Assessment of Protein Content and Phosphorylation Level in Synechocystis sp. PCC 6803 under Various Growth Conditions Using Quantitative Phosphoproteomic Analysis.

The photosynthetic apparatus and metabolic enzymes of cyanobacteria are subject to various controls, such as transcriptional regulation and post-translational modifications, to ensure that the entire cellular system functions optimally. In particular, phosphorylation plays key roles in many cellular controls such as enzyme activity, signal transduction, and photosynthetic apparatus restructuring. Therefore, elucidating the governing functions of phosphorylation is crucial to understanding the regulatory mechanisms underlying metabolism and photosynthesis. In this study, we determined protein content and phosphorylation levels to reveal the regulation of intracellular metabolism and photosynthesis in Synechocystis sp. PCC 6803; for this, we obtained quantitative data of proteins and their phosphorylated forms involved in photosynthesis and metabolism under various growth conditions (photoautotrophic, mixotrophic, heterotrophic, dark, and nitrogen-deprived conditions) using targeted proteomic and phosphoproteomic analyses with nano-liquid chromatography-triple quadrupole mass spectrometry. The results indicated that in addition to the regulation of protein expression, the regulation of phosphorylation levels of cyanobacterial photosynthetic apparatus and metabolic enzymes was pivotal for adapting to changing environmental conditions. Furthermore, reduced protein levels of CpcC and altered phosphorylation levels of CpcB, ApcA, OCP, and PsbV contributed to the cellular response of the photosynthesis apparatus to nitrogen deficiency.

Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms.

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein.

Comparative Targeted Proteomics of the Central Metabolism and Photosystems in SigE Mutant Strains of Synechocystis sp. PCC 6803.

A targeted proteome analysis was conducted to investigate the SigE dependent-regulation of central metabolism in Synechocystis sp. PCC 6803 by directly comparing the protein abundance profiles among the wild type, a sigE deletion mutant (ΔsigE), and a sigE over-expression (sigEox) strains. Expression levels of 112 target proteins, including the central metabolism related-enzymes and the subunits of the photosystems, were determined by quantifying the tryptic peptides in the multiple reaction monitoring (MRM) mode of liquid-chromatography⁻triple quadrupole mass spectrometry (LC⁻MS/MS). Comparison with gene-expression data showed that although the abundance of Gnd protein was closely correlated with that of gnd mRNA, there were poor correlations for GdhA/gdhA and glycogen degradation-related genes such as GlgX/glgX and GlgP/glgP pairs. These results suggested that the regulation of protein translation and degradation played a role in regulating protein abundance. The protein abundance profile suggested that SigE overexpression reduced the proteins involved in photosynthesis and increased GdhA abundance, which is involved in the nitrogen assimilation pathway using NADPH. The results obtained in this study successfully demonstrated that targeted proteome analysis enables direct comparison of the abundance of central metabolism- and photosystem-related proteins.