Molecular biological characterization of assembly apparatus for photosystem I complex

光系统I复合物组装装置的分子生物学表征

• 批准号: 11640649
• 负责人: TAKAHASHI Yuichiro
• 金额: $ 2.24万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11640649/
• 关键词: photosynthesis; photosystem; assembly; tagged protein; affinity chromatography; subunit compositon; chloroplast transformation; Chlamydomonas reinahrdtii; 光化学系複合体; 生合成; 葉緑体遺伝子; Ycf4タンパク; タンパクの精製; 葉緑体; 光化学系1; 形質転換; タンパク質工学; ヒスチジンタグ

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.

光系统 I (PSI) 是一种多蛋白复合物，由十多个亚基和一百多个辅助因子组成。该复合物的功能和结构已被广泛研究，但将组成成分组装成功能复合物的过程很大程度上未知。我们已经报道过，叶绿体编码的 Ycf4 蛋白对于绿藻莱茵衣藻中 PSI 复合物的组装至关重要，并且 Ycf4 是一个大型复合物的组成部分，该复合物暂定为 Ycf4 复合物。我们开发了一种在用十二烷基麦芽糖苷溶解类囊体后使用蔗糖密度梯度离心、DEAE 色谱法和凝胶过滤色谱法纯化 Ycf4-comple 的方法。除了这种经典的生化方法之外，我们还通过衣藻的叶绿体转化生成了 TAP（串联亲和纯化）标记的 Ycf4 复合物。这种 TAP 技术使我们能够通过两步亲和层析纯化 Ycf4 复合物。对独立纯化的 Ycf4 复合物的多肽组成进行的比较研究表明，存在十多个亚基。据估计，Ycf4-复合物的大小高达 1800 kDa。我们目前正在更大规模地纯化 Ycf4 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。

项目成果

Ohnishi, N., Takahashi, Y.: "PsbT polypeptide is required for efficient repair of photodamaged photosystem II reaction center"The Jounral of Biological Chemistry. 276. 33798-33804 (2001)

Ohnishi, N., Takahashi, Y.：“有效修复光损伤的光系统 II 反应中心需要 PsbT 多肽”《生物化学杂志》。

Hatano-Iwasaki, A., Minagawa, J., Inoue, Y and Takahashi, Y.: "Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a photosystem II reaction center protein, D1"Plant Molecular Biology. 42. 353-363 (2

Hatano-Iwasaki, A.、皆川 J.、井上 Y 和高桥 Y.：“光系统 II 反应中心蛋白 D1 加工受损的莱茵衣藻叶绿体 psbA 转化体的表征”植物分子生物学。

Hatano-Iwasaki,A.,Minagawa,J.,Inoue,Y.,and Takahashi,Y.: "Two distinct manganese clusters formed by introducing a mutation in the carboxyl-terminus of a photosystem II reaction center polypeptide, D1, of the green alga Chlamydomonas reinhardtii"Biochimica

Hatano-Iwasaki, A.、皆川 J.、井上 Y. 和高桥 Y.：“通过在光系统 II 反应中心多肽 D1 的羧基末端引入突变而形成两个不同的锰簇。

Ohnishi, N. and Takahashi, Y.: "PsbT polypeptide is required for efficient repair of photodamaged photosystem II reaction center"The Jounral of Biological Chemistry. 276. 33798-33804 (2001)

Ohnishi, N. 和 Takahashi, Y.：“有效修复光损伤的光系统 II 反应中心需要 PsbT 多肽”《生物化学杂志》。

Hatano-Iwasaki, A., Minagawa, J., Inoue, Y., Takahashi, Y.: "Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein, D1"Plant Molecular Biology. 4

Hatano-Iwasaki，A.，皆川，J.，井上，Y.，高桥，Y.：“莱茵衣藻叶绿体 psbA 转化体的表征，光系统 II 反应中心蛋白 D1 前体的加工受损”植物分子生物学