PHYSIOLOGICAL REVIEWS   Vol. 79 No. 1 January 1999, pp. S167-S173
Copyright ©1999 by the American Physiological Society

Biosynthesis and Degradation of CFTR

RON R. KOPITO

Department of Biological Sciences, Stanford University, Stanford, California

I. INTRODUCTION
II. BIOGENESIS OF WILD-TYPE AND MUTANT CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR
    A. Mislocalization of F508 CFTR
    B. Inefficient Intracellular Maturation of CFTR Precursors
    C. Immature CFTR Molecules Are Transiently Associated With Molecular Chaperones
III. DEGRADATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR
    A. Immature CFTR Is Rapidly Degraded
    B. F508 Produces a Conditional Block in Folding of NBD1
    C. Misfolding of NBD1 and NBD2 Have Nonequivalent Effects on CFTR Maturation
    D. Degradation of Misfolded CFTR by the Ubiquitin-Proteasome Pathway
    E. Are Other Proteases Also Involved in ER Degradation of CFTR?
IV. PROSPECTS FOR CYSTIC FIBROSIS THERAPY
REFERENCES

	ABSTRACT

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Kopito, Ron R. Biosynthesis and Degradation of CFTR. Physiol. Rev. 79, Suppl.: S167-S173, 1999.  Many of the mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause cystic fibrosis interfere with the folding and biosynthetic processing of nascent CFTR molecules in the endoplasmic reticulum. Mutations in the cytoplasmic nucleotide binding domains, including the common allele F508, decrease the efficiency of CFTR folding, reduce the probability of its dissociation from molecular chaperones, and largely prevent its maturation through the secretory pathway to the plasma membrane. These mutant CFTR molecules are rapidly degraded by cytoplasmic proteasomes by a process that requires covalent modification by multiubiquitination. The effects of temperature and chemical chaperones on the intracellular processing of mutant CFTR molecules suggest that strategies aimed at increasing the folding yield of this protein in vivo may eventually lead to the development of novel therapies for cystic fibrosis.

	I. INTRODUCTION

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The autosomal recessive human genetic disorder cystic fibrosis (CF) is caused by the loss or dysfunction of a plasma membrane Cl channel known as the CF transmembrane conductance regulator (CFTR) (15, 26, 28). Although the number of disease-associated sequence alterations at the CFTR locus now exceeds 400, a single allele accounts for nearly 70% of CF chromosomes (15). F508 is associated with a severe form of the disease; >90% of CF patients have at least one F508 allele. The subject of intensive scrutiny, the F508 allele encodes an unstable and inefficiently folded CFTR polypeptide that fails to be delivered to its proper cellular location in the plasma membrane. The investigation of the mechanisms that underlie the biosynthesis, trafficking, and degradation of F508 CFTR provides a unique opportunity to understand the pathogenesis of this genetic disease at the molecular and cellular level and is the subject of the present review.

The human CFTR gene encodes an integral membrane glycoprotein composed of 1,440 amino acid residues (26). Sequence analysis indicates that CFTR is a member of the "traffic ATPase" or ABC transporter superfamily (11, 12). Alignment of the CFTR sequence with other members of this family suggests the existence of five distinct structural domains: two membrane-spanning domains (MSD), each composed of six membrane-spanning helices; two nucleotide-binding domains (NBD); and one central regulatory "R domain." Although basic aspects of CFTR transmembrane topology have been confirmed by biochemical analysis, the model remains necessarily crude in the absence of any detailed structural information. Two essential features of CFTR structure are of particular note for the discussions that follow. The bulk of the amino acid residues that comprise the NBD and R domain are predicted to be exposed to the cytoplasmic face of cellular membranes; aside from the "loops" that intervene between transmembrane helices 1 and 2 in MSD1 and between 7 and 8 in the corresponding position in MSD2, very little of the mass of CFTR is likely to be exposed to the luminal or exofacial surface of cellular membranes.

