ORGANIZATION OF ONE-CARBON METABOLISM--A YEAST MODEL

描述：这项建议的长期目标是了解 叶酸介导的一碳代谢的组织和区划 在真核生物中。新陈代谢区隔现象普遍存在于 真核生物，从最高级的哺乳动物到最低的单细胞形式，并且是 正常细胞功能的一个关键方面。该组织， 叶酸介导的单碳代谢的区隔作用和控制 人们对此知之甚少。这笔赠款支持的以前的工作演示了 酿酒酵母作为模式系统的有用性。这个 申请人建议继续使用生化、分子遗传学、 和13C核磁共振技术，以进一步剖析一碳单元的流动 在细胞质、线粒体和内质网之间。他将使用 嘌呤生物合成、线粒体蛋白质合成和甲基 生物发生分别作为一碳代谢的指示物 车厢。提出了四个具体目标：(1)分离和鉴定 酵母编码AICAR转移酶/IMP环水解酶基因的突变 和5，10-亚甲基-四氢呋喃还原酶；(2)研究MIS1、DFR1的作用 线粒体一碳代谢中的ORF YBL03-11基因产物； 研究ADE3基因产物C1-THF的非催化功能 合成酶，在嘌呤生物合成中；(4)确定一碳单位如何 分布在三个主要的池(CHO-THF，CH2-THF，CH3-THF)中 对不同的生长和营养条件做出反应。实验性的 设计涉及：(A)构建特定阻断的酵母突变体 一碳反应；(B)甲硫酰-tRNA水平的生化分析 野生型和突变菌株的线粒体中；(C)合成致死和 双杂交筛选这些通路的其他成分；和(D)核磁共振 ~(13)C标记野生型和突变型菌株的体内分析 底物直接跟随单碳的隔间流动 单位。

DESCRIPTION: The 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. The applicant proposes to continue the 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. He 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.