de Vitry C, Olive J, Drapier D, Recouvreur M, Wollman F A
Service de Photosynthèse, Institut de Biologie Physico-Chimique, Paris, France.
J Cell Biol. 1989 Sep;109(3):991-1006. doi: 10.1083/jcb.109.3.991.
We studied the assembly of photosystem II (PSII) in several mutants from Chlamydomonas reinhardtii which were unable to synthesize either one PSII core subunit (P6 [43 kD], D1, or D2) or one oxygen-evolving enhancer (OEE1 or OEE2) subunit. Synthesis of the PSII subunits was analyzed on electrophoretograms of cells pulse labeled with [14C]acetate. Their accumulation in thylakoid membranes was studied on immunoblots, their chlorophyll-binding ability on nondenaturating gels, their assembly by detergent fractionation, their stability by pulse-chase experiments and determination of in vitro protease sensitivity, and their localization by immunocytochemistry. In Chlamydomonas, the PSII core subunits P5 (47 kD), D1, and D2 are synthesized in a concerted manner while P6 synthesis is independent. P5 and P6 accumulate independently of each other in the stacked membranes. They bind chlorophyll soon after, or concomitantly with, their synthesis and independently of the presence of the other PSII subunits. Resistance to degradation increases step by step: beginning with assembly of P5, D1, and D2, then with binding of P6, and, finally, with binding of the OEE subunits on two independent high affinity sites (one for OEE1 and another for OEE2 to which OEE3 binds). In the absence of PSII cores, the OEE subunits accumulate independently in the thylakoid lumen and bind loosely to the membranes; OEE1 was found on stacked membranes, but OEE2 was found on either stacked or unstacked membranes depending on whether or not P6 was synthesized.
我们研究了莱茵衣藻中几个突变体的光系统II（PSII）组装情况，这些突变体无法合成PSII核心亚基之一（P6 [43 kD]、D1或D2）或放氧增强子（OEE1或OEE2）亚基之一。用[14C]乙酸脉冲标记细胞的电泳图谱分析了PSII亚基的合成。通过免疫印迹研究了它们在类囊体膜中的积累，通过非变性凝胶研究了它们的叶绿素结合能力，通过去污剂分级分离研究了它们的组装，通过脉冲追踪实验和体外蛋白酶敏感性测定研究了它们的稳定性，并通过免疫细胞化学研究了它们的定位。在衣藻中，PSII核心亚基P5（47 kD）、D1和D2以协同方式合成，而P6的合成是独立的。P5和P6在堆叠膜中彼此独立积累。它们在合成后不久或同时结合叶绿素，且与其他PSII亚基的存在无关。对降解的抗性逐步增加：从P5、D1和D2的组装开始，然后是P6的结合，最后是OEE亚基在两个独立的高亲和力位点（一个用于OEE1，另一个用于OEE2，OEE3与之结合）上的结合。在没有PSII核心的情况下，OEE亚基在类囊体腔中独立积累，并与膜松散结合；发现OEE1在堆叠膜上，但OEE2在堆叠或非堆叠膜上，这取决于P6是否合成。
