水稻镁离子螯合酶调控蛋白Gun4羧基末端的功能分析

Functional Analysis of Carboxyl-terminal of Oryza sativa Gun4,a Regulatory Protein of Magnesium Chelatase

植物叶绿素生物合成途径(镁分支)中的第一个酶是镁离子螯合酶,它由I(ChlI)、D(ChlD)和H(ChlH)三个亚基组成.各亚基不仅参与叶绿素的生物合成,而且还与叶绿体到细胞核的反向信号传导有关.Gun4是一个卟啉结合蛋白,它能提高植物镁离子螯合酶的活性,在缺乏Gun4的条件下,镁离子螯合酶整个复合物非常不稳定,不足以开始催化反应.我们以前的研究发现,Gun4的C末端几个氨基酸具有重要作用.在本文中,通过缺失突变,获得了C端缺失了8个氨基酸残基突变的Gun4(Gun4L).酵母双杂交与GST-pull down实验发现,Gun4L仍能与H亚基相互作用,原核表达和纯化Gun4L和镁离子螯合酶各亚基后,重组镁离子螯合酶的酶活分析证实,Gun4L对镁离子螯合酶的活性失去了激活作用.

The first enzyme in the plant chlorophyll biosynthetic pathway （Mg2＋ branch） is magnesium chelatase. It consists of three subunits： I （ChlI）, D （ChlD） and H （ChlH）. Each subunit is involved not only in the chlorophyll biosynthesis, but also in plastid-to-nucleus retrograde signal transduction, Gun4 is a porphyrin-binding protein. It can activate the magnesium chelatase. In the absence of Gun4, the complex of magnesium chelatase is very unstable and cannot start the reaction. Our previous studies found that the C-terminal several amino acids of Gun4 are important. In this study, we removed the C- terminal 8 amino acids residues from Gun4 by deletion mutation and obtained the mutated Gun4L. Yeast two-hybrid system and GST-pull down assays showed that Gun4L can still interact with ChlH. After prokaryotic expression and purification of Gun4L and Mg-chelatase subunits, the reconstituted Mgchelatase activity assay demonstrated that Gun4L lost the activating ability on magnesium chelatase.