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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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Wild-type CFTR but not DAA-CFTR binds COPII. (A) HEK293 stably expressing wild type or ΔF508 were lysed in the presence of 1% Triton X-100, and Sec23/Sec24 complex recovered by immunoprecipitation with a CFTR-specific mAb was quantitated as described in Materials and methods. CFTR (bands B and C) and Sec24 were detected by immunoblotting with specific antibody as described in Materials and methods. (B) As in A except the amount CFTR recovered by immunoprecipitation with a Sec24-specific polyclonal antibody was quantitated as described in Materials and methods using immunoblotting. HEK293 cells not expressing CFTR were used as a control. CFTR (bands B and C) were detected by immunoblotting with specific antibody. (C) HEK293 cells were transfected with pcDNA3.1 plasmids containing either wild-type CFTR or DAA-CFTR as described previously (Yoo et al., 2002). Cells were lysed in the presence of 1% Triton X-100, and the amount of Sec23/Sec24 complex recovered bound to band B of CFTR (expressed as a Sec24/band B ratio) was quantitated as described in Materials and methods. CFTR (bands B and C) and Sec24 were detected by immunoblotting with specific antibodies. (D) HEK293 stably expressing wild type or ΔF508 incubated at 37°C (wild type) or for the indicated time at 30°C (ΔF508) were lysed in the presence of 1% Triton X-100, and the amount of Sec23/Sec24 complex recovered bound to CFTR by immunoprecipitation with a CFTR-specific antibody was quantitated as described in Materials and methods. Error bars indicates the SD for samples generated for each time point in triplicate.
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fig5: Wild-type CFTR but not DAA-CFTR binds COPII. (A) HEK293 stably expressing wild type or ΔF508 were lysed in the presence of 1% Triton X-100, and Sec23/Sec24 complex recovered by immunoprecipitation with a CFTR-specific mAb was quantitated as described in Materials and methods. CFTR (bands B and C) and Sec24 were detected by immunoblotting with specific antibody as described in Materials and methods. (B) As in A except the amount CFTR recovered by immunoprecipitation with a Sec24-specific polyclonal antibody was quantitated as described in Materials and methods using immunoblotting. HEK293 cells not expressing CFTR were used as a control. CFTR (bands B and C) were detected by immunoblotting with specific antibody. (C) HEK293 cells were transfected with pcDNA3.1 plasmids containing either wild-type CFTR or DAA-CFTR as described previously (Yoo et al., 2002). Cells were lysed in the presence of 1% Triton X-100, and the amount of Sec23/Sec24 complex recovered bound to band B of CFTR (expressed as a Sec24/band B ratio) was quantitated as described in Materials and methods. CFTR (bands B and C) and Sec24 were detected by immunoblotting with specific antibodies. (D) HEK293 stably expressing wild type or ΔF508 incubated at 37°C (wild type) or for the indicated time at 30°C (ΔF508) were lysed in the presence of 1% Triton X-100, and the amount of Sec23/Sec24 complex recovered bound to CFTR by immunoprecipitation with a CFTR-specific antibody was quantitated as described in Materials and methods. Error bars indicates the SD for samples generated for each time point in triplicate.

Mentions: Although the results described in the previous section raise the possibility that CFTR may exit the ER through the di-acidic code, it remained to be shown that wild-type CFTR can engage the COPII machinery and whether interaction with COPII can be disrupted by the ΔF508- or DAA-CFTR mutations. For this purpose, we first examined the ability of CFTR to bind the Sec23/Sec24 cargo selection complex in immunoprecipitates recovered from detergent lysates of HEK293 cell lines stably expressing either wild-type CFTR or ΔF508-CFTR. As shown in Fig. 5 A, in cells expressing wild-type CFTR, the protein is largely in the band C mature form with <5% in the band B ER form, an expected result given that wild-type CFTR accumulates in post-Golgi compartments. In contrast, in ΔF508-CFTR–expressing HEK293 cells, the only form is band B reflecting its ER localization, its level of expression reflecting continuous degradation by ERAD (Fig. 5 A). Despite the large difference in the steady-state level of CFTR in wild-type and ΔF508-expressing cells, when we quantitated the recovery of Sec24 with respect to the total amount of wild-type CFTR or ΔF508-CFTR in band B using immunoblotting, we observed that ΔF508-CFTR bound <25% of that observed for wild-type CFTR (Fig. 5 A). Sec24 was not recovered from HEK293 cells that do not express CFTR (unpublished data). Because immunoprecipitations were performed on ice, it is likely that the temperature-sensitive phenotype of ΔF508-CFTR resulted in partial folding after transfer to ice, accounting for the observed level of recovery of Sec24. Although the band B ER form would be expected to be the only form of CFTR that would bind the ER-specific COPII coat machinery, we examined whether or not immunoprecipitation of Sec24 would recover band C, the cell surface form. Despite the large excess of band C in the cell (Fig. 5 A), immunoprecipitation of Sec24 followed by immunoblotting for CFTR only recovered the band B forms of either wild type or ΔF508 (Fig. 5 B).


