Limits...
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.

Show MeSH

Related in: MedlinePlus

CFTR uses a di-acidic code for export from the ER. (A) Location of Phe508 and the di-acidic code in the NBD1 domain of CFTR (Lewis et al., 2004; 1ROX.pdb). Shown in blue is Phe508; shown in red (carbon backbone) and yellow (side-chains) is the orientation of residues Asp565 and Asp567 (di-acidic code). (B) Cells were either transfected or cotransfected with the indicated wild-type or mutant CFTR and the Sar1-GTP mutant as indicated. Processing from band B to band C was quantitated as described in Fig. 1. (C) Effect of BFA on stability of wild-type CFTR, ΔF508-CFTR, or the DAA-CFTR mutant in the ER. White lines indicate that intervening lanes have been spliced out. Cells were pulse-labeled for 30 min with [35S]Met at 37°C. 10 μg/ml BFA was added and incubation continued for 3 h at 37°C. (lanes a, d, and g) Total CFTR found in band B after pulse (expressed as a value of 100%). (lanes b, e, and h) Fraction of CFTR found in band B after 3-h incubation in the absence of BFA. (lanes c, f, and i) Fraction of CFTR found in band B after 3-h incubation in the presence of BFA. The dashed bar in lane b indicates the total B + C at the 3-h time point in the absence of BFA. Results are typical of at least two independent experiments for each condition shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172508&req=5

fig4: CFTR uses a di-acidic code for export from the ER. (A) Location of Phe508 and the di-acidic code in the NBD1 domain of CFTR (Lewis et al., 2004; 1ROX.pdb). Shown in blue is Phe508; shown in red (carbon backbone) and yellow (side-chains) is the orientation of residues Asp565 and Asp567 (di-acidic code). (B) Cells were either transfected or cotransfected with the indicated wild-type or mutant CFTR and the Sar1-GTP mutant as indicated. Processing from band B to band C was quantitated as described in Fig. 1. (C) Effect of BFA on stability of wild-type CFTR, ΔF508-CFTR, or the DAA-CFTR mutant in the ER. White lines indicate that intervening lanes have been spliced out. Cells were pulse-labeled for 30 min with [35S]Met at 37°C. 10 μg/ml BFA was added and incubation continued for 3 h at 37°C. (lanes a, d, and g) Total CFTR found in band B after pulse (expressed as a value of 100%). (lanes b, e, and h) Fraction of CFTR found in band B after 3-h incubation in the absence of BFA. (lanes c, f, and i) Fraction of CFTR found in band B after 3-h incubation in the presence of BFA. The dashed bar in lane b indicates the total B + C at the 3-h time point in the absence of BFA. Results are typical of at least two independent experiments for each condition shown.

Mentions: To understand the requirement for COPII in the export of CFTR from the ER, we examined the CFTR sequence for the presence of the evolutionarily conserved tyrosine-based di-acidic code required for export of VSV-G (Nishimura and Balch, 1997; Nishimura et al., 1999; Sevier et al., 2000) and other transmembrane proteins from the ER in mammalian cells (Bannykh and Balch, 1998; Bannykh et al., 1998) and yeast (Votsmeier and Gallwitz, 2001; Malkus et al., 2002). Notably, CFTR contained a YKDAD motif (residues 563 to 567) in the first nucleotide binding motif (NBD1) that is evolutionarily conserved and similar to that found in VSV-G (YxDxE; Nishimura and Balch, 1997; Sevier et al., 2000). The structure of the NBD1 domain of CFTR (1ROZ.pdb) reveals that the YKDAD motif is located in a surface-exposed loop linking the NH2-terminal helical domain containing Phe508 to the COOH-terminal sheet domain (Lewis et al., 2004; Fig. 4 A). Strikingly, transient expression of CFTR mutants in which either one (DAA-CFTR; Fig. 4 B, inset and bottom panel) or both (AAA-CFTR; unpublished data) conserved Asp residues were mutated to Ala resulted in complete inhibition of export from the ER as measured by the inability of band B to be processed to band C and a morphological distribution consistent with localization to the ER (Bannykh et al., 2000; unpublished data). Like ΔF508-CFTR, cotransfection with Sar1-GTP failed to promote export or stabilize DAA-CFTR from ERAD, suggesting that mutation of the di-acidic motif uncoupled CFTR from COPII (Fig. 4 B). We also examined whether or not, like ΔF508-CFTR, the DAA-CFTR phenotype is temperature-sensitive by examining its rate of export at the lowered temperature (30°C). Whereas transfer of ΔF508-CFTR to 30°C resulted in significant enhancement of transport to the cell surface reflecting its effect on conformational stability of NBD1 as observed previously (Denning et al., 1992; French et al., 1996), we did not observe a similar increase in DAA-CFTR export at 30°C (unpublished data). These results suggest that the DAA mutation does not affect the fold of NBD1 in a similar fashion to the ΔF508 mutation that can be stabilized by incubation at reduced temperature.


