COMPARTMENTATION OF ONE-CARBON METABOLISM--A YEAST MODEL

这项提案的广泛的长期目标是开发酵母 酿酒酵母作为真核生物模型研究 叶酸介导的一碳代谢的区室化。代谢 区室化在真核生物中普遍存在，从最高的 从哺乳动物到最低级的单细胞形式。代谢区室化是一种 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， 非酮性高甘氨酸血症的特征是错误的区室化 一种酶或底物。 细胞区室化也会影响 用药物治疗疾病。叶酸介导的一碳转移 辅酶在几种主要的细胞过程中起重要作用， 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、甲基生物合成和维生素 新陈代谢.然而，很少有人知道的划分， 叶酸介导的一碳代谢。这个基本的代谢问题 将使用酵母分子遗传学和13 C 匪r本建议的具体目标是：（1）完成一套 通过分离酵母， 编码两种丝氨酸羟甲基转移酶基因的突变体 同工酶和甘氨酸裂解系统;（2）将13 C-NMR分析应用于 酵母的野生型和突变株，以表征一种- 细胞质和线粒体之间的碳单位， 控制流量;（3）研究MTD 1基因的作用，编码一种 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区室化。实验 设计涉及（a）经典的遗传和分子遗传方法， 酵母突变体的分离阻断了特定的反应， （B）对体内标记的野生型- 型和突变株与富含13 C的底物孵育。这些 实验应该使我们能够确定每种酶的作用， 在细胞质和线粒体区室中的作用; 线粒体一碳代谢在细胞质过程中起作用;以及 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 vs. 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).