	II. BIOGENESIS OF WILD-TYPE AND MUTANT CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

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On SDS-PAGE gels, "mature" CFTR endogenously expressed in colonic cell lines like T84 migrates as a diffuse band of 150-160 kDa, which has been referred to as "band C" (2). The CFTR sequence contains two potential Asn-linked oligosaccharide chains (at positions 894 and 900). Pulse-chase analysis indicates that CFTR is initially synthesized as a 135- to 140-kDa core-glycosylated precursor containing high-mannose oligosaccharide chains that are distinguishable by their sensitivity to endoglycosidase H (endoH) (2, 18, 35). Thus, like other integral membrane glycoproteins, the initial stages of CFTR biogenesis begin with the formation in the endoplasmic reticulum (ER) membrane of a core-glycosylated 135- to 140-kDa "immature" form that is a precursor to the mature 150- to 160-kDa CFTR that contains complex, endoH-resistant oligosaccharide chains. Sometimes this mature form bearing complex glycans is designated "fully glycosylated." To avoid confusion, the term mature CFTR in this review refers specifically to CFTR that migrates as a diffuse, 150- to 160-kDa band (equivalent to band C; Ref. 2) that is resistant to digestion with endoH and thus presumed to have matured at least to the cis/medial cisternae of the Golgi apparatus. The term immature CFTR specifically refers to the 135- to 140-kDa, endoH-sensitive form (equivalent to band B; Ref. 2), corresponding to nascent CFTR that has not been processed by mannosidases in the cis/medial Golgi. The existence of other bands, including an apparently unglycosylated form (band A; Ref. 2), complicates this analysis and is discussed in section IIID.

The initial suggestion that CFTR "mislocalization" contributes to the pathogenesis of CF was provided by Cheng et al. (2), who observed that COS cells transiently transfected with F508 fail to accumulate immunoreactive CFTR corresponding to the size of mature CFTR. In contrast, both the mature and the immature forms of CFTR accumulate in COS cells transfected with wild-type CFTR. This steady-state distribution of CFTR in transfected cells has been confirmed in different cell lines by other laboratories (18, 35) and is also reflected in the pattern of immunoreactivity observed by light microscopy of transfected cells and human tissues. For example, CFTR antibodies detected immunoreactive protein at the cell periphery in cells stably or transiently transfected with wild-type CFTR (2). In contrast, immunoreactivity was restricted to a perinuclear ER-like distribution in cells expressing F508. Kartner et al. (14) used monoclonal anti-CFTR antibodies to examine CFTR expression in frozen sections of human sweat glands from normal and CF skin biopsies. They observed a strong signal in the apical plasma membrane region of sweat ducts from normal donors, consistent with physiological studies of transepithelial Cl transport. In contrast, only weak signals were detected in duct cells from F508 homozygotes; those signals were restricted to an apparent "cytoplasmic and perinuclear" location and were absent from the plasma membrane region. Yang et al. (39) used electron microscopy coupled with immunoperoxidase staining to localize CFTR in cell lines transformed with recombinant adenovirus vectors, confirming a peripheral distribution for wild-type CFTR. Their data also confirmed that F508 CFTR was localized to the ER region. However, because of the diffusion typical of the immunoperoxidase product, precise localization of CFTR to a specific membrane structure or to a particular face of the ER or plasma membrane was not possible in those studies. Clearly, a thorough electron microscopic study of CFTR localization in tissue and cells expressing mutant and wild-type proteins, using a more precise reporter such as colloidal gold, would help to resolve some important issues regarding the cell biology of CFTR.

