Figure 2. Hsp70 allows Hsp104 to overcome ADP inhibition.
(A) ADP impact on GFP disaggregation by Hsp104 and the Hsp70 system. Heat-aggregated GFP (0.04 mg ml-1) was incubated with Hsp104 (1 μM), Ssa1 (2 μM) and Sis1 (0.4 μM) at 2.6 mM ATP and at the indicated concentrations of ADP. A control experiment was performed without Hsp104 (grey). (B) The relative GFP renaturation rates by Hsp104 and Hsp70-Hsp40 at the indicated ADP concentrations were calculated from the initial slope of the fluorescence curves from (A). (C) ADP effect on ATP hydrolysis by Hsp104 during the Hsp70-assisted disaggregation. ATPase activity was measured for Hsp104 (1 μM) in the presence of Ssa1 (1 μM), Sis1 (0.1 μM) and aggregated GFP (0.2 mg ml-1) (green) or for Hsp104 alone (blue) under the conditions as in (A). For comparison, the ATPase activity of Ssa1, with Sis1 and GFP and without Hsp104 was assessed under the same conditions (grey). Data are the average of three experiments (± SD).