REGULATION & COMPARTMENTATION IN PROLINE METABOLISM

规定

• 批准号: 3298634
• 负责人: MARJORIE C BRANDRISS
• 金额: $ 14.49万
• 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/3298634
• 关键词: Saccharomyces; aminoacid metabolism; biotechnology; gene expression; genes; genetic manipulation; genetic regulation; mitochondria; molecular cloning; molecular genetics; 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.

本研究提案的总体目标是了解 酿酒酵母脯氨酸代谢的方方面面 作为集成一系列复杂系统的模型系统 相互作用涉及到核、细胞质和 细胞内的线粒体隔间。脯氨酸的生物合成 而脯氨酸的利用涉及一系列反应，这些反应具有 一种常见的中间体以及底物和产品池，其 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扮演的角色 蛋白质的作用，隔室分离的重要性，如何 重要的代谢物进入和离开线粒体 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 了解基本新陈代谢过程的工作原理将使 我们要了解和治疗像差的情况 监管就会发生。本提案涉及的问题涉及 脯氨酸降解和生物合成的调控及其作用 脯氨酸代谢中的区隔作用。 脯氨酸利用途径的正向调节元件， PUT3基因，将在分子水平上进行克隆和分析。 这将涉及确定PUT3基因调控的研究 表达、零突变的表型和相互作用 调节因子与途径中的其他元素发生了相互作用。三种类型的 基因融合(PUT1-Galk、PUT1-lacZ和PUT2-lacZ)将用于 分离其他阳性或阴性反式作用的突变 影响脯氨酸利用的调节剂。 脯氨酸生物合成的三个结构基因的克隆 将研究它们的途径，以确定它们是否受到控制 由脯氨酸特异和/或一般氨基酸控制的调控 利用Northern分析野生型mRNAs的成分， 脯氨酸营养缺乏症，一般控制缺陷菌株。 物理相互作用(例如，复杂的形成)和亚细胞 这些酶的定位将通过酶分析进行研究，如 以及通过使用产生的抗体进行免疫沉淀 纯化的蛋白质。 细胞隔室在脯氨酸代谢中的重要性 将通过扰动它们的正常位置来研究，使用基因 利用工程技术移动细胞质中的脯氨酸生物合成 进入线粒体的酶和线粒体的脯氨酸 降解酶进入细胞质。脯氨酸的转运 将通过适应所使用的技术来研究线粒体 在高等细胞中测量代谢产物的通量：渗透性肿胀 线粒体和通过油的离心式过滤。其影响 立体专一性，能量解偶联剂，脯氨酸类似物和 将确定对脯氨酸转运有抑制作用的蛋白质。