The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER.

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Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.

Misprocessing and mislocalization of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been described for the major CF-causing mutation (delta F508) in heterologous expression systems in vitro. We have generated monoclonal antibodies (mAbs) to CFTR with the aim of localizing the protein and its CF variants in vivo. Of the tissues where CFTR was observed, only the sweat gland is readily available and does not undergo secondary changes due to CF disease pathology. Sweat ducts from CF patients homozygous for delta F508 did not show the typical apical membrane staining seen in control biopsies. This demonstrates that the biosynthetic arrest and intracellular retention of delta F508 CFTR initially observed in vitro does occur in vivo and emphasizes the need to focus efforts on understanding the mislocalization.

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The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multi-domain membrane protein which matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32 residue segment termed the regulatory insertion (RI) not present in other ABC transporters. We report here that RI deletion enabled ΔF508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T1/2 of ~14h in cells, similar to the wild-type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermo-stabilizing influence on the channel with gating up to at least 40°C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508 containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remain incomplete. Here we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in the first nucleotide domain (NBD1), including the structurally diverse region (SDR), the gamma phosphate switch loop and the Regulatory Insertion (RI). Molecular Dynamic analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild-type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the SDR, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein’s inherent stability and channel activity returns a near-normal state.

Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve ΔF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37°C (<2-fold), and the mature product remained short-lived (T(1/2)∼4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that ΔF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.