蓝细菌藻胆体蛋白磷酸化动态变化对光合适应的调控机制研究

Protein phosphorylation is discovered in all types of organisms and regulates a staggering number of cellular processes, including cell proliferation, metabolism, signal transduction, environmental acclimation et al. Phycobilisome is the light harvesting complex of cyanobacteria helping to attract light and transmit light. It was reported that most subunits of phycobilisome in Synechocystis PCC 6803 could be phosphorylated. And some other cyanobacterias were also reported to hold the phosphorylation of phycobilisome subunits, which seems to be relative conserved in cyanobacteria...We have ever reported 194 in vivo phosphorylation sites from 149 proteins in Synechocystis PCC 6803. Among all the identified phosphoproteins, the β subunits of phycocyanins (CpcBs) were found to be phosphorylated on four amino acids and the current results demonstrated that the phosphorylation status of CpcBs affect the state transition in Synechocystis PCC 6803. ..However, the conclusion obtained in our study just relied on only the physiology experiment. Actually, whether the phosphorylation affect the state transition in cyanobacteria has been discussed for decades and nothing about the regulation mechanism of the phycobilisome subunits’ phosphorylation level have been known. The phosphorylation of phycobilisome protein has also been reported to be involved in the degradation of phycobilisome complex, although this study depended on only the in vitro results. ..Based on these facts, in this project we will try to explore the role of protein phosphorylation in phycobilisome by focusing on their regulation during high light acclimaiton. We will at first examine the phosphorylation dynamics of phycobilisome protein during high light acclimation. Then to find out which kinase and phosphatase are responsible for the regulation of the phosphorylation dynamics of phycobilisome protein, a phosphatases and kinases mutant pool in Synechocystis PCC 6803 will be constructed. The phosphorylation level of phycobilisome protein in these corresponding mutants will be compared with the wild type. To turn out whether the phosphorylation of phycobilisome protein hold the role on state transition, phycobilisome degradation or the other photosynthetic mechanisms, the performance of these mutants in these aspects will be carefully watched. Moreover, NaF, a phosphatase inhibitor, will also be recruited to explore the function of phycobilisome phosomephosphorylation through inhibiting the dynamics of phosphorylation. Eventually, based on all the results obtained, the regulation as well as the function of the phycobilisome phosphorylation will be found out.

磷酸化修饰是一种普遍的、功能多样的蛋白质翻译后修饰。蓝细菌的捕光复合体，藻胆体上，大部分结构蛋白都被证明有磷酸化修饰，并且这些藻胆体蛋白磷酸化修饰在多种蓝细菌中都存在。申请人曾经在鉴定集胞藻PCC6803的磷酸谱的基础上，对藻胆体蛋白CpcB的磷酸化功能进行了初步研究，生理学研究结果表明CpcB的磷酸化可能影响了状态转化。.目前，关于藻胆体磷酸化参与调节状态转化还缺乏确定的分子生物学证据，并且有关藻胆体蛋白磷酸化的其他生物学功能的研究也存在疑义。本项目中，我们将以研究集胞藻强光后藻胆体蛋白磷酸化动态变化为切入点，构建磷酸酶和激酶突变体库，确定控制藻胆体蛋白磷酸化动态变化的酶，随后通过对比藻胆体磷酸化动态变化异常的突变株与野生型，同时观测添加抑制剂抑制磷酸化动态变化的影响，确定藻胆体蛋白磷酸化对相关光合响应（包括状态转化）的调控，并探索其中的具体机制，最终明确藻胆体蛋白磷酸化的生物学功能。

磷酸化修饰是一种普遍的、功能多样的蛋白质翻译后修饰。蓝细菌的捕光复合体，藻胆体上，大部分结构蛋白都被证明有磷酸化修饰，并且这些藻胆体蛋白磷酸化修饰在多种蓝细菌中都存在。而关于藻胆体磷酸化是否参与调节状态转化还存在较大争议，并且有关藻胆体蛋白磷酸化的其他生物学功能的研究也不清楚。本项目中，我们通过构建磷酸酶和激酶突变体库，随后通过对比藻胆体磷酸化动态变化异常的突变株与野生型，同时添加磷酸酶和激酶抑制剂抑制磷酸化动态变化，明确了在蓝藻中，完全不同于真核光合生物中，藻胆体磷酸化并不参与调节蓝藻的状态转化，未来对蓝藻状态转化分子机制的研究必须跳出真核光合生物状态转化已有结论的限制。我们的结果也显示，藻胆体非光化学淬灭机制也不依赖于藻胆体的磷酸化过程。而我们在探讨藻胆体非光化学淬灭机制与藻胆体磷酸化相互关系的过程中，意外发现了一个对藻胆体非光化学淬灭关键蛋白，OCP，的表达实行负调控的sRNA， 我们命名为ApcZ。ApcZ位于藻胆体编码基因apcABC操纵子3‘末端，能够结合ocp mRNA的RBS序列进而抑制其翻译，最终通过ApcZ的调节作用，蓝藻实现了藻胆体和藻胆体非光化学淬灭之间的协同调控。该发现填补了关于OCP蛋白水平调节机制认识的空白。