Identification And Function Analysis Of A Novel Auxiliary Protein Nrf In Synechocystis Sp. PCC6803

It is well known that four types of photosynthetic membrane protein complexes were identified in the thylakoid membranes, including photosystem I (PSI), photosystem II (PSII), cytochrome b6f(Cytb6f) and ATPase. Two decades ago, the fifth photosynthetic membrane protein complex, NADPH dehydrogenase (NDH-1), was also identified in the cyanob acteial thylakoid membrane. The NDH-1complex is involved in a variety of bio-energy reactions, including cellular respiration, CO2uptake and cyclic electron transport around PSI. A general function of the complex is to transfer electrons from an electron donor to quinone, thereby generating a proton motive force used for ATP synthesis, which is required for the survival of cells under numerous environmental stress conditions. Recently, four types NDH-1complexes NDH-1L,-1L’,-IMS, and-IMS’were identified in cyanobacterial cells using proteomics strategy. All these four types NDH-1complexes are involved in NDH-CET. Desipte many important achievements have been made in subunit compositions and functions of the multuiple NDH-1complexes in cyanobacteira, the assembly of NDH-1complexes and related auxiliary proteins are still poorly understood.To identify the novel subunits or auxiliary proteins of NDH-1complex and assist in understanding the importance of cyanob acterial NDH-1complex, we isolated two high light-insensitive mutants after Synechocystis sp. PCC6803cells were transformed with a transposon-tagged library. Both mutants were tagged in the same nrf gene encoding an unknown protein. To confirm whether mutation of nrf is the result of the high light-resistance phenotype, we constructed an nrf deletion mutant. To test whether the high light-dependent phenotype of the mutants resulted from increased NDH-1-mediated cyclic electron transport around photosystem I (NDH-CET), we monitored the postillumination rise in chlorophyll a fluorescence, P700and P700+. The results confirmed that inactivation of nrf increased the activity of NDH-CET, resulting in the high light-resistance phenotype. The homologous gene of nrf is absent in the genome of Chlamydomonas reinhardtii, which lacks the chloroplast ndh genes using nucleic acid sequence analysis method. On the basis of the Nrf localization results, we conclude that Nrf is a cytoplasmic protein. Therefore, it may function as a novel auxiliary protein for NDH-1complexes. To prove this possibility, we compared the accumulation of Ndh subunits in total proteins or in the soluble fraction in WT and△nrf We also analyzed the assembly of NDH-1L and-1M complexes in the thylakoid membranes of WT and△nrf. The results suggested that inactivation of nrf improved the assembly of NDH-1complexes because of reduced the Ndh subunits amounts in the soluble fractions, but unchanged in the total proteins in△nrf compared with those in WT. Based on the results above, we may conclude that the soluble Nrf protein localizes in the cytoplasm and inhibits the efficient assembly of NDH-1L and-1M complexes. Furthermore, the Nrf protein contains TPR and PRP domains that they are involved in the protein-protein interaction. Therefore, we speculate that the protein Nrf may work in the assembly process of NDH-1complexes. However, the detailed mechanism underlying these reactions needs further investigation.In conclusion, this thesis identified a novel auxiliary protein, Nrf, and its inactivation increased the NDH-CET activity because of increased efficient assembly of NDH-1L and NDH-1M complexes.