Fig. 4: AnkJ inhibits the activity of Ceg14 through direct protein-protein interactions.

a AnkJ suppresses the yeast toxicity of Ceg14. Serially diluted cells of the indicated yeast strains were spotted on the indicated media. Images were captured after incubating at 30 °C for 2 d (left panels). Relevant proteins was detected using specific antibodies against Ceg14 or AnkJ. PGK was probed as a loading control (right panels). 1, uninduced; 2, galactose-induced. Ceg14 was driven by the galactose-inducible promoter. b Interactions between Ceg14 and AnkJ. Lysates from HEK293T cells transfected to express the indicated proteins were divided into three identical samples, which were subjected to immunoprecipitation with the indicated antibodies. Coprecipitated proteins were detected by immunoblotting. c Interactions between Ceg14 and AnkJ determined by GST pulldown. The indicated proteins were mixed at 25 °C for 30 min and GST beads were used to capture potential protein complexes. Interactions were assessed by Coomassie brilliant blue (CBB) staining. d AnkJ and Ceg14 interacts in L. pneumophila. Flag-Ceg14 was expressed in wild-type or the ∆ankJ mutant. Binding between Flag-Ceg14 and AnkJ was determined by immunoprecipitation and copurified proteins were detected by immunoblotting. Data shown in (a–d) each was a representative of three independent experiments with similar results. e AnkJ inhibits Ceg14-catalyzed ATP hydrolysis. Serially diluted AnkJ was preincubated with Ceg14 and actin and the mixtures were then added to reactions containing ATP, the reaction was allowed to proceed for 2 h at 37 °C prior to HPLC analysis. The quantity of AMP was determined using peak areas from standard AMP samples. The quantity of AMP was normalized to samples from reactions without AnkJ. f AnkJ stopped ongoing ATP hydrolysis by Ceg14. Ceg14 and actin were added to 10 identical ATP samples. At 10, 20, 30 min, a pair of samples were processed,one by rapidly freezing and the second by adding AnkJ and were allowed to proceed for an additional 1.5 h at 37 °C before HPLC analysis. AMP levels in each group were normalized to the 0 min control samples incubated at 37 °C for 2 h without AnkJ. For (e, f) data shown were mean ± s.e. from three replicates. Unpaired two-tailed Student’s t-tests were performed. Source data are provided as a Source Data file.