TARGETING OF HUMAN OTC TO THE MITOCHONDRIAL MATRIX

所提出的研究旨在了解 线粒体区室化， 前体，在细胞核中编码并在细胞质中合成 多聚核糖体，通过其NH 2- 末端前导肽，跨一个或两个 膜，并通过蛋白水解加工成其活性形式。 我们将继续使用该系统作为分析模型， 人线粒体基质酶鸟氨酸转氨甲酰酶 (OTC)，其催化尿素循环的第二步， 哺乳动物 这里建议的研究旨在： 和表征的线粒体的组成部分， 酿酒酵母和哺乳动物细胞 具有特异性识别，导入基质隔室，和 OTC前体的蛋白水解加工;测定 这些成分是否与其他蛋白质共享， 线粒体;和分析的高阶结构的 OTC前导肽。 在酵母和哺乳动物细胞中， 将采取方法分离编码输入的基因， 组件：抑制突变将在两个 允许突变OTC的酵母菌和HeLa细胞 到达线粒体的前体;阻止 进口/加工野生型OTC前体并导致 有条件的生长缺陷状态也将被隔离， 酵母菌 将采用生物化学方法来研究 涉及进口的组件。 野生型OTC前体 将在E.通过诱变质粒 程序化产生连接野生型的融合蛋白， OTC前导肽与半乳糖激酶，并测定高- 通过菌落颜色测定法测定酶活性的水平表达。 的 过量生产的前体将被纯化并用于各种用途， 研究，包括旨在 检查细胞器识别和竞争的动力学 用于与其他前体识别;交联反应 设计用于识别与 OTC前体;和结构研究，旨在分析 前导肽的高级结构。 体外合成的 具有反应性氨基酸侧链或 酶活性成熟部分也可用作亲和- 标记试剂，以鉴定额外的相互作用的线粒体 件.

The proposed studies are directed to understanding the system of mitochondrial compartmentation, whereby mitochondrial protein precursors, encoded in the nucleus and synthesized on cytoplasmic polyribosomes, are posttranslationally recognized via their NH2- terminal leader peptides, translocated across one or both membranes, and proteolytically processed to their active forms. We will continue to use as a model for analysis of this system the human mitochondrial matrix enzyme ornithine transcarbamylase (OTC), which catalyzes the second step of the urea cycle in mammals. The studies proposed here are aimed at: identification and characterization of components of the mitochondria of both Saccharomyces cerevisiae and mammalian cells that are involved with specific recognition, import to the matrix compartment, and proteolytic processing of the OTC precursor; determination of whether such components are shared by other proteins destined for mitochondria; and analysis of the higher-order structure of the OTC leader peptide. In both yeast and mammalian cells, genetic approaches will be taken to isolating genes encoding import components: suppressing mutations will be isolated in both Saccharomyces and HeLa cells that permit mutant OTC precursors to reach the mitochondria; mutations that block import/processing of the wild-type OTC precursor and result in a conditional growth-deficient state will also be isolated in Saccharomyces. Biochemical approaches will be taken to studying components involved with import. The wild-type OTC precursor will be overproduced in E. coli by mutagenizing a plasmid programming production of a fusion protein joining the wild-type OTC leader peptide with galactokinase, and assaying for high- level expression of enzyme activity by a colony color assay. The overproduced precursor will be purified and used in a variety of studies, including mitochondrial binding studies designed to examine kinetics of recognition by the organelles and competition for recognition with other precursors; crosslinking reactions designed to identify specific components that interact with the OTC precursor; and structural studies designed to analyze the higher-order structure of the leader peptide. In vitro synthesized precursors with either reactive amino acid side chains or enzymatically active mature portions will also be used as affinity- labeling reagents, to identify additional interacting mitochondrial components.