| Photosynthesis is the most important chemical reaction on the earth.In the light reactions of photosynthesis,however,linear electron transport from water to NADP+does not satisfy the ATP/NADPH production ratio required by the Calvin-Benson cycle and photorespiration.The stoichiometric balancing of ATP and NADPH must occur rapidly because the pool sizes of ATP and NADPH are relatively small and fluxes through primary metabolism are large.Among the mechanisms proposed to account for the balancing of the ATP/NADPH production,PSI cyclic electron flow has been proposed to be the most prominent candidate for the regulator of the ATP/NADPH ratio.In PSI cyclic electron transport,electrons are recycled from the stromal reducing pool to the plastoquinone(PQ)pool,generating ApH and consequently ATP without accumulation of NADPH.Thus,depending on the environmental stress or physiological conditions,the ATP/NADPH ratio can be adjusted to the required levels by tuning the ratio of linear electron transport to PSI cyclic electron transport.In cyanobacteria,the main route for CEF involves type I NADPH dehydrogenase complex,namely,NDH-1 complexes.Cyanobacterial NDH-1 complexes are localized in the thylakoid membrane and belong to the complex I family.On the basis of sequence similarity analysis,the complex I family was suggested to originate from a common ancestor.During evolution,however,respiratory NDH-1 and photosynthetic NDH-1 have developed different catalytic activities.The former has become equipped with a new N module consisting of three subunits and capable of oxidizing NADH,and the latter,as was recently suggested,has retained an original electron input module,which accepts electrons from Fd.Structurally,respiratory NDH-1 and photosynthetic NDH-1 contain a conserved L-shaped skeleton,but numerous oxygenic photosynthesis-specific(OPS)subunits were added to the photosynthetic NDH-1.The addition of these seven OPS Ndh subunits to cyanobacteria during evolution might be closely associated with the participation of NDH-1 in photosynthetic reactions such as NDH-CEF.Then,researching these OPS subunits is important to understand the function and evolution of photosynthetic NDH-1.Additionally,in consideration of the only existence in oxygenic photosynthetic organisms,comprehending the feature of OPS subunits plays a necessary role in understanding the relationship of NDH-1 and photosynthesis and the concept of photosynthetic membrane-bound complexes of NDH-1.In hence,identification and functional analysis of new OPS Ndh subunits will be essential to clarify the catalytic mechanism of this enzyme in cyanobacteria.In this study,we successfully identified a new OPS subunit by screening transposon-bearing library and so on.Major results are summarized as follows:(1)Two growth slowly mutants at high light conditions were isolated and identified from 6803 transformed with a transposon-bearing library.Both mutants had a tag in ndm1 by PCR analysis.Then we constructed ndm1-deletion mutant by replacing the entire ndm1 coding region with a KamR.Deletion of ndm1 impaired NDH-CEF activity and destabilized the NDH-1 complexes.Therefore,we successfully obtained an NDH-CEF activity impaired mutant.(2)The result of bioinformatics analysis of NDM1 and its homologies suggested that NDM1 protein contained two transmembrane helixes and was widespread in oxygenic photosynthetic organisms,except Chlamydomonas reinhardtii,which had no NDH-1 complexes.Also,NDM1 protein was co-migrated with Ndh subunits in BN-PAGE.Therefore,we suggested NDM1 is a new OPS subunit of NDH-1 complexes.(3)The result of yeast two-hybrid system assay revealed that NDM1 interacts with NdhA.In the thylakoid membranes of ?ndm1,the amount of NdhA was decreased markedly.Taken together,we concluded the the new membrane-bound OPS subunit NDM1 stabilize the NDH-1 complex via interacting with NdhA.From the above,we successfully screened and identified a new OPS subunit of cyanobacterial NDH-1 complexes,and uncovered itsfunctions and mechanism. |
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