Conformational and Temperature-sensitive Stability Defects of the ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Post-endoplasmic Reticulum Compartments*

Deletion of phenylalanine at position 508 (ΔF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that ΔF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous ΔF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of ΔF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued ΔF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4–20-fold accelerated degradation rate between 37 and 40 °C is, most likely, due to decreased conformational stability of the rescued ΔF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native ΔF508 CFTR.

Cystic fibrosis (CF) 1 is one of the most prevalent lethal genetic disorders among Caucasian populations (1). The CF gene encodes the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl Ϫ channel and conductance regulator, expressed at the apical membrane of secretory epithelia (2,3). CFTR, a member of the ABC transporter family, consists of two structurally homologous halves, each comprised of six transmembrane (TM) helices and a nucleotide binding domain (NBD1 and NBD2), which are connected by the regulatory (R) domain (3). This complex, multidomain structure conceivably renders the posttranslational folding of wild type (wt) CFTR inefficient. More than 50% of the newly synthesized wt CFTR remains incompletely folded and is degraded at the endoplasmic reticulum (ER), whereas the remaining 25-50% undergoes an ATP-dependent conformational maturation and is exported to the cis/medial-Golgi, where its complex glycosylation can occur (4,5).
The hallmark of CF is the loss of cAMP-activated chloride conductance in the epithelial plasma membrane of airways, intestine, and exocrine glands (6 -8). More than 900 mutations have been identified in the CF gene, leading to impaired biosynthesis, processing, activation, and/or stability of CFTR (7,8). The most frequent mutation, deletion of phenylalanine at position 508 (⌬F508) in the NBD1, is found in Ͼ90% of the patients and detected in ϳ70% of CF chromosomes (1,7). It is believed that deletion of Phe-508 interrupts the posttranslational folding of CFTR (4, 5, 9 -11) and targets the core-glycosylated folding intermediate for degradation, predominantly via the ubiquitin-proteasome pathway at the ER (12,13). Exposure of ER-retention signals may contribute to the inability of folding intermediate(s) to exit the ER (14). Accordingly, negligible expression of ⌬F508 CFTR could be detected at the cell surface by immunochemical techniques in recombinant cells, CF primary airway cells, and CF tissues (9,15,16).
The recognition that the ⌬F508 CFTR channel is functional both in vivo (17)(18)(19) and after its reconstitution into the phospholipid bilayer (20) suggested that the CF phenotype could be alleviated by relocating the mutant CFTR from the ER to the plasma membrane. Reduced temperature (10,21,22), chemical chaperones (23)(24)(25), and down-regulation of Hsp70 (26,27) activity are thought to partially revert the folding defect of ⌬F508 CFTR and promote the accumulation of the functional channel at the cell surface. Importantly, using more sensitive electrophysiological techniques, constitutive accumulation of ⌬F508 CFTR was documented in the plasma membrane of primary epithelia from ⌬F508 homozygous mice (21,28) and in the intestinal and gallbladder epithelia of homozygous ⌬F508 patients (29,30). These studies parallel the results of recent immunolocation reports to some extent and suggest that the processing defect of the ⌬F508 CFTR is tissue-specific (30 -33). If the ER retention of the mutant is not complete, accelerated disposal from the post-ER compartments could contribute to its inability to express at the physiological level in certain tissues. Indeed, based on indirect evidence, we proposed that the rescued ⌬F508 CFTR has a short residence time at the cell surface of Chinese hamster ovary cells (CHO), expressing ⌬F508 CFTR heterologously (34). However, neither this nor any other study has provided direct biochemical or structural information regarding the behavior of ⌬F508 CFTR following its escape from ER.
Here we provide direct biochemical and functional evidence for the biological instability of the complex-glycosylated ⌬F508 CFTR. Furthermore, we show that the stability defect is temperature-sensitive, which is likely due to an attenuated conformational stability of the native state, demonstrated by increased protease susceptibility and the thermoaggregation tendency of the rescued mutant relative to its wt counterpart. The implications of these observations are fundamental with respect to understanding the cellular phenotype and designing more efficient therapeutic strategies in CF.

