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. 1993 Jul;102(3):843–850. doi: 10.1104/pp.102.3.843

Characteristics of an Hsp70 homolog localized in higher plant chloroplasts that is similar to DnaK, the Hsp70 of prokaryotes.

H Wang 1, M Goffreda 1, T Leustek 1
PMCID: PMC158855  PMID: 8278536

Abstract

Members of the 70-kD heat-shock protein (Hsp70) family are important cellular factors that are thought to mediate protein folding and assembly. A chloroplast-localized Hsp70 homolog (Chsp70) was recently identified based on its similarity to DnaK, the Hsp70 homolog of Escherichia coli (D. Amir-Shapira, T. Leustek, B. Dalie, H. Weissbach, N. Brot [1990] Proc Natl Acad Sci USA 87: 1749-1752). To learn more about the function of Chsp70, we purified the protein from Spinacia oleracea chloroplasts by ATP-agarose affinity chromatography. A single, 75,000-D protein was isolated which becomes phosphorylated on a threonine residue when incubated with [gamma-32P]ATP and 10 mM Ca2+, a property similar to DnaK. Chloroplast fractionation and immunoblot analysis showed that Chsp70 is a soluble stromal protein. Chsp70-specific antiserum was used to clone a partial cDNA that shows greater homology with Hsp70 from prokaryotes than with cytoplasmic Hsp70 from eukaryotes. The antiserum and cDNA were used to study Chsp70 expression. Following heat shock of spinach seedlings at 37 degrees C, Chsp70 synthesis increase 12-fold, the level of Chsp70 mRNA increases 5-fold, and the level of Chsp70 protein increases less than 2-fold. Chsp70 is constitutively expressed in all spinach tissues, indicating that it is likely to be localized in all plastid types. The highest levels occur in seeds, leaves, florets, and seedlings grown in the light. Lower levels occur in roots, stems, and etiolated seedlings.

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Selected References

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  1. Brennan S. O., Myles T., Peach R. J., Donaldson D., George P. M. Albumin Redhill (-1 Arg, 320 Ala----Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site. Proc Natl Acad Sci U S A. 1990 Jan;87(1):26–30. doi: 10.1073/pnas.87.1.26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cegielska A., Georgopoulos C. Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis. J Biol Chem. 1989 Dec 15;264(35):21122–21130. [PubMed] [Google Scholar]
  3. Chen Q., Lauzon L. M., DeRocher A. E., Vierling E. Accumulation, stability, and localization of a major chloroplast heat-shock protein. J Cell Biol. 1990 Jun;110(6):1873–1883. doi: 10.1083/jcb.110.6.1873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chitnis P. R., Nelson N. Molecular cloning of the genes encoding two chaperone proteins of the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem. 1991 Jan 5;266(1):58–65. [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Flaherty K. M., DeLuca-Flaherty C., McKay D. B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 1990 Aug 16;346(6285):623–628. doi: 10.1038/346623a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Jolly S. O., Bogorad L. Preferential transcription of cloned maize chloroplast DNA sequences by maize chloroplast RNA polymerase. Proc Natl Acad Sci U S A. 1980 Feb;77(2):822–826. doi: 10.1073/pnas.77.2.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ko K., Bornemisza O., Kourtz L., Ko Z. W., Plaxton W. C., Cashmore A. R. Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope. J Biol Chem. 1992 Feb 15;267(5):2986–2993. [PubMed] [Google Scholar]
  11. 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]
  12. Langer T., Lu C., Echols H., Flanagan J., Hayer M. K., Hartl F. U. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 1992 Apr 23;356(6371):683–689. doi: 10.1038/356683a0. [DOI] [PubMed] [Google Scholar]
  13. Leustek T., Amir-Shapira D., Toledo H., Brot N., Weissbach H. Autophosphorylation of 70 kDa heat shock proteins. Cell Mol Biol. 1992 Feb;38(1):1–10. [PubMed] [Google Scholar]
  14. Liberek K., Skowyra D., Zylicz M., Johnson C., Georgopoulos C. The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein. J Biol Chem. 1991 Aug 5;266(22):14491–14496. [PubMed] [Google Scholar]
  15. 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]
  16. Marshall J. S., Keegstra K. Isolation and characterization of a cDNA clone encoding the major hsp70 of the pea chloroplastic stroma. Plant Physiol. 1992 Oct;100(2):1048–1054. doi: 10.1104/pp.100.2.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McCarty J. S., Walker G. C. DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9513–9517. doi: 10.1073/pnas.88.21.9513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miernyk J. A., Duck N. B., David N. R., Randall D. D. Autophosphorylation of the pea mitochondrial heat-shock protein homolog. Plant Physiol. 1992 Oct;100(2):965–969. doi: 10.1104/pp.100.2.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scaramuzzi C. D., Stokes H. W., Hiller R. G. Heat shock Hsp70 protein is chloroplast-encoded in the chromophytic alga Pavlova lutherii. Plant Mol Biol. 1992 Feb;18(3):467–476. doi: 10.1007/BF00040663. [DOI] [PubMed] [Google Scholar]
  20. Vierling E., Key J. L. Ribulose 1,5-Bisphosphate Carboxylase Synthesis during Heat Shock. Plant Physiol. 1985 May;78(1):155–162. doi: 10.1104/pp.78.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]

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