The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain–binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding–induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain–binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.
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31 January 2005
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January 31 2005
The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum
Nathan N. Alder,
Nathan N. Alder
1Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843
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Ying Shen,
Ying Shen
2Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105
3Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163
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Jeffrey L. Brodsky,
Jeffrey L. Brodsky
4Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
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Linda M. Hendershot,
Linda M. Hendershot
2Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105
3Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163
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Arthur E. Johnson
Arthur E. Johnson
1Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843
5Department of Chemistry, Texas A&M University, College Station, TX 77843
6Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843
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Nathan N. Alder
1Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843
Ying Shen
2Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105
3Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163
Jeffrey L. Brodsky
4Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
Linda M. Hendershot
2Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN 38105
3Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163
Arthur E. Johnson
1Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843
5Department of Chemistry, Texas A&M University, College Station, TX 77843
6Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843
Correspondence to Arthur E. Johnson: [email protected]
Abbreviations used in this paper: I−, iodide ion; KRM, salt-washed ER microsome; pPL, preprolactin; rBiP, recombinant hamster BiP; RNC, ribosome-nascent chain complex; RRM, reconstituted microsome; SBD, substrate-binding domain; SRP, signal recognition particle; XRM, lumen-extracted microsome.
Received:
September 28 2004
Accepted:
December 17 2004
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2005
J Cell Biol (2005) 168 (3): 389–399.
Article history
Received:
September 28 2004
Accepted:
December 17 2004
Connected Content
This article has been corrected
Correction: The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum
Citation
Nathan N. Alder, Ying Shen, Jeffrey L. Brodsky, Linda M. Hendershot, Arthur E. Johnson; The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum . J Cell Biol 31 January 2005; 168 (3): 389–399. doi: https://doi.org/10.1083/jcb.200409174
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