Abstract:[Background] Proteases can degrade the misfolded or nonfunctional proteins in cells. Clp family protein is one of the important protease complexes. ClpP is the core of proteolysis of Clp protease complex. According to genomic data, there are four different ClpP proteins in Synechocystis sp. PCC6803, namely ClpP1–ClpP4. As a vital functional component of proteolytic complex, ClpP in Synechocystis is currently poorly studied. The study on its physiological function and substrate regulation is limited. [Objective] To explore the functions of ClpP2 in Synechocystis and identify potential substrate clusters, thereby providing experimental support for the mechanism research of ClpP2. [Methods] The ClpP2 mutant strain (ΔClpP2) was constructed, and the growth experiment and photosynthetic system characteristic experiment were carried out. Target proteins regulated by ΔClpP2 were identified by isobaric tag for relative absolute quantitation (iTRAQ), and the metabolic pathways involved in the substrate proteins were analyzed via bioinformatics. Finally, parallel reaction monitoring (PRM) was used to verify part of the quantitative data. [Results]ΔClpP2 grew into the logarithmic phase through photoautotrophy under natural conditions, but did not grow normally when met high-light or high-temperature stress. Compared with the wild-type (WT), ΔClpP2 showed significantly reduced photosystem Ⅱ (PSⅡ) electron transport efficiency and circle electron transport activity of photosystemⅠ(PSⅠ). A total of 206 differentially expressed proteins in ΔClpP2 were identified by iTRAQ quantitative proteomics. Among them, 131 were up-regulated and 74 were down-regulated, which provided a rich substrate library. Gene Ontology (GO) analysis showed that ClpP2 was mainly involved in the transport of various substances, and ABC transporter pathway was enriched notably. Thirty-four differentially expressed proteins were successfully verified by PRM technology. [Conclusion] ClpP2 is not necessary for the growth of Synechocystis, but is essential when Synechocystis meets high-temperature or high-light stress. ClpP2 inactivation reduces the activity of the photosynthetic system in Synechocystis. ClpP2 might affect the photosynthetic system by regulating ion transport. ClpP2 is likely to bind with ClpX to form a protease complex.
