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

光系统1（PSI）是一个由10多个亚基和100多个辅因子组成的超复合体，参与光合作用的电子传递。Littte等对光系统1复合体的组装机制有一定的研究，我们发现叶绿体编码的Ycf 3和Ycf 4在PSI复合体的组装中起着重要的作用。在本研究中，我们纯化了含有Ycf 4的大复合物（Ycf 4-comptex），并表征了Ycf 4复合物的多肽组成。由于Ycf 4在类囊体膜中的量是如此之低，我们已经产生了Ycf 4与TAP标签融合的莱茵衣藻chbropbst转化体。采用两步亲和层析法从类胰蛋白酶膜提取液中纯化Ycf 4复合物，并进行SDS-PAGE分析其多肽组成。发现Ycf 4复合物含有大量的31 kDa多肽以及TAP标记的Ycf 4。用凝胶过滤柱层析法测定了Ycf_4复合物的表观分子量，并估算了Ycf_4复合物的表观分子量大于6 MDa。这一发现有力地表明Ycf 4与许多拷贝的32 kDa多肽形成复合物。还需要进一步纯化Ycf 4复合物以鉴定其他次要多肽以阐明Ycf 4复合物的整个结构。本研究首次揭示了PSI复合体组装器的分子结构

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