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

参与光合作用电子传递的光系统1(PSI)是由10多个亚基和100多个辅因子组成的超复合体。我们对光系统1复合体的组装机制知之甚少，到目前为止，我们已经揭示了叶绿体编码的YCF3和YCF4对于PSI复合体的组装是至关重要的。在本研究中，我们纯化了一个含有YCF4(YCF4-comtex)的大型复合体，并对其多肽组成进行了鉴定。由于YCF4在类囊体膜中的含量很低，我们产生了赖氏衣藻的chbropbst转化子，其中Ycf4与TAP-Tag融合积累。用两步亲和层析法从类固醇膜提取液中纯化了YCF4复合体，并对其多肽组成进行了SDS-PAGE分析。结果表明，YCF4复合体中含有大量的31 kDa多肽以及带有TAP标签的YCF4。我们还用凝胶过滤柱层析法测定了YCF4络合物的表观分子尺寸，并估算了YCF4络合物的表观分子质量大于6mda。这一观察结果强烈表明YCF4与多个32 kDa多肽形成了一个复合体，我们将在下一步克隆这一32 kDa多肽的cDNA。此外，还需要进一步纯化YCF4复合体，以鉴定其他次要多肽，以阐明YCF4复合体的完整结构。本研究首次揭示了PSI复合体组装装置的分子结构。

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