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

植物中的四氢吡咯生物合成(TPB)由二十多个酶促步骤组成，由于光反应中间产物的合成及其最终产物叶绿素和血红素的时空需求不同，TPB受到严格控制。该提案旨在控制TPB途径开始时5-氨基乙酰丙酸(ALA)的限速合成，该合成由谷氨酰-tRNA还原酶(GluTR)和谷氨酸-1-半醛氨基转移酶(GSAT)两种酶执行。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、寡聚化和亚胞质分区水平上的平衡代谢流。这些目标是基于TTP1和TTP3的初步鉴定，这两个新的TPB辅助因子源于四肽重复(TPR)蛋白家族，由于与GluTR的相互作用，它们很可能被指定为控制ALA合成的功能。有人提议通过实验来揭示这两种TPR蛋白是否都是叶绿体中ALA合成酶稳定结合所必需的，要么是促进膜上ALA合成的失活(TTP1的假设作用)，要么是通过基质中的反馈控制实现修改的ALA合成(TTP3的建议作用)。这些研究包括TPB酶与其协同调控因子之间的物理相互作用研究，以及ttp1或ttp3单突变体和双突变体的鉴定，这些突变体包含TPB的其他已知调控因子的突变，如流感蛋白。最后，对ttp3和ttp3过表达系进行了生化分析，以寻找丙氨酸合成的额外调节联系，以及卟啉-TPB的合成和ttp3-相互作用的研究。总体而言，这些研究有望获得更多关于白天和夜间以及在光强变化期间ALA合成的复杂控制的知识。

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.