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)

CFTR uses a di-acidic code for export from the ER. (A) Location of Phe508 and the di-acidic code in the NBD1 domain of CFTR (Lewis et al., 2004; 1ROX.pdb). Shown in blue is Phe508; shown in red (carbon backbone) and yellow (side-chains) is the orientation of residues Asp565 and Asp567 (di-acidic code). (B) Cells were either transfected or cotransfected with the indicated wild-type or mutant CFTR and the Sar1-GTP mutant as indicated. Processing from band B to band C was quantitated as described in Fig. 1. (C) Effect of BFA on stability of wild-type CFTR, ΔF508-CFTR, or the DAA-CFTR mutant in the ER. White lines indicate that intervening lanes have been spliced out. Cells were pulse-labeled for 30 min with [35S]Met at 37°C. 10 μg/ml BFA was added and incubation continued for 3 h at 37°C. (lanes a, d, and g) Total CFTR found in band B after pulse (expressed as a value of 100%). (lanes b, e, and h) Fraction of CFTR found in band B after 3-h incubation in the absence of BFA. (lanes c, f, and i) Fraction of CFTR found in band B after 3-h incubation in the presence of BFA. The dashed bar in lane b indicates the total B + C at the 3-h time point in the absence of BFA. Results are typical of at least two independent experiments for each condition shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172508&req=5

fig4: CFTR uses a di-acidic code for export from the ER. (A) Location of Phe508 and the di-acidic code in the NBD1 domain of CFTR (Lewis et al., 2004; 1ROX.pdb). Shown in blue is Phe508; shown in red (carbon backbone) and yellow (side-chains) is the orientation of residues Asp565 and Asp567 (di-acidic code). (B) Cells were either transfected or cotransfected with the indicated wild-type or mutant CFTR and the Sar1-GTP mutant as indicated. Processing from band B to band C was quantitated as described in Fig. 1. (C) Effect of BFA on stability of wild-type CFTR, ΔF508-CFTR, or the DAA-CFTR mutant in the ER. White lines indicate that intervening lanes have been spliced out. Cells were pulse-labeled for 30 min with [35S]Met at 37°C. 10 μg/ml BFA was added and incubation continued for 3 h at 37°C. (lanes a, d, and g) Total CFTR found in band B after pulse (expressed as a value of 100%). (lanes b, e, and h) Fraction of CFTR found in band B after 3-h incubation in the absence of BFA. (lanes c, f, and i) Fraction of CFTR found in band B after 3-h incubation in the presence of BFA. The dashed bar in lane b indicates the total B + C at the 3-h time point in the absence of BFA. Results are typical of at least two independent experiments for each condition shown.
Mentions: To understand the requirement for COPII in the export of CFTR from the ER, we examined the CFTR sequence for the presence of the evolutionarily conserved tyrosine-based di-acidic code required for export of VSV-G (Nishimura and Balch, 1997; Nishimura et al., 1999; Sevier et al., 2000) and other transmembrane proteins from the ER in mammalian cells (Bannykh and Balch, 1998; Bannykh et al., 1998) and yeast (Votsmeier and Gallwitz, 2001; Malkus et al., 2002). Notably, CFTR contained a YKDAD motif (residues 563 to 567) in the first nucleotide binding motif (NBD1) that is evolutionarily conserved and similar to that found in VSV-G (YxDxE; Nishimura and Balch, 1997; Sevier et al., 2000). The structure of the NBD1 domain of CFTR (1ROZ.pdb) reveals that the YKDAD motif is located in a surface-exposed loop linking the NH2-terminal helical domain containing Phe508 to the COOH-terminal sheet domain (Lewis et al., 2004; Fig. 4 A). Strikingly, transient expression of CFTR mutants in which either one (DAA-CFTR; Fig. 4 B, inset and bottom panel) or both (AAA-CFTR; unpublished data) conserved Asp residues were mutated to Ala resulted in complete inhibition of export from the ER as measured by the inability of band B to be processed to band C and a morphological distribution consistent with localization to the ER (Bannykh et al., 2000; unpublished data). Like ΔF508-CFTR, cotransfection with Sar1-GTP failed to promote export or stabilize DAA-CFTR from ERAD, suggesting that mutation of the di-acidic motif uncoupled CFTR from COPII (Fig. 4 B). We also examined whether or not, like ΔF508-CFTR, the DAA-CFTR phenotype is temperature-sensitive by examining its rate of export at the lowered temperature (30°C). Whereas transfer of ΔF508-CFTR to 30°C resulted in significant enhancement of transport to the cell surface reflecting its effect on conformational stability of NBD1 as observed previously (Denning et al., 1992; French et al., 1996), we did not observe a similar increase in DAA-CFTR export at 30°C (unpublished data). These results suggest that the DAA mutation does not affect the fold of NBD1 in a similar fashion to the ΔF508 mutation that can be stabilized by incubation at reduced temperature.

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