Article |
2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada
3 Howard Hughes Medical Institute, Department of Physiology and Biochemistry, University of California, San Francisco, San Francisco, CA 94143
4 Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
Address correspondence to Gergely L. Lukacs, Hospital for Sick Children, Program in Cell and Lung Biology, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: (416) 813-5125. Fax: (416) 813-5771. email: glukacs{at}sickkids.ca
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Abstract |
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Key Words: recycling; sorting; mutation; endocytosis; ubiquitin receptors
Abbreviations used in this paper: CFTR, cystic fibrosis transmembrane conductance regulator; CHX, cycloheximide; ESCRT, endosomal sorting complex required for transport; Hrs, hepatocyte growth factorregulated tyrosine kinase substrate; MVB, multivesicular body; STAM, signal-transducing adaptor molecule; Ub, ubiquitin; Vps, vacuolar protein sorting; wt, wild type.
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Introduction |
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Impaired plasma membrane expression or function of the CFTR, a cAMP-activated chloride channel, accounts for the phenotypic manifestation of CF, the most common inherited disorder in the Caucasian population (Zielenski and Tsui, 1995). Previously, we have identified two naturally occurring pathogenic mutants that display thermosensitive conformational defects in post-Golgi compartments. The COOH-terminally truncated CFTR, lacking the last 70 amino acid residues (70 CFTR), traverses the biosynthetic pathway and undergoes complex glycosylation with the efficiency of wild-type (wt) CFTR (Haardt et al., 1999; Benharouga et al., 2001). In contrast, deletion of Phe508 (
F508, found in 90% of CF patients) imposes an ER processing block on CFTR that could be partially overcome or "rescued" at permissive temperature (<30°C) or by chemical chaperones in cultured cells (Denning et al., 1992; Sato et al., 1996). The conformational defect of the complex-glycosylated rescued
F508 (r
F508) and the
70 CFTR has been demonstrated by their increased protease susceptibility, accounting for the four- to sixfold faster metabolic turnover of the mutants in post-Golgi compartments (Zhang et al., 1998; Benharouga et al., 2001; Sharma et al., 2001).
The present work was undertaken to elucidate the retrieval mechanism of conformationally defective 70 and r
508 CFTR from the cell surface that may represent a paradigm for the peripheral quality control of membrane proteins. The results suggest that misfolding of CFTR dramatically augments the ubiquitination susceptibility of the channel in post-Golgi compartments. In turn, Ub modification serves as a recognition signal for the Ub-dependent endosomal sorting machinery that reroutes the channel from recycling toward the multivesicular body (MVB)/lysosomal degradation.
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Results |
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Misfolding augments the ubiquitination of CFTR in post-Golgi compartments
Although nonnative soluble and ER-associated polypeptides are known substrates of ubiquitination, the susceptibility of poorly folded plasma membrane proteins to Ub conjugation is poorly understood. To assess whether Ub modification is involved in the disposal of nonnative CFTR, the ubiquitination level of wt and mutant CFTR, confined to post-Golgi compartments, was determined. To this end, complete degradation of the core-glycosylated F508,
70, and wt CFTR (and their ubiquitinated adducts) was ensured by treating the cells with CHX for 3 h (Fig. 4 a, lanes 1 and 2, bottom and top, respectively; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200312018/DC1). Then, CFTR was immunoisolated under denaturing conditions with anti-CFTR antibody, and the precipitates were probed with anti-Ub antibody. Detection of ubiquitinated r
F508,
70, and wt CFTR in cells expressing exclusively the complex-glycosylated forms by three different anti-Ub antibodies demonstrated that CFTR is susceptible to ubiquitination in post-Golgi compartments (Fig. 4 a; unpublished data). Importantly, densitometry showed that ubiquitination of the complex-glycosylated r
F508 and
70 CFTR was increased by
20-fold relative to the wt channel at 37°C (Fig. 4 b).
