Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1992 Jan;11(1):63–70. doi: 10.1002/j.1460-2075.1992.tb05028.x

Interconversion of three differentially modified and assembled forms of BiP.

P J Freiden 1, J R Gaut 1, L M Hendershot 1
PMCID: PMC556426  PMID: 1740116

Abstract

The immunoglobulin heavy chain binding protein BiP/GRP78 is post-translationally modified by phosphorylation and ADP ribosylation. In cells induced to synthesize higher levels of BiP, either due to the accumulation of nontransported proteins or to glucose starvation, both BiP phosphorylation and ADP ribosylation are reduced. BiP bound to other proteins is unmodified, suggesting that both phosphorylation and ADP ribosylation are restricted to the unbound BiP pool. In the present study, both modifications were further characterized in terms of their stability, the pool of BiP that harbored these modifications, and the relationship between the modified and unmodified forms of BiP. While levels of BiP synthesis vary according to the physiological state of a cell, we found that both induced and uninduced cells contain similar amounts of free BiP. However, free BiP in uninduced cells was found primarily in an aggregated state, whereas in cells that accumulate nontransported proteins, it was predominantly monomeric. Both phosphorylation and ADP ribosylation were restricted to the aggregated form of free BiP. These post-translational modifications occurred upon release of BiP from associated proteins, and could be reversed upon induction of BiP synthesis. Therefore, BiP exists either (1) complexed to other proteins, (2) as a free unmodified monomer, or (3) as free modified aggregates. Our data suggest that BiP can be interconverted from one state to another, and that the various forms are functionally distinct.

Full text

PDF
63

Images in this article

64Image
on p.64
65Image
on p.65
65Image
on p.65
66Image
on p.66
67Image
on p.67
67Image
on p.67
68Image
on p.68

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amir-Shapira D., Leustek T., Dalie B., Weissbach H., Brot N. Hsp70 proteins, similar to Escherichia coli DnaK, in chloroplasts and mitochondria of Euglena gracilis. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1749–1752. doi: 10.1073/pnas.87.5.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Braell W. A., Schlossman D. M., Schmid S. L., Rothman J. E. Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):734–741. doi: 10.1083/jcb.99.2.734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Carlsson L., Lazarides E. ADP-ribosylation of the Mr 83,000 stress-inducible and glucose-regulated protein in avian and mammalian cells: modulation by heat shock and glucose starvation. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4664–4668. doi: 10.1073/pnas.80.15.4664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dorner A. J., Bole D. G., Kaufman R. J. The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol. 1987 Dec;105(6 Pt 1):2665–2674. doi: 10.1083/jcb.105.6.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dorner A. J., Krane M. G., Kaufman R. J. Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells. Mol Cell Biol. 1988 Oct;8(10):4063–4070. doi: 10.1128/mcb.8.10.4063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  9. Haas I. G. BiP--a heat shock protein involved in immunoglobulin chain assembly. Curr Top Microbiol Immunol. 1991;167:71–82. doi: 10.1007/978-3-642-75875-1_4. [DOI] [PubMed] [Google Scholar]
  10. Haas I. G., Meo T. cDNA cloning of the immunoglobulin heavy chain binding protein. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2250–2254. doi: 10.1073/pnas.85.7.2250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haas I. G., Wabl M. Immunoglobulin heavy chain binding protein. Nature. 1983 Nov 24;306(5941):387–389. doi: 10.1038/306387a0. [DOI] [PubMed] [Google Scholar]
  12. Hendershot L. M. Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition. J Cell Biol. 1990 Sep;111(3):829–837. doi: 10.1083/jcb.111.3.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hendershot L. M., Ting J., Lee A. S. Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function. Mol Cell Biol. 1988 Oct;8(10):4250–4256. doi: 10.1128/mcb.8.10.4250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kearney J. F., Radbruch A., Liesegang B., Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol. 1979 Oct;123(4):1548–1550. [PubMed] [Google Scholar]
  15. Kozutsumi Y., Segal M., Normington K., Gething M. J., Sambrook J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature. 1988 Mar 31;332(6163):462–464. doi: 10.1038/332462a0. [DOI] [PubMed] [Google Scholar]
  16. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  17. Ledford B. E., Jacobs D. F. Translational control of ADP-ribosylation in eucaryotic cells. Eur J Biochem. 1986 Dec 15;161(3):661–667. doi: 10.1111/j.1432-1033.1986.tb10491.x. [DOI] [PubMed] [Google Scholar]
  18. Lee A. S., Delegeane A. M., Baker V., Chow P. C. Transcriptional regulation of two genes specifically induced by glucose starvation in a hamster mutant fibroblast cell line. J Biol Chem. 1983 Jan 10;258(1):597–603. [PubMed] [Google Scholar]
  19. Lee A. S. The accumulation of three specific proteins related to glucose-regulated proteins in a temperature-sensitive hamster mutant cell line K12. J Cell Physiol. 1981 Jan;106(1):119–125. doi: 10.1002/jcp.1041060113. [DOI] [PubMed] [Google Scholar]
  20. Leno G. H., Ledford B. E. ADP-ribosylation of the 78-kDa glucose-regulated protein during nutritional stress. Eur J Biochem. 1989 Dec 8;186(1-2):205–211. doi: 10.1111/j.1432-1033.1989.tb15196.x. [DOI] [PubMed] [Google Scholar]
  21. Lin A. Y., Lee A. S. Induction of two genes by glucose starvation in hamster fibroblasts. Proc Natl Acad Sci U S A. 1984 Feb;81(4):988–992. doi: 10.1073/pnas.81.4.988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Munro S., Pelham H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987 Mar 13;48(5):899–907. doi: 10.1016/0092-8674(87)90086-9. [DOI] [PubMed] [Google Scholar]
  23. Pelham H. R. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. doi: 10.1016/0092-8674(86)90693-8. [DOI] [PubMed] [Google Scholar]
  24. Schlessinger J. Signal transduction by allosteric receptor oligomerization. Trends Biochem Sci. 1988 Nov;13(11):443–447. doi: 10.1016/0968-0004(88)90219-8. [DOI] [PubMed] [Google Scholar]
  25. Schlossman D. M., Schmid S. L., Braell W. A., Rothman J. E. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):723–733. doi: 10.1083/jcb.99.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schmid S. L., Braell W. A., Rothman J. E. ATP catalyzes the sequestration of clathrin during enzymatic uncoating. J Biol Chem. 1985 Aug 25;260(18):10057–10062. [PubMed] [Google Scholar]
  27. Schmid S. L., Rothman J. E. Two classes of binding sites for uncoating protein in clathrin triskelions. J Biol Chem. 1985 Aug 25;260(18):10050–10056. [PubMed] [Google Scholar]
  28. Vogel J. P., Misra L. M., Rose M. D. Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J Cell Biol. 1990 Jun;110(6):1885–1895. doi: 10.1083/jcb.110.6.1885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Watowich S. S., Morimoto R. I. Complex regulation of heat shock- and glucose-responsive genes in human cells. Mol Cell Biol. 1988 Jan;8(1):393–405. doi: 10.1128/mcb.8.1.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Welch W. J., Garrels J. I., Thomas G. P., Lin J. J., Feramisco J. R. Biochemical characterization of the mammalian stress proteins and identification of two stress proteins as glucose- and Ca2+-ionophore-regulated proteins. J Biol Chem. 1983 Jun 10;258(11):7102–7111. [PubMed] [Google Scholar]
  31. Zylicz M., LeBowitz J. H., McMacken R., Georgopoulos C. The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6431–6435. doi: 10.1073/pnas.80.21.6431. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES