Conformational maturation of CFTR but not its mutant counterpart (delta F508) occurs in the endoplasmic reticulum and requires ATP.

Metabolic labeling experiments followed by immunoprecipitation were performed to investigate the kinetics, location and inhibitor sensitivity of degradation of both wild-type (wt) and mutant (delta F508) cystic fibrosis conductance transmembrane regulator (CFTR). At the earliest stages of the biosynthetic process, both wt and delta F508 CFTR were found to be susceptible to degradation by endogenous proteases. Virtually all delta F508 CFTR and 45-80% of wt CFTR were rapidly degraded with a similar half-life (t1/2 approximately 0.5 h). The remaining wt CFTR attained a protease-resistant configuration regardless of whether traffic between the endoplasmic reticulum (ER) and Golgi was operational. Metabolic energy is required for the conformational transition, but not to maintain the stability of the protease-resistant wt CFTR. Intracellular degradation of delta F508 CFTR and of incompletely folded wt CFTR occurs in a non-lysosomal, pre-Golgi compartment, as indicated by the sensitivity of proteolysis to different inhibitors and temperature. Accordingly, products of the degradation of delta F508 CFTR could be detected by immunoblotting in isolated ER, but not in the Golgi. Together, these results suggest a dynamic equilibrium between two forms of wt CFTR in the ER: an incompletely folded, protease-sensitive form which is partially converted by an ATP-dependent process to a more mature form that is protease-resistant and capable of leaving the ER. The inability delta F508 CFTR to undergo such a transition renders it susceptible to complete and rapid degradation in a pre-Golgi compartment.     

Delta F508 CFTR pool in the endoplasmic reticulum is increased by calnexin overexpression

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, Delta F508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of Delta F508 CFTR with calnexin, an ER chaperone, results in the ERAD of Delta F508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of Delta F508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the Delta F508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated Delta F508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of Delta F508 CFTR in the ER.     

Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.

The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-DeltaF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000) Traffic 1, 852-870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNAp REceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells.     

Maintaining low Ca2+ level in the endoplasmic reticulum restores abnormal endogenous F508del-CFTR trafficking in airway epithelial cells

The most common mutation in cystic fibrosis, F508del, results in cystic fibrosis transmembrane conductance regulator protein (CFTR) that is retained in the endoplasmic reticulum (ER). Retention is dependent on chaperone proteins, many of which, like calnexin, require calcium for optimal activity. Here, we show that a limited and a maintained ER calcium level is sufficient to inhibit the F508del-CFTR/calnexin interaction and to restore the cAMP-dependent CFTR chloride transport, thus showing the correction of abnormal trafficking. We used Western blot analysis, iodide efflux and calcium measurement techniques applied to the human airway epithelial cystic fibrosis cell line CF15 (F508del/F508del). The inhibition of ER calcium pump, with thapsigargin, curcumin, 2,5-di(t-butyl)hydroquinone or cyclopiazonic acid, maintains a threshold levels of calcium that is correlated to the recovery of endogenous F508del-CFTR transport activity. In particular, cyclopiazonic acid restores a 2-aminoethyoxydiphenyl borate-sensitive F508del-CFTR trafficking with an EC50 of 915 nm. By contrast, the 1,4,5-trisphosphate or IP3 receptor activators, i.e., ATP and histamine, while transiently emptying the ER intracellular calcium store, did not affect the trafficking of F508del-CFTR. Our data suggest that decreasing the ER calcium level is not sufficient to restore the defective trafficking of F508del-CFTR, whereas decreasing and also maintaining low ER calcium level allow correction of defective biosynthetic pathway of endogenous F508del-CFTR in human airway epithelial cells.     

Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.     

Specific autophagy and ESCRT components participate in the unconventional secretion of CFTR

The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl^? channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.     

Chemical and biological folding contribute to temperature-sensitive DeltaF508 CFTR trafficking

Proteostasis (Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science 2008;319:916-919) refers to the biology that maintains the proteome in health and disease. Proteostasis is challenged by the most common mutant in cystic fibrosis, DeltaF508, a chloride channel [the cystic fibrosis transmembrane conductance regulator (CFTR)] that exhibits a temperature-sensitive phenotype for coupling to the coatomer complex II (COPII) transport machine for exit from the endoplasmic reticulum. Whether rescue of export of DeltaF508 CFTR at reduced temperature simply reflects energetic stabilization of the chemical fold defined by its primary sequence or requires a unique proteostasis environment is unknown. We now show that reduced temperature (30 degrees C) export of DeltaF508 does not occur in some cell types, despite efficient export of wild-type CFTR. We find that DeltaF508 export requires a local biological folding environment that is sensitive to heat/stress-inducible factors found in some cell types, suggesting that the energetic stabilization by reduced temperature is necessary, but not sufficient, for export of DeltaF508. Thus, the cell may require a proteostasis environment that is in part distinct from the wild-type pathway to restore DeltaF508 coupling to COPII. These results are discussed in the context of the energetics of the protein fold and the potential application of small molecules to achieve a proteostasis environment favoring export of a functional form of DeltaF508.     

