Abstract
A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates. These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells.
Similar content being viewed by others
References
Anderson L-O, Borg H, Mikaelsson M: Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity. FEBS Lett 20: 199–202 (1972).
Atkinson BG, Raizada M, Bouchard RA, Frappier JRH, Walden DB: The independent stage-specific expression of the 18-kDa heat shock protein genes during microsporogenesis in Zea mays L. Devel Genet 14: 15–26 (1993).
Baszczynski CL, Walden DB, Atkinson BG: Regulation of gene expression in corn (Zea mays L.) by heat shock. Can J Biochem 60: 569–579 (1982).
Brugge JS: Interaction of the Rous sarcoma virus protein pp60src with the cellular proteins pp50 and pp90. Curr Top Microbiol Immunol 123: 1–22 (1986).
Chen Q, Osteryoung K, Vierling E: A 21-kDa chloroplast heat shock protein assembles into high molecular weight complexes in vivo and in organelle. J Biol Chem 269: 13216–13223 (1994).
Conner TW, Lafayette PR, Nagao RT, Key JL: Sequence and expression of a HSP83 from Arabidopsis thaliana. Plant Physiol 94: 1689–1695 (1990).
Dieckmann CL, Tzagoloff A: Assembly of the mitochondrial membrane system CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem 260: 1513–1520 (1985).
Felsheim RF, Das A: Structure and expression of a heat-shock protein 83 gene of Pharbitis nil. Plant Physiol 100: 1764–1771 (1992).
Hendrick JP, Hartl F-U: Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem 62: 349–384 (1993).
Jackob U, Meyer I, Bugl H, Andre S, Bardwell JCA, Buchner J: Structural organization of procaryotic and eucaryotic hsp90: influence of divalent cations on structure and function. J Biol Chem 270: 14412–14419 (1995).
Koning AJ, Rose R, Comai L: Developmental expression of tomato heat-shock cognate protein 80. Plant Physiol 100: 801–811 (1992).
Krishna P, Sacco M, Cherutti JF, Hill S: Cold-induced accumulation of hsp90 transcripts in Brassica napus. Plant Physiol 107: 915–923 (1995).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Li J, Nagpal P, Vitart V, McMorris TC, Chory J: A role for brassinosteroids in light-dependent development of Arabidopsis. Science 272: 398–401 (1996).
Luan S, Albers MW, Schreiber SL: Light-regulated, tissue-specific immunophilins in a higher plant. Proc Natl Acad Sci USA 91: 984–988 (1994).
Marrs KAE, Casey ES, Capitant SA, Bouchard RA, Dietrich PS, Mettler IJ, Sinibaldi RM: Characterization of two maize hsp90 heat shock protein genes and expression during heat shock, embryogenesis, and pollen development. Devel Genet 14: 27–41 (1993).
Minami Y, Kawasaki H, Miyata Y, Suzuki K, Yahara I: Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90. J Biol Chem 266: 10099–10103 (1991).
Minami Y, Kawasaki H, Suzuki K, Yahara I: The calmodulin-binding domain of the mouse 90-kDa heat shock protein. J Biol Chem 268: 9604–9610 (1993).
Miyata Y, Yahara I: Cytoplasmic 8 S glucocorticoid receptor binds to actin filaments through the 90-kDa heat shock protein moiety. J Biol Chem 266: 8779–8793 (1991).
Miyata Y, Yahara I: The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity. J Biol Chem 267: 7042–7047 (1992).
Parsell DA, Lindquist S: The functions of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet 27: 437–496 (1993).
Perdew GH, Whitelaw ML: Evidence that the 90-kDa heat shock protein (hsp90) exists in cytosol in heteromeric complexes containing hsp70 and three other proteins with Mr of 63,000, 56,000 and 50,000. J Biol Chem 266: 6708–6713 (1991).
Picard D, Khursheed B, Garabedian MJ, Fortin MG, Lindquist S, Yamamoto KR: Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature 348: 166–168 (1990).
Pratt WB: The role of heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor. J Biol Chem 268: 21455–21458 (1993).
Radanyi C, Lombes M, Renoir J-M, Delahaye F, Baulieu E-E: A novel monoclonal anti-rabbit hsp90 antibody: usefulness for studies on hsp90-steroid receptor interaction. J Steroid Biochem Mol Biol 42: 863–874 (1992).
Redmond T, Sanchez ER, Bresnick EH, Schlesinger MJ, Toft DO, Pratt WB, Welsh MJ: Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells. Eur J Cell Biol 50: 66–75 (1989).
Riehl RM, Sullivan WP, Vroman BT, Bauer VJ, Pearson GR, Toft DO: Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species. Biochemistry 24: 6586–6591 (1985).
Rose DW, Welch WJ, Kramer G, Hardesty B: Possible involvement of the 90-kDa heat shock protein in the regulation of protein synthesis. J Biol Chem 264: 6239–6244 (1989).
Sanchez ER, Toft DO, Schlesinger MJ, Pratt WB: Evidence that the 90-kDa phosphoprotein associated with the untransformed L-cell glucocorticoid receptor is a murine heat shock protein. J Biol Chem 260: 12398–12401 (1985).
Stancato LF, Hutchison KA, Krishna P, Pratt WB: Animal and plant-cell lysates share a conserved chaperone system that assembles the glucocorticoid receptor into a functional hererocomplex with hsp90. Biochemistry 35: 554–561 (1996).
Sullivan WP, Vroman BT, Bauer VJ, Puri RK, Riehl RM, Pearson GR, Toft DO: Isolation of steroid receptor binding protein from chicken oviduct and production of monoclonal antibodies. Biochemistry 24: 4214–4222 (1985).
Yabe N, Takahashi T, Komeda Y: Analysis of tissue-specific expression of Arabidopsis thaliana hsp90-family gene HSP81. Plant Cell Physiol 35: 1207–1219 (1994).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Krishna, P., Reddy, R.K., Sacco, M. et al. Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts. Plant Mol Biol 33, 457–466 (1997). https://doi.org/10.1023/A:1005709308096
Issue Date:
DOI: https://doi.org/10.1023/A:1005709308096