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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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ER export is reversibly sensitive to a novel Sar1 temperature-sensitive (Sar1ts) mutant. (A) BHK cells were cotransfected with pcDNA3.1 containing either VSV-G or VSV-G and the indicated Sar1 mutant. The effect of Sar1-GTP (control) or Sar1ts on processing of VSV-G from the endoglycosidase H–sensitive (endo Hs) ER form to the endo H–resistant (endo Hr) Golgi form after a 5-min pulse with [35S]Met at 32°C followed by a 30 min chase at the indicated temperature was determined as described previously (Yoo et al., 2002). (B) BHK cells were cotransfected with pcDNA3.1 containing wild-type CFTR either with vector only (mock) or, where indicated, cotransfected with pcDNA3.1 containing the Sar1ts mutant. The transport and processing of CFTR from band B to C was followed at the indicated temperature as described in Fig. 1. The C/B ratios for the Sar1ts mutant at 32 and 40°C are shown for the 3-h time point. The asterisk in the top panel of B indicates that CFTR is exported very inefficiently from the ER as band C is only weakly processed to early Golgi oligosaccharide forms. White lines indicate that intervening lanes have been spliced out. Results are typical of three independent experiments.
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fig3: ER export is reversibly sensitive to a novel Sar1 temperature-sensitive (Sar1ts) mutant. (A) BHK cells were cotransfected with pcDNA3.1 containing either VSV-G or VSV-G and the indicated Sar1 mutant. The effect of Sar1-GTP (control) or Sar1ts on processing of VSV-G from the endoglycosidase H–sensitive (endo Hs) ER form to the endo H–resistant (endo Hr) Golgi form after a 5-min pulse with [35S]Met at 32°C followed by a 30 min chase at the indicated temperature was determined as described previously (Yoo et al., 2002). (B) BHK cells were cotransfected with pcDNA3.1 containing wild-type CFTR either with vector only (mock) or, where indicated, cotransfected with pcDNA3.1 containing the Sar1ts mutant. The transport and processing of CFTR from band B to C was followed at the indicated temperature as described in Fig. 1. The C/B ratios for the Sar1ts mutant at 32 and 40°C are shown for the 3-h time point. The asterisk in the top panel of B indicates that CFTR is exported very inefficiently from the ER as band C is only weakly processed to early Golgi oligosaccharide forms. White lines indicate that intervening lanes have been spliced out. Results are typical of three independent experiments.

Mentions: To ensure that the aforementioned results with the Sar1 mutants were not a consequence of targeting of either wild-type or mutant CFTR to a nonphysiological pathway, or reflect indirect effects of other cargo in the ER on CFTR export or stability, we developed a temperature-sensitive variant of Sar1. This mutant is dominant-negative at the permissive temperature (32°C), where it is folded properly. It loses this dominant-negative activity when transferred to the restrictive temperature (40°C), where it misfolds. When cotransfected with the type 1 transmembrane protein vesicular stomatitis virus glycoprotein (VSV-G) at 32°C, Sar1ts shows strong inhibition of processing of VSV-G from the endoglycosidase H (endo H)–sensitive ER form to the endo H–resistant Golgi form (Fig. 3 A), indicative of an ER to Golgi transport block. In contrast, at 40°C, a condition that promotes destabilization of the mutant protein, inhibition of export is lost (Fig. 3 A). Thus, Sar1ts shows a thermoreversible temperature-sensitive ER export block.


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)

ER export is reversibly sensitive to a novel Sar1 temperature-sensitive (Sar1ts) mutant. (A) BHK cells were cotransfected with pcDNA3.1 containing either VSV-G or VSV-G and the indicated Sar1 mutant. The effect of Sar1-GTP (control) or Sar1ts on processing of VSV-G from the endoglycosidase H–sensitive (endo Hs) ER form to the endo H–resistant (endo Hr) Golgi form after a 5-min pulse with [35S]Met at 32°C followed by a 30 min chase at the indicated temperature was determined as described previously (Yoo et al., 2002). (B) BHK cells were cotransfected with pcDNA3.1 containing wild-type CFTR either with vector only (mock) or, where indicated, cotransfected with pcDNA3.1 containing the Sar1ts mutant. The transport and processing of CFTR from band B to C was followed at the indicated temperature as described in Fig. 1. The C/B ratios for the Sar1ts mutant at 32 and 40°C are shown for the 3-h time point. The asterisk in the top panel of B indicates that CFTR is exported very inefficiently from the ER as band C is only weakly processed to early Golgi oligosaccharide forms. White lines indicate that intervening lanes have been spliced out. Results are typical of three independent experiments.
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Related In: Results  -  Collection

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fig3: ER export is reversibly sensitive to a novel Sar1 temperature-sensitive (Sar1ts) mutant. (A) BHK cells were cotransfected with pcDNA3.1 containing either VSV-G or VSV-G and the indicated Sar1 mutant. The effect of Sar1-GTP (control) or Sar1ts on processing of VSV-G from the endoglycosidase H–sensitive (endo Hs) ER form to the endo H–resistant (endo Hr) Golgi form after a 5-min pulse with [35S]Met at 32°C followed by a 30 min chase at the indicated temperature was determined as described previously (Yoo et al., 2002). (B) BHK cells were cotransfected with pcDNA3.1 containing wild-type CFTR either with vector only (mock) or, where indicated, cotransfected with pcDNA3.1 containing the Sar1ts mutant. The transport and processing of CFTR from band B to C was followed at the indicated temperature as described in Fig. 1. The C/B ratios for the Sar1ts mutant at 32 and 40°C are shown for the 3-h time point. The asterisk in the top panel of B indicates that CFTR is exported very inefficiently from the ER as band C is only weakly processed to early Golgi oligosaccharide forms. White lines indicate that intervening lanes have been spliced out. Results are typical of three independent experiments.
Mentions: To ensure that the aforementioned results with the Sar1 mutants were not a consequence of targeting of either wild-type or mutant CFTR to a nonphysiological pathway, or reflect indirect effects of other cargo in the ER on CFTR export or stability, we developed a temperature-sensitive variant of Sar1. This mutant is dominant-negative at the permissive temperature (32°C), where it is folded properly. It loses this dominant-negative activity when transferred to the restrictive temperature (40°C), where it misfolds. When cotransfected with the type 1 transmembrane protein vesicular stomatitis virus glycoprotein (VSV-G) at 32°C, Sar1ts shows strong inhibition of processing of VSV-G from the endoglycosidase H (endo H)–sensitive ER form to the endo H–resistant Golgi form (Fig. 3 A), indicative of an ER to Golgi transport block. In contrast, at 40°C, a condition that promotes destabilization of the mutant protein, inhibition of export is lost (Fig. 3 A). Thus, Sar1ts shows a thermoreversible temperature-sensitive ER export block.

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