Molecular biological characterization of assembly apparatus for photosystem I complex

光系统I(PSI)是由10多个亚基和100多个辅因子组成的多蛋白复合体。这种复合体的功能和结构已经被广泛研究，而组成成分组装成功能复合体的情况在很大程度上是未知的。我们已经报道了绿藻Chlamydomonas reinhardtii中编码的YCF4蛋白是PSI复合体组装所必需的，YCF4是一个大的复合体的组成部分，暂定为YCF4-复合体。用蔗糖密度梯度离心法、DEAE柱层析法和凝胶过滤柱层析法对十二烷基麦芽糖苷增溶类囊体后的YCF4-复合体进行了纯化。除了这种经典的生化方法外，我们还通过衣藻的叶绿体转化产生了TAP(串联亲和纯化)标记的YCF4复合体。这一技术使我们能够通过两步亲和层析来纯化YCF4-络合物。对单独纯化的YCF4-络合物的多肽组成进行了比较研究，发现存在10多个亚基。据估计，YCF4-络合物的大小可达1800 kDa。我们目前正在更大规模地提纯YCF4-复合体，并将测定组成多肽的氨基酸序列。这些序列数据将用于寻找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.