REGULATION & COMPARTMENTATION IN PROLINE METABOLISM

规定

• 批准号: 3298637
• 负责人: MARJORIE C BRANDRISS
• 金额: $ 16.85万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298637
• 关键词: Saccharomyces; aminoacid metabolism; biotechnology; gene expression; genes; genetic manipulation; genetic regulation; mitochondria; molecular cloning; molecular genetics; nucleic acid sequence; proline

The overall goal of this research proposal is to understand the various aspects of proline metabolism in Saccharomyces cerevisiae as a model system for the integration of a complex series of interactions that involves the nuclear, cytoplasmic and mitochondrial compartments within the cell. Proline biosynthesis and proline utilization involve a series of reactions which have a common intermediate and pools of substrates and products that must move between the mitochondrial and the cytosolic compartments. It is our objective to learn what roles the identified regulatory proteins play, the importance of the compartmental separation, how the important metabolites enter and leave the mitochondrion and whether the biosynthetic and degradative pathways communicate with one another via regulatory proteins or small molecules. An appreciation for how the basic metabolic processes work will enable us to understand and treat situations in which aberrations in regulation occur. This proposal addresses issues involved in the regulation of proline degradation and biosynthesis and the role of compartmentation in proline metabolism. The positive regulatory element of the proline utilization pathway, the PUT3 gene, will be cloned and analyzed at the molecular level. This will involve studies to determine the regulation of PUT3 gene expression, the phenotype of a null mutation, and interactions the regulator has with other elements in the pathway. Three types of gene fusions (PUTl-galK, PUTl-lacZ and PUT2-lacZ) will be used to isolate mutations in other positive or negative trans-acting regulators that affect proline utilization. Clones of the three structural genes of the proline biosynthetic pathway will be studied to determine whether they are controlled by proline-specific and/or general amino acid control regulatory elements using Northern analysis of mRNAs from the wild-type, proline auxotrophic, and general control defective strains. Physical interactions (e.g. complex formation) and subcellular localization of the enzymes will be studied by enzymatic assays as well as by immunoprecipitation using antibodies generated against purified proteins. The importance of the cellular compartments in proline metabolism will be studied by perturbing their normal locations, using genetic engineering techniques to move a cytoplasmic proline biosynthetic enzyme into the mitochondrion and the mitochondrial proline degradative enzymes into the cytoplasm. The transport of proline into the mitochondrion will be studied by adapting techniques used in higher cells to measure metabolite flux: osmotic swelling of mitochondria and centrifugal filtration through oil. The effects of stereospecificity, energy uncouplers, proline analogs and protein inhibitors on proline transport will be determined.

本研究计划的总体目标是了解 酿酒酵母中脯氨酸代谢的各个方面 作为一个模型系统的一系列复杂的集成， 相互作用，涉及核，细胞质和 细胞内的线粒体区室。 脯氨酸生物合成 和脯氨酸利用涉及一系列反应， 一个共同的中间体和底物和产物的池， 必须在线粒体和胞质之间移动。 我们的目标是了解已确定的监管机构在 蛋白质的作用，房室分离的重要性， 重要的代谢物进入和离开细胞， 生物合成和降解途径是否与 通过调节蛋白或小分子相互作用。 一个 了解基本代谢过程的工作原理， 我们了解和治疗的情况下，畸变， 规则发生。 这项建议涉及的问题， 脯氨酸降解和生物合成的调节以及 脯氨酸代谢的区室化。 脯氨酸利用途径的正调控元件， PUT 3基因将被克隆并在分子水平上进行分析。 这将涉及研究，以确定PUT 3基因的调控 表达，无效突变的表型，以及 调节剂与途径中的其他元素的作用。 三种类型的 基因融合体（PUT 1-galK、PUT 1-lacZ和PUT 2-lacZ）将用于 其他正或负反式作用基因中的孤立突变 影响脯氨酸利用的调节剂。 脯氨酸生物合成三个结构基因的克隆 将研究这些途径，以确定它们是否受到控制 通过脯氨酸特异性和/或一般氨基酸控制调节 使用来自野生型的mRNA的北方分析的元件， 脯氨酸营养缺陷型和一般对照缺陷型菌株。 物理相互作用（例如复合物形成）和亚细胞 酶的定位将通过酶测定法进行研究， 以及通过免疫沉淀，使用针对 纯化的蛋白质。 细胞区室在脯氨酸代谢中的重要性 将通过扰乱它们的正常位置来研究，使用遗传学 工程技术将细胞质脯氨酸生物合成 酶进入线粒体和线粒体脯氨酸 降解酶进入细胞质 脯氨酸的转运 将通过调整所使用的技术来研究 在高等细胞中测量代谢物通量： 线粒体并通过油进行离心过滤。 的影响 立体特异性，能量解偶联剂，脯氨酸类似物和 将测定对脯氨酸转运的蛋白质抑制剂。