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。

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。

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

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》。