Elucidation of pyrimidine salvage and integration into nucleotide metabolism

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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.