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Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions

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Abstract

Previously, it was found that the dnaK1 gene of the halotolerant cyanobacterium Aphanothece halophytica encodes a polypeptide of 721 amino acids which has a long C-terminal region rich in acidic amino acid residues. To understand whether the A. halophytica DnaK1 possesses chaperone activity at high salinity and to clarify the role of the extra C-terminal amino acids, a comparative study examined three kinds of DnaK molecules for ATPase activity as well as the refolding activity of other urea-denatured proteins under various salinity conditions. DnaK1s from A. halophytica and Synechococcus sp. PCC 7942 and the C-terminal deleted A. halophytica DnaK1 were expressed in Escherichia coli and purified. The ATPase activity of A. halophytica DnaK1 was very high even at high salinity (1.0 M NaCl or KCl), whereas this activity in Synechococcus PCC 7942 DnaK1 decreased with increasing concentrations of NaCl or KCl. The salt dependence on the refolding activity of urea-denatured lactate dehydrogenase by DnaK1s was similar to that of ATPase activity of the respective DnaK1s. The deletion of the C-terminal amino acids of A. halophytica DnaK1 had no effect on the ATPase activity, but caused a significant decrease in the refolding activity of other denatured proteins. These facts indicate that the extra C-terminal region of A. halophytica DnaK1 plays an important role in the refolding of other urea-denatured proteins at high salinity. Furthermore, it was shown that DnaK1 could assist the copper binding of precursor apo-plastocyanin as well as that of mature apo-plastocyanin during the folding of these copper proteins.

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References

  • Badcoe, I.G., Smith, C.J., Halsall, D.J., Holbrook, J.J., Lund, P. and Clark, A.R. 1991. Binding of a chaperonin to the folding intermedidates of lactose dehydrogenase. Biochemistry 30: 9195-9200.

    Google Scholar 

  • Bardwell, J.C.A. and Craig, E.A. 1984. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc. Natl. Acad. Sci. USA 81: 848-852.

    Google Scholar 

  • Bhagwat, A.A. and Apte, S.K. 1989. Comparative analysis of proteins induced by heat shock, salinity, and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31. J. Bact. 171: 5187-5189.

    Google Scholar 

  • Boston, R.S., Vitanen, P.V. and Vierling, E. 1996. Molecular chaperones and protein folding in plants. Plant Mol. Biol. 32: 191-222.

    Google Scholar 

  • Bukau, B. and Horwich, A.L. 1998. The Hsp70 and Hsp60 chaperone machines. Cell 92: 351-366.

    Google Scholar 

  • Chitnis, P.R. and Nelson, N. 1991. Molecular cloning of the genes encoding two chaperone proteins of the cyanobacterium Synechocystis sp. PCC 6803. J. Biol. Chem. 266: 58-65.

    Google Scholar 

  • de Boer, A.D. and Weisbeek, P.J. 1991. Chloroplast protein topogenesis: import, sorting and assembly. Biochim. Biophys. Acta. 1071: 221-253.

    Google Scholar 

  • Flaherty, K.M., Deluca-Flaherty, C. and McKay, D.B. 1990. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346: 623-628.

    Google Scholar 

  • Geladopoulos, T.P., Sotiroudis, T.G. and Evangelopoulos, A.E. 1991. A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem. 192: 112-116.

    Google Scholar 

  • Gupta, R.S. and Golding, G.B. 1993. Evolution of Hsp70 gene and its implications regarding relationships between archaebacteria, eubacteria, and eukaryotes. J. Mol. Evol. 37: 573-582.

    Google Scholar 

  • Harrison, C.J., Hayer-Hartl, M., Di Liberto, M., Hartl, F.U. and Kuriyan, J. 1997. Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science 276: 431-435.

    Google Scholar 

  • Hartl, F.U. 1996. Molecular chaperones in cellular protein folding. Nature 381: 571-580.

    Google Scholar 

  • Hibino, T., de Boer, A.D., Weisbeek, P.J. and Takabe, T. 1991. Reconstitution of mature plastocyanin from precursor apoplastocyanin expressed in Escherichia coli. Biochim. Biochys Acta 1058: 107-112.

