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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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CFTR-COPII cargo selection complex. (bottom) The structure of CFTR-NBD1 (1ROX.pdb) is oriented to show its structural homology with the NBD1 structural domain of the bacterial ABC transporter BtuCD (1L7V.pdb). In CFTR-NBD1, Phe508 is shown in blue and residues Asp565 and Asp567 (di-acidic code) are shown in yellow. In BtuCD, the cytosolic domains are shown in green and putative transmembrane spanning domains are shown in red. The short loop shown in red in BtuCD corresponds structurally to the di-acidic code-containing loop in CFTR-NBD1. (middle) Homology structure containing CFTR-NBD1 and BtuCD (Btu-NBD1CFTR) docked to the Sar1/Sec23/24 complex (Bi et al., 2002; Mossessova et al., 2003; see Materials and methods). Gray rectangle illustrates the putative location of the membrane bilayer. (top) The interaction of the CFTR-NBD1 di-acidic code with the binding pocket of Sec23/24 (Bi et al., 2002; Mossessova et al., 2003). Shown in blue are Arg230, 235, 559, and 561 basic residues defining the di-acidic code pocket of Sec24 (Bi et al., 2002; Mossessova et al., 2003). Only a minor steric interference is observed between Asp565 (CFTR-NBD1) and Leu616 (Sec24) in the homology model. See Fig. S2 for a three-dimensional rendering of the interface between CFTR-NBD1 and the Sec23/24 cargo complex. Fig. S2 is available at http://www.jcb.org/cgi/content/full/jcb.200401035/DC1.
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fig6: CFTR-COPII cargo selection complex. (bottom) The structure of CFTR-NBD1 (1ROX.pdb) is oriented to show its structural homology with the NBD1 structural domain of the bacterial ABC transporter BtuCD (1L7V.pdb). In CFTR-NBD1, Phe508 is shown in blue and residues Asp565 and Asp567 (di-acidic code) are shown in yellow. In BtuCD, the cytosolic domains are shown in green and putative transmembrane spanning domains are shown in red. The short loop shown in red in BtuCD corresponds structurally to the di-acidic code-containing loop in CFTR-NBD1. (middle) Homology structure containing CFTR-NBD1 and BtuCD (Btu-NBD1CFTR) docked to the Sar1/Sec23/24 complex (Bi et al., 2002; Mossessova et al., 2003; see Materials and methods). Gray rectangle illustrates the putative location of the membrane bilayer. (top) The interaction of the CFTR-NBD1 di-acidic code with the binding pocket of Sec23/24 (Bi et al., 2002; Mossessova et al., 2003). Shown in blue are Arg230, 235, 559, and 561 basic residues defining the di-acidic code pocket of Sec24 (Bi et al., 2002; Mossessova et al., 2003). Only a minor steric interference is observed between Asp565 (CFTR-NBD1) and Leu616 (Sec24) in the homology model. See Fig. S2 for a three-dimensional rendering of the interface between CFTR-NBD1 and the Sec23/24 cargo complex. Fig. S2 is available at http://www.jcb.org/cgi/content/full/jcb.200401035/DC1.

Mentions: NBD1 domains of a large variety of ABC transporters have a similar structural organization (Schmitt and Tampe, 2002). Structural alignment of the NBD1 domain of CFTR (Lewis et al., 2004) with the NBD1 domain of the ABC transporter BtuCD reveals that the di-acidic code is found in a solvent-exposed loop connecting the NH2-terminal helical domain containing Phe508 with the more COOH-terminal sheet domain (Fig. 4 A and Fig. 6). Given that the structure of Sar1 and the Sec23/24 complex containing a bound di-acidic peptide are available (Huang et al., 2001; Bi et al., 2002; Mossessova et al., 2003), we built a homology model with the NBD1 of CFTR replacing the similar structural fold of NBD1 of BtuCD (Fig. 6). This new structure (BtuCD-NBD1CFTR) was then combined with the structure of the Sec23/24 -Sar1 complex to illustrate the ability of the di-acidic code loop in NBD1CFTR to insert directly into the Sec23/24 di-acidic code binding pocket (Fig. 6, yellow residues; and Fig. S2 for a three-dimensional view, available at http://www.jcb.org/cgi/content/full/jcb.200401035/DC1). Docking reveals little steric interference between residues within the binding pocket, or between residues defining the tertiary structure of BtuCD-NBD1CFTR and the Sar1/Sec24/23 complex. Moreover, the orientation of the NH2 terminus of Sar1 (Fig. 6, red β-strand; residues 25–34), which is linked to the flexible NH2-terminal tail involved in anchoring Sar1 to the membrane (Fig. 6, black dashed line; not observed in the crystal structure; Huang et al., 2001; Bi et al., 2002), is oriented correctly to facilitate recruitment of Sec23/24 by activated Sar1. It is apparent that Sar1 and BtuCD-NBD1CFTR interact with Sec23/Sec24 independently as shown for the holo cargo-selection complex. Thus, the general structural features of the CFTR-NBD1 domain presented within the structure of BtuCD are consistent with the role of the di-acidic code in directing interaction with COPII for exit from the ER.