The absence of F508 from the cell surface could be due either to defective intracellular delivery or maturation of the protein to the plasma membrane, or to instability of the mature F508 protein at the cell surface. Pulse-chase studies have been performed to discriminate between these two possibilities. Although both forms of CFTR are synthesized as initially indistinguishable 140-kDa immature core-glycosylated precursors, only wild-type CFTR is chased to a ~160-kDa, endoH-resistant mature form (18, 35). These results suggest that deletion of Phe-508 interferes with a step in the maturation of immature CFTR from the ER to the Golgi complex. This step is normally accompanied by the acquisition of complex N-linked oligosaccharide chains and is an important checkpoint in the quality control pathway that operates in eukaryotic cells to prevent the deployment of improperly folded membrane and secretory glycoproteins (for review, see Ref. 8). Interestingly, maturation of wild-type CFTR is also inefficient. Less than 30% of newly synthesized 140-kDa immature CFTR molecules ever acquire complex oligosaccharides indicative of maturation to post-ER compartments, even in cells expressing low levels of endogenous CFTR (18, 35). Therefore, this inefficiency in CFTR maturation is not likely to be the consequence of overexpression, as originally suggested by Cheng et al. (2). It is possible that CFTR is inefficiently processed because of the absence of a limiting subunit or assembly factor. However, mature CFTR appears to be monomeric (19). Moreover, processing efficiency is low even in polarized epithelial cell lines that express endogenous CFTR and should also express additional putative interacting proteins. Possibly, this low efficiency in CFTR processing is a consequence of the protein's expression in transformed, immortalized cell lines such as HEK 293 or COS. Maturation of CFTR has never been examined in native tissue or in primary cell culture. Interestingly, the efficiency of assembly of the pentameric nicotinic acetylcholine receptor, which approaches 100% in primary chick muscle cell culture (29), is reduced to ~20% in established muscle cell lines (21) or in transfected cells (6a). Moreover, the inefficiency of acetylcholine receptor assembly in transfected cells can be partially reversed by overexpression of the molecular chaperone calnexin (6a). On the other hand, the maturation of P-glycoprotein, an ABC transporter bearing significant structural similarity to CFTR, is nearly 100% in HEK 293 cells (Ref. 17; C. Ward and R. Kopito, unpublished data), indicating that inefficient processing is not a universal property of transformed cell lines. These data underscore the need to characterize more fully the pathway of CFTR folding and, in particular, the role of cellular assembly factors and chaperones in this process.

The folding of proteins, particularly oligomeric or multidomain proteins, occurs in cells in association with molecular chaperones (9, 20). Transient interaction of nascent polypeptides with such chaperone molecules helps reduce the probability of inappropriate interaction of incomplete partially folded intermediates and increases the overall yield of correctly folded product. Molecular chaperones are present in all cellular compartments including the ER. Yang et al. (39) reported that immature CFTR was immunoprecipitated from pulse-labeled cells with antibodies to cytoplasmic 70-kDa heat shock protein (HSP70), but not with antibodies against the ER chaperone BiP (39). The apparent lack of association of CFTR with BiP may reflect the fact that little of CFTR's mass is likely to be accessible to the ER lumen or that BiP association may be fleeting. No association was detected between HSP70 and the mature form of CFTR (after chase), suggesting that the interaction with HSP70 is transient and is specific for newly synthesized, core-glycosylated chains. Likewise, immature F508 was also present in HSP70 immunoprecipitates from cells expressing the mutant construct. The amount of labeled F508 associated with HSP70 was observed to decrease in parallel with the disappearance and probable degradation (see sect. IIIB) of the mutant protein during the chase period. Although the site(s) of HSP70-CFTR interaction with CFTR or F508 have not been identified, it is reasonable to speculate that HSP70 interacts with cytoplasmically exposed portions, most likely the cytoplasmic loops and the NBD. Significantly, many CF-associated mutations, including F508, map to the putatively cytoplasmic portions of CFTR (see sect. IIIC).

