The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum

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.

[1]  Arthur E Johnson,et al.  Cotranslational Membrane Protein Biogenesis at the Endoplasmic Reticulum* , 2004, Journal of Biological Chemistry.

[2]  Peter J McCormick,et al.  Nascent Membrane and Secretory Proteins Differ in FRET-Detected Folding Far inside the Ribosome and in Their Exposure to Ribosomal Proteins , 2004, Cell.

[3]  Michael W. Morrow,et al.  Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP. , 2003, Molecular biology of the cell.

[4]  J. Tyedmers,et al.  The Sec61p complex is a dynamic precursor activated channel. , 2003, Molecular cell.

[5]  J. Reinstein,et al.  Modulation of the ATPase cycle of BiP by peptides and proteins. , 2003, Journal of molecular biology.

[6]  H. Garside,et al.  An in vitro assay using overexpressed yeast SRP demonstrates that cotranslational translocation is dependent upon the J-domain of Sec63p. , 2003, Biochemistry.

[7]  Kenji Kohno,et al.  Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins. , 2003, Molecular biology of the cell.

[8]  L. Hendershot,et al.  BAP, a Mammalian BiP-associated Protein, Is a Nucleotide Exchange Factor That Regulates the ATPase Activity of BiP* , 2002, The Journal of Biological Chemistry.

[9]  Xi Chen,et al.  ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. , 2002, Developmental cell.

[10]  G. Blatch,et al.  A novel type of co‐chaperone mediates transmembrane recruitment of DnaK‐like chaperones to ribosomes , 2002 .

[11]  R. Wek,et al.  Dimerization and Release of Molecular Chaperone Inhibition Facilitate Activation of Eukaryotic Initiation Factor-2 Kinase in Response to Endoplasmic Reticulum Stress* , 2002, The Journal of Biological Chemistry.

[12]  L. Hendershot,et al.  Identification and Characterization of a Novel Endoplasmic Reticulum (ER) DnaJ Homologue, Which Stimulates ATPase Activity of BiP in Vitro and Is Induced by ER Stress* , 2002, The Journal of Biological Chemistry.

[13]  L. Hendershot,et al.  Unassembled Ig heavy chains do not cycle from BiP in vivo but require light chains to trigger their release. , 2001, Immunity.

[14]  M. Wiedmann,et al.  In Vitro Binding of Ribosomes to the β Subunit of the Sec61p Protein Translocation Complex* , 2001, The Journal of Biological Chemistry.

[15]  Barry P. Young,et al.  Sec63p and Kar2p are required for the translocation of SRP‐dependent precursors into the yeast endoplasmic reticulum in vivo , 2001, The EMBO journal.

[16]  D. Haslam,et al.  HEDJ, an Hsp40 Co-chaperone Localized to the Endoplasmic Reticulum of Human Cells* , 2000, The Journal of Biological Chemistry.

[17]  D. Raden,et al.  Role of the Cytoplasmic Segments of Sec61α in the Ribosome-Binding and Translocation-Promoting Activities of the Sec61 Complex , 2000, The Journal of cell biology.

[18]  S. Blond,et al.  Interaction of Murine BiP/GRP78 with the DnaJ Homologue MTJ1* , 2000, The Journal of Biological Chemistry.

[19]  W. Nastainczyk,et al.  Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Anne Bertolotti,et al.  Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response , 2000, Nature Cell Biology.

[21]  R. Kraft,et al.  Mammalian Sec61 Is Associated with Sec62 and Sec63* , 2000, The Journal of Biological Chemistry.

[22]  T. Rapoport,et al.  Interaction of BiP with the J-domain of the Sec63p Component of the Endoplasmic Reticulum Protein Translocation Complex* , 1999, The Journal of Biological Chemistry.

[23]  T. Rapoport,et al.  BiP Acts as a Molecular Ratchet during Posttranslational Transport of Prepro-α Factor across the ER Membrane , 1999, Cell.

[24]  C. Gross,et al.  Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  J. Brodsky,et al.  Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER. , 1998, Molecular biology of the cell.

[26]  T. Rapoport,et al.  J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences. , 1998, Molecular cell.

[27]  J. Brodsky,et al.  Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum , 1998, FEBS letters.

[28]  S. Landry,et al.  Role of the J-domain in the cooperation of Hsp40 with Hsp70. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[29]  L. Hendershot,et al.  BiP Maintains the Permeability Barrier of the ER Membrane by Sealing the Lumenal End of the Translocon Pore before and Early in Translocation , 1998, Cell.

[30]  Bernd Bukau,et al.  The Hsp70 and Hsp60 Chaperone Machines , 1998, Cell.

[31]  J Frank,et al.  Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex. , 1997, Science.

[32]  Jialing Lin,et al.  Both Lumenal and Cytosolic Gating of the Aqueous ER Translocon Pore Are Regulated from Inside the Ribosome during Membrane Protein Integration , 1997, Cell.

[33]  R. Schekman,et al.  The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae , 1997, The Journal of cell biology.

[34]  A. Johnson,et al.  The Aqueous Pore through the Translocon Has a Diameter of 40–60 Å during Cotranslational Protein Translocation at the ER Membrane , 1997, Cell.

[35]  S. White,et al.  Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin. , 1997, Biophysical journal.

[36]  B. Jungnickel,et al.  Oligomeric Rings of the Sec61p Complex Induced by Ligands Required for Protein Translocation , 1996, Cell.

[37]  J. Reinstein,et al.  The second step of ATP binding to DnaK induces peptide release. , 1996, Journal of molecular biology.

[38]  Craig M. Ogata,et al.  Structural Analysis of Substrate Binding by the Molecular Chaperone DnaK , 1996, Science.

[39]  L. Hendershot,et al.  Inhibition of immunoglobulin folding and secretion by dominant negative BiP ATPase mutants. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[40]  F. Hartl,et al.  Hip, a novel cochaperone involved in the eukaryotic hsc70/hsp40 reaction cycle , 1995, Cell.

[41]  L. Hendershot,et al.  In Vitro Dissociation of BiP-Peptide Complexes Requires a Conformational Change in BiP after ATP Binding but Does Not Require ATP Hydrolysis (*) , 1995, The Journal of Biological Chemistry.

[42]  R. Schekman,et al.  BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[43]  G. Reinhart,et al.  Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore , 1994, Cell.

[44]  G. Reinhart,et al.  The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation , 1993, Cell.

[45]  G. Blobel,et al.  Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation , 1993, Cell.

[46]  L. Hendershot,et al.  Mutations within the nucleotide binding site of immunoglobulin-binding protein inhibit ATPase activity and interfere with release of immunoglobulin heavy chain. , 1993, The Journal of biological chemistry.

[47]  G. Blobel,et al.  A protein-conducting channel in the endoplasmic reticulum , 1991, Cell.

[48]  J. Rothman,et al.  Peptide binding and release by proteins implicated as catalysts of protein assembly. , 1989, Science.

[49]  R. Freedman,et al.  Defective co-translational formation of disulphide bonds in protein disulphide-isomerase-deficient microsomes , 1988, Nature.

[50]  G. Blatch,et al.  A novel type of co‐chaperone mediates transmembrane recruitment of DnaK‐like chaperones to ribosomes , 2002, The EMBO journal.

[51]  A. Johnson,et al.  The translocon: a dynamic gateway at the ER membrane. , 1999, Annual review of cell and developmental biology.

[52]  G. Blobel,et al.  Preparation of microsomal membranes for cotranslational protein translocation. , 1983, Methods in enzymology.

[53]  E. King,et al.  The colorimetric determination of phosphorus. , 1932, The Biochemical journal.