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-COPII cargo selection complex. (bottom) The structure of CFTR-NBD1 (1ROX.pdb) is oriented to show its structural homology with the NBD1 structural domain of the bacterial ABC transporter BtuCD (1L7V.pdb). In CFTR-NBD1, Phe508 is shown in blue and residues Asp565 and Asp567 (di-acidic code) are shown in yellow. In BtuCD, the cytosolic domains are shown in green and putative transmembrane spanning domains are shown in red. The short loop shown in red in BtuCD corresponds structurally to the di-acidic code-containing loop in CFTR-NBD1. (middle) Homology structure containing CFTR-NBD1 and BtuCD (Btu-NBD1CFTR) docked to the Sar1/Sec23/24 complex (Bi et al., 2002; Mossessova et al., 2003; see Materials and methods). Gray rectangle illustrates the putative location of the membrane bilayer. (top) The interaction of the CFTR-NBD1 di-acidic code with the binding pocket of Sec23/24 (Bi et al., 2002; Mossessova et al., 2003). Shown in blue are Arg230, 235, 559, and 561 basic residues defining the di-acidic code pocket of Sec24 (Bi et al., 2002; Mossessova et al., 2003). Only a minor steric interference is observed between Asp565 (CFTR-NBD1) and Leu616 (Sec24) in the homology model. See Fig. S2 for a three-dimensional rendering of the interface between CFTR-NBD1 and the Sec23/24 cargo complex. Fig. S2 is available at http://www.jcb.org/cgi/content/full/jcb.200401035/DC1.
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fig6: CFTR-COPII cargo selection complex. (bottom) The structure of CFTR-NBD1 (1ROX.pdb) is oriented to show its structural homology with the NBD1 structural domain of the bacterial ABC transporter BtuCD (1L7V.pdb). In CFTR-NBD1, Phe508 is shown in blue and residues Asp565 and Asp567 (di-acidic code) are shown in yellow. In BtuCD, the cytosolic domains are shown in green and putative transmembrane spanning domains are shown in red. The short loop shown in red in BtuCD corresponds structurally to the di-acidic code-containing loop in CFTR-NBD1. (middle) Homology structure containing CFTR-NBD1 and BtuCD (Btu-NBD1CFTR) docked to the Sar1/Sec23/24 complex (Bi et al., 2002; Mossessova et al., 2003; see Materials and methods). Gray rectangle illustrates the putative location of the membrane bilayer. (top) The interaction of the CFTR-NBD1 di-acidic code with the binding pocket of Sec23/24 (Bi et al., 2002; Mossessova et al., 2003). Shown in blue are Arg230, 235, 559, and 561 basic residues defining the di-acidic code pocket of Sec24 (Bi et al., 2002; Mossessova et al., 2003). Only a minor steric interference is observed between Asp565 (CFTR-NBD1) and Leu616 (Sec24) in the homology model. See Fig. S2 for a three-dimensional rendering of the interface between CFTR-NBD1 and the Sec23/24 cargo complex. Fig. S2 is available at http://www.jcb.org/cgi/content/full/jcb.200401035/DC1.
Mentions: NBD1 domains of a large variety of ABC transporters have a similar structural organization (Schmitt and Tampe, 2002). Structural alignment of the NBD1 domain of CFTR (Lewis et al., 2004) with the NBD1 domain of the ABC transporter BtuCD reveals that the di-acidic code is found in a solvent-exposed loop connecting the NH2-terminal helical domain containing Phe508 with the more COOH-terminal sheet domain (Fig. 4 A and Fig. 6). Given that the structure of Sar1 and the Sec23/24 complex containing a bound di-acidic peptide are available (Huang et al., 2001; Bi et al., 2002; Mossessova et al., 2003), we built a homology model with the NBD1 of CFTR replacing the similar structural fold of NBD1 of BtuCD (Fig. 6). This new structure (BtuCD-NBD1CFTR) was then combined with the structure of the Sec23/24 -Sar1 complex to illustrate the ability of the di-acidic code loop in NBD1CFTR to insert directly into the Sec23/24 di-acidic code binding pocket (Fig. 6, yellow residues; and Fig. S2 for a three-dimensional view, available at http://www.jcb.org/cgi/content/full/jcb.200401035/DC1). Docking reveals little steric interference between residues within the binding pocket, or between residues defining the tertiary structure of BtuCD-NBD1CFTR and the Sar1/Sec24/23 complex. Moreover, the orientation of the NH2 terminus of Sar1 (Fig. 6, red β-strand; residues 25–34), which is linked to the flexible NH2-terminal tail involved in anchoring Sar1 to the membrane (Fig. 6, black dashed line; not observed in the crystal structure; Huang et al., 2001; Bi et al., 2002), is oriented correctly to facilitate recruitment of Sec23/24 by activated Sar1. It is apparent that Sar1 and BtuCD-NBD1CFTR interact with Sec23/Sec24 independently as shown for the holo cargo-selection complex. Thus, the general structural features of the CFTR-NBD1 domain presented within the structure of BtuCD are consistent with the role of the di-acidic code in directing interaction with COPII for exit from the ER.

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