EXPERIMENTAL PROCEDURES
Cell Lines-A mixture of stably transfected baby hamster kidney (BHK) cells, expressing human wt and ⌬F508 CFTR with a carboxylterminal hemagglutinin (HA) epitope, was generated and maintained as described (4). Characterization of the HA-tagged CFTR variants will be described elsewhere.
Isolation of Microsomes-Isolation of ER, Golgi, and plasma membrane-enriched microsomes from BHK cells was performed using nitrogen cavitation and differential centrifugation as described (11). Where specified, the core-glycosylated wt or mutant CFTR was eliminated from the cells during a 3-h incubation in the presence of cycloheximide (CHX, 100 g/ml). The microsomal pellet was resuspended in HSE medium (10 mM sodium HEPES, 0.25 M sucrose, pH 7.6) and used either immediately or after being snap-frozen in liquid nitrogen.
Limited Proteolysis and Glycosidase Digestion-Microsomes were isolated from ⌬F508 and wt CFTR expressor BHK cells and incubated at a protein concentration of 1.3-1.5 and 0.8 -1.0 mg/ml, respectively, in the presence of trypsin or proteinase K for 15 min at 4°C in digestion buffer (phosphate-buffered saline) as described. Proteolysis was terminated by the addition of phenylmethylsulfonyl fluoride to 1 mM, and samples were immediately denatured in 2ϫ Laemmli sample buffer at 37°C for 20 min.
To distinguish between high mannose and complex-type N-linked oligosaccharide modification of CFTR, cell lysates were incubated with endoglycosidase H (7 g/ml) and peptide N-glycosidase F (31 g/ml) at 37°C for 3 h. The mobility shift of deglycosylated CFTR was visualized with immunoblotting using anti-HA mAb.
Electrophoresis and Immunoblotting-CFTR immunoblotting was performed with anti-HA mAb (Babco). Proteolytic digestion patterns were visualized with the mouse monoclonal M3A7 and L12B4 anti-CFTR Abs. L12B4 localizes to the region of the cytoplasmic NBD1 of CFTR (epitope within the range of amino acid positions 386 and 412), and M3A7 localizes to the region of the cytoplasmic NBD2 of CFTR (epitope within the range of amino acid positions 1365 and 1395) (11). Immunoblotting of ERP72 and GRP78 was performed with the rabbit polyclonal (SPA720) and mouse monoclonal (SPA826, Stressgen) antibodies, respectively. Immunoblots, with multiple exposures, were quantified using DuoScan transparency scanner and N.I.H. Image 6.1 software (developed by the National Institutes of Health and available at their web site).
Metabolic Labeling and Immunoprecipitation-Metabolic labeling and immunoprecipitation were performed essentially as described (4). Monolayer cells were pulse-labeled under defined conditions and chased at the indicated temperature for the time intervals specified for each experiment. Membrane proteins were solubilized in 1 ml of RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate, pH 8.0) supplemented with protease inhibitors (10 g/ml leupeptin and pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, and 10 mM iodoacetamide). Immunoprecipitates, obtained with anti-HA or M3A7 and L12B4 anti-CFTR mAbs, were analyzed by SDSpolyacrylamide gel electrophoresis and fluorography. The radioactivity incorporated was quantified using a PhosphorImager (Molecular Dynamics) with ImageQuant software (Molecular Dynamics) (4).
Determination of cAMP-stimulated Iodide Conductance-The plasma membrane cAMP-dependent halide conductance of transfected BHK cells was determined with iodide efflux as described (35). In brief, the chloride content was replaced with iodide by incubating the cells in loading buffer (136 mM NaI, 3 mM KNO 3 , 2 mM Ca(NO 3 ) 2 , 11 mM glucose, 20 mM HEPES, pH 7.4) for 60 min at room temperature. Iodide efflux was initiated by replacing the loading buffer with efflux medium (composed of 136 mM nitrate in place of iodide). The extracellular medium was replaced every minute with efflux buffer (1 ml). After a steady-state was reached, the intracellular cAMP level was raised by agonists (10 M forskolin, 0.2 mM CTP-cAMP, and 0.2 mM isobutylmethylxanthine) to achieve maximal phosphorylation of ⌬F508 CFTR, and collection of the efflux medium resumed for an additional 6 -9 min. The amount of iodide in each sample was determined with an iodideselective electrode (Orion).
