Fig 1. DSF stimulates the autokinase activity of full-length RpfC.

(A) Schematic view of the secondary structure of full-length RpfC. The 22 amino acid residues of the N-terminal that are putatively located within the periplasmic space are depicted. Grey quadrangles (numbered 1–5) within the inner-membrane represent transmembrane helices. Domain names are according to the pfam database. (B) Truncated RpfC without input domain did not show autokinase activity. RpfC inverted membrane vesicles (IMV) (lane 1, 1 μg) and truncated histidine kinase VgrS (soluble) fused with a MBP (maltose binding protein, lanes 6–9, 10.7 μM for each) were used as positive controls. Lanes 2–5, samples containing 25.0 μM soluble RpfC^Δinput. (C) DSF-stimulated autokinase activity of RpfC liposome. Lanes 6–10, 10 μM DSF was added together with ATP to the reaction mixture. The lower panels show proteins stained with Coomassie brilliant blue, which served as loading controls. (D) Full-length RpfC embedded in IMV exhibited autokinase activity that could be stimulated by DSF. All lanes, samples containing 1.4 μg RpfC^FL IMV. Lanes 7–9, 10 μM DSF was added to the reaction mixture of each sample together with ATP. (E-F) Quantification of the dose-dependent DSF stimulation of RpfC autokinase activity. (E) Autokinase activity of RpfC stimulated by different concentrations of DSF; (F) Quantification the band intensity of (E). Black bars represent the standard deviation (n = 3). Data points were fitted using a logarithmic model. Intensities of the autophosphorylation bands were estimated using Quantity One software. (B-E) Upper panels show the results of autokinase assays. Each lane contains 1.4 μg RpfC^FL liposome or 1.4 μg RpfC^FL IMV that was co-incubated with 100 μM ATP, including 10 μCi [γ-³²P]-ATP, for indicated times. All reactions were immediately stopped and separated by 12% SDS-PAGE prior to autoradiography. Each experiment was independently repeated three times, and a representative experiment is shown.