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.

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ΔF508 CFTR Pool in the Endoplasmic Reticulum Is Increased by Calnexin Overexpression

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0379 ◽

2004 ◽

Vol 15 (2) ◽

pp. 563-574 ◽

Cited By ~ 74

Author(s):

Tsukasa Okiyoneda ◽

Kazutsune Harada ◽

Motohiro Takeya ◽

Kaori Yamahira ◽

Ikuo Wada ◽

...

Keyword(s):

Cystic Fibrosis ◽

Endoplasmic Reticulum ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Δf508 Cftr ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Cystic Fibrosis Transmembrane ◽

Erad Pathway

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, ΔF508 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 ΔF508 CFTR with calnexin, an ER chaperone, results in the ERAD of ΔF508 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 ΔF508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the ΔF508 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 ΔF508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of ΔF508 CFTR in the ER.

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Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator

The Journal of Cell Biology ◽

10.1083/jcb.200210086 ◽

2003 ◽

Vol 160 (2) ◽

pp. 157-163 ◽

Cited By ~ 51

Author(s):

Lianwu Fu ◽

Elizabeth Sztul

Keyword(s):

Cystic Fibrosis ◽

Proteasomal Degradation ◽

Degradation Pathway ◽

Restrictive Temperature ◽

Transmembrane Conductance Regulator ◽

Nucleotide Exchange ◽

Transmembrane Conductance ◽

Yeast Saccharomyces Cerevisiae ◽

Nucleotide Exchange Factor ◽

Cystic Fibrosis Transmembrane

Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.

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