Thermoaggregation Assay-Following the solubilization of BHK cells in Laemmli sample buffer, the aggregation tendency of wt and mutant CFTR was compared by exposing the lysate to temperatures ranging from 37 to 100°C for 5 min. SDS-resistant macromolecular aggregates were sedimented with centrifugation (17,000 ϫ g for 15 min). Monomeric mutant and wt CFTR, ERP72, GRP78, and Na ϩ /K ϩ -ATPase remaining in the supernatant were measured with quantitative immunoblotting using the appropriate Ab and ECL.

Deletion of Phe-508 Compromises the Stability of CFTR in the Post-ER Compartments-
To attain high sensitivity immunodetection, the influenza HA epitope-tagged ⌬F508 and wt CFTR were expressed stably in BHK cells. Immunoblot analysis showed that the HA-tagged ⌬F508 CFTR appears as an endoglycosidase H (endo-H)-and peptide N-glycosidase F (PN-Gase-F)-sensitive, core-glycosylated polypeptide with an apparent molecular mass Ϸ140 -150 kDa (Fig. 1a, empty arrowhead) at 37°C (9). The processing defect of the ⌬F508 CFTR could be partially overcome by the combination of glycerol and low temperature treatment, similarly to its nontagged counterpart, as reported in a number of cultured cells (10,23,24). Following the optimization of the rescue conditions, accumulation of the complex-glycosylated ⌬F508 CFTR was indicated by the appearance of endo-H-resistant immunoreactive polypeptide with an apparent molecular mass Ϸ170 kDa (Fig. 1a, black arrowhead) (9). The N-linked oligosaccharide modification and the apparent molecular mass of the rescued ⌬F508 CFTR are virtually identical to that of the HA-tagged complex-glycosylated wt CFTR (Fig. 1).
The biological stability of the complex-glycosylated wt and rescued ⌬F508 CFTR was determined upon inhibition of protein biosynthesis with CHX or vesicular transport from ER to Golgi with brefeldin A (BFA). After the accumulation of the complex-glycosylated ⌬F508 CFTR at 26°C, cells were incubated in the presence of CHX or BFA at 37°C, and the remaining CFTR was measured with quantitative immunoblotting, using anti-HA mAb as a function of incubation time (Fig. 1, b  and c). Densitometry revealed that the half-life of the complexglycosylated ⌬F508 CFTR (t1 ⁄2 Ϸ 5 h) is at least four times shorter than that of the wt CFTR (t1 ⁄2 Ϸ 22 h) at 37°C, regardless of the inhibitor (Fig. 1d). Similarly fast disposal of the ⌬F508 CFTR (t1 ⁄2 Ϸ 5 h) could be observed in the absence of CHX or BFA, after shifting the temperature from 26 to 37°C during the chase (Fig. 1c).
