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

光系统I复合体组装装置的结构与功能分析

• 批准号: 13640651
• 负责人: TAKAHASHI Yuichiro
• 金额: $ 2.11万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13640651/
• 关键词: photosyrthesis; photosystem 1; assembly; chloroplast; Ycf4 protein; tagged protein; affinity chromatography; green alga Chlamydomonas reinahrdtii; Ycf4タンパク質; 緑藻クラミドモナス; 光化学系; 形質転換; タグ融合タンパク質

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

参与光合作用电子传递的光系统1（PSI）是一个由十多个亚基和一百多个辅助因子组成的超级复合体。 Littte 熟知光系统 1 复合体的组装机制，到目前为止，我们已经揭示了叶绿体编码的 Ycf3 和 Ycf4 对于 PSI 复合体的组装至关重要。在本研究中，我们纯化了一个含有Ycf4的大型复合物（Ycf4-comptex），并表征了Ycf4复合物的多肽组成。由于类囊体膜中Ycf4的含量如此低，我们产生了莱茵衣藻的chbropbst转化体，其中与 TAP 标签融合的 Ycf4 不断积累。随后使用两步亲和色谱法从 tnytekoid 膜提取物中纯化 Ycf4 复合物，并进行 SDS-PAGE 分析其多肽组成。结果发现Ycf4复合物含有大量31 kDa的多肽以及TAP标记的Ycf4。我们还通过凝胶过滤柱色谱测定了Ycf4复合物的表观分子大小，并估计Ycf4复合物的表观分子量超过6MDa。这一观察结果强烈表明，Ycf4 与许多 32 kDa 多肽拷贝形成复合物。我们将在下一步中克隆该 32 kDa 多肽的 cDNA。还需要进一步纯化Ycf4复合物以鉴定其他次要多肽以阐明Ycf4复合物的完整结构。本研究首次揭示了PSI comptex.e组装装置的分子结构

项目成果

Yuichiro Takahashi: "UNESCO-EOLSS(Encyclopedia of Life Support System)"Genome science of cyanobacteria, green algae and plants (Genome science of green algae)(印刷中).

高桥雄一郎：《UNESCO-EOLSS（生命支持系统百科全书）》蓝藻、绿藻和植物的基因组科学（绿藻基因组科学）（出版中）。

Michael Hippler, Blandine Rimbault, Yuichiro Takahashi: "Photosynthetic complex assembly in Chlamydomonas reinhardtii"Protist. 153. 197-220 (2002)

Michael Hippler、Blandine Rimbault、Yuichiro Takahashi：“莱茵衣藻中的光合复合体组装”原生生物。