Elucidation of pyrimidine salvage and integration into nucleotide metabolism

核苷酸是每个生物体的中心分子，因为它们是核酸的基石，起着能量提供者、辅因子和信号的作用。核苷酸可以从头形成，并可以通过回收途径进行循环。因此，这两个过程基本上决定了细胞的核苷酸水平。尽管嘧啶从头合成的每一步都是由单个基因编码的，但尿嘧啶和尿苷的挽救是由蛋白质家族的成员促进的，而且只有很少的蛋白质被分析过。尿苷激酶和尿嘧啶磷酸核糖转移酶的可塑性异构体的突变体表现出严重的表型，强调了这些挽救反应的高度重要性。然而，来自胞质回收酶的结果是有些初步的，部分与自己的结果相矛盾，表明了胞质回收的主要作用，尽管到目前为止还完全没有被探索。因此，我们的目标是深入分析嘧啶在拟南芥中的挽救作用。为此，我们将在纯化蛋白的水平上揭示所有可能的抢救酶亚型的生化性质，并确定它们的亚细胞定位。此外，在存在或不存在有毒底物类似物的情况下，对无菌培养中相应突变体的分析将表明每种酶对嘧啶挽救的相对贡献。可能的蛋白质-蛋白质相互作用的可塑性抢救酶将分析共IP和随后的质谱学。由于作为一个整体，嘧啶代谢的亚细胞组织，中间产物的运输，特别是跨叶绿体被膜是必不可少的。然而，几个必需的转运蛋白仍然未知，因此，这些缺失的转运蛋白的鉴定和生化特性代表了这一提议的另一个目标。最后，我们将遵循一个假设，即嘧啶从头合成和挽救反应的基因在空间表达模式上不同，这意味着中间产物的细胞间运输。因此，我们将分析这两条通路中关键基因的细胞类型特异性表达。这一假说旨在解释在核苷酸从头合成中所谓的网状突变体的表型，并可能有助于我们理解为什么在拟南芥质膜上发现如此多的核苷酸碱基和核苷运输蛋白质。

Nucleotides are central molecules in every living organism as they represent building blocks for nucleic acids and function as energy providers, cofactors and signals. Nucleotides can be formed de novo and can be recycled by the salvage pathway. Thus, both processes essentially determine cellular nucleotide levels. Whereas each step in pyrimidine de novo synthesis is encoded by a single gene, salvage of uracil and uridine is facilitated by members of a protein family and only few of them have been analyzed. Mutants of plastidic isoforms of uridine kinases and uracil posphoribosyl transferases show severe phenotypes, underlining a high importance of these salvage reactions. However, results from plastidic salvage enzymes are somewhat preliminary and in part contradicting and own results indicate a major role for cytosolic salvage in addition, which is albeit completely unexplored up to now. Therefore, we aim to analyze pyrimidine salvage in Arabidopsis in depth. For this, we will unravel the biochemical properties of all putative salvage enzyme isoforms at the level of the purified proteins and determine their subcellular localization. In addition the analysis of corresponding mutants in axenic culture in presence or absence of toxic substrate analogs will be indicative for the relative contribution of each enzyme to pyrimidine salvage. Putative protein-protein interactions of plastidic salvage enzymes will be analyzed by co-iP and subsequent mass spectrometry. Due to the subcellular organization of pyrimidine metabolism as a whole, transport of intermediates especially across the plastid envelope is indispensable. However, several required transporters are still unknown and thus, the identification and biochemical characterization of these missing transport proteins represents a further goal of this proposal. Finally, we will follow a hypothesis where genes of pyrimidine de novo synthesis and salvage reactions differ in their spatial expression patterns implying intercellular transport of intermediates. Therefore, the cell type specific expression of key genes in both pathways will be analyzed. This hypothesis aims to explain the phenotype of so called reticulate mutants in nucleotide de novo synthesis and may foster our understanding why so many proteins for nucleobase and nucleoside transport are found at the Arabidopsis plasma membrane.