To verify that the deletion of Phe-508 destabilizes the complex-glycosylated CFTR, metabolic pulse-chase experiments were performed on transfectants expressing wt or rescued ⌬F508 CFTR (Fig. 2a). Phosphorimage analysis confirmed that the complex-glycosylated ⌬F508 was eliminated four times faster (t1 ⁄2 Ϸ 4.5 h) than wt CFTR (t1 ⁄2 Ϸ 18 h) at 37°C (Fig. 2b). Similar turnover rates were obtained upon rescuing the mu-FIG. 1. Stability of the complex-glycosylated ⌬F508 and wt CFTR cellular pool. The expression of HA-tagged wt and ⌬F508 CFTR in stably transfected BHK cells was monitored with immunoblotting, using anti-HA mAb and ECL. Black arrowhead, complex-glycosylated (band C); empty arrowhead, core-glycosylated (band B); gray arrowhead, unglycosylated CFTR (band A). a, the processing defect of the ⌬F508 CFTR was partially reverted by incubating the cells in medium supplemented with 10% glycerol at 26°C for overnight. Endo-H and PNGase F digestions were performed as described under "experimental Procedures." To facilitate immunodetection of the rescued mutant, cells were incubated for 1 h with cycloheximide. 50, 100, and 20 g of protein were separated from ⌬F508, rescued ⌬F508, and wt CFTR expressors, respectively. b, the disappearance of complex-glycosylated wt CFTR was monitored in the presence of BFA (5 g/ml) or CHX (100 g/ml). Subsequent to the indicated chase, cells were solubilized and equal amounts of protein (20 g) were immunoblotted with anti-HA mAb. c, a similar approach was used to determine the in vivo stability of the complex-glycosylated ⌬F508 CFTR, after a 24-h rescue treatment (see a). Expression of Na ϩ /K ϩ -ATPase was monitored with anti-Na ϩ /K ϩ -ATPase mAb. For reference, total protein from untreated wt (20 g) and ⌬F508 CFTR expressors (100 g) were loaded (two lanes on the far right). d, half-life determination of the wt and mutant CFTR. The complex-glycosylated wt and ⌬F508 CFTR persisting in the cell was calculated from densitometry of immunoblots shown on b and c. The chase was performed in the absence or presence of CHX or BFA, as indicated at 37°C. Data are expressed as percentage of the initial complex-glycosylated wt or ⌬F508 CFTR. Mean Ϯ S.E. (n ϭ 3-8). tant with either glycerol or reduced temperature alone, and on mock-treated wt CFTR, ruling out that osmotic or cold stress can account for the difference (data not shown). The accelerated disappearance of ⌬F508 CFTR also cannot be attributed to the loss of the carboxyl-terminal epitope, because similar halflives were measured with antibodies to epitopes located in NBD1, NBD2, or at the amino-terminal tail (data not shown). Finally, preliminary results obtained on pancreatic ductal epithelia expressing the ⌬F508 CFTR constitutively showed that the mutation impairs the stability of CFTR in polarized epithelia to a degree comparable with that found in nonpolarized cells (data not shown). These results collectively support the notion that the instability is an intrinsic property of the rescued ⌬F508 CFTR rather than epitope-or cell-specific.
Biochemical and Functional Stability of ⌬F508 CFTR at the Cell Surface-To examine whether the turnover of the plasma membrane-associated mutant CFTR is similar to that of the complex-glycosylated ⌬F508 CFTR pool, which comprises the trans-Golgi network, secretory vesicles, and endosomes as well, the fate of the rescued ⌬F508 CFTR was followed with cell surface biotinylation and iodide efflux measurements.
The plasma membrane proteins of BHK cells, expressing ⌬F508, rescued ⌬F508, or wt CFTR, were covalently tagged with NHS-SS-biotin, affinity-isolated on streptavidin beads, and immunoblotted with anti-HA mAb. Both the rescued ⌬F508 and the wt CFTR are amenable to biotinylation, in contrast to the ER-resident, core-glycosylated ⌬F508 CFTR (Fig. 3a). Densitometric analysis revealed that Ϸ50% of the biotinylated ⌬F508 CFTR disappeared after 4 h and became undetectable by 10 h of chase at 37°C (Fig. 3b). In contrast, the turnover of biotinylated wt CFTR was more than 4-fold slower (t1 ⁄2 Ϸ 18 h, data not shown) than the rescued ⌬F508 CFTR but comparable with the complex-glycosylated wt CFTR pool ( Fig.  1d and 2b).
The lack of endogenous cAMP-dependent anion conductance of BHK cells permitted us to monitor the arrival of functional ⌬F508 CFTR to the plasma membrane by the iodide efflux assay. At permissive temperatures, the cAMP-stimulated iodide release was proportional with the length of the rescue period up to 8 h (Fig. 4a, inset). After allowing ⌬F508 CFTR to accumulate at the cell surface for 5 h, raising the temperature to 37°C evoked a rapid disappearance of the mutant. The amount of iodide released by cAMP-dependent protein kinase stimulation decreased by 50% after 4 h and became undetectable after 10 h of chase at 37°C (Fig. 4, a and b) in cells expressing rescued ⌬F508, whereas no decrease was apparent over 10 h in wt CFTR expressors. Because the cAMP-activated iodide release could not be detected in rescued mock-transfected and parental BHK cells (data not shown), the functional and the biotinylation studies jointly indicate that the ⌬F508 CFTR channels are as unstable at the cell surface as in the post-ER compartments.
