Identification Of PPIase Protein Mutants And Functional Characterization Of Immunophillin CYN28 In Chlamydomonas

Photosynthesis,the process by which photosynthetic organisms transform light energy into chemical energy.Light energy is captured and used to convert water and carbon dioxide into oxygen and energy-rich organic compounds.This process is the key to the survival of all organisms and an important means for maintaining the carbon-oxygen cycle on the earth.Peptidyl-prolyl cis-trans isomerase(PPIase)is thought to be essential for protein folding during protein synthesis in cells.PPIase proteins play certain roles in plant growth and development,stress resistance and photosynthesis.PPIase proteins are also involved in the assembly and repair of photosystem ?(PS?).In Arabidopsis thaliana,PPIase proteins such as At CYP20-3,At CYP38,At FKBP20-2 have been reported to participate in the biogenesis of PS?,but the specific mechanism is unclear.Chlamydomonas reinhardtii is a single-celled green algae with a fast growth cycle,clear genetic background,and capable of photosynthetic heterogeneous growth.These are the advantages of studying eukaryotic photosynthesis,but PPIase proteins in Chlamydomonas reinhardtii have not systematically been researched.The target protein in this study involves three family members of PPIase proteins: CYPs,FKBPs and parvulins.The selected immunophilin proteins in this article are located in the thylakoid lumen of chloroplast,including A(CYN28),B,C,D,E and F is selected as the parvulin.Firstly,we identify the inserted position of the PPIase mutants selected above,next identify the accumulation of transcripts of the mutants,and select the non-expressed or weakly expressed mutants for phenotypic analysis;finally we find that growth phenotype in cyn28 is the most obvious under the high light.So this mutant is highly light-sensitive,which is used for a focus material for the further research.The main findings are as follows:(1)It was further confirmed that cyn28 is a mutant which was significantly inhibited under high light.The results of western blot and BN-2D experiment showed that PS? core subunit D1 and PS? supercomplex were significantly reduced in cyn28.The analysis of the complemention can preliminarily determine that CYN28 is involved in the biogenesis of PS?.Immunophilin protein CYN28 is conserved in the evolutionary process.In vitro PPIase activity assay showed that CYN28 still has enzyme activity even when it contains a reduced number of PPIase key residues.(2)We constructed the complementary vector of CYN28 with GFP tag,and the GFP antibody was used to capture the interacting protein of CYN28 from Chlamydomonas cells via Co-IP.The Co-IP samples of CYN28-GFP were analyzed by mass spectrometry.It was found that FtsH1/2,a key protease involved in the repair of PS?,were significantly enriched.Co-IP,pull down,and yeast two-hybrid experiments all confirmed that the N-terminal sequences of FtsH1/2 are interacted with CYN28 on the side of thylakoid lumen.(3)To explore the physiological significance of the interaction between the CYN28 and FtsH,our results showed that the accumulation of FtsH1/2 in the cyn28 was significantly decreased under high light.To further explore the reasons for the decrease of FtsH content in cyn28 under high light,the cytoplasmic translation inhibitor cycloheximide was used to treat the whole cell protein under high light to observe whether there is any difference in FtsH stability between cyn28 and wild type cells.The results showed that CYN28 protein participates in the turnover of FtsH under high light to repair damaged photosystem ? and further helps Chlamydomonas cells to resist high light stress.(4)The other PPIase mutants,b,c,d,e,and f were also preliminarily analyzed in this study,among which mutant b was weakly expressed and the others were not expressed.So far,B,D,E and F have completed the constructions of complemention with their native promoter successfully,and B and E have been screened for the transformants.