Animal and Plant Cell Lysates Share a Conserved Chaperone System That Assembles the Glucocorticoid Receptor into a Functional Heterocomplex with hsp90

Abstract

The hormone-binding domain of the glucocorticoid receptor must be bound to heat shock protein (hsp) 90 for it to have a high-affinity steroid-binding conformation. Cell-free assembly of a glucocorticoid receptor-hsp90 heterocomplex is brought about in reticulocyte lysate by a preformed protein-folding complex containing hsp90, hsp70, and other proteins [Hutchison, K.A., Dittmar, K. D., & Pratt, W.B. (1994) J. Biol. Chem. 269, 27894-27899]. In this "foldosome" system, hsp70 is required for assembly of the receptor-hsp90 complex and concomitant activation of steroid-binding activity [Hutchison, K.A., Dittmar, K.D., Czar, M.J., & Pratt, W.B. (1994) J. Biol. Chem. 269, 22157-22161]. All previous experiments involving cell-free assembly of both receptor-hsp90 and protein kinase-hsp90 heterocomplexes have been carried out with the protein-folding system in rabbit reticulocyte lysate. In this work, we show that concentrated lysates of receptor-free mouse (L cells) and insect (Sf9) cells and also a plant (wheat germ) lysate fold the immunopurified glucocorticoid receptor into a functional (i.e., steroid binding) heterocomplex with hsp90. Receptor heterocomplex formation in animal lysates and in the plant lysate are not identical in that the dynamics of complex assembly are different, but both systems produce a functional complex that binds steroid. Also, in contrast to animal and insect complexes, receptor-plant hsp90 complexes are not stabilized by molybdate. When added to the other lysate, purified plant and animal hsp90s show partial complementarity, in that a receptor-hsp90 complex is formed but the receptor is not converted to the steroid-binding conformation. When added to rabbit reticulocyte lysate that has been depleted of endogenous hsp70, purified wheat germ and mouse hsp70's are equally active in promoting both assembly of receptor-hsp90 heterocomplexes and conversion of receptor to the steroid-binding conformation. Thus, hsp70 from the plant kingdom has conserved the ability to interact functionally with chaperone proteins of the animal kingdom to cooperate in protein folding as evidenced by formation of a functional receptor-hsp90 heterocomplex.

Modification, Development, Application and Prospects of Tandem Affinity Purification Method

“…By using a TAP-tagged hybrid transcription factor as bait, HSP70 and HSP60 were co-purified. This result was verified by former reports (Dittmar et al, 1997;Stancato et al, 1996). Through the TAP strategy, Liu et al demonstrated that Hsp90 associated with the plant resistance protein N , which meant that Hsp90 plays an important role in plant defense (Kanzaki et al, 2003;Takahashi et al, 2003).…”

A Small Heat Shock Protein Cooperates with Heat Shock Protein 70 Systems to Reactivate a Heat-Denatured Protein

“…In some cellular processes, such as activation of steroid hormone receptors, Hsp70 is known to act in a large chaperone complex involving Hsp90 and a variety of co-chaperones including Hop (Hsp70-Hsp90 organizing protein) (Frydman and Hö hfeld, 1997; Pratt and Toft, 1997). Hsp90 and Hop are found in plants (Hernandez Torres et al, 1995;Boston et al, 1996), and the same complex of chaperones exists in plants, as evidenced by the substitution of wheat germ lysate for reticulocyte lysate in steroid receptor activation reactions (Stancato et al, 1996). However, addition of human Hsp90 had no effect on the yield or rate of refolding, and supplementation of the system with Hop plus Hsp90 only moderately increased the apparent rate constant of refolding (Table I).…”

Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development

“…The second pathway involves an intracellular steroid-activated receptor complex to directly regulate the transcription of genes by binding to the promoter [45]. In plants, although a chaperon heterocomplex similar to that of intracellular steroid receptor in animal has been identified [46][47][48], neither the candidate gene encoding a putative intracellular receptor of BR has been found in the Arabidopsis genome [49], nor evidence suggested the existence of BR intracellular receptor. However, three BRI1 homologue were identified in Arabidopsis genome [49].…”