Extended Data Fig. 3: The IQ motif of SidJ is required for its optimal response to CaM.
From: Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase
a, b, The IQ motif is required for the optimal activity of SidJ in response to CaM. Serially diluted CaM was preincubated with SidJ (a) or the SidJ(I841D/Q842A) mutant (b) and SdeA in the glutamylation buffer at 37 °C for 2 h. A cocktail containing 4×Flag–Rab33b, NAD+ and ubiquitin was added to the reactions. After incubation for another 2 h at 37 °C, proteins separated by SDS–PAGE were assessed using the indicated antibodies. In each panel, data shown are one representative from at least three independent experiments with similar results. c, The SidJ(I841D/Q842A) mutant complements the intracellular growth defect of the ΔsidJ mutant. A. castellanii was infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points. Experiments on each strain were performed in triplicate and similar results were obtained in two independent experiments. Results are from one representative experiment performed in triplicate from three independent experiments; error bars represent s.e.m. (n = 3).
