Organization of the Pathway of Urea Synthesis In Situ

University Of Southern California, Los Angeles CA

Investigators

Abstract

The cell is a highly structured complex system, in which ultrastructural elements form scaffolds for the attachments of organized arrays of functionally-related enzymes and other proteins. The purpose of this project is to identify mechanisms underlying the intracellular organization of soluble enzyme systems, with the urea cycle as a model. The pathway of urea synthesis in mammalian liver consists of five enzymes in two cellular compartments; the first two enzymes are in the mitochondrial matrix, and the next three are in the cytoplasm. Although all the enzymes are soluble (they go into solution when cells or organelles are disrupted in the absence of detergent), biochemical studies have demonstrated that the pathway is highly organized in situ, behaving as a functional unit within which intermediates are channeled between enzymes and compartments. Those studies showed that the three cytoplasmic enzymes are sequentially organized at the mitochondrial membrane. It was also shown that, like their respective proteins, the mRNA's of two of the cytoplasmic enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), are localized next to the mitochondria. The 3'untranslated region (3'UTR) of ASS mRNA binds specifically to a 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 these messages around liver mitochondria in situ, and the functional role of the protein-binding sequences of the 3'UTR's. This will be done by constructing vectors containing various mRNA sequences fused to the coding sequence of green fluorescent protein (GFP), transfecting these into hepatocytes in culture, and determining the location of the GFP in the cells by standard fluorescence and confocal microscopy. 2. To characterize the ASS mRNA 3'UTR binding complex and/or peptides. The complex and 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 with endogenous mRNA for localization. After inducing increased endogenous expression of the urea 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 and ASL proteins to maintain the localization of the latter in situ, and to begin to identify the interacting domains of ASS and ASL. The two-hybrid method will be used to screen a liver cDNA library for components interacting with ASS or ASL. These will be cloned, sequenced, and overexpressed, and the proteins characterized as described above for Aim 2. Site-directed mutagenesis of selected regions of ASS and ASL, and analysis of the effects of the mutations on two-hybrid interactions will be used to identify interacting regions. The intracellular organization of soluble enzyme systems is a significant and basic feature of cells. Identification of the underlying mechanisms is an important matter of general interest in 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.