Skip to main content
SpringerLink
Log in

Functional mode of NtHSP17.6, a cytosolic small heat-shock protein fromNicotiana tabacum

  • Published:
Journal of Plant Biology Aims and scope Submit manuscript

Abstract

Small heat-shock proteins (sHsps) are ubiquitous stress proteins with molecular chaperone activity. They share characteristic homology with the α-crystallin protein of the mammalian eye lens as well as being ATP-independent in their chaperone activity. We isolated a clone for a cytosolic class I sHsp,NtHSP17.6, fromNicotiana tabacum, and analyzed its functional mode for such activity. Following its transformation intoEscherichia coli and its over-expression, NtHSPI 7.6 was purified and examinedin vitro. This purified NtHSPI 7.6 exhibited typical chaperone activity in a light-scattering test. It was enable to protect a model substrate, firefly luciferase, from heat-induced aggregation. Non-denaturing PAGE showed that NtHSP17.6 formed a dodecamer in its native conformation, and was bound to its substrate under heat stress. A labeling test with bis-ANS indicated that this binding might be linked to newly exposed hydrophobic sites of the NtHSPI 7.6 complexes during heat shock. Based on these data, we suggest that NtHSP17.6 is a molecular chaperone that functions as a dodecamer in a heat-induced manner.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Literature Cited

  • Abdulle R, Mohindra A, Fernando P, Heikkia JJ (2002)Xenopus small heat shock proteins, Hsp30C and Hsp 30D, maintain heat- and chemically denatured luciferase in a folding competent state. Cell Stress Chaper 7: 6–16

    Article  CAS  Google Scholar 

  • Agashe VR, Hartl F-U (2000) Roles of molecular chaper-ones in cytoplasmic protein folding. Cell Dev Biol 11: 15–25

    Article  CAS  Google Scholar 

  • Anderson LO, Borg H, Mikaelsson M (1972) Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity. FEBS Lett 20: 199–202

    Article  Google Scholar 

  • Basha E, Lee GJ, Breci LA, Hausrath AC, Buan NR, Giese KC, Vierling E (2004a) The identity of proteins associated with a small heat shock protein during heat stressin vivo indicates that these chaperones protect a wide range of cellular functions. J Biol Chem 279: 7566–7575

    Article  PubMed  CAS  Google Scholar 

  • Basha E, Lee GJ, Demeler B, Vierling E (2004b) Chaperone activity of cytosolic small heat shock proteins from wheat. FEBS Lett 271: 1426–1436

    CAS  Google Scholar 

  • Caspers GJ, Leunissen JAM, de Jong WW (1995) The expanding small heat-shock protein family and structure predictions of the conserved “alpha-crystalline domain”. J Mol Evol 40: 238–248

    Article  PubMed  CAS  Google Scholar 

  • Cho EK, Hong CB (2004) Molecular cloning and expression pattern analyses of heat shock protein 70 genes fromNicotiana tabacum. J Plant Biol 47: 149–159

    Article  CAS  Google Scholar 

  • Das KP, Surewicz WK (1995) Temperature-induced exposure of hydrophobic surfaces and its effect on the chap-erone activity of a-crystalline. FEBS Lett 369: 321–325

    Article  PubMed  CAS  Google Scholar 

  • de Jong WW, Caspers GJ, Leunissen JAM (1998) Genealogy of the a-crystallin/small heat-shock protein super-family. Intl J Biol Macromol 22: 151–162

    Article  Google Scholar 

  • de Jong WW, Leunissen JAM, Voorter CEM (1993) Evolution of the α-crystalline/small heat shock protein family. Mol Biol Evol 10: 103–126

    PubMed  Google Scholar 

  • Ehrnsperger M, Graber S, Gaestel M, Buchner J (1997) Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation. EMBO J 16: 221–229

    Article  PubMed  CAS  Google Scholar 

  • Ehrnsperger M, Hergersberg C, Wienhues U, Nichtl A, Buchner J (1998) Stabilization of proteins and peptides in diagnostic immunological assays by the molecular chaperone Hsp25. Anal Biochem 259: 218–225

    Article  PubMed  CAS  Google Scholar 

  • Ehrnsperger M, Lilie H, Gaestel M, Buchner J (1999) The dynamics of Hsp25 quaternary structure. J Biol Chem 274: 14867–14874

    Article  PubMed  CAS  Google Scholar 

  • Ellis RJ (1997) Molecular chaperones: Avoiding the crowd. Curr Biol 7: R531-R533

