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. 1990 Mar;87(5):1749–1752. doi: 10.1073/pnas.87.5.1749

Hsp70 proteins, similar to Escherichia coli DnaK, in chloroplasts and mitochondria of Euglena gracilis.

D Amir-Shapira 1, T Leustek 1, B Dalie 1, H Weissbach 1, N Brot 1
PMCID: PMC53560  PMID: 2106681

Abstract

The heat-shock response of Euglena gracilis was studied by pulse-labeling cells with [35S]sulfate at both the normal growth temperature (21 degrees C) and an elevated temperature (36 degrees C). Analysis of the labeled proteins by polyacrylamide gel electrophoresis indicated that the rate of synthesis of at least 3 major and 15 minor polypeptides increased in cells grown at the higher temperature. Three of the proteins appear to be immunologically related to the ubiquitous approximately 70-kDa heat-shock protein (Hsp70) family. One protein of 68 kDa was found in the cytoplasm (P68cyt) and was the major heat-shock protein in Euglena gracilis. Two other proteins, 68 and 70 kDa, were localized in mitochondria (P68mit) and chloroplasts (P70chl), respectively, and they crossreacted with a polyclonal antibody raised against the Escherichia coli heat-shock protein DnaK. Like DnaK, P68mit and P70chl could be phosphorylated in vitro with [gamma-32P]ATP in a reaction that was stimulated by Ca2+. A protein with characteristics similar to those of P70chl was also found in chloroplasts isolated from maize and spinach.

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Selected References

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

  1. 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]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Bukau B., Walker G. C. Delta dnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures. J Bacteriol. 1989 Nov;171(11):6030–6038. doi: 10.1128/jb.171.11.6030-6038.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheng M. Y., Hartl F. U., Martin J., Pollock R. A., Kalousek F., Neupert W., Hallberg E. M., Hallberg R. L., Horwich A. L. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 1989 Feb 16;337(6208):620–625. doi: 10.1038/337620a0. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Craig E. A., Kramer J., Kosic-Smithers J. SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4156–4160. doi: 10.1073/pnas.84.12.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Craig E. A., Kramer J., Shilling J., Werner-Washburne M., Holmes S., Kosic-Smithers J., Nicolet C. M. SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein. Mol Cell Biol. 1989 Jul;9(7):3000–3008. doi: 10.1128/mcb.9.7.3000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deshaies R. J., Koch B. D., Schekman R. The role of stress proteins in membrane biogenesis. Trends Biochem Sci. 1988 Oct;13(10):384–388. doi: 10.1016/0968-0004(88)90180-6. [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. Eilers M., Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986 Jul 17;322(6076):228–232. doi: 10.1038/322228a0. [DOI] [PubMed] [Google Scholar]
  11. Engman D. M., Kirchhoff L. V., Donelson J. E. Molecular cloning of mtp70, a mitochondrial member of the hsp70 family. Mol Cell Biol. 1989 Nov;9(11):5163–5168. doi: 10.1128/mcb.9.11.5163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ezaki B., Ogura T., Mori H., Niki H., Hiraga S. Involvement of DnaK protein in mini-F plasmid replication: temperature-sensitive seg mutations are located in the dnaK gene. Mol Gen Genet. 1989 Aug;218(2):183–189. doi: 10.1007/BF00331267. [DOI] [PubMed] [Google Scholar]
  13. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  14. Heschl M. F., Baillie D. L. Characterization of the hsp70 multigene family of Caenorhabditis elegans. DNA. 1989 May;8(4):233–243. doi: 10.1089/dna.1.1989.8.233. [DOI] [PubMed] [Google Scholar]
  15. Krishnasamy S., Mannan R. M., Krishnan M., Gnanam A. Heat shock response of the chloroplast genome in Vigna sinensis. J Biol Chem. 1988 Apr 15;263(11):5104–5109. [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. Leustek T., Dalie B., Amir-Shapira D., Brot N., Weissbach H. A member of the Hsp70 family is localized in mitochondria and resembles Escherichia coli DnaK. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7805–7808. doi: 10.1073/pnas.86.20.7805. [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. Liberek K., Georgopoulos C., Zylicz M. Role of the Escherichia coli DnaK and DnaJ heat shock proteins in the initiation of bacteriophage lambda DNA replication. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6632–6636. doi: 10.1073/pnas.85.18.6632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Loomis W. F., Wheeler S., Schmidt J. A. Phosphorylation of the major heat shock protein of Dictyostelium discoideum. Mol Cell Biol. 1982 May;2(5):484–489. doi: 10.1128/mcb.2.5.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lynch M. J., Buetow E. D. Isolation of intact nuclei from Euglena Grancilis. Exp Cell Res. 1975 Mar 15;91(2):344–348. doi: 10.1016/0014-4827(75)90113-5. [DOI] [PubMed] [Google Scholar]
  23. Marshall J. S., DeRocher A. E., Keegstra K., Vierling E. Identification of heat shock protein hsp70 homologues in chloroplasts. Proc Natl Acad Sci U S A. 1990 Jan;87(1):374–378. doi: 10.1073/pnas.87.1.374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McMullin T. W., Hallberg R. L. A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene. Mol Cell Biol. 1988 Jan;8(1):371–380. doi: 10.1128/mcb.8.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McMullin T. W., Hallberg R. L. A normal mitochondrial protein is selectively synthesized and accumulated during heat shock in Tetrahymena thermophila. Mol Cell Biol. 1987 Dec;7(12):4414–4423. doi: 10.1128/mcb.7.12.4414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Orozco E. M., Jr, Mullet J. E., Hanley-Bowdoin L., Chua N. H. In vitro transcription of chloroplast protein genes. Methods Enzymol. 1986;118:232–253. doi: 10.1016/0076-6879(86)18076-1. [DOI] [PubMed] [Google Scholar]
  28. Osafune T., Schiff J. A. W10BSmL, a mutant of Euglena gracilis var. bacillaris lacking plastids. Exp Cell Res. 1983 Oct 15;148(2):530–535. doi: 10.1016/0014-4827(83)90176-3. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Pfanner N., Neupert W. Transport of F1-ATPase subunit beta into mitochondria depends on both a membrane potential and nucleoside triphosphates. FEBS Lett. 1986 Dec 15;209(2):152–156. doi: 10.1016/0014-5793(86)81101-2. [DOI] [PubMed] [Google Scholar]
  31. SMITH L. A study of some oxidative enzymes of baker's yeast. Arch Biochem Biophys. 1954 Jun;50(2):285–298. doi: 10.1016/0003-9861(54)90044-2. [DOI] [PubMed] [Google Scholar]
  32. Skelly S., Fu C. F., Dalie B., Redfield B., Coleman T., Brot N., Weissbach H. Antibody to sigma 32 cross-reacts with DnaK: association of DnaK protein with Escherichia coli RNA polymerase. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5497–5501. doi: 10.1073/pnas.85.15.5497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tilly K., Yarmolinsky M. Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication. J Bacteriol. 1989 Nov;171(11):6025–6029. doi: 10.1128/jb.171.11.6025-6029.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Zylicz M., Georgopoulos C. Purification and properties of the Escherichia coli dnaK replication protein. J Biol Chem. 1984 Jul 25;259(14):8820–8825. [PubMed] [Google Scholar]
  39. 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]

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