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Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii^[W]

^a Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
^b School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, United Kingdom
^c Lehrstuhl für Pflanzenphysiologie, Friedrich-Schiller-Universität Jena, 07743 Jena, Germany
^d Institut für Biochemie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany
^e Department of Life of Sciences, Faculty of Science and Engineering, Chuo University, Bunkyo, Tokyo 112-8551, Japan
^f Departments of Molecular Biology and Plant Biology, University of Geneva, CH-1211, Geneva, 14 Switzerland

² Address correspondence to taka{at}cc.okayama-u.ac.jp.

Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of >1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography–tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wild-type cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures 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 polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.