Figure 1
Phosphorylation of eYfnI and the extracellular domains of other LTA synthases by PrkC. (A) In vitro phosphorylation assays of eYfnI and eYfnI variants by PrkC. The catalytic domain of PrkC, PrkCc, was incubated with [γ-33P] ATP, MBP and eYfnI-WT or the eYfnI-T297A, eYfnI-S298A and eYfnI-T303A variants at 37 °C for 15 min. Samples were separated by SDS-PAGE and visualized by autoradiography. The upper bands correspond to the phosphorylated form of eYfnI, the band below to the autophosphorylated PrkCc and the lower band to the phosphorylated MBP. (B) In vitro phosphorylation of eYfnI by PrkCc and dephosphorylation by PrpC. PrkCc was incubated with [γ-33P] ATP, MBP and eYfnI at 37 °C for 10 min (lane 1) then PrpC was added to the reaction and incubated for 10 min at 37 °C (lane 2). (C) In vitro phosphorylation assays of eYfnI by YabT. The cytoplasmic domain of YabT was incubated with [γ-33P] ATP, MBP and eYfnI at 37 °C for 15 min. Samples were separated by SDS-PAGE and visualized by autoradiography. The upper bands correspond to the autophosphorylated YabT and the lower band to MBP-P. (D) In vitro phosphorylation assays of the extracellular domains of LTA synthases by PrkCc. PrkCc was incubated with [γ-33P] ATP, MBP and the extracellular catalytic domain of YfnI, LtaS, YvgJ and YqgS at 37 °C for 15 min. Samples were separated by SDS-PAGE and visualized by autoradiography. The upper bands correspond to the phosphorylated eLTA synthases, the band below to PrkCc-P and the lower band to MBP-P. Full-length autoradiograms are presented in the supplemental data. Coomassie stained gels showing the amount of all purified proteins used in the phosphorylation tests are presented in Fig. S2.
