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1.
Fig. 6.

Fig. 6. From: Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Schematic model illustrating the different roles of Hsp70 in Hsp104-mediated protein disaggregation. Hsp70 is required to activate the Hsp104 motor to extract polypeptides from aggregates (Upper) and often binds or colocalizes with aggregates together with Hsp40 (Lower).

Jungsoon Lee, et al. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8513-8518.
2.
Fig. 4.

Fig. 4. From: Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Hsp104MI is activated by Strep-Tactin and recovers functional protein in the absence of Hsp70/40. (A–C) Strep-Tactin–dependent protein disaggregation by Hsp104 Strep-tag variants. Strep-Tactin (tetramer; 4 × 15 kDa) and BSA (monomer; 67 kDa) were added in n-fold molar excess of Hsp104 monomer. Recovered enzymatic activities are expressed relative to the wild-type bichaperone system. Averages of three independent measurements ± SD are shown. (D) Cartoon illustrating the proposed model by which Strep-Tactin unlocks the protein disaggregating activity of Hsp104MI. In this model, Hsp104 binds aggregated proteins in an ATP-dependent manner, but requires Strep-Tactin to initiate protein disaggregation. The unfolding and translocation of substrates requires ATP hydrolysis.

Jungsoon Lee, et al. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8513-8518.
3.
Fig. 3.

Fig. 3. From: Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Functional analysis of engineered Hsp104 Strep-tag variants. (A) Schematic diagram of Hsp104MI, Hsp104MR1, Hsp104MR2, and Hsp104MR3 showing the location of the inserted Strep-tag (boxed) with the Strep-Tactin binding motif in bold. Residues that were replaced are shown in gray. (B) Induced thermotolerance assay. Δhsp104 yeast, expressing the indicated wild-type or mutant protein, were heat-shocked at 50 °C for 20 min as described in SI Text. Shown are 10-fold serial dilutions. (C) ATPase activities of Hsp104 wild-type and Strep-tag variants without and with 4× Strep-Tactin (T) or κ-casein (C). (D and E) Coupled chaperone activities of Hsp104 wild-type and variants in the presence of Hsp70/40. Averages of three independent measurements ± SD are shown.

Jungsoon Lee, et al. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8513-8518.
4.
Fig. 2.

Fig. 2. From: Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Hsp70 binds to the M-domain via its nucleotide-binding domain. (A) Domain structure of Hsp70 (52). The ATP-binding domain (NBD) is colored orange, the substrate-binding pocket (SBDΔC) purple, and the C-terminal domain (CTD) gray. The N and C termini are indicated. (B) Bead-binding assay using immobilized Hsp104 hexamer to capture full-length Hsp70, Hsp70ΔC, Hsp70NBD, Hsp70SBDΔC, or Hsp40. Input, final wash, and eluate were analyzed by Western blotting. The eluate from a negative control using beads only (no Hsp104) is also shown. (C) The M-domain consists of four α-helices (H1–H4), which make up motif 1 (cyan) and motif 2 (gold). The locations of the introduced cysteine mutations are shown as purple spheres. (D) Photo-activated cross-linking of BPIA-labeled Hsp104* variants and Hsp70ΔC (ΔC), Hsp70NBD (N), and Hsp70SBDΔC (S) in the presence of ADP. Arrows indicate the cross-linked products with Hsp70ΔC (red) and Hsp70NBD (black).

Jungsoon Lee, et al. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8513-8518.
5.
Fig. 5.

Fig. 5. From: Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Hsp70 is a potent activator of the Hsp104 motor. (AD) Hsp70 (monomer) or Strep-Tactin (tetramer) was added in n-fold excess over Hsp104 monomer. Averages of three independent measurements ± SD are shown. (A) Hsp70 activates the Hsp104 protein disaggregating activity in a concentration-dependent manner. Recovered enzymatic activities of heat-aggregated β-gal are expressed relative to the complete Hsp104/Hsp70/40 system. (B) Initial rate of β-gal recovery by mixtures of Hsp104 wild-type and Trap mutant at the indicated ratios without and with Hsp70/40. (Inset) An enlarged view of the innate protein remodeling activity of Hsp104. (C) Relative recovery rate of β-gal by Hsp104/Trap and their respective Hsp104MI variants with Hsp70/40, 10× Hsp70, or 4× Strep-Tactin. The dashed gray line represents the linear decrease expected if the activity of the Hsp104 hexamer is proportional to the number of wild-type subunits present. (D) Relative ATPase activities of Hsp104MI/TrapMI without and with Hsp70/40, 10× Hsp70, or 4× Strep-Tactin.

Jungsoon Lee, et al. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8513-8518.
6.
Fig. 1.

Fig. 1. From: Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Identifying the Hsp70:Hsp104 binding sites using peptide array technology. (A) 3D structure of Hsp104 based on the cryoEM fit (6). The NTD is colored yellow, the D1-domain red, the M-domain green, and the D2-domain blue. The symbol indicates the sixfold symmetry axis. (B) Hsp104 peptide array probed with His6-Hsp70ΔC and (C) with Hsp70ΔC (without His6-tag), which functions as a negative control. Overlapping peptides containing at least two consecutive spots are boxed and colored according to domain location. Peptides that function as either positive or negative internal binding controls, including His6- and poly-alanine–containing peptides, are highlighted in gray. (D) Sequence of Hsp70ΔC-binding peptides. Consensus motifs are boxed in gray. (E) Binding of His6-Hsp70ΔC to mutated Hsp70-binding motifs in the Hsp104 M-domain. Mutated residues are shown in red. (F) FFL activity recovered by Hsp104 mutants together with Hsp70/40. Averages of three independent measurements ± SD are shown.

Jungsoon Lee, et al. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8513-8518.

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