Organization of the Pathway of Urea Synthesis In Situ

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The cell is a highly structured complex system, in which ultrastructural elements formscaffolds for the attachments of organized arrays of functionally-related enzymes and otherproteins. The purpose of this project is to identify mechanisms underlying the intracellularorganization of soluble enzyme systems, with the urea cycle as a model. Thepathway of urea synthesis in mammalian liver consists of five enzymes in two cellularcompartments; the first two enzymes are in the mitochondrial matrix, and the next three are inthe cytoplasm. Although all the enzymes are soluble (they go into solution when cells ororganelles are disrupted in the absence of detergent), biochemical studies have demonstratedthat the pathway is highly organized in situ, behaving as a functional unit within which intermediates are channeled between enzymes and compartments. Those studies showed thatthe three cytoplasmic enzymes are sequentially organized at the mitochondrial membrane. Itwas also shown that, like their respective proteins, the mRNA's of two of the cytoplasmicenzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), are localizednext to the mitochondria. The 3'untranslated region (3'UTR) of ASS mRNA binds specifically toa protein complex that is located on the outside of liver mitochondria. The specific aims of this project focus on studies of the mechanisms of ASS and ASL mRNA localization and of the macromolecular interactions that maintain the localization of the proteins in situ. There are four aims:1. To determine the sequence(s) in ASS and ASL mRNA's responsible for localizing thesemessages around liver mitochondria in situ, and the functional role of the protein-bindingsequences of the 3'UTR's. This will be done by constructing vectors containing variousmRNA sequences fused to the coding sequence of green fluorescent protein (GFP), transfectingthese into hepatocytes in culture, and determining the location of the GFP in the cells bystandard fluorescence and confocal microscopy.2. To characterize the ASS mRNA 3'UTR binding complex and/or peptides. The complexand any other specifically-binding peptides will be purified on RNA affinity columns, and characterized by SDS-PAGE, isoelectric focusing, and N-terminal sequencing. This will be followed by library screening and cloning and sequencing of the genes.3. To determine if ASS and ASL mRNA localization is required for urea cycle function.The mRNA targeting sequences will be overexpressed in cultured hepatocytes, to compete withendogenous mRNA for localization. After inducing increased endogenous expression of theurea cycle enzymes, the ability of these cells to synthesize urea will be measured.4. To identify and characterize other cellular components that may interact with ASS andASL proteins to maintain the localization of the latter in situ, and to begin to identify theinteracting domains of ASS and ASL. The two-hybrid method will be used to screen a livercDNA library for components interacting with ASS or ASL. These will be cloned, sequenced, andoverexpressed, and the proteins characterized as described above for Aim 2. Site-directedmutagenesis of selected regions of ASS and ASL, and analysis of the effects of the mutationson two-hybrid interactions will be used to identify interacting regions.The intracellular organization of soluble enzyme systems is a significant and basic featureof cells. Identification of the underlying mechanisms is an important matter of general interestin the fields of metabolic biochemistry, cell biology, differentiation, and signal transduction.These studies will increase our knowledge and understanding of the regulation of urea synthesis, a major function of mammalian liver. The studies will also provide basic information directly relevant to other cytoplasmic enzymes known to be associated with the mitochondrial outer membrane, and to other metabolic pathways whose function may be dependent on specific enzyme organization and localization.