Organization of the Pathway of Urea Synthesis In Situ

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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. ***