Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Diphosphate / metabolism
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Adenosine Diphosphate / pharmacology
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Adenosine Triphosphatases / drug effects
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Adenosine Triphosphatases / metabolism
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Adenosine Triphosphate / metabolism
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Adenosine Triphosphate / pharmacology
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Animals
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Binding Sites / genetics
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Binding Sites / physiology
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Binding, Competitive
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Cricetinae
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Endoplasmic Reticulum / metabolism
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Endoplasmic Reticulum / physiology*
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Endoplasmic Reticulum Chaperone BiP
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Fluorescence Polarization
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Fluorescent Dyes / chemistry
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Fungal Proteins / metabolism
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HSP70 Heat-Shock Proteins / metabolism
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Heat-Shock Proteins / genetics
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Heat-Shock Proteins / metabolism
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Heat-Shock Proteins / physiology*
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Iodine / chemistry
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Membrane Proteins / metabolism
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Membrane Proteins / physiology*
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Microsomes / metabolism
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Models, Biological
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Molecular Chaperones / genetics
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Molecular Chaperones / metabolism
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Molecular Chaperones / physiology*
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Mutation
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Peptides / chemistry
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Peptides / pharmacology
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Prolactin / chemistry
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Prolactin / metabolism
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Protein Binding
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Protein Conformation / drug effects
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Protein Precursors / chemistry
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Protein Precursors / metabolism
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Protein Structure, Tertiary / physiology
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Protein Transport / physiology
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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SEC Translocation Channels
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Spectrometry, Fluorescence
Substances
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Endoplasmic Reticulum Chaperone BiP
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Fluorescent Dyes
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Fungal Proteins
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HSP70 Heat-Shock Proteins
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Heat-Shock Proteins
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KAR2 protein, yeast
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Membrane Proteins
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Molecular Chaperones
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Peptides
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Protein Precursors
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Recombinant Proteins
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SEC Translocation Channels
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Adenosine Diphosphate
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preprolactin
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Adenosine Triphosphate
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Prolactin
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Iodine
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Adenosine Triphosphatases