    Article  PubMed  CAS  Google Scholar 

  • Fink AL (1999) Chaperone-mediated protein folding. Physiol Rev 79: 425–449

    PubMed  CAS  Google Scholar 

  • Fu X, Chang Z (2004) Temperature-dependent subunit exchange and chaperone-like activities of Hsp16.3, a small heat shock protein fromMycobacterium tuberculosis. Biochem Biophys Res Comm 316: 291–299

    Article  PubMed  CAS  Google Scholar 

  • Haslbeck M (2000) sHsps and their role in the chaperone network. Cell Mol Life Sci 59: 1649–1657

    Article  Google Scholar 

  • Haslbeck M, Walke S, Stromer T, Ehrnsperger M, White HE, Chen S, Saibil HR, Buchner J (1999) Hsp 26: A temperature-regulated chaperone. EMBO J 18: 6744–6751

    Article  PubMed  CAS  Google Scholar 

  • Horwich AL, Weissman JL (1997) Deadly conformations-protein misfolding in prion disease. Cell 74: 909–917

    Article  Google Scholar 

  • Horwitz J (1992) Alpha-crystalline can function as a molecular chaperone. Proc Natl Acad Sci USA 89: 10449–10453

    Article  PubMed  CAS  Google Scholar 

  • Jaenicke R (1995) Folding and association versus misfolding and aggregation of proteins. Phil Trans R Soc Lond B Biol Sci 348: 97–105

    Article  CAS  Google Scholar 

  • Jakob U, Gaestel M, Engel K, Buchner J (1993) Small heat shock proteins are molecular chaperones. J Biol Chem 277: 38468–38475

    Google Scholar 

  • Jinn TL, Yeh YC, Lin CY (1989) Stabilization of soluble proteinsin vitro by heat shock protein-enriched ammonium sulfate fraction from soybean seedlings. Plant Cell Physiol 30: 463–469

    CAS  Google Scholar 

  • Joe MK, Park SM, Lee YS, Hwang DS, Hong CB (2000) High temperature stress resistance ofEscherichia coli induced by a tobacco class I low molecular weight heat-shock protein. Mol Cells 5: 519–524

    Article  Google Scholar 

  • Kim KK, Kim R, Kim SH (1998) Crystal structure of a small heat shock protein. Nature 394: 595–599

    Article  PubMed  CAS  Google Scholar 

  • Kim KR, Joe MK, Hong CB (2004) Tobacco small heat-shock protein, NtHSP18.2, has broad substrate range as a molecular chaperone. Plant Sci 167: 1017–1025

    Article  CAS  Google Scholar 

  • Lee GJ, Vierling E (2000) A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein. Plant Physiol 122: 189–198

    Article  PubMed  CAS  Google Scholar 

  • Lee GR, Roseman HR, Vierling E (1997) A small heat shock protein stably binds heat denatured model substrates and can maintain a substrate in a folding-competent state. EMBO J 16: 659–671

    Article  PubMed  CAS  Google Scholar 

  • Levy EJ, McCarty J, Bukau B, Chirico WJ (1995) Conserved ATPase and luciferase refolding activities between bacteria and yeast Hsp70 chaperones and modulators. FEBS Lett 368: 435–440

    Article  PubMed  CAS  Google Scholar 

  • Mogk A, Deuerling E, Vorderwillbecke S, Vierling E, Bernd B (2003) Small heat shock proteins, CIpB and the DnaK system, form a functional triad in reversing protein aggregation. Mol Microbiol 50: 585–595

    Article  PubMed  CAS  Google Scholar 

  • Mogk A, Tomoyasu T, Goloubinoff P, Rüdiger S, Röder D, Langen H, Bukau B (1999) Identification of thermo-labileEscherichia coli proteins: Prevention ind reversion of aggregation by DnaK and CIpB. EMBO J 18: 6934–6949

    Article  PubMed  CAS  Google Scholar 

  • Narberhaus F (2002) a-Crystalline-type heat shock proteins: Socializing minichaperones in the context of a multichaperone network. NMBR 66: 64–93

    CAS  Google Scholar 

  • Nover L (1990) Heat Shock Response. CRC Press,

  • Boca Raton Park SM (2002) Structural and functional diversity of small heat shock proteins inNicotiana tabacum. Ph.D. thesis, Seoul National University, Seoul

