(A) Sup35 (2.5μM) was incubated for 8h with rotation (80rpm) to generate fibers and then incubated with Hsp104 (0.01–20μM) plus ATP (5mM) for 60min at 25°C (black) or 10°C (blue). Disassembly was assessed by the amount of SDS-resistant Sup35. Values represent means±SD (n=3).
(B) Kinetics of Hsp104 (2μM) induced Sup35 fiber (2.5μM monomer) disassembly in the presence of ATP (5mM) or the non-hydrolyzable analog AMP-PCP (5mM) (an alternative to AMP-PNP) at 25°C. Disassembly was monitored as in (A). Values represent means±SD (n=3).
(C) Sup35 fibers (2.5μM monomer) were treated at 25°C with the indicated Hsp104 proteins (2μM) plus ATP (5mM) or Hsp104 plus AMP-PNP (5mM), ATP (5mM) and GdmCl (20mM), ADP (5mM), or no nucleotide. Wt denotes wild type Hsp104. Disassembly was monitored by the amount of SDS-resistant Sup35. Values represent means±SD (n=3).
(D, E) Sup35 fibers (2.5μM monomer) were incubated with Hsp104 (2μM) plus ATP (5mM) for 0–60min at 25°C, and at various times reactions were processed for EM. Scale bars: 2μm (D), 0.25μm (E).