Figure 5. Hsp70 does not support Hsp104 in processing of disordered, non-aggregated proteins.
Proteolysis of fRCMLa (5 μM) by HAP (1 μM) and ClpP (1.8 μM), carried out at 2.6 mM ATP with or without 1 mM ADP and in the presence or absence of Ssa1 (2 μM) and Ydj1 (0.5 μM), as indicated. Grey line shows a control experiment, in which HAP was omitted. a. u. – arbitrary units.
Figure 5—figure supplement 1. Hsp70 interaction with fRCMLa.
Ssa1 (2 μM) and Ydj1 (0.5 μM) were injected to the reaction mixture containing fRCMLa (5 μM) at 2.6 mM ATP. Complex formation was monitored by following fluorescence anisotropy. a. u. – arbitrary units.
Figure 5—figure supplement 2. Hsp70 does not support Hsp104 in processing of α-casein.
α-casein (20 μM) was incubated with HAP (1 μM) and ClpP (3.6 μM) at 2.6 mM ATP in the presence of, optionally, 1 mM ADP, 2 μM Ssa1 and 0.5 μM Ydj1, as indicated, or at 10 mM ATP with an ATP regeneration system comprising 0.2 mg ml-1 creatine kinase and 20 mM creatine phosphate (two last lines). In a control experiment α-casein was incubated with ClpP only (two first lines). Proteolysis of α-casein was assessed after 1 hour with SDS-PAGE.
Figure 5—figure supplement 3. Derepressed HAP D484K is efficient in translocation of disordered proteins at the physiological ATP and ADP concentrations.
fRCMLa (5 μM) proteolysis by HAP (1 μM) or HAP D484K (1 μM) and ClpP (1.8 μM), was carried out at 2.6 mM ATP and 1 mM ADP and in the presence of the Hsp70 chaperone system comprising Ssa1 (2 μM) and Ydj1 (0.5 μM). In a control experiment HAP was omitted (grey).