Screening And Identification Of A Novel Subunit NdhS In The Cyanobacterial NDH-1Complex

Two decades ago, the fifth cyanobacterial photosynthetic membrane proteincomplex, NADPH dehydrogenase (NDH-1), was discovered in the thylakoidmembrane. This complex is involved in many biological energy reactions, andessential to CO2uptake, cyclic electron transport around photosystem I and cellularrespiration. The structure of cyanobacterial NDH-1complex is similar to E. coli andthe mitochondrial respiratory chain NADPH oxidoreductase (complex I), and all ofthem are "L-type". However, the homologous genes to three active subunits incatalytic center of the E. coli complex I are absent in cyanobacterial genome. Up tonow, therefore, little is know regarding the enzymatic characteristic of NDH-1complex in cyanobacteria. In the past few years, many significant progresses havebeen made, including the identification of17subunits (NdhA-Q) in cyanobacterialNDH-1L complex. However, the entire subunit composition of cyanobacterialNDH-1complex still remains elusive.To identify the novel subunits of NDH-1complex and understand the molecularcomposition and the importance of cyanobacterial NDH-1complex, we first obtainedtwo high light-sensitive mutants by screening a transposon-tagged library ofSynechocystis sp. strain PCC6803(hereafter Synechcoysitis6803). The reusltsshowed that both mutants impaired the rate of NDH-1-mediated CET (NDH-CET)and were tagged in the same ssl0352gene that encodes an unknown protein. To testwhether mutation of ssl0352is the result of the high light-sensitive phenotype, weconstructed and obtained its deletion mutant. The results of physiological activitesconfirmed that ssl0352mutation impaired the activity of NDH-CET, therebyresulting in the high light-sensitive phyenotype. Secondly, the homologous gene ofssl0352is absent in the genome of Chlamydomonas reinhardtii, which lacks the chloroplast ndh genes by using nucleic acid sequence analysis method. Further, therehas a co-migration of Ssl0352protein with the NDH-1L and NDH-1M complexes.Additionally, the Ssl0352protein possess of the characteristics of NDH-1subunit, forexamples, the thylakoid membrane localization, low abundance, independent ofphotosystem activities. Taking all these results together, we conclude that Ssl0352isa novel subunit of cyanobacterial NDH-1complexes and designate it NdhS. Finally,the assembly of the NDH-1L and NDH-1M complexes and their content in the cellswere not affected in the ndhS mutant. Furtehr, the NdhS protein contains a Srchomology3-like domain and might be involved in the interaction of NDH-1complexes with active domain. Therefore, it seems likely that deletion of NdhSprotein influences the interaction of NDH-1complex with active domain, therebyreducing the activity of NDH-CET. However, the detailed mechanism underlyingthese reactions needs further investigation.In conclusion, this thesis identified a novel subunit, NdhS, in NDH-1L andNDH-1M complexes of Synechocystis6803, and its inactivation impaired theNDH-CET activity. Such impairment does not the result from reduction in totalNDH-1proteins or disassociation of the NDH-1complexes.