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. 2020 May 29;9:e56048. doi: 10.7554/eLife.56048

Figure 4. The PrkA/PrpC pair controls the phosphorylation status of ReoM.

(A–B) Non-denaturing, native PAGE analysis of the phosphorylation (A) and dephosphorylation (B) of ReoM in vitro. The components of each lane in the Coomassie-stained gel are annotated above the image and the position and identity of relevant bands is marked to the side. (C) LC-MS analysis of intact ReoM. The deconvoluted mass spectrum for non-phosphorylated ReoM (black) is overlaid over the equivalent spectrum for mono-phosphorylated ReoM, P-ReoM (red). (D) LC-MS/MS was used to perform peptide mapping analysis that revealed that Thr7 is the sole phosphosite of ReoM. The MS/MS fragmentation spectra of the phosphorylated peptide encompassing Asp5-Lys22 is presented with b-ion fragmentation in blue and y-ion fragmentation shown in red, whilst the precursor ion (m/z 1116.86, z = 2+) is represented by a blue diamond.

Figure 4.

Figure 4—figure supplement 1. LC-MS analysis of intact ReoM.

Figure 4—figure supplement 1.

ESI-QTOF raw data for (A) unmodified ReoM and (B) mono-phosphorylated ReoM.
Figure 4—figure supplement 2. LC-MS analysis of ReoM T7A.

Figure 4—figure supplement 2.

ESI-QTOF raw data for (A) unmodified ReoM T7A and (B) ReoM T7A following incubation with PrkA-KD. (C) Overlaid deconvoluted mass spectrum demonstrating the lack of phosphorylation of ReoM T7A in the absence (black) and presence of PrkA-KD (red).
Figure 4—figure supplement 3. Dephosphorylation of P-ReoM by PrpC.

Figure 4—figure supplement 3.

LC-MS analysis of ReoM after PrpC incubation in the presence of manganese. (A) ESI-QTOF raw data and (B) deconvoluted mass spectrum demonstrating the presence of non-phosphorylated ReoM.
Figure 4—figure supplement 4. Dephosphorylation of P-PrkA-KD by PrpC.

Figure 4—figure supplement 4.

LC-MS analysis of intact PrkA-KD following incubation with PrpC (A) ESI-QTOF raw data and (B) deconvoluted mass spectrum indicating the presence of non-phosphorylated PrkA-KD.