Figure 2. ATPase stimulation of Hsp82p:Hsp82p^LL heterodimers.

(A) Bar graph showing the intrinsic ATPase rates of wildtype Hsp82p and Hsp82p mutants (Hsp82p^V391E, Hsp82p^D79N, Hsp82p^E33A, and Hsp82p^LL). Reactions contained 4 μM of Hsp82p or Hsp82p mutants. ATPase rate shown in micromolar ATP hydrolyzed per minute per micromolar of enzyme (1/min). (B) The addition of Hsp82p^LL potently stimulates the ATPase activity of wildtype Hsp82p. Hsp82p^LL was titrated into reactions containing 2 μM of wildtype Hsp82p. The resulting stimulated ATPase rate is shown as a fold stimulation of the intrinsic Hsp82p rate. (C) Titration of Aha1p and Aha1p^N stimulates the ATPase activity of Hsp82p:Hsp82p^LL heterodimers. Titration of Hch1p inhibits the ATPase activity of Hsp82p:Hsp82p^LL heterodimers at low concentrations (by ~16% at 2.5 μM Hch1p and ~11% at 5 μM Hch1p). Heterodimers are formed by mixing 1 μM Hsp82p and 5 μM of Hsp82^LL. ATPase rates are shown as a fold stimulation the intrinsic rate of Hsp82p:Hsp82p^LL heterodimers (stippled line). (D) ATPase stimulation of wildtype Hsp82p by Aha1p, Hch1p and Aha1p^N. The V_max values for co-chaperone stimulation by each of these co-chaperones are 3.8 ± 0.4 min⁻¹ (Aha1p), 0.4 ± 0.1 min⁻¹ (Hch1p), and 0.8 ± 0.1 min⁻¹. Reactions contained 2 μM of Hsp82p with indicated concentrations of co-chaperones. All Aha1p, Hch1p, and Aha1p^N titrations are shown as black triangles, blue circles, and blue squares, respectively. The ATPase rate is shown as a fold stimulation of the intrinsic Hsp82p rate (stippled line).