Extended Data Fig. 4: Protein kinase assay of purified HRI.
From: Mitochondrial stress is relayed to the cytosol by an OMA1–DELE1–HRI pathway
a, Purified recombinant HRI, DELE1 and eIF2α. A total of 800 ng of each recombinant protein was subjected to SDS–PAGE and stained with Coomassie blue (n = 1 gel). b, c, HRI kinase reactions were performed with 1 μM recombinant yeast eIF2α and various amounts of purified recombinant HRI protein. Reactions were stopped at the time points indicated by removing 5-μl aliquots of the kinase reaction mixture and mixing with an equal volume of 2× SDS loading buffer. The SDS samples were then dotted on nitrocellulose blots and subjected to immunoblotting analysis with antibodies against phosphorylated eIF2α. b, Representative dot blot. c, Densitometric quantification of dot blots expressed as the average value from n = 2 individual experiments. To enable a reaction in a linear range, 25 nM HRI and a 5-min incubation time were used for all the subsequent experiments. d, Enzyme kinetic constants for HRI activity with or without DELE1 in the presence or absence of haemin (mean ± s.e.m., n = 3 individual reactions, fit for data shown in Fig. 4f). Kinetic constants were determined by fitting to the Michaelis–Menten equation using a least-squares fit in Prism v.6.07. The constants calculated from HRI + haemin (marked with asterisks) are not accurate because at this range of substrate concentrations, the enzymatic reaction is first order and never reaches Vmax. But higher substrate concentrations cannot be used to obtain Vmax conditions, as purified eIF2α protein will precipitate at higher concentrations.