Taken together, the immunoblot, metabolic pulse-chase, biotinylation, and iodide measurements provide the first direct evidence that the functional and biochemical half-life of the complex-glycosylated ⌬F508 CFTR is 4 -5-fold shorter than its wt counterpart at 37°C, suggesting that structural differences may persist between the rescued ⌬F508 and wt CFTR at the plasma membrane.
The Thermostability of Rescued ⌬F508 CFTR in Vivo-To test whether the stability defect of the complex-glycosylated ⌬F508 CFTR at 37°C is related to its thermolability, pulselabeled cells were chased at temperatures ranging from 28 to 40°C. Although the t1 ⁄2 of wt and rescued ⌬F508 CFTR converged at 28°C (Ϸ50 h), a striking difference became apparent at temperatures above 30°C. Rescued ⌬F508 CFTR was at least 20-fold more unstable (t1 ⁄2 Ϸ 0.8 h) than its wt counterpart (t1 ⁄2 Ϸ 18 h) at 40°C (Fig. 5, a and b). In sharp contrast, no difference could be resolved in the relative turnover rates of the core-glycosylated (ER-resident) wt and ⌬F508 CFTR between 28 and 40°C (Fig. 5b), consistent with the notion that the core-glycosylated form represents a common folding intermediate, which is less susceptible to thermal denaturation (4, 5, 11). Considering that unfolding of soluble and membrane pro-FIG. 2. Turnover of metabolically labeled complex-glycosylated ⌬F508 and wt CFTR. A, wt and ⌬F508 CFTR expressors were metabolically labeled for 20 min and 3 h, respectively, under the conditions indicated. Conversion of the core-glycosylated into the complex-glycosylated form was ensured during a 2-h chase, prior to the stability of the complex-glycosylated form being assessed. After the chase at 37°C for the indicated time, CFTR was immunoprecipitated and visualized with fluorography. b, radioactivity associated with the complex-glycosylated wt and ⌬F508 CFTR was measured with phosphorimage analysis and expressed as a percentage of the initial incorporation at time 0 (means Ϯ S.E., n ϭ 3-4).
teins upon heat shock accelerates their cellular degradation (36), our results suggest that the thermal resistance of the complex-glycosylated ⌬F508 CFTR toward unfolding is substantially lower than its wt counterpart. Because large quantities of purified CFTR are not available to test this hypothesis directly, the protease susceptibility of native and the thermoaggregation propensity of solubilized CFTR variants were measured as indirect indicators of their structural stability.
The Impact of ⌬F508 Mutation on the Thermoaggregation and Protease Susceptibility of CFTR-SDS-solubilized cell lysates, obtained from BHK cells expressing ⌬F508, rescued ⌬F508, or wt CFTR, were heat-denatured at temperatures ranging between 37 and 100°C. Insoluble aggregates were sedimented by centrifugation, and monomeric CFTR remaining in the supernatant was quantified by immunoblotting. The thermostability of solubilized CFTR was characterized by measuring the aggregation temperature (T a ), at which 50% of monomeric CFTR is converted into SDS-resistant aggregates (Fig. 6a).
