Most F508del-CFTR Is Targeted to Degradation at an Early Folding Checkpoint and Independently of Calnexin

Abstract

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.

ACKNOWLEDGMENTS

We are grateful to Luís Reis and Mário Neto for helping with the production of CFTR glycosylation mutants and iodide efflux assays, respectively. We thank K. Kirk (Birmingham, Ala.) for N-terminal anti-CFTR antibody, M. B. Brenner (Boston, Mass.) for the anti-calnexin AF8 antibody and cDNA, J. R. Riordan (Scottsdale, Ariz.) for the CHO cell lines stably expressing wt and F508del-CFTR, G. Lukacs (Toronto, Canada) for the BHK cell line stably expressing wt CFTR, and K. Nagata (Kyoto, Japan) for anti-EDEM antibody and cDNA.

This work was supported by Praxis XXI research grants POCTI/35737/MGI/2000 and POCTI/47382/MGI/2002 and Praxis Ph.D. fellowship (C.M.F.) BD/11094/97.