Radicicol

The C. reinhardtii wild-type strain was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection, Chinese Academy of Science, Wuhan, China). C. reinhardtii cells were cultured in liquid tris-acetate phosphate media (TAP) under 100 μmol (photons) m⁻² s⁻¹ white light with a 12 h photoperiod at 25 °C. Three-day-old cells in the logarithmic growth phase were collected for further experiments [26 (link)].
Fumagillin, mevastatin, radicicol, wortmannin, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), dimethyl sulfoxide (DMSO), and other chemical agents were purchased from Sigma-Aldrich (Shanghai, China). Four mycotoxins and DCMU stock solutions were dissolved in 100% DMSO and further diluted with sterile water. The final concentration of DMSO in the working solutions of all chemical agents was less than 1% (v/v).

ATPase assays were performed by a spectrophotometric assay using an enzymatic ATP regenerating system^{89 (link)}. 10 μM of human Hsp90 and 3 μM of yeast Hsp90 were used in 40 mM Hepes (pH 7.5), 150 mM KCl, 5 mM MgCl₂. Measurements were performed at 30 °C for the yeast proteins and at 37 °C for the human homologues. To substract the background activity, 100 μM radicicol (Sigma, Germany) were added at the end of the measurement. The data were analyzed by linear regression using Origin 8.0 and for ATP-binding, data were fitted according to the Michaelis–Menten equation. For the activation by Aha1, a low salt assay buffer (40 mM Hepes (pH 7.5), 20 mM KCl, 5 mM MgCl₂) was used, as the affinity between Hsp90 and Aha1 is salt sensitive^{57 (link)}.

ATPase activities were measured using a regenerating ATPase assay as described before25 (link). In this coupled enzyme assay, the reduction of NADH is detected by the decrease of absorbance at 340 nm using a Cary 50 Bio UV/VIS spectrophotometer (Varian). The assays were performed in 50 mM Hepes (pH 7.5), 50mM KCl, 5mM MgCl₂ and 2 mM ATP. ATPase activity was measured at 30 °C using 2 μM yHsp90 and 2 μM Sti1 variant in a total volume of 150 μl. The ATPase reaction was started by adding 2 mM ATP. 100 μM Radicicol (Sigma Aldrich) was used to inhibit Hsp90 ATPase. The remaining ATPase activity in the presence of Radicicol was subtracted from the total activity.

The human embryonic kidney cell line 293T (Graham et al., 1977 (link)) was grown in high-glucose DMEM supplemented with 10 % fetal bovine serum (FBS). DNA transfection in 293T cells was performed using Lipofectamine 2000 reagent (no. 11,668,019; Thermo Fisher Scientific) (Dalby et al., 2004 (link)) according to the manufacturer's protocols. Generally, cells were harvested 24 h after transfection to analyze the expression and the protein localization of transfected genes. MG132 (no. C2211; Sigma-Aldrich), chloroquine diphosphate (no. ab142116; Abcam), and radicicol (no. R2146; Sigma-Aldrich) were purchased commercially.

All yeast strains are isogeneic to W303, but contain the additional met2 and lys2 mutations [5] (link), [57] (link) (Table 1). Yeast cells were grown in either yeast extract-peptone-dextrose (YPD- 1% Bacto yeast extract, 2% peptone, and 2% dextrose) or defined synthetic complete media supplemented with 2% dextrose and were transformed by lithium acetate methods. Growth was examined by spotting 10-fold serial dilutions of yeast cultures on appropriate media, followed by incubation for two days at 25°C, 30°C or 37°C. Yeast strains containing deletions or mutation of SSA1-4 and SSE1 genes were generous gifts from Dr. Elizabeth Craig (University of Wisconsin-Madison), and Kevin Morano (University of Texas Medical School at Houston), respectively. Radicicol was obtained from Sigma.