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. 1992 Aug 1;118(3):541–549. doi: 10.1083/jcb.118.3.541

Transient aggregation of nascent thyroglobulin in the endoplasmic reticulum: relationship to the molecular chaperone, BiP

PMCID: PMC2289550  PMID: 1353499

Abstract

Because of its unusual length, nascent thyroglobulin (Tg) requires a long time after translocation into the endoplasmic reticulum (ER) to assume its mature tertiary structure. Thus, Tg is an ideal molecule for the study of protein folding and export from the ER, and is an excellent potential substrate for molecular chaperones. During the first 15 min after biosynthesis, Tg is found in transient aggregates with and without interchain disulfide bonds, which precede the formation of free monomers (and ultimately dimers) within the ER. By immunoprecipitation, newly synthesized Tg was associated with the binding protein (BiP); association was maximal at the earliest chase times. Much of the Tg released from BiP by the addition of Mg-ATP was found in aggregates containing interchain disulfide bonds; other BiP- associated Tg represented non-covalent aggregates and unfolded free monomers. Importantly, the immediate precursor to Tg dimer was a compact monomer which did not associate with BiP. The average stoichiometry of BiP/Tg interaction involved nearly 10 BiP molecules per Tg molecule. Cycloheximide was used to reduced the ER concentration of Tg relative to chaperones, with subsequent removal of the drug in order to rapidly restore Tg synthesis. After this treatment, nascent Tg aggregates were no longer detectable. The data suggest a model of folding of exportable proteins in which nascent polypeptides immediately upon translocation into the ER interact with BiP. Early interaction with BiP may help in presenting nascent polypeptides to other helper molecules that catalyze folding, thereby preventing aggregation or driving aggregate dissolution in the ER.

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Selected References

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  1. 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]
  2. 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]
  3. 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]
  4. Flynn G. C., Chappell T. G., Rothman J. E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989 Jul 28;245(4916):385–390. doi: 10.1126/science.2756425. [DOI] [PubMed] [Google Scholar]
  5. Flynn G. C., Pohl J., Flocco M. T., Rothman J. E. Peptide-binding specificity of the molecular chaperone BiP. Nature. 1991 Oct 24;353(6346):726–730. doi: 10.1038/353726a0. [DOI] [PubMed] [Google Scholar]
  6. Freedman R. B., Bulleid N. J., Hawkins H. C., Paver J. L. Role of protein disulphide-isomerase in the expression of native proteins. Biochem Soc Symp. 1989;55:167–192. [PubMed] [Google Scholar]
  7. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  8. Gething M. J., Sambrook J. Transport and assembly processes in the endoplasmic reticulum. Semin Cell Biol. 1990 Feb;1(1):65–72. [PubMed] [Google Scholar]
  9. Gibson R., Schlesinger S., Kornfeld S. The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures. J Biol Chem. 1979 May 10;254(9):3600–3607. [PubMed] [Google Scholar]
  10. 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]
  11. Hurtley S. M., Bole D. G., Hoover-Litty H., Helenius A., Copeland C. S. Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). J Cell Biol. 1989 Jun;108(6):2117–2126. doi: 10.1083/jcb.108.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kassenbrock C. K., Garcia P. D., Walter P., Kelly R. B. Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature. 1988 May 5;333(6168):90–93. doi: 10.1038/333090a0. [DOI] [PubMed] [Google Scholar]
  13. Kim P. S., Arvan P. Folding and assembly of newly synthesized thyroglobulin occurs in a pre-Golgi compartment. J Biol Chem. 1991 Jul 5;266(19):12412–12418. [PubMed] [Google Scholar]
  14. Lazzarino D. A., Gabel C. A. Biosynthesis of the mannose 6-phosphate recognition marker in transport-impaired mouse lymphoma cells. Demonstration of a two-step phosphorylation. J Biol Chem. 1988 Jul 25;263(21):10118–10126. [PubMed] [Google Scholar]
  15. Lodish H. F., Kong N., Snider M., Strous G. J. Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature. 1983 Jul 7;304(5921):80–83. doi: 10.1038/304080a0. [DOI] [PubMed] [Google Scholar]
  16. Machamer C. E., Doms R. W., Bole D. G., Helenius A., Rose J. K. Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein. J Biol Chem. 1990 Apr 25;265(12):6879–6883. [PubMed] [Google Scholar]
  17. Machamer C. E., Rose J. K. Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding. J Biol Chem. 1988 Apr 25;263(12):5955–5960. [PubMed] [Google Scholar]
  18. Malthièry Y., Marriq C., Bergé-Lefranc J. L., Franc J. L., Henry M., Lejeune P. J., Ruf J., Lissitzky S. Thyroglobulin structure and function: recent advances. Biochimie. 1989 Feb;71(2):195–209. doi: 10.1016/0300-9084(89)90057-6. [DOI] [PubMed] [Google Scholar]
  19. Nguyen T. H., Law D. T., Williams D. B. Binding protein BiP is required for translocation of secretory proteins into the endoplasmic reticulum in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1565–1569. doi: 10.1073/pnas.88.4.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Novick P., Ferro S., Schekman R. Order of events in the yeast secretory pathway. Cell. 1981 Aug;25(2):461–469. doi: 10.1016/0092-8674(81)90064-7. [DOI] [PubMed] [Google Scholar]
  21. Pelham H. R. Control of protein exit from the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:1–23. doi: 10.1146/annurev.cb.05.110189.000245. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  24. Rothman J. E. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell. 1989 Nov 17;59(4):591–601. doi: 10.1016/0092-8674(89)90005-6. [DOI] [PubMed] [Google Scholar]
  25. Saraste J., Kuismanen E. Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell. 1984 Sep;38(2):535–549. doi: 10.1016/0092-8674(84)90508-7. [DOI] [PubMed] [Google Scholar]
  26. Singh I., Doms R. W., Wagner K. R., Helenius A. Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly and secretion of anchor-free influenza hemagglutinin. EMBO J. 1990 Mar;9(3):631–639. doi: 10.1002/j.1460-2075.1990.tb08155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Suzuki C. K., Bonifacino J. S., Lin A. Y., Davis M. M., Klausner R. D. Regulating the retention of T-cell receptor alpha chain variants within the endoplasmic reticulum: Ca(2+)-dependent association with BiP. J Cell Biol. 1991 Jul;114(2):189–205. doi: 10.1083/jcb.114.2.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tartakoff A. M. Temperature and energy dependence of secretory protein transport in the exocrine pancreas. EMBO J. 1986 Jul;5(7):1477–1482. doi: 10.1002/j.1460-2075.1986.tb04385.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Wägar G. Effect of actinomycin D on TSH-induced stimulation of thyroidal protein synthesis in the rat. Acta Endocrinol (Copenh) 1974 Sep;77(1):64–70. [PubMed] [Google Scholar]

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