Fig 1.

The predicted kinase motif in PikA is critical for its toxicity to eukaryotic cells. (A) Alignment of the sequences of the putative catalytic center in PikA with those of CTKA, LepB_NTD, MavQ, Risk1, and A1G_01070 using the MUSCLE method. (B) The predicted His-Asn­Asp motif is essential for yeast toxicity of PikA. Cells of yeast strains expressing PikA or the indicated mutants from the galactose­inducible promotor were serially diluted and spotted on the indicated media. The plates were incubated at 30°C for 3 days before image acquisition. The expression of PikA and its derivatives was detected by immunoblotting with the anti-Flag antibody. The metabolic enzyme phosphoglycerate kinase (PGK1) was detected as a loading control. (C) Representative fluorescence and bright field images of HeLa cells transfected to express mCherry, mCherry-tagged PikA, and its enzymatically inactive mutants. Cells transfected for 18 h were used for image acquisition. Scale bars, 50 µm. (D) Quantitation of cell rounding in samples transfected to express PikA and its mutants. For each sample, at least 300 cells were scored and results (mean ± s.e.) shown were from three independent experiments. ***, P < 0.001.