拟南芥叶绿体ATP合酶beta亚基的MAPK磷酸化及磷酸化对ATP合酶功能调节的机理研究

Adenosine 5’-triphosphate (ATP), the universal metabolic energy carrier that links catabolic and anabolic pathways, is involved in the regulation of the most biological processes during growth, development and response to environment in all organisms. ATP also acts as an extracellular signaling molecule. The majority of ATP in cells is synthesized by the enzymes of F1Fo-ATP synthase. ATP synthase is a protein complex with multiple subunits. The beta subunit of ATP synthase is responsible for catalyzing the synthesis of ATP. Several reports have revealed that the beta subunit of the mitochondrial and chloroplast ATP synthases can be phosphorylated. However, the kinase that involves in the phosphorylation of its beta subunit and the function of the beta subunit phosphorylation are currently unknown. Our preliminary data show that, MKK9-MPK3/MPK6 mediated MAPK cascade is involved in the phosphorylation of the beta subunit of chloroplast ATP synthase (namely as ATPB), and subsequently regulates ATP synthesis in Arabidopsis. In this project, we proposed to identify and analyze the upstream MKKKx of MKK9-MPK3/MPK6 during the phosphorylation of ATPB, and study the regulation of ATPB phosphorylation on function of chloroplast ATP synthase. The results obtained from this project will facilitate us to explore the kinase that phosphorylates ATPB and understand the mechanism of chloroplast ATP synthase regulation by ATPB phosphorylation.

ATP 是生物体重要的高能化合物，参与了细胞新陈代谢和生物体生长发育的几乎所有过程； ATP还是重要的胞外信号分子。细胞中绝大部分ATP是由ATP合酶(F1Fo-ATP synthase)合成。ATP合酶由多亚基组成，其中仅beta亚基具有催化活性。线粒体和叶绿体ATP合酶的beta亚基均有磷酸化修饰，但负责其磷酸化的激酶及磷酸化对其功能调控的研究很缺乏。项目组前期在拟南芥中的研究结果发现，MKK9-MPK3/MPK6参与的MAPK级联系统调控了叶绿体ATP合酶beta亚基（ATPB）的磷酸化和ATP的合成。本项目拟研究：（1）MKK9-MPK3/MPK6的上游MKKKx，构建一条参与ATPB磷酸化调控的完整MAPK三激酶级联系统；（2）该级联系统对ATPB磷酸化和磷酸化对叶绿体ATP合酶的调控。揭示磷酸化叶绿体ATPB的激酶及磷酸化对ATP合酶功能调控的机理。

ATP 是生物体重要的高能化合物，参与了细胞新陈代谢和生物体生长发育的几乎所有过程； ATP还是重要的胞外信号分子。细胞中绝大部分ATP是由ATP合酶(F1Fo-ATP synthase)合成，调控其合成的信号有待研究。ATP合酶由多亚基组成，其中仅beta亚基具有催化活性。线粒体和叶绿体ATP合酶的beta亚基均有磷酸化修饰，但负责其磷酸化的激酶及磷酸化对其功能调控的研究很缺乏。项目组有关拟南芥MKK9、MPK3和MPK6转基因和突变体植株的研究发现，MKK9-MPK3/MPK6的激活可以显著增加植株ATP的生成。酵母互作、生化分析及转基因植株分析发现，RAF22是激活MKK9-MPK3/MPK6的上游MKKK，该三激酶组成一个完整的MAPK级联参与调控ATP的生成。利用光系统II抑制剂及离体叶绿体的分析表明，该MAPK级联激活所诱导的ATP产生来源于叶绿体。通过酵母互作、Pull down及定位等分析，证明MPK3和MPK6具有叶绿体定位，能与ATPB互作；磷酸化分析显示，该MAPK级联激活能磷酸化叶绿体ATP合酶的ATPB亚基，鉴定出了ATPB上能被激活的MPK3和MPK6磷酸化的氨基酸位点；利用点突变方法获得模拟磷酸化和非磷酸化的ATPB，发现模拟ATPB磷酸化增强了其与相关亚基的互作；多亚基共表达分析发现，模拟磷酸化的ATPB表达促进CF1重组；植物中表达模拟磷酸化的ATPB促使叶绿体ATP合酶复合体的形成，提高ATP合酶的活性。根据项目研究结果我们提出：RAF22-MKK9-MPK3/MPK6组成完整级联是调控ATP合成的型号级联，该级联的激活通过磷酸化叶绿体ATP合酶的ATPB亚基，促进ATPB与相关亚基的作用和合酶复合体的形成，增强合成ATP的活性。项目的完成，为全面理解ATP合成信号调控及ATP合酶活性调节机制提供了新的理论依据。

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