Nascent CFTR also interacts with the transmembrane ER chaperone calnexin (23). This chaperone is unusual in that its selectivity for newly synthesized chains in the ER is mediated, at least in part, by its lectinlike binding to monoglucosylated high-mannose oligosaccharides (10). Pulse-labeled immature CFTR was coimmunoprecipitated with calnexin from cells transfected with CFTR or F508, and from epithelial cells expressing endogenous CFTR (23). No mature CFTR bands were detected in calnexin coimmunoprecipitates, even after a prolonged chase, consistent with the hypothesis that calnexin is a chaperone that interacts only with incompletely folded, ER forms of nascent polypeptides. Thus, like HSP70, calnexin appears to interact transiently with newly synthesized forms of CFTR; dissociation of immature CFTR from calnexin is accompanied by its conversion to the mature form.

These data suggest that at least two chaperones, HSP70 and calnexin, participate in CFTR biogenesis. Both chaperones appear to form transient complexes with nascent, immature CFTR molecules. Dissociation of these complexes correlates temporally with the maturation of CFTR molecules to a post-ER compartment and with a large increase in the metabolic stability of CFTR molecules. It remains to be established, however, whether the two chaperones act sequentially or simultaneously during CFTR biogenesis.

A key unresolved question is how, or whether, these chaperone interactions contribute to the retention or degradation of misfolded CFTR (or F508) chains in the ER. Both HSP70 and calnexin also interact with immature forms of F508; the amount of F508 associated with either chaperone appears to decrease in parallel with the total amount of immature F508 molecules. These data have been interpreted to indicate that, compared with CFTR, the interaction of HSP70 with F508 is "prolonged." However, to determine whether these chaperones have a role in targeting misfolded F508 molecules for degradation, it would be important to determine whether the chaperones dissociate from misfolded chains before degradation. Although previous studies have established a correlation between the amount of chaperone-associated, pulse-labeled F508 and the total amount of F508 in cells, they have not directly addressed the question of whether all immature F508 is chaperone associated. For example, future studies might assess whether the fraction of F508 that is not associated with chaperones changes during a chase.

How can the "quality control" machinery discriminate between misfolded CFTR molecules and those that are in the process of folding? Are there other chaperones that participate in folding and degradation of CFTR? Pulse-chase and coimmunoprecipitation studies suggest that at least two ER luminal chaperones, BiP and GRP94, do not interact detectably with either F508 or CFTR. However, the participation of other chaperones, including those of the TRiC, HSP90, HSP40, and low-molecular-weight families, may also contribute to CFTR maturation; investigation of these interactions may provide answers to some of the questions posed above.

	III. DEGRADATION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

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The data presented above suggest the following model: CFTR and F508 are synthesized in the rough ER as immature core-glycosylated 140-kDa precursors, which are associated with cytoplasmic HSP70 and calnexin. A fraction (20-40%) of CFTR matures to the Golgi apparatus, coincident with its dissociation from HSP70 and calnexin and with its conversion to a stable (half-life = 7.5 h) ~160-kDa form bearing endoH-resistant complex type oligosaccharides. The majority (60-80%) of immature CFTR and nearly all immature F508 molecules fail to mature and are quantitatively and rapidly (half-life = 20-40 min) degraded. Thus maturation of CFTR precursor from ER to Golgi is accompanied by an ~20-fold increase in stability. Inhibiting ER-to-Golgi traffic with brefeldin A prevents the formation of the high-molecular-weight CFTR species (18, 35). Under these conditions, a fraction (20-40%) of immature-sized CFTR molecules nevertheless becomes resistant to degradation, indicating that escape from the ER is not a prerequisite for the increased stability characteristic of the mature protein. This stabilization likely reflects the acquisition of a correct tertiary structure (see below) that is accompanied by chaperone release and, in the absence of brefeldin A, maturation to the Golgi apparatus. The F508 mutation evidently interferes with this folding step. The F508 molecules, therefore, remain in an unfolded or partially folded state, are not correctly released from interaction with chaperones, and are rapidly delivered to the degradation machinery. It is not, strictly speaking, correct to assert that the F508 mutation causes CFTR to be retained in the ER, because "retained" implies a stability that is not normally observed. This view is also more consistent with the observation that CFTR immunoreactivity is not simply redirected from plasma membrane to ER in sweat duct cells from F508 homozygotes (as is implicit in designating F508 a "trafficking" mutation) but is reduced to nearly undetectable levels (14). What little immunoreactive F508 there is in the ER of mutant cells probably represents molecules that have yet to be degraded; thus it is possible to produce the appearance of ER retention by overexpressing F508 to levels that exceed the capacity of the cells to degrade the protein.

