Fig. 5
Identification of the VgrS cleavage site by the Prc protease. a MALDI–TOF–MS/MS analysis revealed that Prc cleaves the VgrS sensor at the Ala9↓Gln10 site. Upper panel: proteolysis of the VgrS sensor with active Prc. Middle panel: proteolysis of the VgrS sensor with inactive PrcS475A, which was used as a negative control. Lower panel: proteolysis of the recombinant VgrSA9G-Q10A sensor with active Prc. Digested products were detected in positive ion reflectron mode over an m/z range of 700–3500. Spectra showed relative intensities in the mass range of m/z 2000–3400. b Schematic view of the secondary structure of VgrS and the cleavage site in the sensor region. TM transmembrane helix. c Identification of the Prc cleavage site in the VgrS sensor. A MALDI–TOF/TOF MS/MS spectral analysis of m/z 2771.5 from a Prc + VgrS sensor sample. The magnified MS/MS spectra showed the fragment patterns of peptides. d Substitution of VgrSA9G-Q10A resulted in resistance to Prc cleavage. Bacterial strains that encoded recombinant VgrSA9G-Q10A fused with an HA-tag between the 6th and 7th residues were constructed independently in the ΔvgrS and ΔvgrSΔprc backgrounds. The bacterial strains were 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 a 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 an internal control. The experiment was repeated three times. e Detection of the N-terminal short peptide of VgrS generated by Prc cleavage. Full-length VgrS embedded in inverted membrane vesicles was treated by Prc, and the proteolytic products were analysed by QTRAP LC–MS/MS at different time points. A chemically synthesized peptide, NRNIDFFA, was used as standard. Details of the QTRAP LC–MS/MS analysis are shown in Supplementary Fig. 6. f and g Deletion of the N-terminal sequence of VgrS decreased its autophosphorylation level. f An in vitro phosphorylation assay was conducted to measure the autophosphorylation levels of the truncated VgrSΔ9, VgrSΔ58, VgrSΔ72 and VgrSΔ84 embedded in the IMVs (10 μM). The reaction was performed as described in Fig. 4a. g Substitution of VgrSA9G-Q10A did not affect its autokinase activity. Upper panels: autophosphorylation assay. Lower panels: western blotting of the proteins or Coomassie brilliant blue staining was used to check the amount of loaded protein. Two experimental repeats were performed
