METABOLIC COMPARTMENTATION IN NEUROSPORA

神经孢子虫的代谢区室

• 批准号: 3307112
• 负责人: RICHARD Louis WEISS
• 金额: $ 15.7万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307112
• 关键词: Neurospora; aminoacid biosynthesis; aminoacid metabolism; antibody; arginine; carbon nitrogen ligase; citrulline; cytoplasm; enzymes; eukaryote; gene complementation; gene deletion mutation; genetic recombination; intracellular transport; mitochondria; molecular cloning; mutant; organelles; ornithine; ornithine carbamoyltransferase; protein signal sequence; protein transport; urea cycle

The goal of this research project is to characterize the role of compartmentation in the regulation of amino acid metabolism in Neurospora crassa. The organellar localization of metabolic pathways requires considerable expenditure of metabolic energy for protein targeting, organelle assembly, and movement of substrates and products across intracellular membranes. Such expenditures must result in biological efficiencies commensurate with the investment of biological resources. However, it is not always clear what advantages are conferred by such compartmentation. Some general hypotheses have been advanced to explain this compartmentation phenomenon. These include the achievement of high localized concentrations of intermediary metabolites, the isolation of potentially reactive intermediates, the separation of potentially competing reactions and the conservation of solvent capacity. In N. crassa, the biosynthesis of arginine originates in the mitochondria but culminates in the cytosol. Intermediary metabolites (ornithine and citrulline) and the end product (arginine) cross both mitochondrial membranes: ornithine to support polyamine synthesis in the cytosol; citrulline to complete arginine synthesis in the cytosol; and arginine to support mitochondrial protein synthesis. In order to evaluate various hypotheses for the role of metabolic compartmentation, the metabolic and physiological consequences of relocating the arginine biosynthetic enzymes from the mitochondrial matrix to the cytosol will be investigated. Genes for the arginine biosynthetic enzymes leading to citrulline synthesis will be cloned and characterized. Amino acid sequences responsible for targeting the cytosolic precursors to the mitochondria will be identified. The cloned genes will be altered in vitro so that the catalytically active enzymes that they encode will be confined to the cytosol. Where possible, the functionality of the altered genes will be tested by inserting them downstream of a yeast promoter and testing their ability to complement mutations in the homologous yeast genes whose products normally function in the cytosol. The altered genes, arg*, or the homologous yeast (or bacterial) genes inserted downstream of a Neurospora promoter, will be introduced into the appropriate neurospora mutants. Strains carrying multiple altered genes will be constructed by genetic recombination. The consequences of enzyme relocation will be determined by examining the effects of enzyme mislocation on the efficiency of arginine biosynthesis, the channeling of carbamyl-phosphate and ornithine, and the operation of the potentially futile "urea cycle". The results will contribute to our understanding of the function of metabolic compartmentation in eukaryotic cells. Impairment or alterations in compartmentation are the basis of a variety of human disorders.

该研究项目的目标是表征 脉孢菌氨基酸代谢调节的区室划分 粗糙。 代谢途径的细胞器定位需要 蛋白质靶向消耗大量代谢能量， 细胞器组装以及底物和产物的移动 细胞内膜。 此类支出必须导致生物 效率与生物资源的投资相称。 然而，并不总是清楚这种方式带来了哪些优势 划分。 提出了一些一般性假设来解释 这种划分现象。 其中包括取得高成就 中间代谢物的局部浓度，分离 潜在的反应中间体，潜在的分离 竞争反应和溶剂容量的保存。 客栈。 crassa，精氨酸的生物合成起源于线粒体，但 在细胞质中达到顶峰。 中间代谢物（鸟氨酸和 瓜氨酸）和最终产物（精氨酸）穿过两个线粒体 膜：鸟氨酸支持细胞质中多胺的合成； 瓜氨酸在细胞质中完成精氨酸合成；和精氨酸 支持线粒体蛋白质合成。 为了评价各种 代谢区室作用的假设，代谢和 重新定位精氨酸生物合成的生理后果 从线粒体基质到细胞质的酶将 调查了。 精氨酸生物合成酶的基因导致 瓜氨酸合成将被克隆和表征。 氨基酸 负责将胞质前体靶向的序列 线粒体将被识别。 克隆的基因将被改变 体外，以便它们编码的催化活性酶将被 局限于细胞质中。 在可能的情况下， 改变的基因将通过将其插入酵母下游进行测试 启动子并测试它们补充突变的能力 同源酵母基因，其产物通常在细胞质中发挥作用。 改变的基因 arg* 或同源酵母（或细菌）基因 插入脉孢菌启动子的下游，将被引入 适当的神经孢子突变体。 携带多个改变基因的菌株 将通过基因重组构建。 酶的后果 将通过检查酶的影响来确定搬迁 错位对精氨酸生物合成效率的影响 氨甲酰磷酸和鸟氨酸，以及潜在的操作 徒劳的“尿素循环”。 结果将有助于我们的理解 真核细胞代谢区室的功能。 区隔的损伤或改变是多种疾病的基础 人类疾病。