Overexpression of F508 cDNA in mammalian cells leads to the appearance of functional CFTR Cl channels in the plasma membrane (4), suggesting that the F508 mutation is "leaky." Because most people afflicted with CF have at least one copy of F508, the observation of residual function in F508 CFTR raises the possibility of developing a therapeutic strategy based on remediation of the F508 phenotype. In principle, this leakiness could result if one or more components of the quality control apparatus were saturated due to overexpression (a mass-action effect), or if some small proportion of nascent F508 molecules was able to fold correctly, thereby escaping degradation (a kinetic effect). Several lines of evidence argue in favor of the kinetic model. First, F508 molecules can be detected at the cell surface of Xenopus oocytes (6) or mammalian cells (5) when cultured at reduced (20-30°C) temperature, suggesting that the block in maturation is temperature sensitive. Second, F508 maturation can be increased by treating cells with high concentrations of glycerol, a small membrane-permeant polyol that can function as a "chemical chaperone" in stabilizing protein structure (1, 31). Third, studies of the refolding of denatured, isolated, bacterial-expressed NBD1 in vitro suggest that the F508 mutation stabilizes a folding intermediate that is prone to self-aggregation (24, 25). Thus, although the yield of NBD1 refolding is reduced by deletion of Phe-508, neither the kinetics of refolding nor the stability of the refolded protein is affected by this mutation. Moreover, the refolding yield is increased both by reduced temperature and by the presence of chemical chaperones like glycerol (24). These data suggest that the misprocessing phenotype of F508 results from a decrease in the efficiency of one or more steps in the NBD1 folding pathway, with consequent self-aggregation of nascent F508 polypeptides. Whether the particular kinetic step(s) that are affected by the F508 mutation are the same as those that reduce the overall efficiency in wild-type CFTR folding remains to be determined.

The "misfolding" phenotype is not restricted to the F508 allele, although F508 is by far the most common disease-associated allele. Indeed, many missense mutations in CFTR, either naturally occurring (CF associated) or produced through site-directed mutagenesis, appear to interfere with the maturation of nascent CFTR chains to the plasma membrane. These include mutations in the MSD (34), the NBD (7), and the cytoplasmic loops (3, 32, 33). Strikingly, as first noted by Gregory et al. (7), maturation of CFTR appears to be more sensitive to mutations in NBD1 than to mutations at homologous positions in NBD2. For example, of 15 missense or deletion mutations in NBD1, only 3 result in a protein that is able to mature. In contrast, of 13 mutations in NBD2, only 1 has been reported to be misprocessed. This is all the more striking if one considers that of five homologous pairs of residues at the two NBD, all at NBD2 are correctly processed and only one substitution at one NBD1 position is processed. Given the degree of sequence homology between the CFTR NBD and NBD of other nucleotide hydrolyzing proteins, it is surprising that identical mutations at homologous positions in the two CFTR NBD have such different consequences for the overall fate of the protein. One possibility for this discrepancy is that assumptions based on structural similarity are incorrect, or that the folding pathways for these similar domains differ. This issue could be addressed by comparing the effect of mutations at parallel positions in NBD1 and NBD2 in the in vitro refolding assay described above. Alternatively, this phenomenon could suggest that the quality control machinery is more tolerant of subtle mutations at the second NBD compared with the first NBD. This kind of discrimination might result if the commitment to degrade misfolded CFTR occurred early in its biosynthesis, possibly even cotranslationally, as the COOH-terminal domain is synthesized immediately before release of CFTR from the ribosome. It is possible that release from the ribosome or from the translocation apparatus serves as an early "checkpoint" in the quality control processes that monitor protein folding in the ER. Indeed, misfolded CFTR and F508 molecules are degraded rapidly, without apparent lag after synthesis (35). Moreover, at least in vitro, nascent CFTR polypeptides are multiubiquitinated cotranslationally, before release from the ribosome (30). Thus nascent CFTR chains that lack correctly folded NBD1 are more likely to interact with components of the quality control apparatus before the completion of synthesis than are chains that contain misfolded NBD2. Clearly, additional studies will be needed to resolve this puzzling and interesting observation.