The T a of rescued ⌬F508 CFTR was 10°C lower (T a Ϸ 65°C) than its wt counterpart (T a Ϸ 75°C) but 10°C higher than the core-glycosylated ⌬F508 CFTR (T a Ϸ 55°C) (Fig. 6b). The following observations suggest that the progressively decreasing thermostability of the rescued and core-glycosylated ⌬F508 CFTR is likely the consequence of structural differences. Firstly, distinct aggregation pattern was also measured on immunoprecipitated wt and mutant CFTR (data not shown), implying that their aggregation propensity is independent of other polypeptides. Secondly, no difference could be documented in the thermoaggregation of the polytopic Na ϩ /K ϩ -ATPase, in parental BHK cells, or in cells expressing wt, ⌬F508, or rescued ⌬F508 CFTR (Fig. 6c). Conversely, ERP72 FIG. 3. The turnover of ⌬F508 CFTR at the cell surface. a, the turnover of biotinylated ⌬F508 CFTR. Rescued ⌬F508 CFTR were covalently labeled with NHS-SS-biotin and chased for 0 -10 h at 37°C. Biotinylated ⌬F508 CFTR was affinity-isolated on streptavidin beads and immunoblotted with anti-HA mAb. For comparison, biotinylated and nonbiotinylated wt CFTR are also shown. The core-glycosylated forms of neither the wt nor the mutant CFTR are susceptible to biotinylation. A fraction of cell lysate from the corresponding samples was directly probed with anti-HA mAb (lower panel). b, disappearance kinetic of biotinylated ⌬F508 CFTR at 37°C. The amount of biotinylated ⌬F508 CFTR remaining was measured with densitometry of immunoblots, obtained in experiments shown on a, and expressed as the percentage of biotinylated ⌬F508 CFTR before the chase (means Ϯ S.E., n ϭ 3).

FIG. 4.
Residence time of functional ⌬F508 CFTR at the plasma membrane. a, expression level of ⌬F508 CFTR at the cell surface was inferred based on the amount of cAMP-stimulated iodide release from BHK cells. Iodide release was proportional to the duration of the rescue period up to 8 h (inset). To avoid saturation of iodide release assay, subsequent measurements were performed on cells exposed to 26°C and glycerol for 5 h and to 37°C for 2 h (no chase). Continuous incubation at 37°C up to 10 h progressively eliminated the cAMP-stimulated iodide release (4 h and 10 h chase). cAMP-dependent protein kinase-agonist mixture was added at 0 min (arrow). Data points are averages of triplicate determinations in a representative experiment. b, the disappearance kinetic of functional ⌬F508 and wt CFTR from the cell surface. The magnitude of mean cAMP-stimulated iodide release was determined on ⌬F508 and wt CFTR expressors before and after the rescue as described in a. CHX (100 g/ml) was added to the chase medium for the wt CFTR-expressing cells. The iodide efflux was measured after 1 and 2 min of cAMP-dependent protein kinase stimulation for the wt and mutant CFTR, respectively, and expressed as percentage of that in the absence of chase (mean Ϯ S.E., n ϭ 3-4). and GRP78, soluble ER proteins, were resistant to aggregation, presumably due to their fully denatured state in SDS, in contrast to polytopic membrane proteins, which tend to preserve their aggregation tendency following detergent solubilization (37) (Fig. 6c). Finally and most importantly, no significant difference between the thermoaggregation tendency of ⌬F508, rescued ⌬F508, and wt CFTR was detected (T a Ϸ 75°C) when heat-denaturation was performed in Laemmli sample buffer supplemented with 8 M urea (Fig. 6, a and b); suggesting that urea denaturation of residual structural elements, prevailing in SDS micelles (37), abolishes the differences in the thermostability of CFTR variants.
Limited proteolysis in conjunction with immunoblot analysis was used as an alternative and more direct method to demonstrate that the rescued ⌬F508 CFTR is structurally distinct from its wt counterpart. This approach was instrumental in revealing the distinct conformation of the cytosolic domains (representing more than 70% of the CFTR polypeptide) in the complex-glycosylated wt and the core-glycosylated ⌬F508 CFTR and to monitor the folding of wt CFTR (11). Microsomes were isolated with differential centrifugation, and the cleavage patterns of wt, ⌬F508, and rescued mutant CFTR, obtained with limited trypsin and proteinase K digestions, were visualized with immunoblotting. CHX treatment of the cells ensured that the core-glycosylated CFTR was degraded prior to the isolation of microsomes, enriched in the complex-glycosylated wt or rescued ⌬F508 CFTR (Fig. 7a).