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The CFTR-Ub chimera has recycling and stability defects
If Ub conjugation plays a primary role in the recycling and cell surface stability defect of the mutants, fusing Ub to the wt channel (CFTR-Ub) may mimic the consequences of destabilizing mutations. In-frame fusion of Ub to the COOH terminus of CFTR indeed inhibited the channel recycling by 90% (Fig. 5 a), reduced its cell surface T1/2 from
16 to
1.3 h (Fig. 5 b), and diminished the steady-state expression of the mature CFTR (Fig. 5 c). These observations indicate that the chimera reproduces the peripheral trafficking defects of the r
F508 and the
70 CFTR. Fusing a Ub molecule harboring arginines in place of all its lysine residues resulted in a similar expression and recycling defect to that of the CFTR-Ub (unpublished data), suggesting that the lysine residues of Ub do not serve as acceptor sites for poly-Ub chain formation in the chimera. The unaltered internalization rates of CFTR-Ub relative to wt CFTR (4.7 ± 1.3%/min; Fig. 5 d) is consistent with the notion that the Ub-dependent recycling defect is the primary cause for the rapid down-regulation of the chimera from the cell surface, as is for
70 and r
508 CFTR.
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Association of misfolded CFTR and CFTR-Ub chimeras with the Ub-dependent endosomal sorting machinery
It is well established that targeting of certain ubiquitinated cell surface receptors for lysosomal degradation requires the recruitment of Ub-binding adaptor proteins (e.g., Hrs, STAM, and eps15, or their yeast orthologues Vps27, HSE1, and Ede1) to the cytosolic surface of early endosomes (Katzmann et al., 2002; Bonifacino and Lippincott-Schwartz, 2003). Because the hepatocyte growth factorregulated tyrosine kinase substrate (Hrs) and signal-transducing adaptor molecule (STAM) are the primary Ub-interacting motif-containing adaptors that form the sorting complex involving components of the endosomal sorting complex required for transport I (ESCRT I; Bilodeau et al., 2002, 2003; Bishop et al., 2002; Katzmann et al., 2002; Raiborg et al., 2002; Shih et al., 2002; Mizuno et al., 2003; Schnell and Hicke, 2003), the association of Hrs and STAM-2 with CFTR was assessed first. Immunoprecipitation of endogenous Hrs pulled down the complex-glycosylated rF508 and
70 CFTR as well as the CFTR-Ub, but neither the ER-associated
F508 CFTR nor the complex-glycosylated wt CFTR (Fig. 7 a). Selective association of STAM-2 (Fig. 7 b) and the TSG101 (Fig. 7 c), a component of ESCRT I, with the conformationally defective CFTR variants was also documented by the immunoprecipitation technique. Finally, similar results were obtained by probing for the association of CFTR variants with hVps25 and hVps32, components of the ESCRT II and III, respectively (Fig. 7 d). These observations are consistent with our hypothesis that the interaction of destabilized and preferentially ubiquitinated CFTR with the Ub-dependent sorting machinery is responsible for the recycling defect and accelerated degradation of the mutants.
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Rescuing the folding defect or preventing the Ub recognition restores the recycling of CFTR
To demonstrate the conformation-dependent endosomal sorting of CFTR, the subcellular destination of the mutants was determined as a function of the ambient temperature. CFTR containing endocytic compartments were identified based on their characteristic pH and protein composition (Mukherjee et al., 1997). Antimouse Fab fragments conjugated to the pH-sensitive fluorophore FITC were complexed to anti-HA antibody and internalized with CFTR to measure the lumenal pH of CFTR-containing vesicles by ratiometric fluorescence video image analysis (Gagescu et al., 2000). Although recycling endosomes have a pHre of 6.5 ± 0.05 and accumulate transferrin in BHK cells, the lysosomes have a pHly <5.0 (measured by FITC-EGF) and harbor LAMP-1 (Fig. 8 a and Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200312018/DC1).
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Immunostaining of internalized CFTR variants with markers of recycling endosomes (transferrin) and lysosomes (FITC-dextran and Lamp-1) confirmed that internalized rF508 and
70 CFTR were diverted to MVB/lysosomes, whereas the wt CFTR was associated with recycling endosomes at 37°C (Fig. 8 b; unpublished data). Lysosomal proteolysis of the wt and
70 CFTR was also supported by the detection of immunoreactive degradation intermediates in purified lysosomes (Benharouga et al., 2001).
Kinetic model of CFTR peripheral trafficking
Besides sequestration of the mutant CFTR at the early endosomes, accelerated targeting into MVB/lysosomes could enhance the efficiency of the channel degradation (Katzmann et al., 2002; Hicke and Dunn, 2003). We used multicompartment kinetic analysis to estimate the intercompartmental transfer rates of the wt and rF508 channels. Endocytic rate constants (ken) were calculated from the internalization rates. Based on ken, turnover of the cell surface and the complex-glycosylated pools, degradation (kdeg), and recycling rate (kre) constants were computed by the SAAM II multicompartment analysis program. The kdeg of the r
F508 CFTR was increased by twofold relative to wt CFTR (Fig. 8 c). The kre of r
F508 CFTR was attenuated by nearly fivefold as compared with the wt (Fig. 8 c). These results, together with the recycling measurements, suggest that misfolding has a major impact on the sequestration of CFTR at early endosomes. Endosomal retention in concert with modestly accelerated transfer rates into MVB/lysosomal compartments is sufficient to attenuate the cell surface stability of the r
F508 CFTR by 10-fold (Fig. 1 c).
