(A) Ure2 (2.5μM) was incubated for 16h with rotation (80rpm) to generate fibers and then incubated with Hsp104 (0.01–10μM) plus ATP (5mM) for 60min at 25°C. Disassembly was assessed by CR binding. Values represent means±SD (n=3). (B, C) Kinetics of Hsp104 (2μM) induced Ure2 fiber (2.5μM monomer) disassembly in the presence of either ATP (5mM), ATP (5mM) plus GdmCl (20mM), or the non-hydrolyzable analog AMP-PCP (5mM) (an alternative to AMP-PNP). Disassembly was monitored by CR binding (B) or sedimentation analysis (C). Values represent means±SD (n=3).
(D) Ure2 fibers (2.5μM monomer) were incubated with Hsp104 (2μM) plus ATP (5mM) for 0–60min, and at various times reactions were processed for EM. Scale bar, 0.25μm.