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COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code.

Wang X, Matteson J, An Y, Moyer B, Yoo JS, Bannykh S, Wilson IA, Riordan JR, Balch WE - J. Cell Biol. (2004)

Bottom Line: In contrast, COPII is not used to deliver CFTR to ER-associated degradation.Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24.We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in DeltaF508-expressing patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Cystic fibrosis (CF) is a childhood hereditary disease in which the most common mutant form of the CF transmembrane conductance regulator (CFTR) DeltaF508 fails to exit the endoplasmic reticulum (ER). Export of wild-type CFTR from the ER requires the coat complex II (COPII) machinery, as it is sensitive to Sar1 mutants that disrupt normal coat assembly and disassembly. In contrast, COPII is not used to deliver CFTR to ER-associated degradation. We find that exit of wild-type CFTR from the ER is blocked by mutation of a consensus di-acidic ER exit motif present in the first nucleotide binding domain. Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24. We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in DeltaF508-expressing patients.

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Export of CFTR from the ER is COPII dependent. BHK cells were transfected with pcDNA3.1 plasmids containing either wild-type CFTR or ΔF508-CFTR or cotransfected with pcDNA3.1 containing the indicated Sar1 mutants as described previously (Yoo et al., 2002). After a 30-min pulse with [35S]Met, CFTR was immunoprecipitated at the indicated chase time and quantitated using a Molecular Dynamics PhosphoImager as described previously (Yoo et al., 2002). Expression levels of Sar1 mutants (∼5–10-fold endogenous) were monitored by immunoblotting with specific antibody as described previously (Yoo et al., 2002; not depicted). (A and B, top panels) Quantitation of CFTR in band B (ER form) reported as a percentage of total B plus C at the 0 time point. (A and B, bottom panels) Quantitation of the CFTR band C glycosylated form reported as a percentage of total B plus C at the 0 time point. Insets in A and B show representative autoradiographs containing band B and C (arrows). In A (top right panel), a significant increase in amount of band B remaining in six independent experiments for cells transfected with Sar1-GTP compared with Sar1-GDP at 3 h is shown (P < 0.05). The error bars represent SEM. The dashed horizontal line indicates the level of band B remaining in the wild-type (mock) control. In A (bottom right panel), the C/B ratios are shown for the 3-h time point. Results in A and B are typical of at least three independent experiments.
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fig1: Export of CFTR from the ER is COPII dependent. BHK cells were transfected with pcDNA3.1 plasmids containing either wild-type CFTR or ΔF508-CFTR or cotransfected with pcDNA3.1 containing the indicated Sar1 mutants as described previously (Yoo et al., 2002). After a 30-min pulse with [35S]Met, CFTR was immunoprecipitated at the indicated chase time and quantitated using a Molecular Dynamics PhosphoImager as described previously (Yoo et al., 2002). Expression levels of Sar1 mutants (∼5–10-fold endogenous) were monitored by immunoblotting with specific antibody as described previously (Yoo et al., 2002; not depicted). (A and B, top panels) Quantitation of CFTR in band B (ER form) reported as a percentage of total B plus C at the 0 time point. (A and B, bottom panels) Quantitation of the CFTR band C glycosylated form reported as a percentage of total B plus C at the 0 time point. Insets in A and B show representative autoradiographs containing band B and C (arrows). In A (top right panel), a significant increase in amount of band B remaining in six independent experiments for cells transfected with Sar1-GTP compared with Sar1-GDP at 3 h is shown (P < 0.05). The error bars represent SEM. The dashed horizontal line indicates the level of band B remaining in the wild-type (mock) control. In A (bottom right panel), the C/B ratios are shown for the 3-h time point. Results in A and B are typical of at least three independent experiments.

Mentions: To follow export of CFTR from the ER, baby hamster kidney (BHK) cells were transfected with CFTR using a vaccinia-transient expression system, pulse-labeled with [35S]methionine, and CFTR transport to the Golgi detected by processing of the band B N-linked core-oligosaccharide form to the band C complex form by Golgi-associated glycosyltransferases that has reduced migration on SDS-PAGE (Yoo et al., 2002; Fig. 1 A, inset, CFTR mock). In most tissue culture cell lines engineered to express CFTR, wild-type CFTR is inefficiently exported, resulting in processing of only 20–30% of the ER-associated band B to the Golgi-and post-Golgi mature band C form (Fig. 1 A, inset and bottom left panel; Riordan, 1999). CFTR failing to exit the ER is degraded by ERAD indicated by the loss of band B (Fig. 1 A, inset and top panel; Xiong et al., 1999; Gelman et al., 2002; Lenk et al., 2002).


COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code.

