TARGETING OF HUMAN OTC TO THE MITOCHONDRIAL MATRIX

拟议的研究旨在了解 线粒体区划，线粒体蛋白 前体，在细胞核中编码，在细胞质上合成 多核糖体，翻译后识别通过它们的NH2- 末端前导肽，跨一个或两个转位 膜，并对其进行蛋白质水解性处理，使其具有活性。 我们将继续使用这个模型来分析这个系统 人线粒体基质酶鸟氨酸转氨甲基酶 (OTC)，它催化尿素循环的第二步 哺乳动物。这里提出的研究的目的是：识别 以及对两者线粒体成分的表征 涉及酿酒酵母和哺乳动物细胞 通过特定的识别，导入到矩阵隔间，以及 非处方药前体的蛋白质分解处理.测定 这些成分是否被其他蛋白质所共享 以及对线粒体的高阶结构的分析 非处方药先导肽。在酵母和哺乳动物细胞中，基因 将采取方法分离编码进口的基因 组件：抑制突变将在两者中隔离 允许突变OTC的酵母菌和HeLa细胞 到达线粒体的前体；阻断的突变 进口/加工野生型OTC前体并导致 条件生长缺陷状态也将被隔离在 酵母菌。将采用生化方法进行研究 与导入相关的组件。野生型非处方药前体 将通过诱变一种质粒而在大肠杆菌中过量生产 野生型融合蛋白的程序化生产 OTC前导肽与半乳糖激酶结合，并测定高... 用菌落比色法进行酶活性水平表达。这个 过量生产的前体将被提纯并用于各种 研究，包括线粒体结合研究，旨在 检测细胞器识别和竞争的动力学 用于与其他前体的识别；交联反应 旨在标识与 场外交易前体；以及结构研究，旨在分析 先导肽的高阶结构。体外合成 具有反应性氨基酸侧链或 具有酶活性的成熟部分也将用作亲和力- 标记试剂，以识别额外的相互作用的线粒体 组件。

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.