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. 1996 Jul;111(3):931–939. doi: 10.1104/pp.111.3.931

Purification and characterization of chaperonin 60 and heat-shock protein 70 from chromoplasts of Narcissus pseudonarcissus.

M Bonk 1, M Tadros 1, J Vandekerckhove 1, S Al-Babili 1, P Beyer 1
PMCID: PMC157912  PMID: 8754688

Abstract

In chromoplast differentiation during flower formation in Narcissus pseudonarcissus, the molecular chaperones chaperonin 60 (Cpn60; alpha and beta) and heat-shock protein 70 (Hsp70) greatly increase in abundance. Both were purified and shown to be present in a functional form in chromoplasts, indicating their requirement in the extensive structural rearrangements during the chloroplast-to-chromoplast transition. The purified proteins, sequenced N terminally and from internal peptides, showed strong homology to plastid Cpn60 and Hsp 70 representatives from other plant species. During chromoplast differentiation, the carotenoid biosynthetic pathway is strongly induced. The corresponding enzymes are all nuclear encoded and form a large, soluble, hetero-oligomeric protein complex after import but prior to their membrane attachment. By immunoprecipitations we have shown that the plastid Hsp70 is a structural constituent of a soluble entity also containing phytoene desaturase.

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Selected References

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  1. Al-Babili S., von Lintig J., Haubruck H., Beyer P. A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70-complexed and competent for flavinylation, membrane association and enzymatic activation. Plant J. 1996 May;9(5):601–612. doi: 10.1046/j.1365-313x.1996.9050601.x. [DOI] [PubMed] [Google Scholar]
  2. Bauw G., De Loose M., Inzé D., Van Montagu M., Vandekerckhove J. Alterations in the phenotype of plant cells studied by NH(2)-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4806–4810. doi: 10.1073/pnas.84.14.4806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bertsch U., Soll J., Seetharam R., Viitanen P. V. Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8696–8700. doi: 10.1073/pnas.89.18.8696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beyer P., Kröncke U., Nievelstein V. On the mechanism of the lycopene isomerase/cyclase reaction in Narcissus pseudonarcissus L. chromoplasts. J Biol Chem. 1991 Sep 15;266(26):17072–17078. [PubMed] [Google Scholar]
  5. Beyer P., Mayer M., Kleinig H. Molecular oxygen and the state of geometric isomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts. Eur J Biochem. 1989 Sep 1;184(1):141–150. doi: 10.1111/j.1432-1033.1989.tb15000.x. [DOI] [PubMed] [Google Scholar]
  6. Braig K., Otwinowski Z., Hegde R., Boisvert D. C., Joachimiak A., Horwich A. L., Sigler P. B. The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature. 1994 Oct 13;371(6498):578–586. doi: 10.1038/371578a0. [DOI] [PubMed] [Google Scholar]
  7. Buchner J., Schmidt M., Fuchs M., Jaenicke R., Rudolph R., Schmid F. X., Kiefhaber T. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry. 1991 Feb 12;30(6):1586–1591. doi: 10.1021/bi00220a020. [DOI] [PubMed] [Google Scholar]
  8. Craig E. A., Weissman J. S., Horwich A. L. Heat shock proteins and molecular chaperones: mediators of protein conformation and turnover in the cell. Cell. 1994 Aug 12;78(3):365–372. doi: 10.1016/0092-8674(94)90416-2. [DOI] [PubMed] [Google Scholar]
  9. Fourie A. M., Sambrook J. F., Gething M. J. Common and divergent peptide binding specificities of hsp70 molecular chaperones. J Biol Chem. 1994 Dec 2;269(48):30470–30478. [PubMed] [Google Scholar]
  10. Gatenby A. A., Ellis R. J. Chaperone function: the assembly of ribulose bisphosphate carboxylase-oxygenase. Annu Rev Cell Biol. 1990;6:125–149. doi: 10.1146/annurev.cb.06.110190.001013. [DOI] [PubMed] [Google Scholar]
