Posttranslational Regulation of Pep-Carboxylase Activity in Higher Plants

9315928 Chollet该实验计划由三个研究领域组成，即通过调节磷酸化和变构代谢物效应物控制高等植物体内磷酸烯醇式丙酮酸羧化酶（PEPC）活性。 具体的研究目标包括（i）通过使用整合的生物化学、免疫学和/或分子遗传学方法对完整的C4和CAM植物进行原位研究，继续仔细检查高度调节的PEPC激酶信号转导链，以及通过对完整的C4叶肉细胞和原生质体进行原位研究，进一步阐明复杂的C4 PEPC激酶信号转导链。 这些目标的结果可能会强烈影响我们对L-苹果酸（负变构效应物）调节PEPC活性的基本理解。 相反，项目（ii）将集中于由正磷酸化效应物（例如，葡萄糖6-P，丙糖-P），通过用光亲和标记和定点诱变技术定位该激活剂结合结构域。 最后，项目（iii）将严格评估可能的监管磷酸化的非光合PEPC在叶片和根瘤C3植物的综合体内（分离的叶片和根瘤），在原位（完整的叶原生质体），并在体外（分离的PEPC和PEPC激酶）分析。 从这三个项目的结果将不仅描绘的重要细节的控制C4-和CAM-光合作用，和一般的C/N代谢在植物细胞质PEPC水平的细节，但在同一时间将提供急需的洞察酶调节高等植物可逆的蛋白磷酸化。 本项目主要研究高等植物中二氧化碳固定酶磷酸烯醇丙酮酸羧化酶（PEPC）的翻译后调节。 虽然这种酶最为人所知的是其在C4光合作用和作物如玉米、高粱、甘蔗和菠萝（CAM）中的景天科酸代谢（CAM）期间的CO2固定中的作用，但它也在一般植物碳/氮代谢中起作用。 除了这些重要的生理和功能考虑之外，PEPC酶代表了高等植物中由蛋白磷酸酶和蛋白丝氨酸激酶的相反作用控制的酶的少数熟知的实例之一。 因此，这种复杂的调节磷酸化循环形成了我们目前许多研究的中心焦点。 了解这个循环是如何运作的，对于调节CO2同化和植物的新陈代谢都很重要。 它可以形成植物生物技术工程的基础，以提高生长和生产力。 该项目由MCB代谢生物化学计划和IBN植物综合生物学计划共同支持。 ***

9315928 Chollet The experimental plan is composed of three areas of research on the control of higher plant phosphoenolpyruvate carboxylase (PEPC) activity in vivo by regulatory phosphorylation and allosteric metabolite effectors. Specific research objectives include (i) the continued scrutiny of the highly regulated PEPC-kinase signal- transduction chain by in situ studies with intact C4 and CAM plants using an integrated biochemical, immunological and/or molecular- genetic approach, and the further elucidation of the complex C4 PEPC-kinase signal-transduction chain by in situ studies with intact C4-mesophyll cells and protoplasts. The outcome of these objectives could strongly affect our basic understanding of the regulation of PEPC activity by L-malate, the negative allosteric effector. Conversely, project (ii) will focus on the opposing control of C4/CAM PEPC activity exerted by positive phosphorylated effectors (e.g., glucose 6-P, triose-P) by mapping this activator binding domain with photoaffinity-labeling and site-directed mutagenesis techniques. Finally, project (iii) will critically assess the possible regulatory phosphorylation of nonphotosynthetic PEPC in leaves and root-nodules from C3 plants by integrative in vivo (detached leaves and nodules), in situ (intact leaf protoplasts), and in vitro (isolated PEPC and PEPC-kinase) analysis. Results from these three projects will not only delineate important details of the control of C4- and CAM- photosynthesis, and details of general C/N metabolism in plants at the level of cytosolic PEPC, but at the same time will provide much needed insight into enzyme regulation in higher plants by reversible protein phosphorylation. %%% This project is focused on the posttranslational regulation of the carbon dioxide (CO2)-fixing enzyme phosphoenolpyruvate carboxylase (PEPC) in higher plants. While this enzyme is best known for its role in CO2 fixation during C4 photosynthesis and Crassulacean acid metabolism (CA M) in crops as maize, sorghum, sugarcane, and pineapple (CAM), it also functions in general plant carbon/nitrogen metabolism. Besides these important physiological and functional considerations, the PEPC enzyme represents one of the few well- known examples in higher plants of an enzyme that is controlled by the opposing action of a protein phosphatase and a protein-serine kinase. Consequently, this complex regulatory phosphorylation cycle forms the central focus of many of our present studies. Understanding about how the cycle operates is important in regulation of CO2 assimilation and in plant metabolism in general. It can form the basis for biotechnological engineering of plants to improve growth and productivity. This project is supported jointly by Metabolic Biochemistry Program, MCB and the Plant Integrative Biology Program, IBN. ***