basal conditions to approximately 80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, beta and epsilon. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3beta and epsilon and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.

Regulation of CFTR trafficking by its R domain.

Phosphorylation of the R domain is required for cystic fibrosis transmembrane conductance regulator (CFTR) channel gating, and cAMP/protein kinase A (PKA) simulation can also elicit insertion of CFTR into the plasma membrane from intracellular compartments (Bertrand, C. A., and Frizzell, R. A. (2003) Am. J. Physiol. 285, C1-C18). We evaluated the structural basis of regulated CFTR trafficking by determining agonist-evoked increases in plasma membrane capacitance (Cm) of Xenopus oocytes expressing CFTR deletion mutants. Expression of CFTR as a split construct that omitted the R domain (Deltaamino acids 635-834) produced a channel with elevated basal current (Im) and no DeltaIm or trafficking response (DeltaCm) upon cAMP/PKA stimulation, indicating that the structure(s) required for regulated CFTR trafficking are contained within the R domain. Additional deletions showed that removal of amino acids 817-838, a 22-amino acid conserved helical region having a net charge of -9, termed NEG2 (Xie, J., Adams, L. M., Zhao, J., Gerken, T. A., Davis, P. B., and Ma, J. (2002) J. Biol. Chem. 277, 23019-23027), produced a channel with regulated gating that lacked the agonist-induced increase in CFTR trafficking. Injection of NEG2 peptides into oocytes expressing split DeltaNEG2 CFTR prior to stimulation restored the agonist-evoked DeltaCm, consistent with the concept that this sequence mediates the regulated trafficking event. In support of this idea, DeltaNEG2 CFTR escaped from the inhibition of wild type CFTR trafficking produced by overexpression of syntaxin 1A. These observations suggest that the NEG2 region at the C terminus of the R domain allows stabilization of CFTR in a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA stimulation.

An obligatory heterodimer of 14-3-3beta and 14-3-3epsilon is required for aldosterone regulation of the epithelial sodium channel.

Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323-16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3beta with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3beta alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3beta knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3epsilon, a second aldosterone-induced isoform. As observed previously for 14-3-3beta knockdown, small interfering RNA-induced reduction of 14-3-3epsilon markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3beta and 14-3-3epsilon were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3beta or 14-3-3epsilon reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, beta and epsilon, interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport.

The epithelial sodium channel (ENaC) traffics to apical membrane in lipid rafts in mouse cortical collecting duct cells.

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.

Identification and membrane localization of electrogenic sodium bicarbonate cotransporters in Calu-3 cells.

Cystic fibrosis (CF) is a severely life-shortening genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). Impaired bicarbonate secretion is a key component of CF-related pancreatic disease, but the role of impaired bicarbonate secretion in CF lung disease is less well understood. The submucosal glands of the conducting airways produce and secrete a complex airway surface liquid that lines the airway epithelium and plays a significant role in mucociliary clearance. The serous cell is the predominant cell type of the submucosal gland and a predominant site of CFTR expression. Calu-3 cells are a model of airway submucosal gland serous cells that demonstrates vectorial bicarbonate secretion in response to elevations in cAMP. Based on previously published measurements of unidirectional ion flux, pharmacological inhibition of short-circuit current and ion substitution studies, one can hypothesize the existence of an electrogenic sodium bicarbonate cotransporter (NBC) in the basolateral membrane of Calu-3 cells that mediates bicarbonate entry from the interstitium. To test this hypothesis, we performed reverse-transcriptase PCR, western blotting, and surface biotinylation to identify and localize electrogenic NBCs in Calu-3 cells. Our data demonstrate that both pNBC1 and NBC4 mRNAs can be identified and that their protein products are expressed at the basolateral membrane of polarized Calu-3 cells. These data suggest that these transporters contribute to regulated bicarbonate secretion across Calu-3 cells and perhaps human airway submucosal glands.

Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hyperabsorption in cystic fibrosis.

Efficient clearance of mucus and inhaled pathogens from the lung is dependent on an optimal airway surface liquid (ASL) volume, which is maintained by the regulated transport of sodium and chloride across the airway epithelium. Accumulating evidence suggests that impaired mucus clearance in cystic fibrosis (CF) airways is a result of ASL depletion caused by excessive Na(+) absorption through the epithelial sodium channel (ENaC). However, the cellular mechanisms that result in increased ENaC activity in CF airways are not completely understood. Recently, proteases were shown to modulate the activity of ENaC, but the relevance of this mechanism to the physiologic regulation of ASL volume is unknown. Using primary human airway epithelial cells, we demonstrate that: (i) protease inhibitors are present in the ASL and prevent the activation of near-silent ENaC, (ii) when the ASL volume is increased, endogenous protease inhibitors become diluted, allowing for proteolytic activation of near-silent channels, and (iii) in CF, the normally present near-silent pool of ENaC is constitutively active and the alpha subunit undergoes increased proteolytic processing. These findings indicate that the ASL volume modulates the activity of ENaC by modification of the serine protease-protease inhibitor balance and that alterations in this balance contribute to excessive Na(+) absorption in cystic fibrosis.

