Regulation and Assembly of Pyruvate Dehydrogenase Complexes

光合作用、光呼吸作用和线粒体呼吸作用的一个主要相互作用点是由线粒体丙酮酸脱氢酶复合物（mtPDC）催化的反应。这种多酶复合物是碳进入克雷布斯循环的主要入口，用于能源生产和生物合成中间体的产生。PDC的质体异构体为脂肪酸生物合成、支链氨基酸、某些类异戊二烯和生长调节剂提供前体。mtPDC部分受一种新的特异性组氨酸样蛋白激酶（PDK）催化的多个丝氨酸残基的可逆磷酸化调控。目的：确定植物PDC蛋白E1亚基的磷酸化位点；在效应器和动力学方面表征重组PDKs；建立PDK异构体的表达与植物发育和环境影响的关系；在PDK一级序列中建立催化和结合必需的氨基酸；利用PDK过表达和过低表达的转基因植物，以及在玉米和拟南芥中鉴定PDK的“基因敲除”，确定PDK的生理作用；研究C4和C3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；研究PDC组份酶的体外组装及其进口组装。实验方法：1)从具有不同活性状态的复合物中纯化32p标记的E1亚基，并鉴定磷酸化丝氨酸。2)确定重组PDK的效应物和动力学。PDKisoforms的特异性杂交探针和抗体将用于测量发育和空间表达，以及对环境和营养线索的反应。4)将使用标准的定点诱变方法系统地改变PDK的特定氨基酸残基，并使用激酶缺失的PDC检测其活性。5)拟南芥将发生转化；将使用t -DNA标记的基因组DNA池来完成PDK和PDH的下调。6)体外组装研究将利用组分酶的亚基与各种伴侣系统相结合；进口和组装将使用纯化的豌豆线粒体和亚基前体。7)影响mtPDC可逆磷酸化的条件和代谢物将使用纯化的功能性线粒体、原生质体或整个组织来确定。意义：获得的结果将有助于阐明多种不同的分解代谢和合成代谢途径之间相互作用的控制。将确定一类独特的蛋白激酶的机制和结构要求。将确定影响PDK和PDC亚单位表达的因素。详细的合成，细胞器的进口，组装的PDC组件将被解决。最后，探讨了调控质体和mtPDC活性的机制。质体PDC可能是脂肪酸生物合成所必需的，并且在这个亚细胞区室中需要独特的调节特性。

A major point of interaction among photosynthesis, photorespiration and mitochondrial respiration is the reaction catalyzed by the mitochondrial pyruvate dehydrogenase complex (mtPDC). This multi-enzyme complex is the primary entry point for carbon into the Krebs cycle for energy production and the generation of biosynthetic intermediates. The plastid isoform of PDC provides the precursor for fatty acid biosynthesis, branched-chain amino acids, some isoprenoids and growth regulators. The mtPDC is regulated in part by reversible phosphorylation of multiple serine residues catalyzed by a novel and specific histidine-like protein kinase (PDK). Objectives: To establish the phosphorylation sites on the E1 subunits of plant PDC; to characterize recombinant PDKs with regard to effectors and kinetics; to establish the expression of PDK isoforms as function of plant development and environmental influences; to establish amino acids essential for catalysis and binding in the PDK primary sequence; to establish the physiological role of PDKs using transgenic plants over- and underexpressing PDK and by identifying "gene knockouts" of PDK in maize and Arabidopsis thaliana; to characterize events, mechanisms and modulators of PDC activity during development and photosynthesis in C4 and C3 plants; to investigate in vitro assembly of PDC component enzymes and their import and assembly. Experimental approach: 1) 32P-labeled E1 subunit will be purified from the complexes exhibiting various states of activity and the phosphorylated serines identified. 2) Effectors and kinetics of recombinant PDK will be determined. 3) Specific hybridization probes and antibodies for PDKisoforms will be used to measure developmental and spatial expression, andresponses to environmental and nutritional cues. 4) Standard site-directed mutagenesis methods will be used to systematically change specific amino acid residues of PDK, and activities will be assayed using kinase-depleted PDC. 5) A. thaliana will be transformed; down regulation of PDK and PDH will be accomplished usingT-DNA tagged genomic DNA pools. 6) In vitro assembly studies will utilize subunits of component enzymes in conjunction with various chaperone systems; import and assembly will utilize purified pea mitochondria and subunit precursors. 7) Conditions and metabolites that affect reversible phosphorylation of mtPDC will be determined using purified functional mitochondria, protoplasts or whole tissues. Significance: The results obtained will help clarify control of interactions among multiple distinct catabolic and anabolic pathways. The mechanisms and structural requirements of a unique class of protein kinases will be determined. Factors that affect expression of PDK and of the PDC subunits will be identified. Details of the synthesis, organellar import, and assembly of the components of PDC will be resolved. Finally, mechanisms for controlling the activities of plastid and mtPDC will be ascertained. Plastid PDC is likely to be essential for fatty acid biosynthesis, and unique regulatory properties are required in this subcellular compartment.