The in situ protease resistance of rescued ⌬F508 CFTR to trypsin and proteinase K was consistently 2-fold lower than that of the wt CFTR, regardless of whether the NBD2-or the NBD1-specific mouse monoclonal M3A7 and L12B4 anti-CFTR antibody was used (Fig. 7, b and c, and data not shown). Differences in the banding patterns between the rescued ⌬F508 and wt CFTR could also be recognized, which was more obvious with the NBD2-specific M3A7 than with the NBD1specific L12B4 mAb. This suggests that the altered accessibility of the proteolytic cleavage sites are not restricted to the NBD1, but encompasses the NBD2 in the rescued ⌬F508 CFTR (Fig. 7, b and c). A more substantial difference was observable in the protease resistance and proteolytic digestion patterns of the rescued and core-glycosylated ⌬F508 CFTR (Fig. 7, b and  c). Because neither the location, N-linked glycosylation, nor the association with peripheral membrane proteins alters the protease susceptibility of the wt CFTR (11), these results imply that the conformation and/or conformational stability of the rescued ⌬F508 CFTR lies between that of the core-glycosylated ⌬F508 CFTR and the fully mature wt CFTR. DISCUSSION While "saturation" of the ER quality control cannot be precluded in heterologous systems overexpressing ⌬F508 CFTR, this is not the case in the homozygous knock-in transgenic mouse, expressing ⌬F508 CFTR under its endogenous promoter (21,28). Short circuit current measurements have shown that at least 4% of the wt CFTR activity is present in the apical membrane of the intestinal epithelium and approximately 1-2% in cultured gall bladder epithelium of the homozygous ⌬F508 mouse, which could be augmented by 16 -18-fold at the reduced temperature (21). Extrapolating from our results, stabilization of the ⌬F508 CFTR could be responsible for a 4-fold increase of the plasma membrane chloride conductance, whereas the rest of the difference could be attributed to increased folding efficiency. Conversely, at physiological temperature, the short residence time of the spontaneously or pharmacologically rescued ⌬F508 CFTR compromises its ability to accumulate at the plasma membrane, thus providing additional explanation for the difficulties in detecting spontaneously escaped ⌬F508 CFTR both in native tissues and in cultured cells (9,15,16).
A number of mechanisms, or the combination thereof, could explain the biological instability of the complex-glycosylated ⌬F508 CFTR. First, structural alterations may accelerate the endocytosis and inhibit the recycling of the ⌬F508 CFTR from the endosomes to the cell surface, promoting the proteolytic degradation in the endolysosome. Second, preferential delivery of the mutant from post-ER compartments to the lysosomes may occur, similarly to the progressive aggregation of furin in the trans-Golgi network (38). Because repeated attempts failed to detect aggregated ⌬F508 CFTR in the detergent-insoluble fractions and the cell surface appearance of the mutant was FIG. 5. The stability defect of the rescued ⌬F508 CFTR is temperature-sensitive. a, after the pulse labeling of wt (20 min at 37°C) and ⌬F508 CFTR (3 h at 26°C in the presence of 10% glycerol) expressors, the conversion of core-to complex-glycosylated form was allowed to occur for 2 h at 37°C. Subsequent chase was performed at the indicated temperatures. For comparison, both wt and rescued ⌬F508 CFTR were loaded. b, the turnover of the core-and complex-glycosylated ⌬F508 and wt CFTR was determined with phosphorimage analysis from experiments shown on a (means Ϯ S.E., n ϭ 3-4, inset and data not shown). The ratio of complex-glycosylated wt and ⌬F508 CFTR half-lives (filled circle) is plotted as a function of temperature. In contrast, the relative turnover rate of the core-glycosylated wt and mutant CFTR (empty circles) is insensitive to the same temperature range. verified with both biochemical and functional assays, this scenario is unlikely to be the case. Finally, deletion of Phe-508 may promote the exposure of a dispersed degradation signal, comprised of hydrophobic patches and flexible loops in the rescued mutant. These motifs, similar to those described in the hydroxymethylglutaryl-CoA reductase (39) and conceivably exposed in the structurally destabilized G-protein-coupled receptor (40), can be recognized by the cellular proteolytic mechanisms and are perhaps responsible for the degradation. Whether a global structural destabilization, affecting the cytosolic and the transmembrane domains, is exclusively responsible for the accelerated degradation of the complex-glycosylated ⌬F508 CFTR or altered targeting mechanisms are also involved remains to be established. Based on the observations that structural destabilization of carboxyl-terminally truncated CFTR and G-protein-coupled receptors coincides with their accelerated disposal, and in the case of the mutant CFTR this process depends upon the activity of the ubiquitin-protea-some degradation pathway, 2 we favor the third scenario.
