Elucidation of pyrimidine salvage and integration into nucleotide metabolism

阐明嘧啶补救和整合到核苷酸代谢中

• 批准号: 273384021
• 负责人: Dr. Torsten Möhlmann
• 金额: --
• 依托单位: Fachgebiet Pflanzenphysiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/273384021?language=en
• 关键词: Elucidation; pyrimidine; salvage; integration; into

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.

核苷酸是每个生物体中的中心分子，因为它们代表核酸的构建模块，并作为能量提供者，辅因子和信号发挥作用。核苷酸可以从头形成，并且可以通过补救途径再循环。因此，这两个过程基本上决定细胞核苷酸水平。而嘧啶从头合成的每一步都是由一个单一的基因编码，挽救尿嘧啶和尿苷是由一个蛋白质家族的成员，只有少数人进行了分析。尿苷激酶和尿嘧啶核糖转移酶的质体异构体突变体表现出严重的表型，强调了这些补救反应的高度重要性。然而，从质体补救酶的结果是有点初步的，部分矛盾和自己的结果表明，胞质补救的主要作用，此外，这是完全未探索到现在。因此，我们的目标是深入分析拟南芥中的嘧啶补救。为此，我们将在纯化的蛋白质水平上揭示所有推定的补救酶同工型的生化特性，并确定其亚细胞定位。此外，在存在或不存在毒性底物类似物的情况下，在无菌培养物中对相应突变体的分析将指示每种酶对嘧啶补救的相对贡献。将通过co-iP和随后的质谱法分析质体补救酶的推定蛋白质-蛋白质相互作用。由于整个嘧啶代谢的亚细胞组织，中间体的运输，特别是穿过质体包膜是必不可少的。然而，几种所需的转运蛋白仍然是未知的，因此，这些缺失的转运蛋白的鉴定和生化表征代表了本提案的进一步目标。最后，我们将遵循一个假设，即嘧啶从头合成和补救反应的基因在其空间表达模式不同，这意味着中间体的细胞间运输。因此，将分析两种途径中关键基因的细胞类型特异性表达。这一假说旨在解释核苷酸从头合成中所谓的网状突变体的表型，并可能促进我们理解为什么在拟南芥质膜上发现如此多的核碱基和核苷转运蛋白。