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.

该研究项目的目的是表征 在神经孢子中氨基酸代谢调节中的隔室 克拉萨。 代谢途径的细胞器定位需要 用于蛋白质靶向的代谢能量的大量支出， 细胞器组件，以及跨越底物和产品的运动 细胞内膜。 这样的支出必须导致生物学 效率与生物资源的投资相称。 但是，并不总是清楚这种优势得到了哪些优势 隔室。 提出了一些一般假设来解释 这种隔室现象。 这些包括高高的成就 中间代谢物的局部浓度，分离 潜在的反应性中间体，潜在的 竞争反应和溶剂能力的保护。 客栈。 Crassa，精氨酸的生物合成起源于线粒体，但 最终在细胞质中。 中间代谢物（鸟氨酸和 瓜氨酸）和最终产物（精氨酸）交叉两个线粒体 膜：鸟嘌呤以支持细胞质中的多胺合成； 瓜氨酸以完成细胞质中精氨酸的合成；和精氨酸 支持线粒体蛋白的合成。 为了评估各种 假设代谢区室的作用，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是 调查。 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 负责靶向胞质前体的序列 线粒体将被识别。 克隆的基因将在 体外，它们编码的催化活性酶将是 局限于细胞质。 在可能的情况下， 改变的基因将通过将酵母的下游插入来测试 促进者并测试其补充突变的能力 同源酵母基因的产物通常在细胞质中起作用。 改变的基因，Arg*或同源酵母（或细菌）基因 将插入神经孢子启动子的下游，将引入 适当的神经孢子突变体。 携带多个改变基因的菌株 将通过遗传重组构建。 酶的后果 搬迁将通过检查酶的作用来确定 精氨酸生物合成效率的错误分配，通道 氨基甲酰磷酸和鸟氨酸的作用，以及潜在的 徒劳的“尿素周期”。 结果将有助于我们的理解 真核细胞中代谢区分室的功能。 隔室的损害或变化是多样性的基础 人类疾病。