Standardized ATPase Assay Protocol

All ATPase assays were carried out using the PK/LDH regenerating system as previously described (Panaretou et al., 1998 (link); Armstrong et al., 2012 (link); Horvat et al., 2014 (link); Wolmarans et al., 2016 (link)). Average values of the experiments are shown with error expressed as standard deviation using Prism GraphPad. The ATPase rates are either shown as μm ATP hydrolyzed per minute per μm of Hsp82p (1/min) or as a percentage of the maximally Aha1p-stimulated ATPase rate of Hsp82p, Hsp82p^K178C, Hsp82p^K178C* (derivatized), or Hsp82p^K178C-Smt3p^Cys. Fit lines were calculated according to the following equation: (Y=((B_max*X)/(K_app+X))+X0). The final conditions of all the reactions are 25 mm Hepes (pH 7.2), between 1 and 25 mm NaCl, 5 mm MgCl₂, 1–2 mm DTT, 0.3 mm NADH, 2 mm ATP, 1 mm phosphoenol pyruvate (PEP), 2.5 μl of pyruvate kinase/lactate dehydrogenase (PK/LDH) (Sigma), and 0.5% DMSO. Identical reactions were quenched with 50–100 μm NVP-AUY922 and subtracted from unquenched reactions to correct for contaminating ATPase activity.