Expression of a wild-type CFTR maintains the integrity of the biosynthetic/secretory pathway in human cystic fibrosis pancreatic duct cells.

The structural integrity of the Golgi complex is essential to its functions in the maturation, sorting, and transport of plasma membrane proteins. Previously, we demonstrated that in pancreatic duct CFPAC-1 cells, which express DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator), the intracellular trafficking of carbonic anhydrase IV (CA IV), a membrane protein involved in HCO(3)(-) secretion, was impaired. To determine whether these abnormalities were related to changes in the Golgi complex, we examined the ultrastructure and distribution of Golgi compartments with regard to the microtubule cytoskeleton in CFPAC-1 cells transfected or not with the wild-type CFTR. Ultrastructural and immunocytochemical analysis showed that in polarized CFPAC-1 cells, Golgi stacks were disconnected from one another and scattered throughout the cytoplasm. The colocalization of CA IV with markers of Golgi compartments indicated the ability of stacks to transfer this enzyme. This Golgi dispersal was associated with abnormal microtubule distribution and multiplicity of the microtubule-organizing centers (MTOCs). In reverted cells, the normalization of Golgi structure, microtubule distribution, and MTOC number was observed. These observations suggest that the entire biosynthetic/secretory pathway is disrupted in CFPAC-1 cells, which might explain the abnormal intracellular transport of CA IV. Taken together, these results point to the fact that the expression of DeltaF508 CFTR affects the integrity of the secretory pathway.     

Insulin-Like Growth Factor 1 (IGF-1) Enhances the Protein Expression of CFTR

Low levels of insulin-like growth factor 1 (IGF-1) have been observed in the serum of cystic fibrosis (CF) patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR), whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL)- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.     

FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability

Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.     

Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

The lethal genetic disease cystic fibrosis is caused predominantly by in‐frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide‐binding domain (NBD1) of CFTR, which functions as an ATP‐gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light‐scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg‐ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second‐site mutations that increase the solubility of isolated F508del‐NBD1 in vitro and suppress the trafficking defect of intact F508del‐CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del‐CFTR.     

Most F508del-CFTR is targeted to degradation at an early folding checkpoint and independently of calnexin

Biosynthesis and folding of multidomain transmembrane proteins is a complex process. Structural fidelity is monitored by endoplasmic reticulum (ER) quality control involving the molecular chaperone calnexin. Retained misfolded proteins undergo ER-associated degradation (ERAD) through the ubiquitin-proteasome pathway. Our data show that the major degradation pathway of the cystic fibrosis transmembrane conductance regulator (CFTR) with F508del (the most frequent mutation found in patients with the genetic disease cystic fibrosis) from the ER is independent of calnexin. Moreover, our results demonstrate that inhibition of mannose-processing enzymes, unlike most substrate glycoproteins, does not stabilize F508del-CFTR, although wild-type (wt) CFTR is drastically stabilized under the same conditions. Together, our data support a novel model by which wt and F508del-CFTR undergo ERAD from two distinct checkpoints, the mutant being disposed of independently of N-glycosidic residues and calnexin, probably by the Hsc70/Hsp70 machinery, and wt CFTR undergoing glycan-mediated ERAD.     

