Fig. 4

Prc cleaves the sensor region of VgrS to inhibit its autokinase activity. a–c Prc inhibited full-length VgrS autophosphorylation in a protease activity-dependent manner. a Full-length VgrS embedded in the inverted membrane vesicles (IMV, 10 μM) was phosphorylated by [γ-³²P]ATP. Before the addition of 10 μCi [γ-³²P]ATP, active Prc or inactive PrcS^475A (2 μM) was added into the mixture. VgrS^H186A IMV was used as a negative control of phosphorylation. b Prc did not affect the autophosphorylation of a soluble, cytosolic fragment of VgrS. In total, 10 μM soluble VgrS containing the transmitter region (MBP-VgrS^cyto) was used in the assay. c Prc inhibited the phosphorylation level of VgrR. After VgrS autophosphorylation, 10 μM VgrR was added into the mixture to elicit the VgrS–VgrR phosphotransfer reaction. In a, b and c: Upper panels show autophosphorylation assays; lower panels show Coomassie bright blue-stained gels used to check the amount of loaded protein. Aliquots were removed from the mixture at the indicated time points. The reaction was stopped by 6X SDS buffer, separated by SDS-PAGE and analysed by autoradiography. d and e Prc degraded the VgrS sensor in vitro. The purified VgrS sensor (100 μM) was co-incubated with 5 μM active Prc d or inactive Prc^S475A e in enzymatic reaction buffer at 28 °C for the indicated time. Reactions were stopped, and the products were analysed by SDS-PAGE together with silver staining. f Western blotting revealed that Prc degraded N-terminal HA-tags in vivo. A bacterial strain that encoded recombinant VgrS fused with an HA-tag between the 6th and 7th residues was constructed independently in the ΔvgrS background. The bacterial strain was stimulated by 1.0 M sorbitol for different time periods. Total proteins were extracted, separated by SDS-PAGE, and analysed by western blotting. A polyclonal antibody of VgrS (α-VgrS) was used to measure the amount of VgrS protein, while monoclonal HA antibody (α-HA) was used to detect the N-terminal region of VgrS that was potentially cleaved by Prc, and the polyclonal antibody of RNAP (α-RNAP) was used as internal control. a–f, the experiments were repeated three times