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Discussion |
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Misfolding promotes the ubiquitination of CFTR in post-Golgi compartments
Compelling evidence suggests that Ub conjugation is a prerequisite for the disposal of misfolded rF508 and
70 CFTR from the cell surface.
First, we demonstrated a correlation between the conformational destabilization and the ubiquitination propensity of rF508 and
70 CFTR in post-Golgi compartments. Rescuing the folding defect at the permissive temperature (28°C) diminished, whereas thermo-denaturation (40°C) further increased the ubiquitination of the mutants, leading to the preferential recycling or MVB/lysosomal targeting, respectively (Fig. 4 c). Neither accelerated biosynthesis nor the saturation of the degradation machinery can explain the nearly 20-fold increased level of ubiquitinated mutants as compared with that of the wt CFTR. The translational rates of the
70 and
F508 CFTR were comparable to that of the wt CFTR in BHK cells (Zhang et al., 1998; Haardt et al., 1999), and the copy number of wt CFTR at the cell surface is estimated to be only a few thousand molecules per cell.
Second, covalent attachment of Ub to the COOH-terminal tail of the wt CFTR reproduced the recycling as well as the cell surface stability defects of the mutants (Fig. 5). The genetically fused Ub mimics the cellular consequences of post-translational ubiquitination of CFTR rather than provoking misfolding. This conclusion is supported by the largely preserved processing efficiency of the CFTR-Ub (unpublished data), and the proportionally decreased PKA-stimulated whole-cell current and cell surface density of the chimera (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200312018/DC1). Furthermore, the Ile44Ala point mutation in Ub was able to restore the recycling and stability of the chimera to that of the wt CFTR (Fig. 5).
Finally, the most direct evidence for the role of Ub-conjugation was provided by the fact that heat inactivation of the E1 Ub-activating enzyme prevented the disappearance of 70 and r
F508 CFTR from the plasma membrane in ts20 cells without influencing their endocytosis rates (Fig. 6). Collectively, these observations support the pivotal role of Ub modification in the down-regulation of the misfolded r
F508 and
70 CFTR from the cell surface via post-endocytic mechanism(s).
Ubiquitination is known to effectively down-regulate receptors and transporters from the plasma membrane of yeast and mammalian cells (Rotin et al., 2000; Sorkin and Von Zastrow, 2002; Bonifacino and Traub, 2003; Hicke and Dunn, 2003). Ligand-induced (e.g., EGF and ß-adrenergic agonist) or constitutive (e.g., ENaC) ubiquitination of membrane proteins requires substrate recognition by Ub-conjugating enzymes and Ub protein ligases (E2/E3s; Pickart, 2001). This process is usually mediated by proteinprotein interaction domains of the relevant E3s (e.g., SH2, WW, and PDZ domains in Cbl, Nedd4/Rsp5, and LNX, respectively) and their cognate binding sites (e.g., phospho-Tyr, PPXY, phospho-Ser/Thr, or PDZ-binding motif; Weissman, 2001; Hicke and Dunn, 2003). Although it cannot be precluded that exposure of similar signals is involved in the ubiquitination of the two CFTR mutations, we favor the scenario that Ub conjugation is promoted by the unfolding of the channel and involves solvent-exposed hydrophobic protein surfaces. A similar mechanism has been invoked in the degradation of misfolded polypeptides at the ER (Laney and Hochstrasser, 1999; Cyr et al., 2002; Ellgaard and Helenius, 2003). Structural destabilization of the rF508 and
70 CFTR nucleotide-binding domains was indeed demonstrated (Zhang et al., 1998; Benharouga et al., 2001). This mechanism would also be reminiscent of the degradation signal identified in random peptides and in the Mat
2 transcription factor (Sadis et al., 1995; Gilon et al., 1998, 2000; Johnson et al., 1998).
