REGULATION AND COMPARTMENTATION IN PROLINE METABOLISM

这个项目的长期目标是了解分子 水稻氮同化过程中基因表达调控机制的研究进展 真核生物。这些包括调节蛋白的作用， 酶和底物的分隔，以及代谢产物的通量 代谢途径的整体功能。脯氨酸在土壤中的利用 酿酒酵母作为本研究的模型系统。 脯氨酸利用酶由核基因编码，翻译成 在细胞质中，并定位于脯氨酸所在的线粒体 转化为谷氨酸。Pro、氧和一个官能团电子 运输链对这一过程至关重要。的表达 结构基因受一种名为PUT3的激活剂的调控。这种蛋白质 在构成上与这些基因的启动子结合，但激活 只有在没有首选氮源的情况下才能转录 有脯氨酸存在。这项研究的重点是确定 脯氨酸可供利用的信号通过 PUT3蛋白与转录机制的关系。功能界别 激活蛋白的结构域将通过对突变的分析来确定 编码激活剂缺陷蛋白的PUT3基因的构建 基因特定区域的定点突变，交换 结构域与GAL4的结构域，GAL4是一种特性良好的激活剂，并且 测量这些改变对DNA结合的影响， 转录激活、脯氨酸反应性和二聚化 蛋白质。PUT3蛋白将使用DNA亲和力进行纯化 层析并检查翻译后修饰 (磷酸化、糖基化、脯氨酸结合和与 其他蛋白质)使用标准的生物化学方法。一个新的阶层 控制这一途径的负调控因子的突变 将通过遗传和分子方法进行鉴定和表征。 这个类别中的一个成员，PUT6基因的产物，显然是有效的 通过PUT3控制脯氨酸的利用，但不是特定的 这条路。与PUT3相互作用的基因将由 PUT3和PUT3c高拷贝和外源抑制基因的分离 突变。这个系统提供了一个有用的模型来理解变化 在细胞环境中向新陈代谢的元素发出信号 真核生物中的途径。

The long term objective of this project is to understand the molecular mechanisms that regulate gene expression in nitrogen assimilation in eukaryotic organisms. These include the roles of regulatory proteins, compartmentation of enzymes and substrates, and metabolite flux in the overall function of a metabolic pathway. Proline utilization in the yeast Saccharomyces cerevisiae serves as the model system for this study. The proline utilization enzymes are encoded by nuclear genes, translated in the cytosol, and targeted to the mitochondrion where proline is converted to glutamate. Proline, oxygen and a functional electron transport chain are essential for this process. Expression of the structural genes is regulated by an activator called PUT3. This protein is constitutively bound to the promoters of these genes, but activates their transcription only when preferred nitrogen sources are absent and proline is present. The focus of this study is to determine how the signal that proline is available for utilization is transmitted through the PUT3 protein to the transcriptional machinery. The functional domains of the activator protein will be defined by an analysis of mutant PUT3 genes that encode activator-defective proteins, construction of site-directed mutations in specific domains of the gene, exchange of domains with those of GAL4, a well-characterized activator, and measurement of the effects of these alterations on DNA binding, transcriptional activation, proline-responsiveness and dimerization of the protein. The PUT3 protein will be purified using DNA-affinity chromatography and examined for post-translational modifications (phosphorylation, glycosylation, proline-binding and association with other proteins) using standard biochemical methods. A new class of mutations in negative regulators controlling this pathway has been identified and will be characterized by genetic and molecular methods. One member of this class, the product of the PUT6 gene, apparently works through PUT3 in controlling proline utilization, but is not specific to this pathway. Genes that interact with PUT3 will be identified by the isolation of high copy and extragenic suppressors of put3 and PUT3c mutations. This system provides a useful model to understand how changes in the cellular environment are signalled to the elements of a metabolic pathway in eukaryotic organisms.