Turkina Maria V, Kargul Joanna, Blanco-Rivero Amaya, Villarejo Arsenio, Barber James, Vener Alexander V
Division of Cell Biology, Linköping University, SE-581 85 Linköping, Sweden.
Mol Cell Proteomics. 2006 Aug;5(8):1412-25. doi: 10.1074/mcp.M600066-MCP200. Epub 2006 May 2.
Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.
莱茵衣藻光合膜中体内蛋白质磷酸化位点的图谱显示，磷酸化过程中主要的环境依赖性变化集中在光系统II（PSII）核心与其捕光天线（LHCII）之间的界面处。从暴露于影响光合作用的四种不同环境条件下的藻类细胞中分离出光合膜：（i）黑暗需氧，对应于光合状态1；（ii）氮气气氛下的黑暗，对应于光合状态2；（iii）适度光照；以及（iv）高光。通过胰蛋白酶从膜上切割表面暴露的磷酸化肽段，进行甲基酯化，通过固定化金属亲和色谱法富集，并通过纳米喷雾四极杆飞行时间质谱法测序。在莱茵衣藻中，对应于15个基因的蛋白质中共绘制了19个体内磷酸化位点。同时测定了7种蛋白质的氨基末端乙酰化。6种成熟LHCII蛋白质的测序氨基末端与预测的不同。从状态1到状态2的转变诱导了PSII核心成分D2和PsbR的磷酸化以及次要LHCII天线亚基CP29的四重磷酸化，以及主要LHCII复合物Lhcbm1和Lhcbm10成分的磷酸化。将藻类细胞暴露于适度或高光下会导致PSII核心的D1和CP43蛋白质发生额外的磷酸化。高光处理导致CP29在7个不同残基处发生特异性超磷酸化，另一个次要LHCII成分CP26在单个苏氨酸处发生磷酸化，以及包括Lhcbm4、Lhcbm6、Lhcbm9和Lhcbm11在内的主要LHCII复合物的其他亚基发生双重磷酸化。PSII核心与相关天线蛋白界面处的环境诱导蛋白质磷酸化，特别是CP29连接蛋白的多重差异磷酸化，提示了光合状态转变的控制机制以及在高光胁迫下LHCII与PSII解偶联以实现热能耗散的机制。