The indistinguishable in vivo and in vitro protease susceptibility profile of the core-glycosylated ⌬F508 and the early folding intermediate of wt CFTR, with the absence of aggregated ⌬F508 CFTR in the detergent-insoluble fraction, suggested that ⌬F508 favors the formation of folding intermediate(s) (11). This may occur by imposing a kinetic block on the folding reaction of CFTR, by energetically destabilizing the native form, or a combination of these. Whereas previous data obtained on recombinant NBD1 domain are compatible with a kinetic block (41,42), the present results and data derived using synthetic peptides (43) suggest that deletion of Phe-508 has multiple effects. The mutation not only interferes with the posttranslational folding in a temperature-dependent manner, as reported by a number of laboratories (4,5,10,11,21), but FIG. 6. Thermoaggregation of ⌬F508 and wt CFTR. a, wt, ⌬F508, and rescued ⌬F508 CFTR-expressing cells were solubilized in 2ϫ Laemmli sample buffer, and samples were incubated at the indicated temperature for 5 min. Aggregates were sedimented, and wt, ⌬F508, and rescued ⌬F508 CFTR remaining in the supernatant were visualized with anti-HA mAb and ECL. To eliminate the core-glycosylated forms in wt and rescued ⌬F508 CFTR expressors, cells were treated with CHX (100 g/ml) for 2 h before solubilization. ERP72 and GRP78 were detected with antibodies described under "Experimental Procedures." A significant fraction of the SDS-resistant aggregates, containing CFTR variants, could be recovered in Laemmli sample buffer comprising 8 M urea at room temperature (data not shown). Where indicated, heat denaturation was performed in 2ϫ Laemmli sample buffer supplemented with 8 M urea. b and c, quantitative assessment of the thermoaggregation tendencies. b, the monomeric complex-glycosylated wt and ⌬F508 CFTR (rescued ⌬F) and the core-glycosylated ⌬F508 CFTR (⌬F) were quantified with densitometry on immunoblots shown on a and expressed as the percentage detected without heat denaturation (means Ϯ S.E., n ϭ 3-7). c, the thermoaggregation of ERP72 (solid symbols) and Na ϩ /K ϩ -ATPase (open symbols) was determined as described for CFTR in untransfected BHK cells (triangles) and wt (diamonds), rescued ⌬F508 (squares), or core-glycosylated ⌬F508 CFTR (circles) expressors (mean Ϯ S.E., n ϭ 3-7). also renders temperature-sensitive stability defect to the complex-glycosylated (or native) ⌬F508 CFTR. According to the classical terminology the distinctive feature of the temperature-sensitive folding mutants is the absence of detectable defects in the protein formed at the permissive temperature (44). Therefore, ⌬F508 CFTR appears not to belong to this category of mutations. Intriguingly, while glycerol could partially rescue the folding defect of the ⌬F508 CFTR at 37°C, it was unable to restore the impaired stability of the rescued form, 3 suggesting that distinct structural alterations are responsible for the folding and the stability defect.
In summary, the in vivo turnover measurements together with the protease susceptibility and thermoaggregation results indicate that whereas the ⌬F508-imposed folding defect can be partially overcome, the biological and structural characteristics of the rescued ⌬F508 1 CFTR, generated at permissive temper-ature, remain distinct from its wt counterpart. Development of novel strategies to stabilize the native ⌬F508 CFTR would complement the present therapeutic approaches aiming to correct the folding defect at the ER, in tissue culture models (10,(22)(23)(24)(25)45), in CF mice (21), and in human trials (46,47).