Results: 5

1.
Figure 4

Figure 4. Hsp90Ec mutants are defective in client binding. From: Uncovering a region of Hsp90 important for client binding in E. coli and chaperone function in yeast.

(A) The ATPase activity of Hsp90Ec wild type or mutants with or without L2 was measured. Data from 3 replicates are presented as mean ± SEM.
(B) Binding of Hsp90Ec wild type or mutants to IAEDANS-labeled Δ131Δ was measured by fluorescence anisotropy. Binding curves are the average of two independent measurements.
See also Figure S4.

Olivier Genest, et al. Mol Cell. ;49(3):464-473.
2.
Figure 3

Figure 3. The Hsp90Ec mutant proteins exhibit defective chaperone activity in vitro. From: Uncovering a region of Hsp90 important for client binding in E. coli and chaperone function in yeast.

(A) Reactivation of heat-denatured luciferase by Hsp90Ec wild type or mutants in conjunction with DnaK, CbpA and GrpE (K/A/E) was measured over time. The value obtained with K/A/E alone was subtracted. It was previously shown that only the soluble fraction of the denatured luciferase is reactivated by Hsp90Ec and the DnaK system and that this soluble fraction corresponds to about 20% of the total luciferase (Genest et al., 2011). Thus Hsp90Ec wild type in combination with the DnaK system reactivates about 50% of the soluble inactive luciferase in this experiment.
(B) Initial linear rates of luciferase reactivation (red) by Hsp90Ec wild type or mutants in the presence of K/A/E were calculated from Figure 3A and the rate of wild type reactivation set to 100%. The ATPase activity of Hsp90Ec wild type or mutants was measured and is represented in blue.
In (A) and (B), data from three replicates are presented as mean ± SEM.
See also Figure S3.

Olivier Genest, et al. Mol Cell. ;49(3):464-473.
3.
Figure 2

Figure 2. Hsp90Ec mutations in the middle and C-terminal domains block the overproduction phenotypes. From: Uncovering a region of Hsp90 important for client binding in E. coli and chaperone function in yeast.

(A) E. coli MG1655ΔhtpG cells overexpressing plasmid-encoded Hsp90Ec wild type (WT) or the indicated mutants were incubated at 37°C overnight on LB-ampicillin plates containing 0.2% arabinose and 1% SDS.
(B) Strains used in (A) were grown overnight at 37°C in the presence of 0.2% arabinose and analyzed by DIC microscopy. Size bars are 10 μm.
(C) Strains used in (A) were grown overnight at 37°C in the presence of 0.2% arabinose and protein expression determined by Western blot analysis using Hsp90Ec antibody. Detection of EF-TU was used as a loading control.
(D) Model of the Hsp90Ec dimer made from the X-ray structures of Hsp90Ec in the apo form (pdb: 2ioq) and the C-terminal domain of Hsp90Ec (pdb: 1sf8) visualized using PYMOL (www.pymol.org). In one protomer, the N-terminal domain is colored tan, the middle domain is light blue and the C-terminal domain is green. Residues that were mutated are represented as CPK models.
See also Figure S2 and Table S1.

Olivier Genest, et al. Mol Cell. ;49(3):464-473.
4.
Figure 1

Figure 1. Hsp90Ec overproduction leads to decreased cell survival, filamentation and SDS sensitivity in E. coli. From: Uncovering a region of Hsp90 important for client binding in E. coli and chaperone function in yeast.

(A) E. coli MG1655 cells harboring the Hsp90Ec overexpression plasmid (pHsp90Ec) or vector were grown at 37°C in the presence of 0.2% arabinose to induce Hsp90Ec production. The number of viable cells was determined as described in Supplemental Information and plotted as colony forming units (CFU) per mL.
(B) Cells described in (A) were incubated at 37°C in the presence of 0.2% arabinose and growth was followed by measuring OD600.
(C–D) Cells expressing Hsp90Ec from pHsp90Ec (C) or carrying the empty vector
(D) were grown overnight at 37°C in the presence of 0.2% arabinose and visualized by DIC microscopy. Size bars are 10 μm.
(E) Cells expressing Hsp90Ec from pHsp90Ec or carrying the empty vector were grown without arabinose to early stationary phase and aliquots of 10-fold serial dilutions were spotted on LB-ampicillin plates (Amp) containing 0.2% arabinose (Ara) and 1% SDS as indicated. Plates were incubated overnight at 37°C.
In (A) and (B), data from three replicates are presented as mean ± SEM.
See also Figure S1.

Olivier Genest, et al. Mol Cell. ;49(3):464-473.
5.
Figure 5

Figure 5. Several amino acids residues important for Hsp90Ec function are also important for yeast Hsp82 function. From: Uncovering a region of Hsp90 important for client binding in E. coli and chaperone function in yeast.

(A) Alignment of E. coli Hsp90Ec, S. cerevisiae Hsp82 and human Hsp90A showing mutated residues.
(B) Model of the Hsp82 dimer made from the X-ray structure of Hsp82 in the closed conformation (pdb: 2cg9) visualized using PYMOL (www.pymol.org). In one protomer, the N-terminal domain is colored tan, the middle domain is light blue and the C-terminal domain is green. Residues that were mutated are represented as CPK models.
(C) Ability of Hsp82 mutants to support growth was assessed via a plasmid shuffle assay as described in Supplemental Information. S. cerevisiae G612 lacks chromosomal copies of HSC82 and HSP82 and is supported by wild type HSP82 on a URA3 plasmid. Strains with HSP82 alleles on LEU2 plasmids that support viability grow on FOA, which prevents growth of cells carrying the URA3-based HSP82 plasmid. Three individual transformants are shown.
(D) Overnight cultures of strains expressing Hsp82 wild type or mutants that supported growth were diluted to equal density, and aliquots of five-fold serial dilutions were spotted onto YPAD plates and incubated for three days at the indicated temperatures. The cells expressing the Hsp82 defective mutants looked normal by microscopy at the permissive temperature.
(E) Glucocorticoid receptor maturation. Strains expressing Hsp82 wild type or mutants that supported growth were transformed with plasmids encoding GR and a downstream LacZ reporter (Louvion et al., 1996). After growth in the presence of deoxycorticosteroid, β-galactosidase activity was measured. Values are averages of three independent measurements reported as relative to wild type. Error bars indicate standard deviations.
(F) Binding of Hsp82 wild type or mutants to IAEDANS-labeled Δ131Δ was measured by fluorescence anisotropy. Binding curves are the average of two independent measurements.
(G) Reactivation of heat-denatured luciferase by Hsp82 wild type or mutants in conjunction with Hsp70 and Ydj1 (70/40), and Sti1 was measured over time. Data from three replicates are presented as mean ± SEM.
(H) The ATPase activity of Hsp82 wild type or mutants was measured. Data from three replicates are presented as mean ± SEM.
See also Figure S5.

Olivier Genest, et al. Mol Cell. ;49(3):464-473.

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