Analysis of further regulatory effects on the synthesis of 5-aminolevulinic acid by auxiliary factors in Arabidopsis thaliana

植物四吡咯生物合成（TPB）由20多个酶促步骤组成，由于光反应中间体的合成及其终产物叶绿素和血红素的不同时空需求，TPB受到严格控制。该提案解决了在TPB途径开始时控制5-氨基乙酰丙酸（ALA）的限速合成，该途径由两种酶谷氨酰-tRNA还原酶（GluTR）和谷氨酸-1-半醛氨基转移酶（GSAT）进行。ALA合成的翻译后控制在概念上是基于不同的机制，以确保TPB中参与酶的活性、稳定性、寡聚化和亚质体区室化水平上的平衡代谢流。这些目标是基于初步表征的TTP 1和TTP 3，TPB的两个新的辅助因子，来自家庭的tetratricopeptide-repeat（TPR）蛋白，这可能是最有可能被分配功能的ALA合成的控制，因为已被证明的相互作用与GluTR。实验提出，以解开是否TPR蛋白所需的ALA合成酶在叶绿体中的稳定协会，无论是促进失活的ALA合成在膜（一个假设的作用TTP 1）或实施修改后的ALA合成的反馈控制在基质中（一个建议的作用TTP 3）。这些研究包括TPB酶与其协同调节因子之间的物理相互作用研究，以及单ttp 1或ttp 3突变体和双突变体的表征，其中包含TPB的其他已知调节因子（如FLU蛋白）的突变。最后，对TTP 3和TTP 3过表达株系进行生物化学分析，以了解ALA合成与TPB和TTP 3-相互作用研究的卟啉合成的初始步骤之间的额外调节连接。总的来说，这些研究有望获得更多关于ALA合成在白天和夜间以及在变化的光强度期间的复杂控制的知识。

Tetrapyrrole biosynthesis (TPB) in plants consists of more than twenty enzymatic steps and is tightly controlled because of the synthesis of photoreactive intermediates and the different spatio-temporal demands of their end-products chlorophyll and heme. The proposal addresses the control of the rate-limiting synthesis of 5-aminolevulinic acid (ALA) at the beginning of the TPB pathway, which is carried out by the two enzymes glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase (GSAT). The posttranslational control of ALA synthesis is conceptually based on different mechanisms ensuring the balanced metabolic flow in TPB on the level of activity, stability, oligomerisation and subplastidal compartmentation of the participating enzymes. The objectives are based on the preliminary characterization of TTP1 and TTP3, two novel auxiliary factors of TPB derived from the family of tetratricopeptide-repeat (TPR) proteins, which can most likely be assigned functions in the control of ALA synthesis because of the proven interaction with GluTR. Experiments are proposed to unravel whether both TPR proteins are required for stable association of ALA synthesis enzymes in the chloroplasts, either to promote inactivation of ALA synthesis at the membrane (a hypothesized role for TTP1) or to implement a modified ALA synthesis by feedback-control in the stroma (a proposed role for TTP3). The studies include physical interaction studies between the TPB enzymes and their cooperating regulatory factors as well as the characterization of the single ttp1 or ttp3 mutants and double mutants, which contain a mutation for other known regulatory factors of TPB, such as the FLU protein. Finally, ttp3 and TTP3-overexpressing lines are biochemically analyzed for the additional regulatory connection of ALA synthesis with the initial steps of the porphyrin-synthesis of TPB and TTP3-interaction studies are intended. In general, these studies are expected to gain more knowledge on the complex control of ALA synthesis during day and night-time as well as during changing light intensities.