ATPase activity measurement (Malachite green phosphate assay)

The Drg1 ATPase activity was measured using the Malachite green phosphate assay (ref. ⁵⁷, Bioassay Systems) as reported previously^19,21. Essentially, purified proteins (Drg1 wild-type and mutant variants) were eluted in 20 mM HEPES-KOH, 150 mM KOAc, 5 mM Mg(OAc)₂, 0.1% Tween-20, 1 mM DTT, pH 6.8). HIS-tagged Rlp24C was heterologously expressed in E. coli and purified exactly as described in ref. ²¹. Purified Drg1 was incubated with 1 mM ATP and the released phosphate was quantified using the malachite green phosphate assay kit. The absorbance of the samples at 600 nm was measured at a GeniusPro TECAN^TM plate reader with an associated Microsoft Excel plugin (XFluor4 v4.51) for data collection. The activity of 10 µg Drg1 (wild type) with 1 mM ATP was measured either alone (=basal activity) or in the presence of 2 µg (1.68 µM) His-Rlp24C. To indicated samples (+Dia), diazaborine was added to a final concentration of 100 µg/ml (370 µM). The specific activity (µmol ATP/h/mg Drg1) of all samples was normalized to the Drg1 basal activity in Microsoft Excel 2019 to display relative activities. For each protein at least two biological replicates were measured with three technical replicates each to determine the mean and standard deviation.