Figure 3. The derepressed D484K variant of Hsp104 is inhibited by ADP.

(A) The ATPase activity of Hsp104 D484K is strongly affected by ADP. ATPase activity of D484K variant was measured at 10 mM ATP and at the indicated concentrations of ADP. Values are the average of three independent experiments (± SD). (B) ADP inhibits the disaggregation activity of Hsp104 D484K in the absence of Hsp70. Disaggregation of heat-aggregated GFP (0.04 mg ml^-1) by Hsp104 D484K (0.5 μM) at 10 mM ATP (blue). After 60 s of the reaction, ADP was added to 2 mM concentration (red). (C) ATP regeneration system or (D) The Hsp70 system restores the disaggregation activity of Hsp104 D484K. The experiment was initiated as in (B), and after 5 min (C) an ATP regeneration system comprising PK (0.1 mg ml^-1) and PEP (40 mM) (purple) or (D) the Hsp70 chaperone system: Ssa1 (2 μM) and Ydj1 (0.5 μM) (green) was added to the reaction mixture.

DOI: http://dx.doi.org/10.7554/eLife.15159.007

Figure 3—figure supplement 1. Hsp104 D484K is affected by ADP similarly as the WT Hsp104.

ATPase activity of WT and D484K variant was measured at the physiological ATP concentration (2.6 mM) in the absence (black) or in the presence of 1 mM ADP (grey). Values are the average of three experiments (± SD).

DOI: http://dx.doi.org/10.7554/eLife.15159.008

Figure 3—figure supplement 2. Protein translocation activity of HAP D484K is inhibited by ADP.

Proteolysis of fRCMLa (5 μM) by HAP D484K (0.5 μM) and ClpP (0.5 μM) was carried out at 10 mM ATP (purple) or in the absence of nucleotide (grey). Two other reactions were performed at 10 mM ATP and either in the presence of extra 2 mM ADP (red) or with an ATP regeneration system (0.2 mg ml^-1 creatine kinase and 120 mM creatine phosphate) (blue). a. u. – arbitrary units.

DOI: http://dx.doi.org/10.7554/eLife.15159.009

Figure 3—figure supplement 3. Hsp70 allows efficient disaggregation at low ATP:ADP ratio.

Disaggregation of heat-aggregated GFP (0.04 mg ml^-1) (black) by Hsp104 D484K (1 μM) measured (A) in the absence or (B) in the presence of the Hsp70 system: Ssa1 (2 μM) and Ydj1 (0.5 μM). The total concentration of adenine nucleotides was 10 mM. At the indicated time points samples were taken from the reaction mixture and subjected to HPLC analysis to assess ADP concentration (grey).

DOI: http://dx.doi.org/10.7554/eLife.15159.010

Figure 3—figure supplement 4. In the absence of ADP the derepressed Hsp104 D484K is independent of Hsp70 in disaggregation.

Disaggregation of heat-aggregated GFP (0.04 mg ml^-1) by Hsp104 D484K (0.5 μM) was carried out at 10 mM ATP in the presence or absence of Ssa1 (2 μM) and Ydj1 (0.5 μM) or an ATP regeneration system (0.2 mg ml^-1 creatine kinase and 120 mM creatine phosphate), as indicated. In a control experiment GFP was incubated with Ssa1 (2 μM), Ydj1 (0.5 μM), without Hsp104.

DOI: http://dx.doi.org/10.7554/eLife.15159.011