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

(A) Refolding of aggregated luciferase by Ssa1-Sis1 +/- 0.1 µM Sse1 (left) or Ssa1-Ydj1 +/- 0.1 µM Sse1 (right). Error bars show SD from three independent repeats. Luciferase activity was measured at indicated time points and normalised to the native activity. (B) Sensor-bound luciferase aggregates incubated with Ssa1-Sis1 +/- 0.1 µM Sse1 or Ssa1-Ydj1 +/- 0.1 µM Sse1, with or without ATP. (C) Binding of Hsc70- DNAJB4 or Hsc70-DNAJA2 with or without Hsp105 to the heat-aggregated luciferase immobilised on the BLI biosensor. (B)-(C) 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 yeast Hsp70 system, followed by the addition of the Hsp104 D484K F508A variant. Dashed lines indicate the beginning of the incubation with the Hsp104 variant. (B) Fluorescence microscopy images of FLUC-EGFP monitored upon addition of Ssa1-Sis1 +/- 0.1 µM Sse1 or Ssa1-Ydj1 +/- 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) Sse1 titration in the refolding of aggregated luciferase by Ssa1-Sis1 (red) or Ssa1-Ydj1 (blue). Activity of luciferase was measured after 1 h and normalised to the native protein. (B) Incubation of Hsc70-DNAJB4 (green) or Hsc70-DNAJA2 (orange) with increasing concentrations of Hsp105 and luciferase aggregates. Luciferase activity was measured after 4 h and normalised to the activity of native protein. (C) Spontaneous folding of non-aggregated luciferase diluted from 5 M GuHCl (grey) alone or with addition of the Hsp70 system comprising Ssa1-Sis1 (red) or Ydj1-Ssa1 (blue) at increasing concentrations of Sse1. Activity of luciferase was measured after 2 h and normalised to the native protein. (D) Binding of Ssa1-Sis1 or Ssa1-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 of Ssa1-Sis1 (red) or Ssa1-Ydj1 (blue) with concentrations of Sse1. (E) Renaturation of heat-aggregated GFP by Ssa1-Sis1 in the presence of Sse1 or Sse1-2 at the indicated concentrations. Right panel shows the plot of GFP activity after 2 h of incubation in the presence of Sse1 (orange) or Sse1-2 (red). 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.

Hsp110 and class B JDP show apparent competition for Hsp70.

(A) Upper panel shows the scheme of the BLI experiment. Binding of Ssa1 in the presence of increasing concentrations of Sse1 to Sis1 immobilised on the BLI sensor through the His6–SUMO tag. Lines are the average of three replicates and the shades designate standard deviation. Dashed lines indicate addition of chaperones to sensor-bound Sis1 and dissociation step. (B) Titration of Sse1 in renaturation of heat-aggregated GFP by Ssa1-Sis1, where Sis1 was used at 0.1 µM (left) or 1 µM (right) concentration. (C) Plot of GFP activity after 3h processed by Ssa1 and simultaneously increasing concentration of Sis1 and Sse1 (left). IC50 of Sse1 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.

Hsp110 impact on Hsp70-dependent disaggregation.

Hsp110 increases Hsp70 binding to the aggregate surface specifically in the presence of class B JDP and improves aggregate remodelling into smaller assemblies but does not aid final protein folding. Hsp110 role at the two former stages significantly stimulates protein disaggregation (dark green shades). By slightly facilitating Hsp70 dissociation from the substrate and JDP, the sub-stoichiometric level of Hsp110 might gradually uncover new Hsp70-binding sites, e. g. buried under the chaperone complex, potentially leading to more abundant and effective Hsp70 recruitment (dashed grey arrows).