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

丙酮酸脱氢酶复合体（PDC）是光合作用、光呼吸作用和线粒体呼吸作用相互作用的主要控制点。本研究的目的是表征C3、C4和CAM植物发育和光合作用过程中PDC活性的事件、机制和调节剂；扩展PDC的生化表征；建立体外和原位调节机制；检查PDC的长期调节；研究PDC组分酶的表达、进口和复合物组装。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、功能性的线粒体结合抑制剂、改变的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定量酶的形式。将使用标准技术克隆PDC的PDH组件。进口和组装研究将利用我们的模型蛋白和Hsc70伴侣系统的知识。该项目将有助于评估以下概念：1)PDC的可逆磷酸化是碳进入克雷布斯循环的主要控制机制；2) PDC调控机制在发育过程中以及光合与非光合组织间存在差异；3)体外和原位调控机制在体内的作用；4) PDC的进口和装配都需要陪护人员。

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.