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Isolation of proteins that speifically interact with the ATPase domain of mammalian ER chaperone, BiP

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Abstract

BiP, immunoglobulin binding protein, is an ER homologue of Hsp 70. However, unlike other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demonstrated the presence of potential regulatory proteins for BiP using a pull-down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull-down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein. Eight proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp 170 and BiP where identified, while the others were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for Bip was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.

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References

  1. Ellgaard, L., M. Molinari, and A. Helenius (1999) Setting the standards: quality control in the secretory pathway.Science 286: 1882–1888.

    Article  CAS  Google Scholar 

  2. Hammond, C. and A. Helenius (1995) Quality control in the secretory pathway.Curr. Opin. Cell Biol. 7: 523–529.

    Article  CAS  Google Scholar 

  3. Haas, I. G. and M. Wabl (1983) Immunoglobulin heavy chain binding protein.Nature 306: 387–389.

    Article  CAS  Google Scholar 

  4. Gething, M. J. and J. Sambrook (1992) Protein folding in the cell.Nature 355: 33–45.

    Article  CAS  Google Scholar 

  5. Hamman, B. D., L. M. Hendershot, and A. E. Johnson (1998) BiP maintains the permeability barrier of the ER membrane by sealing the luminal end of the translocon pore before and early in translocation.Cell 92: 747–758.

    Article  CAS  Google Scholar 

  6. Brodsky, J. L., E. D. Werner, M. E. Dubas, J. L. Goeckeler, K. B. Kruse, and A. A. McCracken (1999) The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct.J. Biol. Chem. 274: 3453–3460.

    Article  CAS  Google Scholar 

  7. Bertolotti, A., Y. Zhang, L. M. Hendershot, H. P. Harding, and D. Ron (2000) Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.Nat. Cell Biol. 2: 326–332.

    Article  CAS  Google Scholar 

  8. Boorstein, W. R., T. Ziegelhoffer, and E. A. Craig (1994) Molecular evolution of the Hsp 70 multigene family.J. Mol. Evol. 38: 1–17.

    Article  CAS  Google Scholar 

  9. Liberek, K., J. Marszalek, D. Ang, C. Georgopoulos, and M. Zylicz (1991)Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.Proc. Natl. Acad. Sci. USA 88: 2874–2878.

    Article  CAS  Google Scholar 

  10. Szabo, A., T. Langer, H. Schroder, J. Flanagan, B. Bukau, and F. U. Hartl (1994) The ATP hydrolysis-dependent reaction cycle of theEscherichia coli Hsp 70 system DnaK, DnaJ, and GrpE.Proc. Natl. Acad. Sci. USA 91: 10345–10349.

    Article  CAS  Google Scholar 

  11. Minami, Y., J. Hohfeld, K. Ohtsuka, and F. U. Hartl (1996) Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40.J. Bio.. Chem. 271: 19617–19624.

    Article  CAS  Google Scholar 

  12. Bimston, D., J. Song, D. Winchester, S. Takayama, J. C. Reed, and R. I. Morimoto (1998) BAG-1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release.EMBO J. 17: 6871–6878.

    Article  CAS  Google Scholar 

  13. Hohfeld, J. and S. Jentsch (1997) GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1.EMBO J. 16: 6209–6216.

    Article  CAS  Google Scholar 

  14. Raynes, D. A. and V. Guerriero, Jr. (1998) Inhibition of Hsp70 ATPase activity and protein renaturation by a novel Hsp70-binding protein.J. Biol. Chem. 273: 32883–32888.

    Article  CAS  Google Scholar 

  15. Hohfeld, J., Y. Minami, and F. U. Hartl (1995) Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle.Cell 83: 589–598.

    Article  CAS  Google Scholar 

  16. Chen, S. and D. F. Smith (1998) Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery.J. Biol. Chem. 273: 35194–35200.

    Article  CAS  Google Scholar 

  17. Johnson, B. D., R. J. Schumacher, E.D. Ross, and D. O. Toft (1998) Hop modulates hsp70/hsp90 interactions in protein folding.J. Biol. Chem. 273: 3679–3686.

    Article  CAS  Google Scholar 

  18. Ballinger, C. A., P. Connell, Y. Wu, Z. Hu, L. J. Thompson, L. Y. Yin, and C. Patterson (1999) Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.Mol. Cell. Biol. 19: 4535–4545.

    CAS  Google Scholar 

  19. Bies, C., S. Guth, K. Janoschek, W. J. Nastainczyk, Volkmer, and R. Zimmermann (1999) A Scj1p homolog and folding catalysts present in dog pancreas microsomes.Biol. Chem. 380: 1175–1182.

    Article  CAS  Google Scholar 

  20. Brightman, S. E., G. L. Blatch, and B. R. Zetter (1995) Isolation of a mouse cDNA encoding MTJ1, a new murine member of the DnaJ family of proteins.Gene 153: 249–254.

