Analyses on structure and function of the assembly apparatus of photosystem I complex

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Phototsystem 1 (PSI) involved in photosynthetic electron transport is a supercomptex consisting of more than ten subunits and one hundred cofactors. Littte is known on mechanisms for assembly of photosystem 1 complex, we have thus far revealed that the chtoroplast-encoded Ycf3 and Ycf4 are crucial for the assembly of PSI complex. In the present study, we purified a large complex containing Ycf4 (Ycf4-comptex) and characterized the polypeptide composition of the Ycf4 complex.Since the amount of Ycf4 in the thylakoid membranes is so low, we have generated chbropbst transformants of Chlamydomonas reinhardtii in which Ycf4 fused with TAP-tag accumulates. Using two-step affinity chromatogiaphy, Ycf4 complex was subsequently purified from the extracts of tnytekoid membranes and was subjected to SDS-PAGE to analyze its porypeptide composition. It was found that Ycf4 complex contains a large amount of 31 kDa polypeptide as well as TAP-tagged Ycf4. We have also determined the apparent molecular size of the Ycf4 complex by gel filtration column chromatography and estimated that Ycf4 complex has apparent molecular mass of more than 6 MDa. This observation strongly suggests that Ycf4 forms a complex with a number of copies of 32 kDa polypeptide.We will in the next step clone cDNA for this 32 kDa polypeptide. It is also necessary to purify furthermore Ycf4 complex to identify other minor polypeptide to elucidate entire structure of Ycf4 complex. The present research has revealed for the first time the molecular architecture of the assembly apparatus of PSI comptex.e