Towards a rational combination therapy of cystic fibrosis

Cystic fibrosis (CF) is most frequently due to homozygous ΔF508-CFTR mutation. The ΔF508-CFTR protein is unstable in the plasma membrane (PM), even if it is rescued by pharmacological agents that prevent its intracellular retention and degradation. Restoring defective autophagy in CF airways by proteostasis regulators (such as cystamine and its reduced form, cysteamine) can rescue and stabilize ΔF508-CFTR at the PM, thus enabling the action of CFTR potentiators, which are pharmacological agents that stimulate the function of CFTR as an ion channel. The effects of cystamine extend for days (in vitro) and weeks (in vivo) beyond washout, suggesting that once peripheral proteostasis has been re-established, PM-resident ΔF508-CFTR sustains its own stability. We demonstrated that the pharmacological inhibition of wild-type CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette subfamily C, member 7)], in bronchial epithelial cells decreases the stability of the CFTR protein by inhibiting autophagy, elevating the abundance of SQSTM1/p62 and its interaction with CFTR at the PM, increasing the ubiqutination of CFTR, stimulating the lysosomal degradation of CFTR and avoiding its recycling. All these effects could be inhibited by cystamine. Moreover, CFTR-sufficient epithelia generate permissive conditions for incorporating ΔF508-CFTR into the PM and stabilizing it at this location. These results provide the rationale for a combination therapy of CF in which pretreatment with cystamine or cysteamine enables the later action of CFTR potentiators.     

F508del disturbs the dynamics of the nucleotide binding domains of CFTR before and after ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is an ion channel responsible for chloride transport in epithelia and it belongs to the class of ABC transporters. The deletion of phenylalanine 508 (F508del) in CFTR is the most common mutation responsible for cystic fibrosis. Little is known about the effect of the mutation in the isolated nucleotide binding domains (NBDs), on dimer dynamics, ATP hydrolysis and even on nucleotide binding. Using molecular dynamics simulations of the human CFTR NBD dimer, we showed that F508del increases, in the prehydrolysis state, the inter-motif distance in both ATP binding sites (ABP) when ATP is bound. Additionally, a decrease in the number of catalytically competent conformations was observed in the presence of F508del. We used the subtraction technique to study the first 300 ps after ATP hydrolysis in the catalytic competent site and found that the F508del dimer evidences lower conformational changes than the wild type. Using longer simulation times, the magnitude of the conformational changes in both forms increases. Nonetheless, the F508del dimer shows lower C-α RMS values in comparison to the wild-type, on the F508del loop, on the residues surrounding the catalytic site and the portion of NBD2 adjacent to ABP1. These results provide evidence that F508del interferes with the NBD dynamics before and after ATP hydrolysis. These findings shed a new light on the effect of F508del on NBD dynamics and reveal a novel mechanism for the influence of F508del on CFTR.     

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR

Induction of endoplasmic reticulum (ER)‐to‐Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)‐mediated, Golgi‐independent unconventional cell‐surface trafficking of the folding‐deficient ΔF508‐cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation‐dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508‐CFTR, such as induction of ER‐to‐Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C‐terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation‐mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation‐inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER‐retained ΔF508‐CFTR and mediates the ER stress‐induced unconventional secretion pathway.      

Rescue of ΔF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway

The most prevalent disease-causing mutation of CFTR is the deletion of Phe508 (ΔF508), which leads to defects in conventional Golgi-mediated exocytosis and cell surface expression. We report that ΔF508-CFTR surface expression can be rescued in vitro and in vivo by directing it to an unconventional GRASP-dependent secretion pathway. An integrated molecular and physiological analysis indicates that mechanisms associated with ER stress induce cell surface trafficking of the ER core-glycosylated wild-type and ΔF508-CFTR via the GRASP-dependent pathway. Phosphorylation of a specific site of GRASP and the PDZ-based interaction between GRASP and CFTR are critical for this unconventional surface trafficking. Remarkably, transgenic expression of GRASP in ΔF508-CFTR mice restores CFTR function and rescues mouse survival without apparent toxicity. These findings provide insight into how unconventional protein secretion is activated, and offer a potential therapeutic strategy for the treatment of cystic fibrosis and perhaps diseases stemming from other misfolded proteins.     

Control of cystic fibrosis transmembrane conductance regulator expression by BAP31

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is stringently controlled by molecular chaperones participating in formation of the quality control system. It has been shown that about 75% of all CFTR protein and close to 100% of the [DeltaPhe(508)] CFTR variant are rapidly degraded before leaving the endoplasmic reticulum (ER). B cell antigen receptor-associated proteins (BAPs) are ubiquitously expressed integral membrane proteins that may control association with the cytoskeleton, vesicular transport, or retrograde transport from the cis Golgi to the ER. The present study delivers evidence for cytosolic co-localization of both BAP31 and CFTR and for the control of expression of recombinant CFTR in Chinese hamster ovary (CHO) cells and Xenopus oocytes by BAP31. Antisense inhibition of BAP31 in various cell types increased expression of both wild-type CFTR and [DeltaPhe(508)]CFTR and enabled cAMP-activated Cl(-) currents in [DeltaPhe(508)]CFTR-expressing CHO cells. Coexpression of CFTR together with BAP31 attenuated cAMP-activated Cl(-) currents in Xenopus oocytes. These data therefore suggest association of BAP31 with CFTR that may control maturation or trafficking of CFTR and thus expression in the plasma membrane.     