Several lines of evidence suggest that misfolded CFTR chains are substrates for the ubiquitin-proteasome pathway. Treatment of cells expressing CFTR or F508 with proteasome inhibitors, including the reportedly specific drug lactacystin, causes the accumulation of multiubiquitinated forms of CFTR that can be detected as a characteristic 7-kDa "ladder," suggesting that CFTR and F508 degradation is mediated by the proteasome (36). Moreover, in proteasome-inhibited cells, CFTR antibodies immunoprecipitate a high-molecular-weight "smear" that is recognized with antibodies to ubiquitin or to an epitope tag on ubiquitin, demonstrating that undegraded CFTR is modified by ubiquitin (36). Finally, CFTR-immunoreactive protein accumulates in cells in which multiubiquitination has been impaired by coexpression of dominant negative ubiquitin, demonstrating that multiubiquitination is not only a consequence of the accumulation of undegraded CFTR, but is a prerequisite for its degradation (36). Although these data establish a role for the ubiquitin-proteasome pathway in CFTR and F508 degradation, they do not exclude the possibility that other proteases may also participate in this process, either independent of or cooperatively with the proteasome.

Proteasomes are abundant in the cytoplasm and nucleus of eukaryotic cells, where they mediate the degradation of ubiquitinated substrates. Although proteasomes have been reported to be associated with the cytoplasmic surface of the ER membrane (22, 27), there is no evidence for the presence of these proteolytic particles within the ER lumen. Because the bulk of CFTR's mass is predicted to be exposed to the cytoplasm, extensive portions of CFTR are potentially accessible to degradation by membrane-associated proteasomes. However, roughly one-third of CFTR is predicted to be embedded in the lipid bilayer. How can cytoplasmic proteasomes degrade these membrane-protected portions of CFTR? One possible mechanism, suggested initially by studies of rapid degradation of newly synthesized myosin heavy chain class I heavy chains (37, 38), may involve dislocation of the translocated and membrane-integrated polypeptide from the ER membrane. Several membrane and secretory proteins have now been shown to be degraded by cytoplasmic proteasomes after their dislocation to the cytoplasm and deglycosylation by cytoplasmic N-glycanases (16). Inhibition of proteasome activity leads to the accumulation of deglycosylated species that are present in the cytosol or at the cytoplasmic side of microsomal vesicles. Although there is presently no direct evidence that such a mechanism also participates in CFTR degradation by the proteasome, it should be noted that, in addition to the characteristic smear of high-molecular-weight aggregated and ubiquitinated CFTR, inhibition of proteasome activity also leads to the accumulation of a detergent-insoluble form of CFTR that migrates at the size of the core protein lacking any N-linked glycans (band A) (36), suggesting that deglycosylation of CFTR does accompany its degradation by the proteasome. Further studies will be required to ascertain whether or not membrane dislocation is required for CFTR degradation. If CFTR is not dislocated, then it is difficult to imagine how proteasomes at the cytoplasmic face of the membrane can degrade all three topological domains of this integral membrane protein.

