Albanese Pascal, Nield Jon, Tabares Jose Alejandro Muñoz, Chiodoni Angelica, Manfredi Marcello, Gosetti Fabio, Marengo Emilio, Saracco Guido, Barber James, Pagliano Cristina
Applied Science and Technology Department - BioSolar Lab, Politecnico di Torino, Viale T. Michel 5, 15121, Alessandria, Italy.
Department of Biology, University of Padova, Via Ugo Bassi 58 B, 35121, Padova, Italy.
Photosynth Res. 2016 Dec;130(1-3):19-31. doi: 10.1007/s11120-016-0216-3. Epub 2016 Jan 9.
In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-D-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12-39), as (CS) and (CS + CSM) megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.
在高等植物中，光系统II（PSII）是一种嵌入叶绿体类囊体膜中的多亚基色素蛋白复合体，在基粒中它大多以二聚体形式存在。其捕光天线系统LHCII由三聚体和单体复合体组成，它们可以不同数量与二聚体PSII核心复合体结合，从而形成不同类型的PSII-LHCII超级复合体。此外，PSII-LHCII超级复合体可以在类囊体膜平面内横向结合，进而形成更高分子量的复合体，即PSII-LHCII巨型复合体（Boekema等人，1999年a，发表于《生物化学》38卷：2233 - 2239页；Boekema等人，1999年b，发表于《欧洲生物化学杂志》266卷：444 - 452页）。在本研究中，通过快速单步增溶，使用去污剂正十二烷基-α-D-麦芽糖苷，从堆叠的豌豆类囊体膜中直接分离出纯的PSII-LHCII巨型复合体，随后进行蔗糖梯度超速离心。对这些巨型复合体进行了生化和结构分析。对负染色样品进行透射电子显微镜观察，随后进行单颗粒分析，结果显示与之前的研究（Boekema等人，1999年a，发表于《生物化学》38卷：2233 - 2239页；Boekema等人，1999年b，发表于《欧洲生物化学杂志》266卷：444 - 452页）相比，PSII-LHCII巨型复合体呈现出一种新的形式，它由两个在膜平面内并排排列的PSII-LHCII超级复合体组成，并与另一个拷贝夹在一起。这个巨型复合体的第二个拷贝很可能来自基粒堆叠的相对膜。观察到两种主要形式的完整夹心巨型复合体，并根据（Dekker和Boekema，2005年，《生物化学与生物物理学报》1706卷：12 - 39页）将其命名为（CS）和（CS + CSM）巨型复合体。通过应用基于凝胶的蛋白质组学方法，对分离出的巨型复合体的蛋白质组成进行了全面表征。总之，分离出的巨型复合体的新结构形式以及相关的建模研究为PSII-LHCII超级复合体不仅在膜平面内而且在叶绿体内的基粒堆叠之间如何相互结合提供了新的见解。
