Mitochondrial Carriers for Serine, Glycine, Formate and Folate from Saccharomyces

应用这一建议的长期目标是了解真核生物中叶酸介导的一碳代谢的组织和区划。代谢区隔在真核生物中普遍存在，从最高级的哺乳动物到最低的单细胞形式，是正常细胞功能的关键方面。叶酸介导的一碳代谢的组织、划分和控制还知之甚少。由这笔赠款支持的以前的工作证明了酿酒酵母作为模型系统的实用性。我建议继续使用生化、分子遗传学和13C核磁共振技术，进一步剖析细胞质、线粒体和内质网之间一碳单位的流动。我们将分别使用嘌呤生物合成、线粒体蛋白质合成和甲基生物合成作为每个隔室中一碳代谢的指标。提出了四个具体的目标：(1)分离和鉴定编码AICAR转移酶/IMP环水解酶和5，10-亚甲基-四氢呋喃还原酶的酵母基因突变株。(2)研究MIS1、DFR1和ORF YBL03-11基因产物在线粒体一碳代谢中的作用。(3)研究ADE3基因产物c1-THF合成酶在嘌呤生物合成中的非催化功能。(4)确定一碳单位如何根据不同的生长和营养条件分配到三个主要池(CHO-THF、CH2-THF、CH3-THF)中。实验设计涉及(A)构建在特定的单碳反应中被阻断的酵母突变体；(B)对野生型和突变型菌株线粒体中甲酰甲硫基-tRNA水平的生化分析；(C)合成致死和双杂交筛选这些途径的其他成分；以及(D)对体内标记有富13C底物的野生型和突变型菌株进行核磁共振分析，以直接跟踪一碳单位的间隔流动。

ApplingThe long-term objective of this proposal is to understand the organization and compartmentation of folate-mediated one-carbon metabolism in eukaryotes. Metabolic compartmentation is found universally in eukaryotes, from the highest mammals to the lowest unicellular forms, and is a critical aspect of normal cell function. The organization, compartmentation, and control of folate-mediated one-carbon metabolism is poorly understood. Previous work supported by this grant demonstrated the usefulness of the yeast Saccharomyces cerevisiae as a model system. I propose to continue our use of biochemical, molecular genetic, and 13C NMR techniques to further dissect the flow of one-carbon units between the cytoplasm, mitochondria, and endoplasmic reticulum. We will use purine biosynthesis, mitochondrial protein synthesis, and methyl group biogenesis, respectively, as indicators of one-carbon metabolism in each compartment. Four Specific Aims are proposed: (1) Isolate and characterize mutants in the yeast genes encoding AICAR transformylase/IMP cyclohydrolase and 5,10-methylene-THF reductase. (2) Investigate the roles of MIS1, DFR1, and ORF YBL03-11 gene products in mitochondrial one-carbon metabolism (3) Investigate the noncatalytic function of the ADE3 gene product, C1-THF synthase, in purine biosynthesis. (4) Determine how one-carbon units are distributed into the three major pools (CHO-THF, CH2-THF, CH3-THF) in response to varying growth and nutritional conditions. The experimental design involves (a) construction of yeast mutants blocked in specific one-carbon reactions; (b) biochemical assays of formylmethionyl-tRNA levels in mitochondria from wild-type and mutant strains (c) synthetic-lethal and two-hybrid screens for additional components of these pathways; and (d) NMR analysis of wild-type and mutant strains labeled in vivo with 13C-enriched substrates to directly follow the intercompartmental flow of one-carbon units.