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>Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii
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Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii
n nnnYcf4 is a thylakoid protein essential for the accumulation ofphotosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandemaffinity purification tagged Ycf4 was used to purify a stableYcf4-containing complex of >1500 kD. This complex also containedthe opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC,PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquidchromatography–tandem mass spectrometry) and immunoblotting.Almost all Ycf4 and COP2 in wild-type cells copurified by sucrosegradient ultracentrifugation and subsequent ion exchange columnchromatography, indicating the intimate and exclusive associationof Ycf4 and COP2. Electron microscopy revealed that the largeststructures in the purified preparation measure 285 x 185 Å;these particles may represent several large oligomeric states.Pulse-chase protein labeling revealed that the PSI polypeptidesassociated with the Ycf4-containing complex are newly synthesizedand partially assembled as a pigment-containing subcomplex.These results indicate that the Ycf4 complex may act as a scaffoldfor PSI assembly. A decrease in COP2 to 10% of wild-type levelsby RNA interference increased the salt sensitivity of the Ycf4complex stability but did not affect the accumulation of PSI,suggesting that COP2 is not essential for PSI assembly.展开▼
机译：ABSTRACTn n
n TOP n <字体颜色= 464c53>抽象 n 介绍 n 结果 n 讨论 n 方法 n 参考文献 n n nnnYcf4是类囊体蛋白，对于积累 照片是必不可少的莱茵衣藻中的系统I（PSI）。在这里，串联的 亲和纯化标记的Ycf4被用于纯化> 1500 kD的稳定的 Ycf4的复合物。该复合物还包含 与视蛋白相关的COP2和PSI亚基PsaA，PsaB，PsaC， PsaD，PsaE和PsaF（通过质谱鉴定（液体 （SUP>色谱-串联质谱）和免疫印迹。 蔗糖 梯度超速离心和随后的离子交换柱共同纯化的野生型细胞中几乎所有的Ycf4和COP2 色谱，表明Ycf4和COP2的紧密和排他性关联 。电子显微镜显示，纯化的制剂中最大的 结构为285 x 185Å; ，这些颗粒可能代表几个 脉冲追踪蛋白标记显示，与含Ycf4的复合物相关的PSI多肽 是新合成的 ，并部分组装为这些结果表明，Ycf4复合物可能充当PSI组装的支架 。 RNA干扰将COP2降低至野生型水平的10％，从而提高了Ycf4 复合物稳定性的盐敏感性，但不影响PSI，的积累建议COP2对于PSI组装不是必需的。展开▼
Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan;
School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, United Kingdom;
Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan;
Lehrstuhl für Pflanzenphysiologie, Friedrich-Schiller-Universit?t Jena, 07743 Jena, Germany;
Lehrstuhl für Pflanzenphysiologie, Friedrich-Schiller-Universit?t Jena, 07743 Jena, Germany|Institut für Biochemie und Biotechnologie der Pflanzen, Westf?lische Wilhelms-Universit?t Münster, 48149 Münster, Germany;
Department of Life of Sciences, Faculty of Science and Engineering, Chuo University, Bunkyo, Tokyo 112-8551, Japan;
Departments of Molecular Biology and Plant Biology, University of Geneva, CH-1211, Geneva, 14 Switzerland;
Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan;