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)

Wild-type CFTR but not DAA-CFTR binds COPII. (A) HEK293 stably expressing wild type or ΔF508 were lysed in the presence of 1% Triton X-100, and Sec23/Sec24 complex recovered by immunoprecipitation with a CFTR-specific mAb was quantitated as described in Materials and methods. CFTR (bands B and C) and Sec24 were detected by immunoblotting with specific antibody as described in Materials and methods. (B) As in A except the amount CFTR recovered by immunoprecipitation with a Sec24-specific polyclonal antibody was quantitated as described in Materials and methods using immunoblotting. HEK293 cells not expressing CFTR were used as a control. CFTR (bands B and C) were detected by immunoblotting with specific antibody. (C) HEK293 cells were transfected with pcDNA3.1 plasmids containing either wild-type CFTR or DAA-CFTR as described previously (Yoo et al., 2002). Cells were lysed in the presence of 1% Triton X-100, and the amount of Sec23/Sec24 complex recovered bound to band B of CFTR (expressed as a Sec24/band B ratio) was quantitated as described in Materials and methods. CFTR (bands B and C) and Sec24 were detected by immunoblotting with specific antibodies. (D) HEK293 stably expressing wild type or ΔF508 incubated at 37°C (wild type) or for the indicated time at 30°C (ΔF508) were lysed in the presence of 1% Triton X-100, and the amount of Sec23/Sec24 complex recovered bound to CFTR by immunoprecipitation with a CFTR-specific antibody was quantitated as described in Materials and methods. Error bars indicates the SD for samples generated for each time point in triplicate.
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fig5: Wild-type CFTR but not DAA-CFTR binds COPII. (A) HEK293 stably expressing wild type or ΔF508 were lysed in the presence of 1% Triton X-100, and Sec23/Sec24 complex recovered by immunoprecipitation with a CFTR-specific mAb was quantitated as described in Materials and methods. CFTR (bands B and C) and Sec24 were detected by immunoblotting with specific antibody as described in Materials and methods. (B) As in A except the amount CFTR recovered by immunoprecipitation with a Sec24-specific polyclonal antibody was quantitated as described in Materials and methods using immunoblotting. HEK293 cells not expressing CFTR were used as a control. CFTR (bands B and C) were detected by immunoblotting with specific antibody. (C) HEK293 cells were transfected with pcDNA3.1 plasmids containing either wild-type CFTR or DAA-CFTR as described previously (Yoo et al., 2002). Cells were lysed in the presence of 1% Triton X-100, and the amount of Sec23/Sec24 complex recovered bound to band B of CFTR (expressed as a Sec24/band B ratio) was quantitated as described in Materials and methods. CFTR (bands B and C) and Sec24 were detected by immunoblotting with specific antibodies. (D) HEK293 stably expressing wild type or ΔF508 incubated at 37°C (wild type) or for the indicated time at 30°C (ΔF508) were lysed in the presence of 1% Triton X-100, and the amount of Sec23/Sec24 complex recovered bound to CFTR by immunoprecipitation with a CFTR-specific antibody was quantitated as described in Materials and methods. Error bars indicates the SD for samples generated for each time point in triplicate.
Mentions: Although the results described in the previous section raise the possibility that CFTR may exit the ER through the di-acidic code, it remained to be shown that wild-type CFTR can engage the COPII machinery and whether interaction with COPII can be disrupted by the ΔF508- or DAA-CFTR mutations. For this purpose, we first examined the ability of CFTR to bind the Sec23/Sec24 cargo selection complex in immunoprecipitates recovered from detergent lysates of HEK293 cell lines stably expressing either wild-type CFTR or ΔF508-CFTR. As shown in Fig. 5 A, in cells expressing wild-type CFTR, the protein is largely in the band C mature form with <5% in the band B ER form, an expected result given that wild-type CFTR accumulates in post-Golgi compartments. In contrast, in ΔF508-CFTR–expressing HEK293 cells, the only form is band B reflecting its ER localization, its level of expression reflecting continuous degradation by ERAD (Fig. 5 A). Despite the large difference in the steady-state level of CFTR in wild-type and ΔF508-expressing cells, when we quantitated the recovery of Sec24 with respect to the total amount of wild-type CFTR or ΔF508-CFTR in band B using immunoblotting, we observed that ΔF508-CFTR bound <25% of that observed for wild-type CFTR (Fig. 5 A). Sec24 was not recovered from HEK293 cells that do not express CFTR (unpublished data). Because immunoprecipitations were performed on ice, it is likely that the temperature-sensitive phenotype of ΔF508-CFTR resulted in partial folding after transfer to ice, accounting for the observed level of recovery of Sec24. Although the band B ER form would be expected to be the only form of CFTR that would bind the ER-specific COPII coat machinery, we examined whether or not immunoprecipitation of Sec24 would recover band C, the cell surface form. Despite the large excess of band C in the cell (Fig. 5 A), immunoprecipitation of Sec24 followed by immunoblotting for CFTR only recovered the band B forms of either wild type or ΔF508 (Fig. 5 B).

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