Importantly, a small amount of ubiquitinated wt, complex-glycosylated CFTR was reproducibly detected by three different anti-Ub antibodies (unpublished data). This cannot be attributed to the presence of other ubiquitinated polypeptides because CFTR was isolated under denaturing conditions. It is tempting to speculate that the low level of ubiquitination of the wt CFTR is caused by its slow, physiological unfolding that eventually terminates its long residence time (T1/2 14 h) at the cellular periphery. The profound difference in the metabolic turnover of protease-resistant, structurally stable wt and the protease-susceptible, conformationally labile mutant CFTR is consistent with the previously proposed hypothesis that the structural stability of soluble polypeptides constitutes one of the determinants of their metabolic stability (McLendon and Radany, 1978; Parsell and Sauer, 1989; Kowalski et al., 1998; Klink and Raines, 2000).
Misfolded CFTR is targeted toward lysosomal degradation by the Ub-dependent endosomal sorting machinery
Recycling of plasma membrane proteins protects polypeptides from degradation and allows them to undergo repeated cycles of endocytosis and exocytosis (Ghosh and Maxfield, 1995; Mellman, 1996). On the other hand, the sorting process at early endosomes offers an efficient mechanism to prevent the accumulation of misfolded membrane proteins at the cell surface. This is exemplified by the second step of the peripheral quality control of CFTR. Using Ub adducts of CFTR, obtained by post-translational ubiquitination or by genetic engineering, we presented three lines of evidence in support of the notion that ubiquitinated channels are selectively retrieved from recycling and are redirected for degradation into MVB/lysosomes. First, ubiquitination efficiently prevented the recycling and dramatically reduced the cell surface density, as well as the stability of the mutant and the chimera (Fig. 1 c, Fig. 3 b, and Fig. 5, a and b). Second, vesicular pH measurements verified that both the mutants and the CFTR-Ub are targeted into lysosomes after their internalization, in contrast to the wt CFTR, which traverses recycling endosomes (Fig. 8 a). The recycling defect provides a plausible explanation for the 422-fold faster metabolic turnover rates of the rescued F508 CFTR at 37 and 40°C, respectively (Sharma et al., 2001). Rescuing the folding defect and thus reducing the ubiquitination of the r
F508 and
70 CFTR restored their constitutive recycling (Fig. 8 a) in parallel to their metabolic stabilization (Benharouga et al., 2001; Sharma et al., 2001). Third, immunocolocalization of endocytosed CFTR with markers of the recycling compartment and lysosomes substantiated the notion that the native and ubiquitinated CFTR are segregated at early endosomes (Fig. 8 b).
Ub-binding adaptor proteins, including Hrs and STAM, have a critical role in the retrieval of ubiquitinated cargo for lysosomal degradation at sorting endosomes (Raiborg et al., 2002; Sachse et al., 2002). Selective binding of destabilized rF508,
70 CFTR, and CFTR-Ub variants, but not the wt CFTR to Hrs, STAM-2, TSG101, Vps25, and Vps32, is consistent with the hypothesis that transport of ubiquitinated CFTR from early endosome to MVB/lysosomes involves its successive association with Hrs/STAM-2 and the human homologues of the ESCRT I, II, and III (Katzmann et al., 2002). Importantly, the I44A mutation in Ub not only prevented the recognition of CFTR-UbA and GST-UbA by Hrs and STAM-2, but also did so with downstream components of the ESCRT I, II, and III complexes (Fig. 7). As a result, CFTR-UbA resumed its constitutive recycling and escaped from MVB/lysosomal degradation (Fig. 5 b and Fig. 8 a), substantiating our working model that Ub recognition and the subsequent association of the misfolded CFTR with components of ESCRT complexes are required for MVB/lysosomal targeting.
Suppressor screens in yeast have identified several class E Vps mutants, including Vps23 and Vps27 (the yeast orthologues of TSG101 and Hrs, respectively), which are either directly or indirectly involved in the degradation of misfolded plasma membrane proteins (Li et al., 1999; Gong and Chang, 2001). Although the role of ubiquitination in the disposal of misfolded membrane proteins has not been established in yeast (Arvan et al., 2002), these observations suggest that some of the components of the peripheral quality control are evolutionarily conserved.
In summary, our analyses demonstrate that the folding state of CFTR is monitored not only during the early stage of its biogenesis in the ER (Brodsky and McCracken, 1999; Ellgaard et al., 1999; Arvan et al., 2002; Hampton, 2002), but is also surveyed in post-Golgi compartments. Our results provide direct evidence for the functional interplay between the ubiquitination machinery recognizing misfolded peripheral membrane proteins and the Ub-dependent endosomal sorting pathway in the elimination of misfolded CFTR, accounting for the cellular and clinical phenotype of the 70 CFTR mutation (Haardt et al., 1999). We propose that similar mechanisms may be involved in the recognition and degradation of other structurally destabilized membrane proteins that escape ER quality control or are generated by environmental stress. Thus, the peripheral quality control may have fundamental significance in the pathogenesis of conformational diseases and in the maintenance of cellular homeostasis.