  • Park SM, Hong CB (2002) Class I small heat-shcck protein gives thermotolerance in tobacco. J Plant Physiol 159: 25–30

    Article  CAS  Google Scholar 

  • Radford SE (2000) Protein folding: Progress made and promises ahead. Trends Biochem Sci 25: 611–618

    Article  PubMed  CAS  Google Scholar 

  • Plesofsky-Vig N, Brambl R (1995) Disruption o the gene for hsp30, an a-crystalline-related heat shock protein ofNeurospora crassa, causes defects in thermotolerance. Proc Natl Acad Sci USA 92: 537–545

    Article  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning; A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Sanger K, Nicklen S, Coulson AR (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74: 5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Scharf KD, Siddique M, Vierling E (2001) Evolution, structure and function of the small heat shock proteins in plants. J Exp Bot 47: 325–338

    Google Scholar 

  • Schlieker C, Bukau B, Mogk A (2002) Prevention and reversion of protein aggregation by molecular chaperones in theE. coli cytosol: Implications for their applicability in biotechnology. J Biotech 96: 13–21

    Article  CAS  Google Scholar 

  • Schumacher RJ, Hurst R, Sullivan WP, McMahon NJ, Toft DO, Matts RL (1994) ATP dependent chaperoning activity of reticulocyte lysate. J Biol Chem 269: 9493–9499

    PubMed  CAS  Google Scholar 

  • Sharma KK, Kaur H, Kumar GS, Kester K (1998) Interaction of 1,1′-bis (4 anilino)naphthalene-5,5′-disulfonic acid with a-crystalline. J Biol Chem 273: 8965–8970

    Article  PubMed  CAS  Google Scholar 

  • Smykal P, Hrdy I, Pechan PM (2000) High-molecular-mass complexes formedin vivo contain small Hsps and Hsp70 and display chaperone-like activity. Eur J Biochem 267: 2195–2207

    Article  PubMed  CAS  Google Scholar 

  • Stromer T, Ehrnsperger M, Gaestel M, Buchner J (2003) Analysis of the interactions of small heat shock proteins with unfolding proteins. J Biol Chem 278: 18015–18021

    Article  PubMed  CAS  Google Scholar 

  • van Montfort R, Slingsby C, Vierling E (2002) Structure and function of the small heat shock protein/α-crystallin family of molecular chaperones.,In AL Horwich, ed, Protein Folding in the Cell. Academic Press, New York, pp 105–156

    Google Scholar 

  • Veinger L, Diamant S, Buchner J, Goloubinoff P (1998) The small heat shock protein IbpB fromEscherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multi-chaperone network. J Biol Chem 273: 11032–11037

    Article  PubMed  CAS  Google Scholar 

  • Vierling E (1991) The role of heat shock-proteins in plant. Annu Rev Plant Physiol Plant Mol Bol 42: 579–620

    Article  CAS  Google Scholar 

  • Wang K, Spector A (2000) a-Crystalline prevents irreversible protein denaturation and acts cooperatively with other heat-shock proteins to renature and stabilize partially denatured protein in an ATP-dependent manner. Eur J Biochem 267: 4705–4712

    Article  PubMed  CAS  Google Scholar 

  • Waters ER, Lee G, Vierling E (1996) Evolution, structure and function of the small heat shock proteins in plants. J Exp Biol 47: 325–338

    CAS  Google Scholar 

  • Waters ER, Vierling E (1999) The diversification of plant cytosolic small heat shock proteins preceded the divergence of mosses. Mol Biol Evol 16: 127–139

    PubMed  CAS  Google Scholar 

  • Yu JH (2004) Functional analyses of cytosolic small heat shock protein and mitochondrial small heat shock protein inNicotiana tabacum. M.S. thesis, Seoul National University, Seoul

Download references

Author information

Authors and Affiliations

  1. Institute of Molecular Biology and Genetics, Seoul National University, 151-742, Seoul, Korea

    Hae-jeong Yoon, Keun Pill Kim, Soo Min Park & Choo Bong Hong

Authors
  1. Hae-jeong Yoon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Keun Pill Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Soo Min Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Choo Bong Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Choo Bong Hong.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yoon, Hj., Kim, K.P., Park, S.M. et al. Functional mode of NtHSP17.6, a cytosolic small heat-shock protein fromNicotiana tabacum . J. Plant Biol. 48, 120–127 (2005). https://doi.org/10.1007/BF03030571

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03030571

Keywords

Search

Navigation