  11. Gatenby A. A. Protein folding and chaperonins. Plant Mol Biol. 1992 Jul;19(4):677–687. doi: 10.1007/BF00026793. [DOI] [PubMed] [Google Scholar]
  12. Glick B. S. Can Hsp70 proteins act as force-generating motors? Cell. 1995 Jan 13;80(1):11–14. doi: 10.1016/0092-8674(95)90444-1. [DOI] [PubMed] [Google Scholar]
  13. Goloubinoff P., Christeller J. T., Gatenby A. A., Lorimer G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature. 1989 Dec 21;342(6252):884–889. doi: 10.1038/342884a0. [DOI] [PubMed] [Google Scholar]
  14. Hemmingsen S. M. The plastid chaperonin. Semin Cell Biol. 1990 Feb;1(1):47–54. [PubMed] [Google Scholar]
  15. 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]
  16. Hurt E., Hauska G., Malkin R. Isolation of the Rieske iron-sulfur protein from the cytochrome b6/f complex of spinach chloroplasts. FEBS Lett. 1981 Nov 2;134(1):1–5. doi: 10.1016/0014-5793(81)80537-6. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Langer T., Pfeifer G., Martin J., Baumeister W., Hartl F. U. Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 1992 Dec;11(13):4757–4765. doi: 10.1002/j.1460-2075.1992.tb05581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lubben T. H., Donaldson G. K., Viitanen P. V., Gatenby A. A. Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone. Plant Cell. 1989 Dec;1(12):1223–1230. doi: 10.1105/tpc.1.12.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MARKHAM R., HITCHBORN J. H., HILLS G. J., FREY S. THE ANATOMY OF THE TOBACCO MOSAIC VIRUS. Virology. 1964 Mar;22:342–359. doi: 10.1016/0042-6822(64)90025-x. [DOI] [PubMed] [Google Scholar]
  21. Madueno F., Napier J. A., Gray J. C. Newly Imported Rieske Iron-Sulfur Protein Associates with Both Cpn60 and Hsp70 in the Chloroplast Stroma. Plant Cell. 1993 Dec;5(12):1865–1876. doi: 10.1105/tpc.5.12.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Martel R., Cloney L. P., Pelcher L. E., Hemmingsen S. M. Unique composition of plastid chaperonin-60: alpha and beta polypeptide-encoding genes are highly divergent. Gene. 1990 Oct 15;94(2):181–187. doi: 10.1016/0378-1119(90)90385-5. [DOI] [PubMed] [Google Scholar]
  24. Mettal U., Boland W., Beyer P., Kleinig H. Biosynthesis of monoterpene hydrocarbons by isolated chromoplasts from daffodil flowers. Eur J Biochem. 1988 Jan 4;170(3):613–616. doi: 10.1111/j.1432-1033.1988.tb13741.x. [DOI] [PubMed] [Google Scholar]
  25. Prasad T. K., Stewart C. R. cDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock. Plant Mol Biol. 1992 Mar;18(5):873–885. doi: 10.1007/BF00019202. [DOI] [PubMed] [Google Scholar]
  26. Schacterle G. R., Pollack R. L. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973 Feb;51(2):654–655. doi: 10.1016/0003-2697(73)90523-x. [DOI] [PubMed] [Google Scholar]
  27. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsugeki R., Nishimura M. Interaction of homologues of Hsp70 and Cpn60 with ferredoxin-NADP+ reductase upon its import into chloroplasts. FEBS Lett. 1993 Apr 12;320(3):198–202. doi: 10.1016/0014-5793(93)80585-i. [DOI] [PubMed] [Google Scholar]
  29. Viitanen P. V., Schmidt M., Buchner J., Suzuki T., Vierling E., Dickson R., Lorimer G. H., Gatenby A., Soll J. Functional characterization of the higher plant chloroplast chaperonins. J Biol Chem. 1995 Jul 28;270(30):18158–18164. doi: 10.1074/jbc.270.30.18158. [DOI] [PubMed] [Google Scholar]
  30. Yalovsky S., Paulsen H., Michaeli D., Chitnis P. R., Nechushtai R. Involvement of a chloroplast HSP70 heat shock protein in the integration of a protein (light-harvesting complex protein precursor) into the thylakoid membrane. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5616–5619. doi: 10.1073/pnas.89.12.5616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yuan J., Henry R., Cline K. Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8552–8556. doi: 10.1073/pnas.90.18.8552. [DOI] [PMC free article] [PubMed] [Google Scholar]

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