Impact of Hsp110 on protein disaggregation by Hsp70 system depends on class of JDP.

(A) Refolding of aggregated luciferase by Ssa1-Sis1 (1 µM Ssa1, 1 µM Sis1) +/- µM Sse1 (0.1 µM) (left) or Ssa1-Ydj1 (1 µM Ssa1, 1 µM Ydj1) +/- Sse1 (0.1 µM) (right). Error bars show SD from three independent repeats. Luciferase activity was measured at indicated time points and normalized to the native activity.

(B) Sensor-bound luciferase aggregates incubated with Ssa1-Sis1 (1 µM Ssa1, 1 µM Sis1) +/- Sse1 (0.1 µM) or Ssa1-Ydj1 (1 µM Ssa1, 1 µM Ydj1) +/- Sse1 (0.1µM), with or without ATP, as indicated.

(C) Binding of Hsc70-DNAJB4 (3 μM Hsc70, 1 μM DNAJB4) or Hsc70-DNAJA2 (3 μM Hsc70, 1 μM DNAJA2) with or without Hsp105 (0.3 μM) to the heat-aggregated luciferase immobilized on the BLI biosensor. (B)-(C) The lines represent the average of three replicates, the shades designate SD and the dashed lines indicate the start of chaperones binding and dissociation steps.

Sse1 promotes modification of aggregates by the Hsp70 system.

(A) Initial incubation of heat-aggregated GFP aggregates with the Hsp70 system, followed by the addition of the Hsp104 D484K F508A variant (0.15 µM). Recovery was initiated by the addition of the mix of indicated chaperones: 1 µM Ssa1, 1 µM Sis1, 1 µM Ydj1 or 0.1 µM Sse1. Dashed lines indicate the beginning of the incubation with the Hsp104 variant. Curves show average values and shades indicate SD from three replicates. (B) Fluorescence microscopy images of luc-GFP monitored upon addition of Ssa1-Sis1 +/- Sse1 or Ssa1-Ydj1 +/- Sse1. Chaperones were used at 1 µM concentration, except for 0.1 µM Sse1. Left panels show controls of the luciferase-GFP aggregates alone and upon the addition of the Hsp70 system without ATP. Quantification of the fraction of aggregates > 2 µm is from three independent replicates. Two-tailed t test was performed: *p < 0.05, ns: not significant.

Susceptibility of Hsp70 to Hsp110 depends on JDP class and phase of disaggregation.

(A) Titration of Sse1 in the refolding of aggregated luciferase by Ssa1-Sis1 (1 µM Ssa1, 1 µM Sis1) (red) or Ssa1-Ydj1 (1 µM Ssa1, 1 µM Ydj1) (blue). Activity of luciferase was measured after 1 h and normalized to that of the native protein. (B) Incubation of Hsc70-DNAJB4 (3 µM Hsc70, 1 µM DNAJB4) (green) or Hsc70-DNAJA2 (3 µM Hsc70, 1 µM DNAJA2) (orange) with luciferase aggregates at increasing concentrations of Hsp105. Luciferase activity was measured after 4 h and normalized to the activity of the native protein. (C) Folding of non-aggregated luciferase diluted from 5 M GuHCl (grey), spontaneous or mediated by the Hsp70 system comprising Ssa1-Sis1 (1 µM Ssa1, 1 µM Sis1) (red) or Ydj1-Ssa1 (1 µM Ssa1, 1 µM Ydj1) (blue) with increasing concentrations of Sse1. Activity of luciferase was measured after 2 h and normalized to the native protein. (D) Binding of Ssa1-Sis1 (1 µM Ssa1, 1 µM Sis1) or Ssa1-Ydj1 Ssa1 (1 µM Ssa1, 1 µM Ydj1) in the presence of Sse1 at the indicated concentrations to the sensor covered with luciferase aggregates. Right panel shows a plot of the binding signal prior to the dissociation step. (E) Renaturation of heat-aggregated GFP by Ssa1-Sis1 (1 µM Ssa1, 1 µM Sis1) in the presence of Sse1 or Sse1-2 at the indicated concentrations. Right panel shows the plot of the recovered GFP activity after 2 h of incubation with the Hsp70 system in the presence of Sse1 (orange) or Sse1-2 (red). (D-E) Dashed lines show the fitting of the [Agonist] vs response model to the data from the stimulation and inhibition phases separately using the GraphPrism Software. (A-E) Error bars and shades indicate SD from 3 repeats.

Hsp110 and class B JDP show apparent competition for Hsp70.

(A) Upper panel shows the scheme of the BLI experiment. Binding of Ssa1 (1µM) in the presence of increasing concentrations of Sse1 to Sis1 immobilized on the BLI sensor through the His6–SUMO tag. Dashed lines indicate the moment of addition of chaperones to the sensor-bound Sis1 and the dissociation step. (B) Renaturation of heat-aggregated GFP by Ssa1-Sis1 (1µM Ssa1, 0.1 µM or 1 µM Sis1) at increasing concentrations of Sse1. (C) Plot of GFP activity after 3 h recovered from aggregates by Ssa1 at different concentrations of Sis1 and Sse1 (left). IC50 was determined by fitting the [Inhibitor] versus response model to the data from three experiments using the GraphPrism Software (dashed lines). Two-tailed t test: *p < 0.05, **p < 0.01. Lines are the average of three replicates and the error bars and shades designate standard deviation.

Impact of Hsp110 on Hsp70-dependent disaggregation.

Hsp110 stimulates aggregate disassembly by Hsp70 with class B JDPs and Hsp104 (green arrow), contrary to the stage of final folding of the solubilized polypeptide (light purple arrow), at which Hsp70 with class A JDP are most effective. Hsp110 improves the disaggregation by increasing Hsp70 binding to the aggregate (upper right panel) and mediates aggregate remodelling into smaller assemblies (middle right panel). By facilitating partial Hsp70 dissociation from the substrate and/or JDP, at the optimal, sub-stoichiometric concentration, Hsp110 might gradually uncover new Hsp70-binding sites (lower right panel, small black arrows), potentially leading to the more abundant and effective Hsp70 recruitment to the aggregate. At higher Hsp110 levels, the destabilisation of Hsp70 interactions with protein substrates and JDPs leads to the inhibition across all stages of protein disaggregation (lower right panel).