(A, B) Urea-denatured luciferase aggregates (50 nM) (A, C) or heat-denatured GFP aggregates (0.45 µM) (B, D) were incubated for 30 min (blue bars), 1 h (green bars), 4 h (black bars), or 6 h (red bars) at 25°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Hsp104 (1 µM), Ssa1 (1 µM), Sis1 (1 µM), RLC (10 mg/ml) (A, B), or SHC (10 mg/ml) (C, D). In the indicated reactions, ATP was omitted or replaced with AMP-PNP. Alternatively, ATP was included but the ATP-regeneration system was replaced with apyrase. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (A, C). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (B, D). Values represent means ± SEM (n = 3).

(A, B) Immunoblots showing depletion of either p97, Hsp70, Hsc70, Hdj1, Hdj2, Hsp105 or Apg-2 from either RLC (A) or SHC (B) as described in Materials and Methods. 20 µg of cytosol was fractionated by SDS-PAGE and processed for immunoblot. (C–F) Urea-denatured luciferase aggregates (50 nM) (C, D) or heat-denatured GFP aggregates (0.45 µM) (E, F) were incubated for 6 h in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated cytosol (10 mg/ml), mock-depleted cytosol (10 mg/ml) or depleted cytosol (10 mg/ml). In the indicated reactions, the small molecule p97 inhibitor, DBeQ (100 µM), or Hsp70 inhibitor, VER 155008 (100 µM), were included. In the indicated reactions, Hsc70-depleted cytosol was supplemented with Hsc70, Hdj1-depleted cytosol was supplemented with Hdj1 and Apg-2-depleted cytosol was supplemented with Apg-2 as described in Materials and Methods. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (C, D). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (E, F). Values represent means ± SEM (n = 3).

(A, B) Urea-denatured luciferase aggregates (50 nM) (A) or heat-denatured GFP aggregates (0.45 µM) (B) were incubated for 30 min (blue bars) or 6 h (red bars) at 37°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Hsp104 (1 µM), Ssa1 (1 µM), Ydj1 (1 µM), RLC (10 mg/ml), SHC (10 mg/ml), Hsc70 (1 µM), Hdj1 (1 µM), Hdj2 (1 µM), Hsp70 (1 µM) and Apg-2 (1 µM). In the indicated reactions, ATP was omitted or replaced with AMP-PNP. Alternatively, ATP was included but the ATP-regeneration system was replaced with apyrase. The small molecule inhibitor VER 155008 (100 µM) was included in the indicated reactions. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (A). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (B). Values represent means ± SEM (n = 3).

(A, B) Urea-denatured luciferase aggregates (50 nM) (A) or heat-denatured GFP aggregates (0.45 µM) (B) were incubated for 30 min (blue bars), 1 h (green bars), 4 h (black bars), or 6 h (red bars) at 25°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Hsp104 (1 µM), Ssa1 (1 µM), Sis1 (1 µM) and Sse1 (1 µM). In the indicated reactions, ATP was either omitted or replaced with AMP-PNP. Alternatively, ATP was included but the ATP-regeneration system was replaced with apyrase. Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (A). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (B). Values represent means ± SEM (n = 3). (C, D) Urea-denatured luciferase aggregates (50 nM) (C) or heat-denatured GFP aggregates (0.45 µM) (D) were incubated for 30 min (blue bars), 1 h (green bars), 4 h (black bars), or 6 h (red bars) at 25°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Ssa1 (1 µM), Sis1 (1 µM), Sse1 (1 µM), Sse1^N572Y:E575A (1 µM, a NEF-defective Sse1 mutant), Ssa1^A300E (1 µM, an Ssa1 mutant unable to interact with Sse1), Fes1 (1 µM, a Ssa1 NEF), Snl1ΔN (1 µM, a Ssa1 NEF), Sse1^K69M (1 µM, an ATPase-dead Sse1 mutant), Ssa1^K69Q (1 µM, an ATPase-dead Ssa1 mutant), Sse1^L433A:N434P (1 µM, a substrate-binding defective Sse1 mutant), Sse1^F439L:M441A (1 µM, a substrate-binding defective Sse1 mutant), Ssa1^L483W (1 µM, a substrate-binding defective Ssa1 mutant), Sis1^H34Q (1 µM, a Sis1 mutant unable to stimulate Ssa1 ATPase activity), Sis1^1–68 (1 µM, the J domain of Sis1), Sis1^1–121 (1 µM, the J and G/F-domains of Sis1) and Ydj1 (1 µM, an Hsp40). Disaggregation and reactivation of luciferase was monitored by luminescence. Luminescence measurements were converted into reactivation yield (% of the maximum recoverable luciferase activity) by comparison to the luminescence of known quantities of soluble, native luciferase (C). Disaggregation and reactivation of GFP was monitored by fluorescence. Fluorescence measurements were converted into reactivation yield (% of the maximum recoverable GFP fluorescence) by comparison to the fluorescence of known quantities of soluble, native GFP (D). Values represent means ± SEM (n = 3).

(A, B) Preformed Sup35 prions (2 µM monomer) were incubated for 6 h at 25°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Ssa1 (2 µM), Sis1 (2 µM), Sse1 (2 µM) (each individual combination is an individual category of the x-axis) in the absence (blue bars) or presence of Hsp104 (0.5 µM, green bars; or 2 µM, black bars). For the 10x(Sse1+Ssa1+Sis1) condition the concentration of Ssa1, Sis1 and Sse1 was increased to 20 µM. Sup35 prion disassembly was assessed by the amount of SDS-resistant Sup35 (A) or by ThT fluorescence (B). Values represent means ± SEM (n = 3). (C, D) Preformed amyloid fibers composed of α-syn (0.5 µM monomer) were incubated for 6 h at 37°C in buffer plus ATP (5 mM) and ATP-regeneration system without or with the indicated combination of Hsc70 (10 µM), Hdj1 (10 µM), Hsp70 (10 µM) and Apg-2 (10 µM) (each individual combination is an individual category of the x-axis) in the absence (blue bars) or presence of Hsp104 (2.5 µM, green bars; or 10 µM, black bars). Fiber integrity was then determined by sedimentation analysis (C) or by ThT fluorescence (D). Values represent means ± SEM (n = 3).