Folding of CFTR Is Predominantly Cotranslational

Kleizen, Bertrand; Vlijmen, Thijs van; Jonge, Hugo R. de; Braakman, Ineke

doi:10.1016/j.molcel.2005.09.007

Cited by 150 publications

(227 citation statements)

References 46 publications

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“…To compare the overall conformation of wild-type Ero1␤ and the mutant proteins, we used a limited proteolysis approach. Partial trypsin digestion has been used to map conformational changes in a number of ER proteins, including polyomavirus (34) and cystic fibrosis transmembrane conductance regulator (35,36). Thus transfected HeLa cell post-nuclear supernatants were treated with a concentration range of TPCK-trypsin for 30 min on ice.…”

Section: Resultsmentioning

confidence: 99%

Mutations in the FAD Binding Domain Cause Stress-induced Misoxidation of the Endoplasmic Reticulum Oxidoreductase Ero1β

Dias-Gunasekara

Lith

Williams

et al. 2006

Journal of Biological Chemistry

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Disulfide bond catalysis is an essential component of protein biogenesis in the secretory pathway, from yeast through to man. In the endoplasmic reticulum (ER), protein-disulfide isomerase (PDI) catalyzes the oxidation and isomerization of disulfide bonds and is re-oxidized by an endoplasmic reticulum oxidoreductase (ERO). The elucidation of ERO function was greatly aided by the genetic analysis of two ero mutants, whose impairment results from point mutations in the FAD binding domain of the Ero protein. The ero1-1 and ero1-2 yeast strains have conditional and dithiothreitol-sensitive phenotypes, but the effects of the mutations on the behavior of Ero proteins has not been reported. Here, we show that these Gly to Ser and His to Tyr mutations do not prevent the dimerization of Ero1␤ or the non-covalent interaction of Ero1␤ with PDI. However, the Gly to Ser mutation abolishes disulfide-dependent PDI-Ero1␤ heterodimers. Both the Gly to Ser and His to Tyr mutations make Ero1␤ susceptible to misoxidation and aggregation, particularly during a temperature or redox stress. We conclude that the Ero FAD binding domain is critical for conformational stability, allowing Ero proteins to withstand stress conditions that cause client proteins to misfold.

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Section: Resultsmentioning

confidence: 99%

Mutations in the FAD Binding Domain Cause Stress-induced Misoxidation of the Endoplasmic Reticulum Oxidoreductase Ero1β

Dias-Gunasekara

Lith

Williams

et al. 2006

Journal of Biological Chemistry

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“…Recognition of misfolded CFTR by ERAD components has been demonstrated to begin as early as co-translational insertion into the ER (43,44). Previous studies indicate that CFTR is co-translationally ubiquitinated, supporting the possibility for proteasomal targeting during the process (6).…”

Section: Uch-l1 Alters the Proteasomal Degradation Of Cftr Duringmentioning

confidence: 99%

Ubiquitin C-terminal Hydrolase-L1 Protects Cystic Fibrosis Transmembrane Conductance Regulator from Early Stages of Proteasomal Degradation

Henderson¹,

Vij²,

Zeitlin³

2010

Journal of Biological Chemistry

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⌬F508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein.We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or ⌬F508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT-or ⌬F508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.

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“…CFTR biogenesis involves several steps that initiate with CFTR's synthesis in the endoplasmic reticulum (ER), where it is cotranslationally folded and core-glycosylated (6,7). To undergo maturation through the Golgi apparatus, CFTR has to pass the ER quality control, which involves a number of chaperones and cochaperones (8)(9)(10)(11)(12), and targets misfolded proteins for ER-associated degradation by the ubiquitin proteasome pathway (13,14).…”

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confidence: 99%

Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms

Roxo-Rosa

Schmidt

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

106

131

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The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V562I were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wtand V562I-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F508del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention͞retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control. endoplasmic reticulum quality control ͉ folding ͉ membrane traffic ͉ arginine-framed motifs ͉ trafficking signals

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Folding of CFTR Is Predominantly Cotranslational

Cited by 150 publications

References 46 publications

Mutations in the FAD Binding Domain Cause Stress-induced Misoxidation of the Endoplasmic Reticulum Oxidoreductase Ero1β

Mutations in the FAD Binding Domain Cause Stress-induced Misoxidation of the Endoplasmic Reticulum Oxidoreductase Ero1β

Ubiquitin C-terminal Hydrolase-L1 Protects Cystic Fibrosis Transmembrane Conductance Regulator from Early Stages of Proteasomal Degradation

Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms

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