Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl ؊ channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTR⌬F508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex. INTRODUCTIONCystic fibrosis transmembrane conductance regulator (CFTR) is a membrane glycoprotein that is localized to the apical surface of epithelial cells that line ducts of glands and airways. CFTR functions as an ATP-gated Cl Ϫ channel that is critical for proper hydration of the mucosal layer that lines lung airways (Welsh and Smith, 1993). Individuals who inherit two mutant forms of CFTR have exceedingly viscous mucous and, due to chronic lung infections, develop cystic fibrosis and often die from lung failure. CFTR is a member of the ATP-binding cassette (ABC) transporter superfamily (Hyde et al., 1990) and is a 1480-amino acid protein that contains two membrane spanning domains (MSDs), MSD1 and MSD2; two cytosolic nucleotide binding domains (NBDs), NBD1 and NBD2; and a regulatory (R) domain (Riordan et al., 1989). The proper folding and assembly of CFTR subdomains in the endoplasmic reticulum (ER) is required for CFTR to engage the COPII machinery and be packaged into vesicles for transport to the plasma membrane (Kopito, 1999;Wang et al., 2004). The folding pathway of this complex polytopic membrane protein has been a topic of great interest, because misfolding results in premature recognition of CFTR by the ER quality control system (ERQC) and degradation by the ubiquitin proteasome system (Skach, 2000). In fact, the most common disease-causing mutation of CFTR, ⌬F508CFTR, results in almost complete degradation of the protein by the ERQC system, which gives rise to a loss of function phenotype and lung disease (Ward and Kopito, 1994).The assembly of CFTR into an ion channel is complicated because it requires the coordinated folding and assembly of its membrane and cytoplasmic domains into a functional unit (Du et al., 2005;Riordan, 2005;Cui et al., 2007). CFTR is a modular protein, and its domains can collapse to a protease-resistant conformation i...

RNF185 Is a Novel E3 Ligase of Endoplasmic Reticulum-associated Degradation (ERAD) That Targets Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

“…Analysis of mutated and truncated forms of CFTR was used to pinpoint the CFTR folding lesions preferentially sensed by RNF5. RNF5 was proposed to sense a defect in both the cooperative folding of the N-terminal regions occurring after synthesis of the R domain and the association of the two MSD domains (27,41). Our preliminary data also indicate that RNF185 preferentially affects the turnover of CFTR proteins truncated after the R domain, 3 suggesting that as for RNF5 the synthesis of MSD2 and NBD2 C-terminal domains is not strictly required for RNF185-dependent quality control.…”

“…CHIP is a cytoplasmic U-box protein that is recruited on Hsc70-bound CFTR and targets it to ubiquitination and degradation after translation (27, 40 -42). RNF5 rather senses CFTR folding defects during translation (27,41) and may be recruited to CFTR lesions in part by the Hsc70-associated Hsp40 DnajB12 (43). RNF5 also associates with Derlin-1 to drive CFTR mutant degradation (27,44).…”

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

“…The answer to this question is not clear, but it appears that the ⌬F508 mutation causes global defects in CFTR that involve misfolding of nucleotide binding domain 1 and failure of nucleotide binding domain 1 to make proper intramolecular contacts required to stabilize the native CFTR structure (7,13,20,22,25). Studies on the mechanism of Corr-4a action suggest that it acts to stabilize the TM regions of CFTR (9,28).…”

Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508

“…However, Corr-4a did not increase the shortened half-life of CFTR 653X⌬F508 or CFTR 837X⌬F508. Yet, Corr-4a did increase the folding efficiency of CFTR 1172X around 2.5-fold and also increased the half-life of CFTR 1172X⌬F508.The extent that deletion of F508 can differentially affect the levels of N-terminal fragments of CFTR are intriguing and have previously been documented (Rosser et al, 2008). Deletion of F508 has only a modest affect on the stability of the MSD1-NBD1 fragment CFTR 653X, but the presence of the R-domain results in a much more pronounced defect.…”

“…To understand why Corr-4a action can be enhanced by inactivation of ERQC machinery, we sought to define the correctable defective folding steps in CFTR and CFTR⌬F508. To accomplish this we determined the effect of Corr-4a on the steady-state levels of CFTR and CFTR⌬F508 fragments that resemble different length biogenic intermediates (Younger et al, 2006;Cui et al, 2007;Rosser et al, 2008; Figure 3). …”