Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Feb 1;108(2):413–423. doi: 10.1083/jcb.108.2.413

Cell cycle-dependent association of HSP70 with specific cellular proteins

PMCID: PMC2115428  PMID: 2645297

Abstract

In asynchronous populations of HeLa cells maintained at control or heat shock temperatures, HSP70 levels and its subcellular distribution exhibit substantial heterogeneity as demonstrated by indirect immunofluorescence with HSP70-specific monoclonal antibodies. Of particular interest is a subpopulation of cells in which the characteristic nuclear accumulation and nucleolar association of HSP70 is not detected after heat shock treatment. This apparent variation in the heat shock response is not observed when synchronized cells are examined. In this study, we demonstrate that three monoclonal antibodies to HSP70, in particular, do not detect nucleolar-localized HSP70 in heat-shocked G2 cells. This is not due to an inability of G2 cells to respond to heat shock as measured by increased HSP70 mRNA and protein synthesis, or due to a lack of accumulation of HSP70 after heat shock in G2. Rather the epitopes recognized by the various antibodies appear to be inaccessible, perhaps due to the association of HSP70 with other proteins. Non-denaturing immunoprecipitations with these HSP70- specific antibodies suggest that HSP70 may interact with other cellular proteins in a cell cycle-dependent manner.

Full Text

The Full Text of this article is available as a PDF (3.7 MB).

Selected References

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

  1. Arrigo A. P., Fakan S., Tissières A. Localization of the heat shock-induced proteins in Drosophila melanogaster tissue culture cells. Dev Biol. 1980 Jul;78(1):86–103. doi: 10.1016/0012-1606(80)90320-6. [DOI] [PubMed] [Google Scholar]
  2. Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Chappell T. G., Welch W. J., Schlossman D. M., Palter K. B., Schlesinger M. J., Rothman J. E. Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell. 1986 Apr 11;45(1):3–13. doi: 10.1016/0092-8674(86)90532-5. [DOI] [PubMed] [Google Scholar]
  7. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  8. Craig E. A. The heat shock response. CRC Crit Rev Biochem. 1985;18(3):239–280. doi: 10.3109/10409238509085135. [DOI] [PubMed] [Google Scholar]
  9. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  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. Hughes E. N., August J. T. Coprecipitation of heat shock proteins with a cell surface glycoprotein. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2305–2309. doi: 10.1073/pnas.79.7.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunt C., Morimoto R. I. Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6455–6459. doi: 10.1073/pnas.82.19.6455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kloetzel P. M., Bautz E. K. Heat-shock proteins are associated with hnRNA in Drosophila melanogaster tissue culture cells. EMBO J. 1983;2(5):705–710. doi: 10.1002/j.1460-2075.1983.tb01488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LaThangue N. B. A major heat-shock protein defined by a monoclonal antibody. EMBO J. 1984 Aug;3(8):1871–1879. doi: 10.1002/j.1460-2075.1984.tb02061.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Levinger L., Varshavsky A. Heat-shock proteins of Drosophila are associated with nuclease-resistant, high-salt-resistant nuclear structures. J Cell Biol. 1981 Sep;90(3):793–796. doi: 10.1083/jcb.90.3.793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lewis M. J., Pelham H. R. Involvement of ATP in the nuclear and nucleolar functions of the 70 kd heat shock protein. EMBO J. 1985 Dec 1;4(12):3137–3143. doi: 10.1002/j.1460-2075.1985.tb04056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lindquist S. The heat-shock response. Annu Rev Biochem. 1986;55:1151–1191. doi: 10.1146/annurev.bi.55.070186.005443. [DOI] [PubMed] [Google Scholar]
  20. Milarski K. L., Morimoto R. I. Expression of human HSP70 during the synthetic phase of the cell cycle. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9517–9521. doi: 10.1073/pnas.83.24.9517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mizzen L. A., Welch W. J. Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression. J Cell Biol. 1988 Apr;106(4):1105–1116. doi: 10.1083/jcb.106.4.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Munro S., Pelham H. R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. doi: 10.1016/0092-8674(86)90746-4. [DOI] [PubMed] [Google Scholar]
  23. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  24. Pelham H. R. Hsp70 accelerates the recovery of nucleolar morphology after heat shock. EMBO J. 1984 Dec 20;3(13):3095–3100. doi: 10.1002/j.1460-2075.1984.tb02264.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Pinhasi-Kimhi O., Michalovitz D., Ben-Zeev A., Oren M. Specific interaction between the p53 cellular tumour antigen and major heat shock proteins. Nature. 1986 Mar 13;320(6058):182–184. doi: 10.1038/320182a0. [DOI] [PubMed] [Google Scholar]
  27. Sakakibara Y. The dnaK gene of Escherichia coli functions in initiation of chromosome replication. J Bacteriol. 1988 Feb;170(2):972–979. doi: 10.1128/jb.170.2.972-979.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sharma S., Rodgers L., Brandsma J., Gething M. J., Sambrook J. SV40 T antigen and the exocytotic pathway. EMBO J. 1985 Jun;4(6):1479–1489. doi: 10.1002/j.1460-2075.1985.tb03806.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Theodorakis N. G., Morimoto R. I. Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability. Mol Cell Biol. 1987 Dec;7(12):4357–4368. doi: 10.1128/mcb.7.12.4357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Velazquez J. M., DiDomenico B. J., Lindquist S. Intracellular localization of heat shock proteins in Drosophila. Cell. 1980 Jul;20(3):679–689. doi: 10.1016/0092-8674(80)90314-1. [DOI] [PubMed] [Google Scholar]
  31. Wang C., Gomer R. H., Lazarides E. Heat shock proteins are methylated in avian and mammalian cells. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3531–3535. doi: 10.1073/pnas.78.6.3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Welch W. J., Feramisco J. R. Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells. J Biol Chem. 1984 Apr 10;259(7):4501–4513. [PubMed] [Google Scholar]
  34. Welch W. J., Feramisco J. R. Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides. Mol Cell Biol. 1985 Jun;5(6):1229–1237. doi: 10.1128/mcb.5.6.1229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Welch W. J., Mizzen L. A. Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes. J Cell Biol. 1988 Apr;106(4):1117–1130. doi: 10.1083/jcb.106.4.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Welch W. J., Suhan J. P. Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J Cell Biol. 1986 Nov;103(5):2035–2052. doi: 10.1083/jcb.103.5.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wu B. J., Morimoto R. I. Transcription of the human hsp70 gene is induced by serum stimulation. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6070–6074. doi: 10.1073/pnas.82.18.6070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wu B., Hunt C., Morimoto R. Structure and expression of the human gene encoding major heat shock protein HSP70. Mol Cell Biol. 1985 Feb;5(2):330–341. doi: 10.1128/mcb.5.2.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zylicz M., Ang D., Georgopoulos C. The grpE protein of Escherichia coli. Purification and properties. J Biol Chem. 1987 Dec 25;262(36):17437–17442. [PubMed] [Google Scholar]
  40. 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 Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES