The Localization And Primary Functional Characterization Of S2P Homologs In Synechocystis Sp. PCC6803

Regulated intramembrane proteolysis (RIP) is a conserved mechanism from bacteria tohumans that regulates signal transduction across the membrane by recruitingmembrane-bound proteases to cleave membrane-spanning regulatory proteins. As animportant protease that performs in RIP, the metalloprotease site-2protease (S2P) hasreceived extensive study during the past decades. SLR0643and SLL0862are two site-2protease homologs in Synechocystis sp. PCC6803, but their functions are unknown.To explore the subcellular localization of two S2P homologs, EGFP was added to the Cterminus of these two proteins as protein tag. Two recombinant plasmids, PsbA2P-0643GFPand PsbA2P-0862GFP, which contain the fusion fragment of egfp and our target genes wereconstructed and transformed to two mutants of Synechocystis sp. PCC6803, slr0643::km andsll0862::km respectively. Two transgenic Synechocystis, psbA2::0643GFP-Cm~r/△0643-Km~rand psbA2::0862GFP-Cm~r/△0862-Km~r, with the fusion genes inserted in their genome, wereobtained after a long period of screening. Transcriptional expression of egfp in two transgenicSynechocystis was detected by RT-PCR, and their GFP fluorescence was detected by LaserScanning Confocal Microscope, both of which confirmed the expression of fusion proteinsunder psbA2promoter. The pictures by Laser Scanning Confocal Microscope imply that bothSLR0643and SLL0862are located in the thylakoid membranes.For functional characterization of gene sll0862, deletion mutant sll0862::cm wasconstructed by homologous recombination. Then the physiological and biochemicalproperties of the mutant sll0862::cm and sll0862::km which had been constructed in our labbefore were characterized. Under normal environment, no obvious difference was foundbetween wild type and two mutants. However, under high temperature, the he growth rate oftwo mutants was much slower than the wild type. Moreover, the WATER-PAM data showedthat the capacity of photosynthesis can hardly recover after30minutes of treatment at50℃or treatment of1.2mM H2O2, while the wild type is able to restore completely in24hours.These results indicate that SLL0862plays a key role in the response to high temperature andoxidative stress in Synechocystis sp. PCC6803, while provide some clues to futher researchthe function of SLL0862.