Extended data Figure 1. Characterization of protein disaggregation/refolding and refolding-only reactions.

a, HSP70–J-protein–HSP110 (HSPA8–J-protein–HSPH2) functional cycle. Concomitant interaction of HSP70 with a J-protein and substrate results in allosteric stimulation of ATP hydrolysis; this traps the substrate in HSP70 (ref. 8). Subsequent NEF (for example, HSP110) promoted ADP dissociation from HSP70, then allows ATP rebinding, which triggers substrate release to complete the cycle^54,55. b, Scheme for in vitro disaggregation/refolding and refolding-only reactions. The aggregates used in disaggregation/refolding assays are preformed by heating luciferase with yeast small heat-shock protein (sHSP) Hsp26 (ref. 4), which is known to co-aggregate with misfolded proteins in vivo^27,28 (see Methods for detailed description). If HSP70, J-protein and HSP110 are instead heated together with substrate and Hsp26, luciferase is denatured into a more easily refoldable, inactive and largely monomeric substrate form used in refolding-only assays. c, SEC profiles of aggregated ³H-labelled luciferase (black; size range 200 kDa to ≥5,000 kDa representing ~2 to >50 aggregated luciferase molecules) and monomeric native luciferase (red; size ~63 kDa). Arrows indicate elution size (kDa). Inset, activity of loaded material. d, SEC profile of partially denatured and largely monomeric luciferase (starting material for refolding-only reactions). Inset, activity of loaded material. e, Chaperone nomenclature. f, Disaggregation and reactivation of preformed luciferase aggregates using human HSP70–HSP110 with human J-proteins JA2, green; JB1, blue; JA2+JB1, magenta or no J-protein, black. Under limiting chaperone (HSP70/HSP110) and increasing J-protein concentrations (A, solid or B, dashed) (n = 3). Data are mean ± s.e.m. Precise concentrations are shown in Extended Data Table 1.