Purification of oxygen-evolving photosystem II complex from a diatom and the binding properties of the extrinsic proteins

硅藻是一种单细胞光合藻类，分布在世界各地的海洋和淡水中，是水生生态系统和全球碳循环中浮游植物群落最重要的生产者之一。尽管它们具有重要意义，但对硅藻中的光系统II （PSII）知之甚少。在这项研究中，我们首次成功地从一种硅藻——薄壁毛藻中制备了类囊体膜和PSII颗粒，这些PSII颗粒保留了高的进化氧活性和所有的外部蛋白质。在没有CaC12的条件下，硅藻类囊体膜和PSII颗粒的出氧活性分别为250 μmol 02/mg Chl/h和850 μmol 02/mg Chl/h。PSII颗粒除含有CP47、CP43、02、D1、细胞色素b559等主要内在蛋白外，还含有岩藻黄素叶绿素a/c结合蛋白，估计每2个叶绿素分子的触角大小为229 Chl a。通过碱性tris处理，从硅藻PSII颗粒中释放出五种外源蛋白。在这5种外源蛋白中，4种蛋白为红藻型外源蛋白，分别为PsbO、PsbQ’、PsbV和PsbU，另1种为一种新型的假设蛋白。克隆并测序了编码新外源蛋白的基因。结果表明，该蛋白含有3个特异性先导序列，分别针对叶绿体内质网、叶绿体包膜和类囊体膜，这表明该蛋白编码于细胞核基因组中，是位于管腔侧的外源蛋白之一。通过外源蛋白与PSII的释放-重构实验，研究这5种外源蛋白的结合特性和功能。

Diatoms are unicellular photosynthetic algae found throughout the world's oceans and freshwater, and constitute one of the most important producers of the phytoplankton communities in aquatic ecosystems and the global carbon cycle. In spite of their significance, little is known about Photosystem II (PSII) in diatoms. In this study, we succeeded for the first time in the preparation of thylakoid membranes and PSII particles retaining a high oxygen-evolving activity and all of the extrinsic proteins from a diatom, Chaetoceros gracilis. The diatom thylakoid membranes and PSII particles showed an oxygen - evolving activity of about 250 and 850 μmol 02/mg Chl/h in the absence of CaC12, respectively. The PSII particles contained fucoxanthin chlotophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, 02, D1, cytochrome b559, and the anntena size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were 〓oichimoterically released from the diatom PSII particles by alkaline Tris-treatment.Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. The gene encoding the novel extrinsic protein was cloned and sequenced. The deduced proteins contained three characteristic leader sequences targeted for chloroplast endoplasmic reticulum, chloroplast envelope and thylakoid membrane, respectively, indicating that the novel protein is encoded in the nuclear genome and constitutes one of the extrinsic proteins located in the lumenal side. The binding properrties and functions of the five extrinsic proteins will be examined by release-reconstitution experiments of their extrinsic proteins with PSII.