14-3-3 isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels.

Aldosterone increases sodium absorption across renal collecting duct cells primarily by increasing the apical membrane expression of ENaC, the sodium entry channel. Nedd4-2, a ubiquitin-protein isopeptide ligase, tags ENaC with ubiquitin for internalization and degradation, but when it is phosphorylated by the aldosterone-induced kinase, SGK1, Nedd4-2 is inhibited and apical ENaC density and sodium absorption increase. We evaluated the hypothesis that 14-3-3 proteins participate in the aldosterone-mediated regulation of ENaC by associating with phosphorylated Nedd4-2. Mouse cortical collecting duct (mCCD) epithelia cultured on filters expressed several 14-3-3 isoforms; this study focused on an isoform whose expression was induced 3-fold by aldosterone, 14-3-3beta. In polarized mCCD epithelia, aldosterone elicited significant, time-dependent increases in the expression of alpha-ENaC, SGK1, phospho-Nedd4-2, and 14-3-3beta without altering total Nedd4-2. Aldosterone decreased the interaction of alpha-ENaC with Nedd4-2, and with similar kinetics increased the association of 14-3-3beta with phospho-Nedd4-2. Short interfering RNA-induced knockdown of 14-3-3beta blunted the aldosterone-induced increase in alpha-ENaC expression, returned alpha-ENaC-Nedd4-2 binding toward prealdosterone levels, and blocked the aldosterone-stimulated increase in transepithelial sodium transport. Incubation of cell extracts with a selective phospho-Nedd4-2 antibody blocked the aldosterone-induced association of 14-3-3beta with Nedd4-2, implicating SGK1 phosphorylation at Ser-328 as the primary site of 14-3-3beta binding. Our studies show that aldosterone increases the expression of 14-3-3beta, which interacts with phospho-Nedd4-2 to block its interaction with ENaC, thus enhancing sodium absorption by increasing apical membrane ENaC density.

Clathrin-mediated endocytosis of the epithelial sodium channel. Role of epsin.

Here we present evidence that the epithelial sodium channel (ENaC), a heteromeric membrane protein whose surface expression is regulated by ubiquitination, is present in clathrin-coated vesicles in epithelial cells that natively express ENaC. The channel subunits are ubiquitinated and co-immunoprecipitate with both epsin and clathrin adaptor proteins, and epsin, as expected, co-immunoprecipitates with clathrin adaptor proteins. The functional significance of these interactions was evaluated in a Xenopus oocyte expression system where co-expression of epsin and ENaC resulted in a down-regulation of ENaC activity; conversely, co-expression of epsin sub-domains acted as dominant-negative effectors and stimulated ENaC activity. These results identify epsin as an accessory protein linking ENaC to the clathrin-based endocytic machinery thereby regulating the activity of this ion channel at the cell surface.

Cysteine string protein interacts with and modulates the maturation of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel whose phosphorylation regulates both channel gating and its trafficking at the plasma membrane. Cysteine string proteins (Csps) are J-domain-containing, membrane-associated proteins that have been functionally implicated in regulated exocytosis. Therefore, we evaluated the possibility that Csp is involved in regulated CFTR trafficking. We found Csp expressed in mammalian epithelial cell lines, several of which express CFTR. In Calu-3 airway cells, immunofluorescence colocalized Csp with calnexin in the endoplasmic reticulum and with CFTR at the apical membrane domain. CFTR coprecipitated with Csp from Calu-3 cell lysates. Csp associated with both core-glycosylated immature and fully glycosylated mature CFTRs (bands B and C); however, in relation to the endogenous levels of the B and C bands expressed in Calu-3 cells, the Csp interaction with band B predominated. In vitro protein binding assays detected physical interactions of both mammalian Csp isoforms with the CFTR R-domain and the N terminus, having submicromolar affinities. In Xenopus oocytes expressing CFTR, Csp overexpression decreased the chloride current and membrane capacitance increases evoked by cAMP stimulation and decreased the levels of CFTR protein detected by immunoblot. In mammalian cells, the steady-state expression of CFTR band C was eliminated, and pulse-chase studies showed that Csp coexpression blocked the conversion of immature to mature CFTR and stabilized band B. These results demonstrate a primary role for Csp in CFTR protein maturation. The physical interaction of this Hsc70-binding protein with immature CFTR, its localization in the endoplasmic reticulum, and the decrease in production of mature CFTR observed during Csp overexpression reflect a role for Csp in CFTR biogenesis. The documented role of Csp in regulated exocytosis, its interaction with mature CFTR, and its coexpression with CFTR at the apical membrane domain of epithelial cells may reflect also a role for Csp in regulated CFTR trafficking at the plasma membrane.