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Materials and methods |
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Expression of recombinant Ub for pull-down assay
Bacterial expression plasmids containing GST-Ub, GST-UbF4A,I44A (GST-Ub2A), and GST-UbF4A (GST-UbA) were constructed by PCR mutagenesis in pGEX4T, and were expressed in HB101 cells. Recombinant proteins were purified, bound to glutathione-sepharose 4B (Amersham Biosciences), and incubated with HeLa cell lysate (3 mg, at 4°C for 2 h). Bound polypeptides were separated by SDS-PAGE and Hrs was visualized by immunoblotting with a polyclonal anti-Hrs antibody (Raiborg et al., 2002).
Cultures of differentiated human primary respiratory epithelia
Nasal polyps were obtained from surgical materials of CF and non-CF individuals with informed consent of the family by a procedure approved by the Research Ethics Board of the Hospital for Sick Children. Two of the CF patients were homozygous F508, and the third one had
F508/R1162X genotype. Explants were grown on collagen-coated dishes in DME/Ham's F12, 20% FBS, gentamycin, streptomycin, and amphotericin-B. Fibroblasts were removed by trypsinization, and epithelial cells were seeded at >80% confluence on collagen-coated filters and were cultured for >3 d. Polarization was demonstrated by the domain-specific CFTR-mediated anion conductance (Fig. S1 a).
Immunoblotting, immunoprecipitation, and electrophysiology
Immunoblotting of CFTR and densitometric analysis was performed with NIH image 1.62 as described previously using ECL (Sharma et al., 2001). Antibodies were used as follows: FK1, FK2 (Affinity BioReagents, Inc.), and P4D1 (Santa Cruz Biotechnology, Inc.) anti-Ub antibodies; a6F anti-Na/K ATPase antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa city, IA), M3A7 and L12B4 anti-CFTR antibody (Chemokine) and a rabbit polyclonal anti-CFTR antibody raised against the COOH-terminal tail of CFTR; 4A10 anti-TSG101 antibody (GeneTex); and anti-Hrs (Raiborg et al., 2002) and anti-E1 enzyme antibodies (Covance). Rabbit pAbs against human Vps25/EAP25 and human Vps32/CHMP4B were raised against maltose-binding protein fusion proteins and were affinity purified on Affi-Gel beads (Bio-Rad Laboratories) containing recombinant proteins. Whole-cell current measurements were performed as described previously (Haardt et al., 1999).
Cell surface density measurements, internalization, and recycling of CFTR
The cell surface density of 3HA-tagged CFTR was measured by the binding of the monoclonal anti-HA antibody (Covance; at 0°C for 1 h) and 125I-labeled goat antimouse secondary antibody (3 µCi/ml, at 0°C for 1 h; Amersham Biosciences). Specific binding was calculated by correcting with the nonspecific antibody adsorption in the presence of 10 µg/ml nonimmune IgG (Santa Cruz Biotechnology, Inc.). Nonspecific antibody binding was usually 35% of the specific signal. Internalization of CFTR was calculated from the removal rate of anti-HA antibody from the cell surface during 25-min internalization at 37°C. Data are expressed as percentage of the specific radioactivity detected before internalization.
To measure CFTR recycling, first the endosomal CFTR was labeled with anti-HA antibody (at 37°C for 30 min). Then the remaining cell surfaceassociated antibody was blocked by biotinylated goat antimouse antibody (KPL) and 10 µg/ml streptavidin (Sigma-Aldrich) on ice. Recycling was initiated by shifting the temperature to 37°C for 510 min. The amount of exocytosed antibodyCFTR complex was measured by biotinylated secondary antibody and 125I-streptavidin (Amersham Biosciences) at 0°C, and was expressed as the percentage of the anti-HA antibody associated with the endosomal compartment before exocytosis. The internalized anti-HA antibody in complex with CFTR was determined in parallel by monitoring the disappearance of cell surface anti-HA antibody with the biotinstreptavidin sandwich technique. The internalized anti-HA antibody was corrected for the degradation of mutant CFTR during the labeling. The radioactivity corresponding to internalized anti-HA antibody was 40,000 and
10,00020,000 cpm for the wt and mutants, respectively. Each data point represents 34 independent experiments, consisting of triplicate measurements.