    Google Scholar 

  • Hibino, T., Lee, B.H. and Takabe, T. 1994. Role of transit peptide seqence of plastocyanin for its expression, processing, and copper-binding activity in Escherichia coli. J. Biochem. 116: 826-832.

    Google Scholar 

  • Hibino, T., Lee, B.H., Takabe, T. and Takabe, T. 1995. Expression and characterization of Met92Gln mutant plastocyanin from Silene pratensis. J. Biochem. 117: 101-106.

    Google Scholar 

  • Iwasak, Y., Ishikawa, H., Hibino, T. and Takabe, T. 1991. Characterization of genes that encode subunits of cucumber PSI complex by N-terminal sequencing. Biochim. Biophys. Acta 1059: 141-148.

    Google Scholar 

  • Kaneko, T., Tanaka, A., Sato, S., Kotani, H., Sazuka, T., Miyajima, N., Sugiura, M. and Tabata, S. 1995. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. I. Sequence features in the 1 Mb region from map positions of 64% to 92% of the genome. DNA Res. 2: 153-166.

    Google Scholar 

  • Lee, B.H., Hibino, T., Jo, J., Viale, A.M. and Takabe, T. 1997. Isolation and characterization of dnaK genomic locus in a halotolerant cyanobacterium Aphanothece halophytica. Plant Mol. Biol. 35: 763-775.

    Google Scholar 

  • McMillin, D.R., Rosenberg, R.C. and Gray, H.B. 1974. Preparation and spectroscopic studies of cobalt(II) derivatives of blue copper proteins. Proc. Natl. Acad. Sci. USA 71: 4760-4762.

    Google Scholar 

  • Nimura, K., Yoshikawa, H. and Takahashi, H. 1994a. Identification of dnaK multigene family in Synechococcus sp. PCC 7942. Biochem. Biophys. Res. Commun. 201: 466-471.

    Google Scholar 

  • Nimura, K., Yoshikawa, H. and Takahashi, H. 1994b. Sequence analysis of the third dnaK homolog gene in Synechococcus sp. PCC 7942.Biochem. Biophys. Res. Commun. 201: 848-854.

    Google Scholar 

  • Nimura, K., Yoshikawa, H. and Takahashi, H. 1996. DnaK3, one of the three DnaK proteins of cyanobacterium Synechococcus sp. PCC7942, is quantitatively detected in the thylakoid membrane. Biochem. Biophys. Res. Commun. 229: 334-340.

    Google Scholar 

  • Pierpaoli, E.V., Sandmeier, E., Baici, A., Schonfeld, H.J., Gisler, S. and Christen, P. 1997. The power stroke of the DnaK/DnaJ/GrpE molecular chaperone system.J. Mol. Biol. 269: 757-768.

    Google Scholar 

  • Rudiger, S., Buchberger, A. and Bukau, B. 1997. Interaction of Hsp70 chaperones with substrates. Nature Struct. Biol. 4: 342-349.

    Google Scholar 

  • Slater, M.R. and Craig, E.A. 1989. The SSA1 and SSA2 genes of the yeast Saccharomyces cerevisiae. Nucl. Acids Res. 17: 805-806.

    Google Scholar 

  • Webb, R. and Sherman, L. 1996. The cyanobacterial heat-shock response and the molecular chaperones. In: D.A. Bryant (Ed.), The Molecular Biology of Cyanobacteria, Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 751-767.

    Google Scholar 

  • Wetzstein, M., Volker, U., Dedio, J., Lobau, S., Zuber, U., Schiesswohl, M.S., Herget, C., Hecker, M. and Schumann, W. 1992. Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J Bact. 174: 3300-3310.

    Google Scholar 

  • Wilbanks, S.M. and McKay, D.B. 1995. How potassium affects the activity of the molecular chaperone Hsc70. II. Potassium binds specifically in the ATPase active site. J. Biol. Chem. 270: 2251-2257.

    Google Scholar 

  • Zhu, X., Zhao, X., Burkholder, W.F., Gragerov, A., Ogata, C.M., Gottesman, M. and Hendrickson, W.A. 1996. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272: 1606-1614.

    Google Scholar 

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Hibino, T., Kaku, N., Yoshikawa, H. et al. Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding under various salinity conditions. Plant Mol Biol 40, 409–418 (1999). https://doi.org/10.1023/A:1006273124726

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