Wang X, Matteson J, An Y, Moyer B, Yoo JS, Bannykh S, Wilson IA, Riordan JR, Balch WE - J. Cell Biol. (2004)

Export of CFTR from the ER is COPII dependent. BHK cells were transfected with pcDNA3.1 plasmids containing either wild-type CFTR or ΔF508-CFTR or cotransfected with pcDNA3.1 containing the indicated Sar1 mutants as described previously (Yoo et al., 2002). After a 30-min pulse with [35S]Met, CFTR was immunoprecipitated at the indicated chase time and quantitated using a Molecular Dynamics PhosphoImager as described previously (Yoo et al., 2002). Expression levels of Sar1 mutants (∼5–10-fold endogenous) were monitored by immunoblotting with specific antibody as described previously (Yoo et al., 2002; not depicted). (A and B, top panels) Quantitation of CFTR in band B (ER form) reported as a percentage of total B plus C at the 0 time point. (A and B, bottom panels) Quantitation of the CFTR band C glycosylated form reported as a percentage of total B plus C at the 0 time point. Insets in A and B show representative autoradiographs containing band B and C (arrows). In A (top right panel), a significant increase in amount of band B remaining in six independent experiments for cells transfected with Sar1-GTP compared with Sar1-GDP at 3 h is shown (P < 0.05). The error bars represent SEM. The dashed horizontal line indicates the level of band B remaining in the wild-type (mock) control. In A (bottom right panel), the C/B ratios are shown for the 3-h time point. Results in A and B are typical of at least three independent experiments.
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fig1: Export of CFTR from the ER is COPII dependent. BHK cells were transfected with pcDNA3.1 plasmids containing either wild-type CFTR or ΔF508-CFTR or cotransfected with pcDNA3.1 containing the indicated Sar1 mutants as described previously (Yoo et al., 2002). After a 30-min pulse with [35S]Met, CFTR was immunoprecipitated at the indicated chase time and quantitated using a Molecular Dynamics PhosphoImager as described previously (Yoo et al., 2002). Expression levels of Sar1 mutants (∼5–10-fold endogenous) were monitored by immunoblotting with specific antibody as described previously (Yoo et al., 2002; not depicted). (A and B, top panels) Quantitation of CFTR in band B (ER form) reported as a percentage of total B plus C at the 0 time point. (A and B, bottom panels) Quantitation of the CFTR band C glycosylated form reported as a percentage of total B plus C at the 0 time point. Insets in A and B show representative autoradiographs containing band B and C (arrows). In A (top right panel), a significant increase in amount of band B remaining in six independent experiments for cells transfected with Sar1-GTP compared with Sar1-GDP at 3 h is shown (P < 0.05). The error bars represent SEM. The dashed horizontal line indicates the level of band B remaining in the wild-type (mock) control. In A (bottom right panel), the C/B ratios are shown for the 3-h time point. Results in A and B are typical of at least three independent experiments.
Mentions: To follow export of CFTR from the ER, baby hamster kidney (BHK) cells were transfected with CFTR using a vaccinia-transient expression system, pulse-labeled with [35S]methionine, and CFTR transport to the Golgi detected by processing of the band B N-linked core-oligosaccharide form to the band C complex form by Golgi-associated glycosyltransferases that has reduced migration on SDS-PAGE (Yoo et al., 2002; Fig. 1 A, inset, CFTR mock). In most tissue culture cell lines engineered to express CFTR, wild-type CFTR is inefficiently exported, resulting in processing of only 20–30% of the ER-associated band B to the Golgi-and post-Golgi mature band C form (Fig. 1 A, inset and bottom left panel; Riordan, 1999). CFTR failing to exit the ER is degraded by ERAD indicated by the loss of band B (Fig. 1 A, inset and top panel; Xiong et al., 1999; Gelman et al., 2002; Lenk et al., 2002).

Bottom Line: In contrast, COPII is not used to deliver CFTR to ER-associated degradation.Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24.We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in DeltaF508-expressing patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Cystic fibrosis (CF) is a childhood hereditary disease in which the most common mutant form of the CF transmembrane conductance regulator (CFTR) DeltaF508 fails to exit the endoplasmic reticulum (ER). Export of wild-type CFTR from the ER requires the coat complex II (COPII) machinery, as it is sensitive to Sar1 mutants that disrupt normal coat assembly and disassembly. In contrast, COPII is not used to deliver CFTR to ER-associated degradation. We find that exit of wild-type CFTR from the ER is blocked by mutation of a consensus di-acidic ER exit motif present in the first nucleotide binding domain. Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24. We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in DeltaF508-expressing patients.

Show MeSH
Related in: MedlinePlus