Cytoplasmic Protein CNAF1 Has A Dual Role In Assembly Of NDH-1 Hydrophilic Arm In Synechocystis Sp. Strain PCC 6803

NADPH dehydrogenase (NDH-1) complex located in thylakoid membrane is involved in various bioenergetic processes including cyclic electron transport around photosystem Ⅰ, cellular respiration and uptake of CO2. Assembly of NDH-1 requires a series of auxiliary proteins due to its complex structure composition. Little is known about these auxiliary factors. To isolate, identify and characterize these proteins can help us understanding molecular mechanism of assembly process of NDH-1 and its functional regulation. Thus, we screened transposon tagged mutant library of Synechocysits 6803 and successfully obtained two mutants which are tagged in the same gene location by transposons, cnaf1. cnaf1 encodes an unknown protein. We performed further research on this protein and results are shown below:1. Δcnaf1 grows more slowly than wild type (WT) whereas there is no evident difference between Δcnaf1 and WT under normal growth light. Chlorophyll fluorescence and kinetis of P700 redox analysis indicated NDH-CET activity was impaired in Δcnaf1. Immunoblotting analysis showed accumulation and assembly of NDH-1 in thylakoid membrane was evidently reduced in Δcnaf1.2. cNAF1 was confined to the soluble cytoplasmic fraction, thus it would not be a subunit of NDH-1, probably rather than an auxiliary protein which is involved in assembly of hydrophilic arm of NDH-1 in cytoplasm. We performed clear native (CN)-PAGE and two dimentional SDS-urea-PAGE to analyze the hypothetical NDH-1 assembly intermediates (NAIs). As in Aradopsis, there were three different NAIs with molecular masses about 300,130,100 kDa. cNAF1 was exclusively localized in NAI300, and NAI300 collapsed in Acnafl. These results indicated cNAF1 stabilized NAI300.3. We further investigated abundance of subunits of hydrophilic arm of NDH-1 in cytoplasm, which turned out that accumulation of NdhH, NdhK, NdhM were evidently increased in Δcnaf1. This was consistent with the above result that absence of cNAF1 collapsed NAI300, which impeded these subunits inserted into thylakoid membrane. Whereas NdhI was significantly reduced, and so was its auxiliary protein Slr1097, which was almost absent from Acnafl. Yeast two hybrid, GST-pull down and Co-IP analysis showed interaction between cNAF1 and Slr1097. These indicated cNAF1 stabilized Slrl 097.Above results showed that cNAF1 played a dual role in assisting assembly of hydrophilic arm of NDH-1...