Figure 5.

The PmrD protein specifically inhibits dephosphorylation of phospho-PmrA by targeting its N-terminal region. (A) Spontaneous dephosphorylation of phospho-PmrA protein in the presence (PmrD) or absence (-) of the PmrD protein: ³²P-labeled phosphorylated GST-PmrB_cT156R beads were used as phosphodonor for the PmrA protein and removed before the assay. The concentrations of the PmrA and PmrD proteins were 2.5 μM. (B) Spontaneous dephosphorylation of phospho-YgiX protein was performed in the presence (PmrD) or absence (-) of the PmrD protein. ³²P-labeled phosphorylated GST-YgiY_c beads were incubated with the YgiX protein for 1 h to produce phospho-YgiX protein and removed before the assay. The concentrations of the YgiX and PmrD proteins were 2.5 μM. (C) Spontaneous dephosphorylation of phospho-PmrA protein was performed in the presence (PmrD) or absence (-) of PmrD protein. All proteins used in this assay were not in contact with PmrB or derivatives, because they were independently overexpressed in E. coli strain EG13796, which lacks the entire pmrAB homolog basRS, and purified as described in Materials and Methods. ³²P-labeled phosphorylated GST-YgiY_c beads were used as phosphodonor for the PmrA protein and removed before the assay. The concentration of the PmrA and PmrD proteins was 2.5 μM. (D) Quantitative analysis of the phosphatase assay of phospho-PmrA protein. ³²P-labeled phosphorylated GST-YgiY_c beads were used as phosphodonor for the PmrA protein and removed before the assay. The concentrations of the PmrA, PmrB_c, and PmrD proteins were 2.5, 5, and 2.5 μM, respectively. The small cationic proteins RNase A and cytochrome C were used as negative controls at a final concentration of 2.5 μM. (E) Quantitative analysis of the phosphatase assay of phospho-YgiX protein. ³²P-labeled phosphorylated GST-PmrB_cT156R beads were incubated with the YgiX protein for 45 min to produce phospho-YgiX protein and removed before the assay. The concentrations of YgiX, YgiY_c, and PmrD proteins were 2.5, 5, and 2.5 μM, respectively. (F) Quantitative analysis of the phosphatase assay of phospho-PmrA-YgiX protein. ³²P-labeled phosphorylated GST-YgiY_c beads were incubated with the PmrA-YgiX protein for 2 h to produce phospho-PmrA-YgiX protein and removed before the assay. The concentrations of the PmrA-YgiX, PmrB_c, and PmrD proteins were 2.5, 5, and 2.5 μM, respectively. (G) Quantitative analysis of the phosphatase assay of phospho-YgiX-PmrA protein. ³²P-labeled phosphorylated GST-PmrB_cT156R beads were incubated with the YgiX-PmrA protein for 2 h to produce phospho-YgiX-PmrA protein and removed before the assay. The concentrations of the YgiX-PmrA, YgiY_c, and PmrD proteins were 2.5, 5 and 2.5 μM, respectively. Samples were analyzed by 10% SDS-PAGE and quantified as described in Materials and Methods.