ORGANIZATION OF ONE-CARBON METABOLISM--A YEAST MODEL

描述：本提案的长期目标是了解 叶酸介导的一碳代谢的组织和区室化 在真核生物中。 代谢区室化普遍存在于 真核生物，从最高的哺乳动物到最低的单细胞形式， 正常细胞功能的一个重要方面。 该组织， 区室化和叶酸介导的一碳代谢的控制， 不太了解。 以前的工作支持这项赠款表明， 作为模型系统的酵母酿酒酵母的有用性。 的 申请人建议继续使用生物化学，分子遗传学， 和13 C NMR技术来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。 他将利用 嘌呤生物合成、线粒体蛋白质合成和甲基 生物起源，分别作为每个碳代谢的指标， 车厢 提出了四个具体目标：（1）分离和表征 编码AICAR转化酶/IMP环化水解酶的酵母基因中的突变体 研究MIS 1、DFR 1、5，10-亚甲基-THF还原酶在大肠杆菌中的作用 和ORF YBL 03 -11基因产物在线粒体一碳代谢中的作用;（3） 研究ADE 3基因产物C1-THF的非催化功能 合成酶，在嘌呤生物合成;（4）确定如何一个碳单位是 分布到三个主要合并液（CHO-THF、CH 2-THF、CH 3-THF）中， 对不同生长和营养条件的反应。 实验 设计包括：（a）构建酵母突变体， 一碳反应;（B）甲酰甲硫氨酰-tRNA水平的生物化学测定 野生型和突变株的线粒体中;（c）合成致死和 双杂交筛选这些途径的其他组分;以及（d）NMR 用13 C富集的13 C标记的野生型和突变体菌株的体内分析 底物直接跟随一碳的室间流动 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.