The ferredoxin 5 (Fd5) phosphoprotein and the S/T protein kinase SpkG are encoded by the slr0148 and slr0152 genes, respectively, which belong to slr0144-slr0152 cluster in Synechocystis 6803. Using targeted proteomic approach, we show that SpkG is responsible for phosphorylation of Fd5 on T18 and T72 residues. Alignment and 3D model of the Fd5 structure suggested a role for phosphorylation in regulation of protein-protein interaction. Further, Fd5 phosphorylation is affected by the Slr0151 protein encoded by the gene preceding spkG in the gene cluster. We propose that Slr0151 functions as auxiliary protein in regulation of the ratio between phosphorylated and non-phosphorylated forms of Fd5. Thus, the S/T protein phosphorylation network in cyanobacteria spreads beyond a triad of phosphoprotein-kinase-phosphatase.

WT and two Synechocystis 6803 knock-out mutants, slr0152(SpkG) and slr0151 were grown in the BG-11 medium in standard conditions, in triplicates. The SRM assays for Fd5 phosphopeptides used in this study were described described in Angeleri et al (2016) J. Prot. Res. 15, 4638-4652. The SRM assays for corresponding non-modified peptides and some other peptides were designed here.

The total protein preparations were reduced, alkylated and digested with trypsin as described in Angeleri et al (2016) J. Prot. Res. 15, 4638-4652. The total peptide mixtures, without TiO2 enrichment, were subjected to the SRM analysis.