Organization of the Pathway of Urea Synthesis In Situ

[601421] Cohen几个实验室的证据表明，细胞是一个高度结构化的系统，其中细胞骨架和胞内膜形成了一个支架，用于连接有组织的功能相关酶阵列。本项目的目的是利用尿素循环作为实验模型，确定细胞内可溶性酶系统组织的一些机制。哺乳动物肝脏的尿素合成途径由5种酶组成，它们在两个细胞区室中起作用；前两个反应是由线粒体基质中的酶催化的，后三个是由细胞质酶催化的。尽管所有的酶都是可溶的（当细胞或细胞器在没有清洁剂的情况下被破坏时，它们会进入溶液），但该途径在原位是高度组织的；它作为一个功能单元，在其中中间体在酶和室之间通道，三种细胞质酶依次组织在线粒体膜上。此外，像它们各自的蛋白质一样，精氨酸琥珀酸合成酶（ASS）和精氨酸琥珀酸裂解酶（ASL）这两种细胞质酶的mrna定位在线粒体附近，表明这些蛋白质的翻译发生在它们最终的细胞内位点。本研究的目的是：1)研究ASS和ASL mrna定位的机制，方法是：a)确定这些mrna是否在特定的多聚体群体中富集，其中一些mrna可能与线粒体和/或细胞骨架的特定成分相关；b)鉴定与靶向相关的mRNA序列；c)识别作为这些mrna靶标的细胞成分。2)确定ASS和ASL蛋白的定位是如何维持的。ASS和ASL之间的蛋白质-蛋白质相互作用，或与线粒体外膜、内质网或线粒体附近的细胞骨架的成分的相互作用，将在原位研究：a)可逆交联试剂和免疫学方法；b)基因表达的双杂交系统。目的是确定参与相互作用的特定蛋白质区域，并检测和表征参与锚定酶的任何其他蛋白质。活细胞是高度结构化的系统，其中某些与整体功能相关的酶蛋白在细胞的特定位置被组织成多组分复合物。因为它允许它们更有效地发挥作用，细胞内的可溶性酶系统组织是细胞的一个重要和基本特征。鉴定这种组织背后的机制对细胞功能的许多方面都是一个重要的普遍关注的问题。这项研究将增加我们对哺乳动物肝脏主要功能尿素合成调控的认识和理解。它还将提供与其他酶途径直接相关的基本信息，这些酶途径的功能可能依赖于特定的细胞内组织和定位。＊＊＊

9601421 Cohen Evidence from several laboratories has shown that the cell is a highly structured system, in which the cytoskeleton and intracellular membranes form a scaffold for the attachment of organized arrays of functionally related enzymes. The purpose of this project is to identify some of the mechanisms underlying the intracellular organization of soluble enzyme systems, using the urea cycle as an experimental model. The pathway of urea synthesis in mammalian liver consists of five enzymes, which operate in two cellular compartments; the first two reactions are catalyzed by enzymes in the mitochondrial matrix, and the next three by cytoplasmic enzymes. Although all the enzymes are soluble (they go into solution when cells or organelles are disrupted in the absence of detergent), the pathway is highly organized in situ; it behaves as a functional unit within which intermediates are channeled between enzymes and compartments, and the three cytoplasmic enzymes are sequentially organized at the mitochondrial membrane. In addition, like their respective proteins, the mRNAs of two of the cytoplasmic enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are localized next to mitochondria, indicating that the translation of these proteins occurs at their final intracellular site. The aims of this research are: 1) To investigate the mechanisms underlying the localization of ASS and ASL mRNAs, by: a) determining if these mRNAs are enriched in a particular polysome population, some of which may be associated in situ with mitochondria, and/or with a specific component of the cytoskeleton; b) identifying the mRNA sequences involved in targeting; and c) identifying the cellular component(s) that serve as the target for these mRNAs. 2) To determine how the localization of ASS and ASL protein is maintained. Protein-protein interactions of ASS and ASL with each other, or with a component of the mitochondrial outer membrane, or of the endoplasmic reticulum or cytoskeleton in the m itochondrial vicinity, will be studied in situ using: a) reversible cross-linking reagents and immunological methods; and b) the two-hybrid system of gene expression. The aim is to identify the specific protein regions participating in the interactions, and to detect and characterize any other proteins involved in anchoring the enzymes. %%% Living cells are highly structured systems, in which certain enzyme proteins that are related in their overall function are organized into multicomponent complexes in specific locations in the cell. Because it allows them to function more efficiently, the intracellular organization of soluble enzyme systems is a significant and basic feature of cells. Identification of the mechanisms underlying that organization is an important matter of general interest for many aspects of cell function. This research will increase our knowledge and understanding of the regulation of urea synthesis, a major function of mammalian liver. It will also provide basic information directly relevant to other enzyme pathways whose function may be dependent on specific intracellular organization and localization. ***