Regulation of the Pyruvate Dehydrogenase Complex in Photosynthetic and Developing Plant Tissue

光合作用、光呼吸和线粒体呼吸之间相互作用的主要控制点是丙酮酸脱氢酶复合体(PDC)。本研究的目的是研究在C3、C4和CAM植物发育和光合作用过程中PDC活性的事件、机制和调节因子；扩展PDC的生化特性；建立体外和原位调节机制；研究PDC的长期调节；以及PDC组成酶的表达、它们的输入和复杂的组装。我们试图建立影响线粒体PDC可逆磷酸化的条件和代谢物，使用纯化的功能线粒体和抑制剂，改变代谢物水平和生理环境。抗体、同位素标记和酶活性测量将用于酶形式的量化。将使用标准技术克隆到PDC的PDH组件。进口和组装研究将利用我们对模型蛋白质和Hsc70伴侣系统的知识。该项目将有助于以下概念的评估：1)PDC的可逆磷酸化是碳进入Krebs循环的主要控制机制；2)PDC在发育过程中以及光合作用和非光合作用组织之间的调节机制不同；3)体外和原位调节机制在体内发挥作用；4)PDC的导入和组装需要监护人。

A major control point for the interaction among photosynthesis, photorespiration and mitochondrial respiration is the pyruvate dehydrogenase complex (PDC). The objectives of this research are to characterize events, mechanisms and modulators of PDC activity during development and photosynthesis in C3, C4, and CAM plants; extend biochemical characterization of PDC; establish in vitro and in situ regulatory mechanisms; examine the long term regulation of PDC; and study the expression of PDC's component enzymes, their import and complex assembly. We seek to establish conditions and metabolites that affect reversible phosphorylation of the mitochondrial PDC using purified, functional mitochondria coupled with inhibitors, altered metabolite levels and physiological environments. Antibodies, isotope labeling and enzyme activity measurements will be used to quantitate enzyme forms. Standard techniques will be used to clone to PDH components of PDC. Import and assembly studies will utilize our knowledge of a model protein and an Hsc70 chaperone system. This project will facilitate evaluation of the following concepts: 1) that reversible phosphorylation of PDC is a primary control mechanism for carbon entry into the Krebs cycle; 2) that PDC regulatory mechanisms differ during development and between photosynthetic and nonphotosynthetic tissues; 3) that the in vitro and in situ regulatory mechanisms function in vivo; 4) that import and assembly of PDC requires chaperones.