The endoplasmic reticulum-associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3-dependent degradation of nascent CFTRDeltaF508

Relative contributions of folding kinetics versus protein quality control (QC) activity in the partitioning of non-native proteins between life and death are not clear. Cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis serves as an excellent model to study this question because folding of nascent CFTR is inefficient and deletion of F508 causes accumulation of CFTRΔF508 in a kinetically trapped, but foldable state. Herein, a novel endoplasmic reticulum (ER)-associated Hsp40, DNAJB12 (JB12) is demonstrated to play a role in control of CFTR folding efficiency. JB12 cooperates with cytosolic Hsc70 and the ubiquitin ligase RMA1 to target CFTR and CFTRΔF508 for degradation. Modest elevation of JB12 decreased nascent CFTR and CFTRΔF508 accumulation while increasing association of Hsc70 with ER forms of CFTR and the RMA1 E3 complex. Depletion of JB12 increased CFTR folding efficiency up to threefold and permitted a pool of CFTRΔF508 to fold and escape the ER. Introduction of the V510D misfolding suppressor mutation into CFTRΔF508 modestly increased folding efficiency, whereas combined inactivation of JB12 and suppression of intrinsic folding defects permitted CFTRΔF508 to fold at 50% of wild-type efficiency. Therapeutic correction of CFTRΔF508 misfolding in cystic fibrosis patients may require repair of defective folding kinetics and suppression of ER QC factors, such as JB12.     

Analysis of the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the cystic fibrosis defect CFTRΔF508

The use of yeast as a model system to study mammalian proteins is attractive, because yeast genetic tools can be utilized if a suitable phenotype is created. STE6, the Saccharomyces cerevisiae a-factor mating pheromone transporter, and CFTR, the mammalian cystic fibrosis transmembrane conductance regulator, are both members of the ATP binding cassette (ABC) superfamily. Teem et al . (1993) described a yeast model for studying a mutant form of the cystic fibrosis protein, CFTRΔF508. The model involved expression of a chimeric molecule in which a portion of yeast STE6 was replaced with the corresponding region from mammalian CFTR. The STE6/CFTR chimera complemented a ste6 mutant strain for mating, indicating that it could export a-factor. However, mating efficiency was dramatically reduced upon introduction of ΔF508, providing a yeast phenotype for this mutation. In human cells, the ΔF508 mutation results in retention of CFTR in the endoplasmic reticulum (ER), and possibly in reduction of its chloride-channel activity. Here we examine the basis for the differences in STE6 activity promoted by the wild-type and mutant STE6/CFTR chimeras. By analysis of protein stability and subcellular localization, we find that the mutant chimera is not ER-retained in yeast. We conclude that the molecular basis for the reduced mating of the STE6/CFTRΔF508 chimera must reflect a reduction in its capacity to transport a-factor, rather than mistrafficking. Thus, STE6/CFTRΔF508 in yeast appears to be a good genetic model to probe certain aspects of protein function, but not to study protein localization.     

Interplay between ER Exit Code and Domain Conformation in CFTR Misprocessing and Rescue

Multiple mutations in cystic fibrosis transmembrane conductance regulator (CFTR) impair its exit from the endoplasmic reticulum (ER). We compared two processing mutants: ΔF508 and the ER exit code mutant DAA. Although both have severe kinetic processing defect, DAA but not ΔF508 has substantial accumulation in its mature form, leading to higher level of processing at the steady state. DAA has much less profound conformational abnormalities. It has lower Hsp70 association and higher post-ER stability than ΔF508. The ER exit code is necessary for ΔF508 residual export and rescue. R555K, a mutation that rescues ΔF508 misprocessing, improves Sec24 association and enhances its post-ER stability. Using in situ limited proteolysis, we demonstrated a clear change in trypsin sensitivity in ΔF508 NBD1, which is reversed, together with that of other domains, by low temperature, R555K or both. We observed a conversion of the proteolytic pattern of DAA from the one resembling ΔF508 to the one similar to wild-type CFTR during its maturation. Low temperature and R555K are additive in improving ΔF508 conformational maturation and processing. Our data reveal a dual contribution of ER exit code and domain conformation to CFTR misprocessing and underscore the importance of conformational repair in effective rescue of ΔF508.