Pure Hsp110, Hsp70 and Hsp40 drive protein disaggregation.

<p>(<b>A, B</b>) Urea-denatured luciferase aggregates (50 nM) (<b>A</b>) or heat-denatured GFP aggregates (0.45 µM) (<b>B</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 (<b>A</b>). 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>B</b>). Values represent means ± SEM (n = 3).