Identification of Hsp90 interactors in Arabidopsis

In all eukaryotes, the 90-kDa cytosol heat shock protein (Hsp90) is essential, abundant at normal temperatures, and induced by stress. Although a critical dependence on Hsp90 has been established for many animal signaling transduction proteins, only disease resistance (R) proteins have been identified as hsp90 client proteins in plants so far.;To identify novel interactors of plant Hsp90, the C-terminal domain and a part of middle region of Brassica napus Hsp90 was used as bait to screen a B. napus pistil cDNA library in a yeast two-hybrid (Y2H) screening. Eight potential novel interactors of plant Hsp90 were identified in this screen and their interaction with full length B. napus Hsp90 was verified by the Y2H assay. An ethylene receptor ERS2 and two tetratricopeptide repeat (TPR) domain containing proteins are among them. Further study revealed that two members of the ethylene receptor family ERS2 and EIN4, and the downstream Raf-like Ser/Thr kinase CTR1 interacted with Hsp90 in the Y2H assay. Results indicated that Hsp90 interacted with the histidine kinase domain of ERS2. The interactions of Hsp90 with ERS2, EIN4 and CTR1 were inhibited by radicicol (RA), a specific inhibitor of Hsp90 in in vitro binding assays. The interaction between ERS2 or EIN4 and hsp90 was more stable in the presence of ADP, whereas the interaction of CTRL with Hsp90 was more stable in the presence of ATP in vitro assay. Data also confirmed association of Hsp90 with ERS2, EIN4 and CTR1 in Arabidopsis thaliana by TAP tagged pull-down assay, respectively. Those results and ethylene hypersensitivity of Hsp90 mutants (lar2-3) to ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) suggest that Hsp90 plays a role in the ethylene signaling pathway.;The newly identified TPR-containing proteins, TPR1 and TPR2, appear to be novel co-chaperones of Hsp90. TPR1 was constitutively expressed in different tissues and localized only in the cytoplasm, on the other hand, TPR2 expression was found to be stress-induced. Both Y2H assay and pull-down assays showed that TPR1 or TPR2 directly interact with Hsp90. Both proteins could enhance the ATPase activity of Hsp90 in vitro, but only TPR1 could facilitate glucocorticoid receptor (GR) maturation in yeast. Taken together, these data suggest that TPR1 and TPR2 are novel co-chaperones of Hsp90 in plants.;Key words. Yeast two-hybrid, Hsp90, Co-chaperone, Ethylene, ERS2, EIN4, CTRL, TPR1, TPR2, ATPase.