COMPARTMENTATION OF ONE-CARBON METABOLISM--A YEAST MODEL

这项提议的广泛、长期目标是发展酵母 以酿酒酵母为真核模型研究酿酒酵母 叶酸介导的一碳代谢的区间化。新陈代谢 区隔作用在真核生物中普遍存在，从最高到最高 哺乳动物到最低的单细胞形式。代谢区隔是一种 正常细胞功能的关键方面：大量的人类疾病， 包括i-细胞病、假性赫勒多角体病、肌氨酸血症和 非酮症性高血糖，以错区化为特征 指酶或底物。细胞区隔也会影响 用药物治疗疾病。叶酸介导的一碳转移 辅酶在几个主要的细胞过程中起着至关重要的作用， 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 新陈代谢。然而，人们对分隔法知之甚少。 叶酸介导的一碳代谢。这个根本性的新陈代谢问题 将使用酵母分子遗传学和13C的组合来解决 核磁共振。这项建议的具体目标是：(1)完成一套 分离酵母菌阻断房室间代谢途径的研究 两种丝氨酸羟甲基转移酶编码基因的突变 同工酶和甘氨酸裂解系统；(2)应用~(13)C-核磁共振分析 野生型和突变型酵母菌株以表征一种- 细胞质和线粒体之间的碳单位以及 控制通量；以及(3)研究MTD1基因的作用，编码一种 NAD依赖的单功能亚甲基四氢叶酸脱氢酶 叶酸介导的一碳单位的区隔。实验性的 设计涉及(A)经典的遗传学和分子遗传学方法 酵母菌特异性反应阻断突变株的分离 以及(B)体内标记的野生型核磁共振分析。 模式菌株和突变菌株与富含13C的底物孵育。这些 实验应该能让我们确定每种酶的作用。 在细胞质和线粒体的隔间中； 线粒体一碳代谢在细胞质过程中发挥作用； 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和NADP)。

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).