COMPARTMENTATION OF ONE-CARBON METABOLISM--A YEAST MODEL

一碳代谢的划分——酵母模型

• 批准号: 3421837
• 负责人: DEAN R APPLING
• 金额: $ 13.92万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3421837
• 关键词: 1 carbon compound; Saccharomyces cerevisiae; alternatives to animals in research; aminoacid metabolism; biological models; cofactor; cytoplasm; folate; formates; fungal genetics; genetic strain; glycine; hydroxymethyltransferases; intracellular transport; isozymes; metabolism; mitochondria; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; purine /pyrimidine metabolism; recombinant DNA; serine; tissue /cell culture

The broad, long-term objective of this proposal is to develop the yeast Saccharomyces cerevisiae as a eukaryotic model to study the compartmentation of folate-mediated one-carbon metabolism. Metabolic compartmentation is found universally in eukaryotes, from the highest mammals to the lowest unicellular forms. Metabolic compartmentation is a critical aspect of normal cell function: a great number of human diseases, including I-cell disease, pseudo-Hurler polydistrophy, sarcosinemia, and nonketotic hyperglycinemia, are characterized by miscompartmentalization of an enzyme or substrate. Cellular compartmentation also impacts the treatment of disease by drugs. One-carbon transfers mediated by folate coenzymes play essential roles in several major cellular processes, including nucleic acid biosynthesis, mitochondrial and chloroplast protein biosynthesis, amino acid metabolism, methyl group biogenesis, and vitamin metabolism. However, very little is known about the compartmentation of folate-mediated one-carbon metabolism. This fundamental metabolic problem will be addressed using a combination of yeast molecular genetics and 13C NMR. The specific aims of this proposal are to: (1) complete the set of metabolic blocks in the intercompartmental path way by isolating yeast mutants in the genes encoding the two serine hydroxymethyltransferase isozymes, and the glycine cleavage system; (2) apply 13C-NMR analysis in wild-type and mutant strains of yeast to characterize the flux of one- carbon units between cytoplasm and mitochondria and the reactions that control the flux; and (3) study the role of the MTD1 gene, encoding an NAD-dependent monofunctional methylenetetrahydrofolate dehydrogenase, in the compartmentation of folate-mediated one-carbon units. The experimental design involves (a) classical genetic and molecular genetic methods for the isolation of yeast mutants blocked in specific reactions of the intercompartmental pathway; and (b) NMR analysis of in vivo labeled wild- type and mutant strains incubated with 13C-enriched substrates. These experiments should allow us to determine the role of each of the enzymes in the cytoplasmic and mitochondrial compartments; the role that mitochondrial one-carbon metabolism plays in cytoplasmic processes; and the roles of the two cytoplasmic methylenetetrahydrofolate dehydrogenases with differing coenzyme specificity (NAD vs. NADP).

该提案的广泛、长期目标是开发酵母 酿酒酵母作为真核模型来研究 叶酸介导的一碳代谢的划分。代谢 区隔在真核生物中普遍存在，从最高层到 哺乳动物到最低的单细胞形式。代谢区室是 正常细胞功能的关键方面：大量人类疾病， 包括 I 细胞病、假性 Hurler 多营养不良、肌氨酸血症和 非酮症高甘氨酸血症，其特点是错室化 酶或底物的。 细胞区隔也会影响 通过药物治疗疾病。叶酸介导的一碳转移 辅酶在几个主要的细胞过程中发挥着重要作用， 包括核酸生物合成、线粒体和叶绿体蛋白 生物合成、氨基酸代谢、甲基生物发生和维生素 代谢。然而，人们对它们的划分却知之甚少。 叶酸介导的一碳代谢。这个基本的代谢问题 将结合酵母分子遗传学和 13C 来解决 核磁共振。本提案的具体目标是： (1) 完成一套 通过分离酵母来阻断室间途径的代谢 编码两种丝氨酸羟甲基转移酶的基因突变 同工酶和甘氨酸裂解系统； (2) 应用 13C-NMR 分析 酵母的野生型和突变株来表征一通量 细胞质和线粒体之间的碳单位以及反应 控制通量； (3) 研究 MTD1 基因的作用，该基因编码 NAD依赖性单功能亚甲基四氢叶酸脱氢酶， 叶酸介导的一碳单位的划分。实验的 设计涉及（a）经典遗传和分子遗传方法 酵母突变体的分离在特定反应中被阻断 室间通路； (b) 体内标记野生型的 NMR 分析 类型和突变菌株与富含 13C 的底物一起孵育。这些 实验应该让我们能够确定每种酶的作用 在细胞质和线粒体区室中；的角色 线粒体一碳代谢在细胞质过程中发挥作用；和 两种细胞质亚甲基四氢叶酸脱氢酶的作用 具有不同的辅酶特异性（NAD 与 NADP）。