Organization of the Pathway of Urea Synthesis In Situ

细胞是一个高度结构化的复杂系统，其中超微结构元件形成支架，用于连接功能相关的酶和其他蛋白质的有序阵列。本项目的目的是以尿素循环为模型，确定可溶性酶系统胞内组织的潜在机制。哺乳动物肝脏中尿素合成的途径由两个细胞室中的五种酶组成;前两种酶在线粒体基质中，接下来的三种在细胞质中。虽然所有的酶都是可溶的（当细胞或细胞器在没有去污剂的情况下被破坏时，它们会进入溶液），但生化研究表明，该途径在原位是高度组织化的，表现为一个功能单元，其中中间体在酶和隔室之间形成通道。这些研究表明，这三种细胞质酶在线粒体膜上顺序排列。研究还表明，两种细胞质酶，乙酰氨基琥珀酸合成酶（ASS）和乙酰氨基琥珀酸裂解酶（ASL）的mRNA，像它们各自的蛋白质一样，位于线粒体附近。ASS mRNA的3 '非翻译区（3' UTR）特异性结合位于肝线粒体外部的蛋白复合物。 该项目的具体目标集中在ASS和ASL mRNA定位的机制和维持蛋白质原位定位的大分子相互作用的研究。有四个目标：1。确定ASS和ASL mRNA中负责将这些信息原位定位在肝脏线粒体周围的序列，以及3 'UTR的蛋白质结合序列的功能作用。这将通过构建含有融合到绿色荧光蛋白（GFP）编码序列的各种smRNA序列的载体，将这些转染到培养的肝细胞中，并通过标准荧光和共聚焦显微镜确定GFP在细胞中的位置.表征ASS mRNA 3 'UTR结合复合物和/或肽。复合物和任何其他特异性结合肽将在RNA亲和柱上纯化，并通过SDS-PAGE、等电聚焦和N-末端测序进行表征。随后将进行文库筛选和基因的克隆和测序。为了确定尿素循环功能是否需要ASS和ASL mRNA定位，mRNA靶向序列将在培养的肝细胞中过表达，以与内源性mRNA竞争定位。在诱导尿素循环酶的内源性表达增加后，将测量这些细胞合成尿素的能力。鉴定和表征可能与ASS和ASL蛋白相互作用的其他细胞组分，以维持后者在原位的定位，并开始鉴定ASS和ASL的相互作用结构域。双杂交方法将用于筛选与ASS或ASL相互作用的组分的肝cDNA文库。这些将被克隆、测序和过表达，并且蛋白质如上文针对目标2所述被表征。通过对ASS和ASL的选择性区域进行定点突变，并分析突变对双杂交相互作用的影响，将用于鉴定相互作用区域。尿素合成是哺乳动物肝脏的重要功能之一，其调控机制的研究是代谢生物化学、细胞生物学、分化和信号转导等领域的重要研究课题。这些研究还将提供与已知与线粒体外膜相关的其他细胞质酶直接相关的基本信息，以及与其功能可能依赖于特定酶组织和定位的其他代谢途径直接相关的基本信息。

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.