The effects of proteasome inhibitors on CFTR degradation have also been evaluated by pulse-chase analysis. Interestingly, proteasome inhibitors have only a modest effect on the half-life of CFTR and, despite a measurable slowing of degradation (after ~1-h chase), do not appear to block CFTR degradation completely (13, 18). For example, even in the presence of proteasome inhibitors, nearly all immature CFTR has disappeared after a 3-h chase. These results have been interpreted by some to suggest that proteasome inhibitors only retard, not block, CFTR degradation, implying that additional nonproteasomal proteolytic systems also contribute to CFTR degradation (13). However, extensive screening of known protease inhibitors in combination with proteasome inhibitors has not revealed any protease inhibitors that further stabilize CFTR (Ward and Kopito, unpublished data). Several observations suggest that an alternative explanation must also be considered. What is measured in these pulse-chase studies is typically the decay in radioactivity migrating at the 140- to 150-kDa size corresponding to immature CFTR, immunoprecipitated from nonionic detergent extracts of labeled cells. Proteasome inhibitors clearly slow this decay. However, this decay does not reflect exclusively degradation. During the chase period, undegraded CFTR molecules gradually shift to a higher molecular weight smear (13) and eventually become insoluble in nonionic detergents (C. Ward, unpublished data). These changes in CFTR mobility could be the result of both multiubiquitination and aggregation, which would be expected if the hydrophobic MSD of this protein were dislocated to the cytosol. It is possible that the "disappearance" of CFTR over time may in fact be due to an increase in molecular weight resulting from ubiquitination and aggregation. Thus it is exceedingly difficult to quantify the fraction of CFTR that is degraded and that which is shifted to sizes that may not even enter the SDS-PAGE gels used for the analyses. It is worth noting that proteasome inhibitors quantitatively inhibit the degradation of other misfolded proteins in the ER including T-cell receptor (40) and myosin heavy chain class I heavy chain (37, 38), suggesting that proteasomes are sufficient for degradation of at least some ER degradation substrates.

	IV. PROSPECTS FOR CYSTIC FIBROSIS THERAPY

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The frequency of the F508 allele among CF patients and the fact that Cl channel activity is not disrupted by loss of Phe-508 suggest the possibility of strategies for CF treatment based on increasing the efficiency of folding and intracellular processing of this mutant. Loss of this amino acid decreases the efficiency of folding of NBD1 in vitro and appears to increase the probability that nascent CFTR chains become committed to degradation by a pathway that involves at least in part the ubiquitin/proteasome pathway. It is unlikely that strategies based on inhibition of intracellular proteases involved in CFTR degradation will be useful therapeutics, because blocking degradation only leads to accumulation of inactive, aggregated forms and has no detectable impact on F508 maturation. Likewise, the data suggest that the ubiquitin pathway is unlikely to be a productive target for therapy, since blocking ubiquitination leads to accumulation of aggregated CFTR and does not appear to increase F508 maturation. These results suggest that even though multiubiquitination of CFTR occurs early in CFTR biogenesis, perhaps cotranslationally, it occurs after the nascent chains have become committed to misfolding. Clearly, the development of strategies aimed at increasing F508 maturation will require a better understanding of the early steps in the folding pathway. In particular, it will be important to characterize more fully the molecular chaperones and cellular factors that interact with nascent CFTR molecules. An exciting prospect is the possibility that chemical chaperones, small, cell-permeant molecules like glycerol, can increase F508 folding yield, possibly by stabilizing an otherwise unstable folding intermediate. The challenge is formidable, however, as glycerol concentrations required for detectable effect on F508 maturation in vivo are in excess of 1 M. Recently, Loo and Clarke (17) reported that misfolding and degradation of another ABC transporter, the human P-glycoprotein, can be corrected in vivo when the protein is bound to transport substrates. The possibility that small molecules will be able to interact specifically with and stabilize a particular CFTR (or F508) folding intermediate, thereby promoting folding, is worthy of consideration.

	FOOTNOTES

   I am indebted to the members of my laboratory for their helpful critique of the manuscript and to Dr. Philip J. Thomas (Univ. of Texas) for sharing data before its publication.

   This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-43994. This manuscript was written during the tenure of an Established Investigatorship of the American Heart Association.

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