Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments

Abstract

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

“…Little is known about the mechanisms responsible for recognition and degradation of abnormal membrane proteins in the distal compartments of the secretory pathway. A post-ER QC mechanism appears to be responsible for recognition and degradation of cytoplasmic domain mutants of CFTR (Benharouga et al, 2001) and the nicotinic acetylcholine receptor ␣-subunit (Keller et al, 2001). Although we cannot exclude the possibility that the mutant TM might perturb the conformation of the short cytoplasmic tail of HA ϩϩ , resulting in its recognition by cytoplasmic chaperones, our data strongly implicate the mutant TM as the primary signal for degradation.…”

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

“…Cell Lines and Transfection-BHK-21 (CCL 10; American Type Culture Collection (ATCC)), COS-1 (CRL-1650; ATTC), MDCK II (CCL-34; ATTC), and CaCo-2 (HTB-37; ATTC) cells were cultured as described previously (10). Pancreatic duct cells PANC-1 (CRL-1469; ATCC) were grown in human tracheal epithelia (9HTE160 Ϫ , designated as HTE), a generous gift of Dr. Dieter Gruenert (Human Molecular Genetics Unit, Department of Medicine, University of Vermont, Burlington, VT), were cultured in Eagle's minimal essential medium and 10% fetal calf serum.…”

“…Soluble proteins were separated on 7-10% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were probed with the mouse monoclonal anti-HA Ab at 1:10,000 dilution, the M3A7 anti-CFTR Ab at 1:1000 dilution (kindly provided by Dr. N. Kartner (41)), or the monoclonal mouse 24 -1 anti-CFTR Ab (Genzyme Inc.) at 1:1000 dilution and visualized by ECL (Amersham Biosciences) as described (10).…”

“…The C-terminal tail is also involved in folding and conformational stabilization of CFTR. Deletion of a hydrophobic patch ( 1413 FLVI) ablates posttranslational folding (9), whereas truncating 70 (⌬70 CFTR) or more amino acid residues destabilizes the mature, complex-glycosylated CFTR (10,11). Finally, high affinity association of multivalent PDZ domain-containing molecules with the conserved C-terminal peptide ( 1476 DTRL) has been demonstrated (12)(13)(14).…”

The CFTR frameshift mutation 3905insT and its effect at transcript and protein level

“…21,36,37 Recent studies indicated that the CFTR C-terminus is not required for the biosynthesis and plasma membrane targeting of CFTR, but indispensable for maintaining the stability of the protein. 38 Benharouga et al 39 could also show that an ERAD-similar mechanism involving the proteasome-ubiquitin pathway may be responsible for the faster turnover and the short residence time at the apical membrane of truncated CFTR. This faster recycling rate of truncated proteins may provide an explanation for the reduced amount of 3905insT CFTR at the apical membrane.…”