Animal and plant cell lysates share a conserved chaperone system that assembles the glucocorticoid receptor into a functional heterocomplex with hsp90.
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.
Stancato
L F
LF
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, 48109, USA.
Hutchison
K A
KA
Krishna
P
P
Pratt
W B
WB
D000818:Animals; D002460:Cell Line; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*; D017510:Protein Folding; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D013045:Species Specificity; D018411:Spodoptera; D014908:Triticum
D018841:HSP90 Heat-Shock Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D011965:Receptors, Glucocorticoid