1. The Plasticity of the Hsp90 Co-chaperone System
Priyanka Sahasrabudhe, Julia Rohrberg, Maximillian M. Biebl, Daniel A. Rutz, Johannes Buchner 
Molecular Cell 
Volume 67, Issue 6, Pages e5 (September 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 67, 947-961.e5DOI: (10.1016/j.molcel.2017.08.004)
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3. Figure 1 The Influence of Hsp90 Co-chaperones on Client Activity
(A–E) Activity of (A) GR, (B) MR, (C) PR, (D) AR, and (E) ER in WT and co-chaperone deletion or knockdown strains normalized to the WT. Shown are mean and SD of at least three independent experiments with biological triplicates in each.
(F) Quantification of v-Src phosphorylation activity in WT and co-chaperone deletion or knockdown strains. Results obtained from 3 biological replicates were used to calculate the mean and SD.
(G) Matrix of client activity in co-chaperone deletion or knockdown strains. Green, reduced client activity (1.5-fold to 2-fold, light green; 2-fold or more, dark green); blue, increased client activity (1.5-fold to 2-fold, light blue; 2-fold or more, darker blue); gray, WT-like activity.
See also Figure S3.
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4. Figure 2 Hsp90 Client Mutant Variants
(A) Activity of GR-1e and GR-5e in WT and co-chaperone deletion or knockdown strains normalized to the WT. Shown are the mean and SD of at least three independent experiments with biological triplicates in each.
(B) Matrix summarizing the results obtained in the activity assays for GR mutants in comparison with WT-GR. Green, reduced client activity (1.5-fold to 2-fold, light green; 2-fold or more, dark green); blue, increased client activity (1.5-fold to 2-fold, light blue; 2-fold or more, darker blue).
(C and D) Activity of v-Src mutants in WT and co-chaperone deletion or knockdown strains. Shown is a survival assay of c-Src3MΔC (C) and c-Src3M (D) (left) and phosphotyrosine activity in uninduced (−) and induced (+) samples (right). #, unspecific band used as a loading control.
(E) Matrix summarizing the results obtained for the mutants in comparison with v-Src. Green, reduced client activity; gray, WT-like activity. The color scheme for c-Src mutants is qualitative, based on results from spot assays and phosphotyrosine blots.
See also Figure S4.
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5. Figure 3 In Vitro Characterization of MR and GR-LBD
(A) Influence of MR- and GR-LBDs on Hsp90’s ATPase activity. The ATPase activity of Hsp90 was measured alone or in the presence of 6 μM MR- or GR-LBD. Shown are the means and SD of three measurements.
(B) Influence of MR-LBD on the closing reaction of Hsp90. The closing kinetics upon addition of ATPγS in the presence and absence of MR-LBD were measured. Both donor and acceptor fluorophores were on the N-terminal domains of separate protomers of Hsp90. Inset: influence of the GR-LBD on the closing reaction of Hsp90 (see also Lorenz et al., 2014).
(C and D) Nucleotide-induced Hsp90 conformations affect MR- and GR binding. AUC sedimentation velocity experiments of (C) Atto-488 labelled MR-LBD (∗MR-LBD) or (D) GR-LBD (∗GR-LBD) with 6 μM Hsp90 and 400 nM LBDs and 2 mM nucleotides; normalized dc/dt values were plotted against the apparent sedimentation coefficient (S).
(E) Co-chaperone complexes with MR-LBD. AUC sedimentation velocity experiments were carried out using labelled MR-LBD to analyze complex formation of the LBD, Hsp90, and the respective co-chaperones. 6 μM Hsp90 and 3 μM co-chaperones were used.
(F) Co-chaperone complexes with GR-LBD. As in (E), AUC sedimentation velocity experiments were carried out using labelled GR-LBD to analyze complex formation of the LBD, Hsp90, and the respective co-chaperones. 6 μM Hsp90 and 3 μM co-chaperones were used.
(G) The fractions of free ∗MR-LBD or ∗GR-LBD were calculated from the AUC experiments normalized to LBD alone.
See also Figure S5.
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6. Figure 4
Protein Levels
(A) Representative blot showing soluble (S) and insoluble (P) hemagglutinin (HA)-GR levels. Phosphoglycerate kinase (PGK) was used as a loading control and to ascertain the complete separation of soluble and pellet fractions.
(B) Comparison of HA-GR level and activity. The soluble HA-GR band intensities were normalized to PGK, and the mean and SD from three independent experiments are shown.
See also Figure S6.
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7. Figure 5 Co-chaperones Influence the Conformation of GR
(A–G) Following pull-down using anti-HA beads, HA-GR was subjected to limited proteolysis with increasing Proteinase K concentrations: (A) WT-ΔSti1, (B) WT-Δp23, (C) WT-ΔCpr6, (D) WT-sgt∗, (E) WT-ΔCpr7, (F) WT-ΔAha1, and (G) WT-ΔHch1 comparison. A polyclonal rabbit anti-GR N499 antibody was used to detect GR. Left: representative western blots. Right: quantification of full-length GR (≈100 kDa) normalized to the WT. Mean and SD from three independent experiments are shown.
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8. Figure 6
Model of Client Processing by the Hsp90 Co-chaperone Machinery
The client proteins may be delivered to the Hsp90 machinery via connector co-chaperones like Sti1, recruiters like Cdc37, transferred from Hsp70, or directly bound by Hsp90. The co-chaperones p23 and Sgt1 together with Hsp90 (all colored) comprise the basic requirement for the maturation of all client proteins. Because the interaction of Sgt1 in the progression of the Hsp90 conformational cycle has not been mapped, its importance has been highlighted by the circular arrow encompassing the cycle. The co-chaperones indicated in gray show client-specific functions. Of note, it is unknown at which stage of the cycle the indicated conformational changes within the client protein occur.
Molecular Cell  , e5DOI: ( /j.molcel )
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