Aggregation of the reaction cenfer-binding protein of photosystem 11 under light stress and cell death

光系统II （PSII）是各种非生物胁迫的目标，包括过量的可见光和高温。PSII的反应中心Dl蛋白在这些压力下容易受损，受损的蛋白要么被特定的蛋白酶降解，要么与附近的多肽如D2蛋白或CP43形成聚集体。在本研究中，我们利用菠菜类囊体和聚胞蓝藻PCC6803分析了光或热损伤Dl蛋白的降解和聚集途径的细节。主要结论如下：(1)菠菜类囊体在40℃高温胁迫30 min后，D1蛋白被破坏，受损的D1蛋白被FtsH蛋白酶降解。我们通过FtsH的溶解和重构实验证明了FtsH蛋白酶的作用。(2)热应激可诱导PSII附近的单线态氧和羟基自由基等活性氧。我们还发现，在热胁迫下，不仅D1蛋白，D2、PsbO和PsbQ蛋白也受到氧化损伤。热胁迫下PSII释放PsbO等外源蛋白。由于热应激在PSII附近引起了显著的脂质过氧化，我们推测热应激引起的脂质过氧化与PSII损伤有关。(3)在缺少FtsH的Synechocystis突变体PCC6803 （slr0228）中，与野生型细胞相比，过量光照诱导D1蛋白显著聚集。这些结果表明，FtsH蛋白酶降解D1聚集物，或者FtsH蛋白酶作为分子伴侣来避免蛋白质聚集。我们也在继续使用其他蛋白酶突变体进行研究。

Photosystem II (PSII) is the target of various abiotic stresses including excess visible light and high temperature. The reaction center Dl protein of PSII is damaged easily under these stresses and the damaged protein is either degraded by a specific protease(s) or forms aggregates with the nearby polypeptides such as D2 protein or CP43. In the present study, we analyzed the details of the degradation and aggregation pathways of the light-or heat-damaged Dl protein, using spinach thylakoids and the cyanobacterium Synechocystis PCC6803. The main points are as follows:(1) When spinach thylakoids were heat-stressed (40℃ for 30 min), the D1 protein was damaged and the damaged D1 protein was degraded by FtsH proteases. We demonstrated the action of FtsH proteases by the solubilization and reconstitution experiments of FtsH.(2) We found that heat stress induces reactive oxygen species, such as singlet oxygen and hydroxyl radicals near PSII. We also hind that under the heat stress, not only the D1 protein but also D2, PsbO and PsbQ proteins are oxidatively damaged. PsbO and other extrinsic proteins were released from PSII under the heat stress. As heat stress induced significant lipid peroxidation near PSII, we speculate that the heat-induced lipid peroxidation is related to the damage to PSII.(3) In the mutant of Synechocystis PCC6803 lacking FtsH (slr0228), excess light induced significant aggregation of the D1 protein, compared with the wild-type cells. These results indicate that the FtsH protease degrades the D1 aggregates, or alternatively that the FtsH protease acts as a molecular chaperone to avoid protein aggregation. We are continuing the study using other protease mutants as well.