Molecular biological characterization of assembly apparatus for photosystem I complex

光系统I（PSI）是一种多蛋白复合体，由10多个亚基和100多个辅因子组成。该复合物的功能和结构已被广泛研究，而组成成分组装成功能复合物在很大程度上是未知的。我们已经报道了绿宝石编码的Ycf 4蛋白对于绿色莱茵衣藻PSI复合物的组装是必需的，并且Ycf 4是一个大复合物的组分，该大复合物暂时被命名为Ycf 4-complex。利用蔗糖密度梯度离心、DEAE-柱层析和凝胶过滤柱层析等方法，对十二烷基麦芽糖苷增溶类囊体后的Ycf 4-comple进行了纯化。除了这种经典的生物化学方法，我们已经产生了TAP（串联亲和纯化）标记的Ycf 4复合体的叶绿体转化衣原体。这种TAP技术使我们能够通过两步亲和层析纯化Ycf 4-复合物。独立纯化的Ycf 4-复合物的多肽组合物的比较研究显示，存在十个以上的亚基。还估计Ycf 4-复合物的大小高达1800 kDa。我们目前正在更大规模地纯化Ycf 4复合物，并将确定组成多肽的氨基酸序列。这些序列数据将用于搜索cDNA克隆。

Photosystem I (PSI) is a multiprotein complex consisting of more than ten subunits and one hundred cofactors. Function and structure of this complex have been extensively studied while the assembly of the constituent components into a functional complex is largely unknown. We have already reported that the chloropalst-encoded Ycf4 protein is essential for the assembly of PSI complex in the green alga Chlamydomonas reinhardtii and that Ycf4 is a component of a large complex that is tentatively designated as Ycf4-complex. We have developed a purification procedure of Ycf4-comple after solubilization of thylakoids with dodecyl-maltoside using sucrose density gradient centrifugation, DEAE-chromatography and gel-filtration chromatography. In addition to this classical biochemical approach, we have generated TAP (tandem affinity purification)-tagged Ycf4 complex by chloroplast transformation of Chlamyodmonas. This TAP-technology enabled us to purify Ycf4-complex by two-step affinity chromatography. Comparative study of polypeptide compositions of Ycf4-complexes purified independently, revealed that more than ten subunits are present. It was also estimated that the size of Ycf4-complex is as large as 1800 kDa. We are currently purifying Ycf4-complex on a much larger scale and will determine amino acid sequence of the constituent polypeptides. These sequence data will be used for searching cDNA clones.