    Article  CAS  Google Scholar 

  21. Shen, Y., L. Meunier, and L. M. Hendershot (2002) Identification and characterization of a novel ER DnaJ homologue, which stimulates BiP's ATPase activity in vitro and is induced by ER stress.J. Biol. Chem. 277: 15947–15956.

    Article  CAS  Google Scholar 

  22. Skowronek, M. H., M. Rotter, and I. G. Haas (1999) Molecular characterization of a novel mammalian DnaJ-like Sec63p homolog.Biol. Chem. 380: 1133–1138.

    Article  CAS  Google Scholar 

  23. Yu, M., R. H. Haslam, and D. B. Haslam (2000) HEDJ, an Hsp40 co-chaperone localized to the endoplasmic reticulum of human cells.J. Biol. Chem. 275: 24984–24992.

    Article  CAS  Google Scholar 

  24. Kabani, M., J. M. Beckerich, and C. Gaillardin (2000) Sls1p stimulates Sec63p-mediated activation of Kar2p in a conformation-dependent manner in the yeast endoplasmic reticulum.Mol. Cell. Biol. 20: 6923–6934.

    Article  CAS  Google Scholar 

  25. Tyson, J. R. and C. J. Stirling (2000) LHS1 and SIL1 provide a lumenal function that is essential for protein translocation into the endoplasmic reticulum.EMBO J. 19: 6440–6452.

    Article  CAS  Google Scholar 

  26. Gaut, J. R. and L. M. Hendershot (1993) The immunoglobulin-binding protein in vitro autophposphylation site maps to a threonine within the ATP binding cleft but is not a detectable site of in vivo phosphorylation.J. Biol. Chem. 269: 12691–12698.

    Google Scholar 

  27. Kassenbrock, C. K. and R. B. Kelly (1989) Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides.EMBO J. 8: 1461–1467.

    CAS  Google Scholar 

  28. Wei, J., J. R. Gaut, and L. M. Hendershot (1995) In vitro dissociation of BiP-peptide complexes requires a conformational change in BiP after ATP binding but does not require ATP hydrolysis.J. Biol. Chem. 270: 26677–26682.

    Article  CAS  Google Scholar 

  29. Chen, X., D. Easton, H. J. Oh, D. S. Lee-Yoon, X. Liu, and J. Subject (1996) The 170 kDa glucose regulated stress protein is a large Hsp7-, Hsp110-like protein of the end oplasmic reticulum.FEBS Lett. 380: 68–72.

    Article  CAS  Google Scholar 

  30. Takayama, S., T. Sato, S. Krajewski, K. Kochel, S. Irie, J. A. Millan, and J. C. Reed. (1995) Cloning and functional analysis of BAG-1: A novel Bcl-2-binding protein with anti-cell death activity.Cell 80: 279–284.

    Article  CAS  Google Scholar 

  31. Takayama, S., D. N. Bimston, S. Matsuzawa, B. C. Freeman, C. Aime-Sempe, Z. Xie, R. I. Morimoto, and J. C. Reed (1997) BAG-1 modulates the chaperone activity of Hsp70/Hsc70.EMBO J. 16: 4887–4896.

    Article  CAS  Google Scholar 

  32. Carlino, A., H. Toledo, D. Skaleris, R. DeLisio, H. Weissbach, and N. Brot (1992) Interactions of liver Grp78 andEscherichia coli recombinant Grp78 with ATP: multiple species and disaggregation.Proc. Natl. Acad. Sci. USA 89: 2081–2085.

    Article  CAS  Google Scholar 

  33. Freiden, P. J., J. R. Gaut, and L. M. Hendershot (1992) Interconversion of three differentially modified and assembled forms of BiP.EMBO J. 11: 63–70.

    CAS  Google Scholar 

  34. Brodsky, J. L., M. Bauerle, M. Horst, and A. J. McClellan (1998) Mictochodrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum. FEBS Lett. 435: 183–186.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Applied Life Sciences, College of Natural Sciences, Dong-Eui University, 614-714, Busan, Korea

    Kyung Tae Chung

  2. Department of Microbiology, College of Natural Sciences, Pusan National University, 609-735, Busan, Korea

    Tae Ho Lee

  3. Department of Pharmaceutical, University of Tennessee, The Health Science Center, 38163, Memphis, TN, USA

    Gyong Suk Kang

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  1. Kyung Tae Chung
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  2. Tae Ho Lee
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Correspondence to Kyung Tae Chung.

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Chung, K.T., Lee, T.H. & Kang, G.S. Isolation of proteins that speifically interact with the ATPase domain of mammalian ER chaperone, BiP. Biotechnol. Bioprocess Eng. 8, 192–198 (2003). https://doi.org/10.1007/BF02935896

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