Vesicular pH measurement of CFTR-containing endocytic organelles
The pH of endocytic vesicles containing CFTR was measured by fluorescence ratio imaging of internalized anti-HA antibody (Covance) complexed with FITC-conjugated goat antimouse Fab antibody (Jackson ImmunoResearch Laboratories). Cells were incubated with the primary and secondary antibodies in tissue culture medium at 37°C for 13 h, washed with NaKH medium (140 mM NaCl, 5 mM KCl, 20 mM Hepes, 10 mM glucose, 0.1 mM CaCl2, and 1 mM MgCl2, pH 7.3), and imaged on a microscope (Axiovert 100; Carl Zeiss MicroImaging, Inc.) at 35°C, equipped with a cooled CCD camera (Princeton Instruments) and a 63x NA 1.4 Planachromat objective. The metabolic stability of CFTR was not influenced by the continuous presence of anti-HA primary and secondary antibody (Fig. S2), indicating that antibody binding did not promote the degradation of CFTR.
Image acquisition and analysis were performed with the MetaFluor® software (Universal Imaging Corp.). Images were acquired at 490 ± 5 nm, and 440 ± 10 nm excitation wavelength, using a 535 ± 25 nm emission filter. In situ calibration curves were obtained by clamping the vesicular pH between 4.5 and 7.0 in K+-rich medium (135 mM KCl, 10 mM NaCl, 20 mM Hepes or 20 mM MES, 1 mM MgCl2, and 0.1 mM CaCl2, containing 10 µM nigericin, 10 µM monensin, and 5 µM carbonyl cyanide p-chlorophenylhydrazon) and recording the fluorescence ratio of cells loaded with FITC-transferrin (Molecular Probes, Inc.), FITC-EGF (followed by a 2-h chase), or FITC-Fab and anti-HA antibody in CFTR-Ub2A expressors. The fluorescence ratios as a function of extracellular pH provided the standard curve for the pH determination of CFTR-containing endosome. In addition, one point calibration was done on each coverslip by clamping the vesicular pH to 6.5. Mono- and multi-peak Gaussian fits for vesicular pH were performed with Origin 7.0 software (OriginLab® Corporation). The average pH of each type of vesicle population was calculated as the arithmetic mean of the data and was identical to the Gaussian mean, based on single-peak distribution fitting, except for 70 CFTR, where two-peak Gaussian distribution analysis was done (Fig. S4).
Kinetic modeling of wt and rF508 CFTR trafficking
The recycling (kre) and degradation (kdeg) rate constants of CFTR were calculated by the SAAM II program (University of Washington, Seattle, WA), using a two-compartmental model (Fig. 8 c) and the Rosenbrock integration method. Data for fitting were obtained from the cell surface decay kinetics of wt and rF508 CFTR complexed to anti-HA antibody. The value of kde was adjusted so that the model reproduced metabolic half-life of the complex-glycosylated r
F508 and wt CFTR measured by the pulse-chase technique (Sharma et al., 2001). It was assumed that degradation of CFTR in the MVB/lysosomes is instantaneous. The rate constants are expressed as fractional transfer of molecules/min.
Statistical analysis
Experiments were repeated at least three times, and graphs include means ± SEM. Two-tailed P values were calculated at 95% confidence level with unpaired t test, using Prism software (GraphPad Software, Inc.).
Online supplemental material
Fig. S1 demonstrates that anti-HA antibody binding does not induce degradation of the wt CFTR. Fig. S2 illustrates the functional polarization of human respiratory epithelia. Elimination of the core-glycosylated CFTR during the CHX chase is documented in Fig. S3. Fig. S4 shows the pH distribution curves of vesicles containing CFTR variants. The electrophysiological characterization of the CFTR-Ub chimera is depicted in Fig. S5. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200312018/DC1.
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Acknowledgments |
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M. Benharouga was supported by a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship. J. So was awarded a summer studentship from the Canadian Cystic Fibrosis Foundation (CCFF). This work was supported by grants from CCFF and the CIHR to G.L. Lukacs. H. Stenmark was supported by the Research Council of Norway, the Novo Nordisk Foundation, and the Norwegian Cancer Society. M. Sharma is a CIHR doctoral student. F. Pampinella is a postdoctoral fellow of the CCFF.
Submitted: 3 December 2003
Accepted: 29 January 2004
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