van Wijk K J, Roobol-Boza M, Kettunen R, Andersson B, Aro E M
Department of Biochemistry, Stockholm University, Sweden.
Biochemistry. 1997 May 20;36(20):6178-86. doi: 10.1021/bi962921l.
In previous studies [van Wijk, K. J., Bingsmark, S., Aro, E.-M., & Andersson, B. (1995) J. Biol. Chem. 270, 25685-25695; van Wijk, K. J., Andersson, B., & Aro, E.-M. (1996) J. Biol. Chem 271, 9627-9636], we have demonstrated that D1 protein synthesized in isolated chloroplasts and thylakoids is incorporated into the photosystem II (PSII) core complex. By pulse-chase experiments in these in vitro systems, followed by sucrose gradient fractionation of solubilized thylakoid membranes, it was shown that this assembly proceeded stepwise; first the D1 protein was incorporated to form a PSII reaction center complex (PSII rc), and through additional assembly steps the PSII core complex was formed. In this study, we have analyzed this assembly process in more detail, with special emphasis on the initial events, through further purification and analysis of the assembly intermediates by nondenaturing Deriphat-PAGE and by flatbed isoelectric focusing. The D2 protein was found to be the dominant PSII reaction center protein initially associating with the new D1 protein. This strongly suggests that the D2 protein is the primary "receptor" or stabilizing component during or directly after synthesis of the D1 protein. After formation of the D1-D2 heterodimer, cyt b559 became attached, whereas the psbI gene product was assembled as a subsequent step, thereby forming a PSII reaction center complex. Subsequent formation of the PSII core occurred by binding of CP47 and then CP43 to the PSII rc. The rapid radiolabeling of a minor population of a PSII core subcomplex without CP43 indicated that an association of newly synthesized D1 protein with a preexisting complex consisting of D2/cyt b55q/psbI gene product/CP47 was possibly occurring, in parallel to the predominant sequential assembly pathway. The kinetics of synthesis and processing of the precursor D1 protein were followed in isolated chloroplasts and were compared with its incorporation into PSII assembly intermediates. No precursor D1 protein was found in PSII core complexes, indicating either that incorporation into the PSII core complex facilitates the cleavage of the C-terminus or, more likely, that processing is more rapid than the assembly into the PSII core.
在之前的研究中[范·维克，K. J.，宾斯马克，S.，阿罗，E.-M.，& 安德森，B.（1995）《生物化学杂志》270，25685 - 25695；范·维克，K. J.，安德森，B.，& 阿罗，E.-M.（1996）《生物化学杂志》271，9627 - 9636]，我们已经证明在分离的叶绿体和类囊体中合成的D1蛋白被整合到光系统II（PSII）核心复合物中。通过在这些体外系统中进行脉冲追踪实验，随后对溶解的类囊体膜进行蔗糖梯度分级分离，结果表明这种组装是逐步进行的；首先D1蛋白被整合形成PSII反应中心复合物（PSII rc），并通过额外的组装步骤形成PSII核心复合物。在本研究中，我们通过非变性十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（Deriphat - PAGE）和平板等电聚焦对组装中间体进行进一步纯化和分析，更详细地分析了这个组装过程，特别关注初始事件。发现D2蛋白是最初与新合成的D1蛋白结合的主要PSII反应中心蛋白。这强烈表明D2蛋白是D1蛋白合成期间或刚合成后主要的“受体”或稳定成分。D1 - D2异二聚体形成后，细胞色素b559附着，而psbI基因产物在后续步骤中组装，从而形成PSII反应中心复合物。随后通过CP47然后CP43与PSII rc结合形成PSII核心。一个没有CP43的PSII核心亚复合物的少数群体的快速放射性标记表明，新合成的D1蛋白可能与由D2/细胞色素b559/psbI基因产物/CP47组成的预先存在的复合物结合，这与主要的顺序组装途径并行发生。在分离的叶绿体中追踪前体D1蛋白的合成和加工动力学，并将其与它整合到PSII组装中间体中的情况进行比较。在PSII核心复合物中未发现前体D1蛋白，这表明要么整合到PSII核心复合物中促进了C末端的切割，要么更有可能的是，加工比组装到PSII核心中更快。
