Identification And Function Analysis Of A Novel Subunit T1 Of Cyanobacterial NDH-1

NDH-1 is a kind of complex,which extensively exists in bacteria,cyanobacteria,higher plants,and mammals.Such complex is involved in the conversion of electric energy into chemical energy.NDH-1 originated from [Ni-Fe] hydrogenase,and its L-type structure is conserved during evolution.NDH-1 is localized in the thylakoid membranes in cyanobacteria,can mediate the cyclic electron transport around photosystem I,and produce an additional amount of ATP,thereby optimizing the ratio of ATP to NADPH and enhancing the efficiency of photosynthesis,which is crucial for cyanobacteria to resist the stresses of high light,high temperature,salt,drought and other environmental stresses.In recent years,a number of important progresses on cyanobacterial NDH-1 have been successfully achieved.Recently,the functions of the catalytically active subunits Ndh S and Ndh V have been identified and characterized.Ndh S contains a ferredoxin(Fd)-binding domain while Ndh V may be involved in stabilization the connection of Ndh S with Fd.It is also suggested that NDH-1 can form an NDH-1-PSI supercomplex with PSI by a linker protein Cpc G2.Besides Ndh S and Ndh V,chloroplast NDH-1also contains Ndh U and Ndh T in the catalytic domain.However,whether there are other components in the catalytic domain of cyanobacteria remains a subject of debate.Whose accepts the electron from Fd in cyanobacterial NDH-1 is not identified yet.In this study,we successfully identified a new component of NDH-1 catalytic domain from Synechocystis 6803 by screening a transposon-bearing library,and carrying out the biochemical and physiological experiments.Major results are summarized as follows:1)Two slow growth mutants under high light conditions were isolated and identified from Synechocystis 6803 transformed with a transposon-bearing library.Both mutants had the same tag in t1 by PCR analysis.Chlorophyll fluorescence measurements indicated that deletion of t1 impaired the NDH-1 activity.Therefore,we successfully obtained an NDH-1 activity-defective mutant.2)T1 co-migrates with NDH-1 in T.Elongate by using blue native(BN)-PAGE and protein blot.According to our previous studies,T1 also co-migrates with NDH-1-PSI supercomplex in Synechocystis 6803.Furthermore,the homologs are present in Chlamydomonas reinhardtii,which lacks the photosynthetic ndh genes.Therefore,we suggest that T1 may be a new subunit of NDH-1 and its absence impairs NDH-1 activity.3)Deletion of t1 significantly affected the accumulation of Ndh S and Ndh V,which were located in active domain of NDH-1,but did not affect the accumulation of hydrophilic arm and membrane arm in the thylakoid membrane by using SDS-PAGE with immunoblotting.However,the assembly of NDH-1L and NDH-1M were not affected in the mutant.It is similar to the results observed in ?ndh S and ?ndh V mutants.Moreover,T1 is easy to degrade,which is also similar to Ndh S and Ndh V.Therefore,we suggest that the T1 may be located in NDH-1 active domain,which is involved in NDH-1 activity via Ndh S and Ndh V subunits.In conclusion,we successfully identified a new component of NDH-1 active domain,its absence impaired NDH-1 activity via Ndh S and Ndh V subunits,thereby resulting in the high light sensitive phenotype.