Organization of the Pathway of Urea Synthesis In Situ

细胞是一个高度结构化的复杂系统，在这个系统中，超微结构元素形成了支架，用于连接功能相关的酶和其他蛋白质的有序阵列。这个项目的目的是以尿素循环为模型，确定细胞内可溶酶系统组织的潜在机制。哺乳动物肝脏的尿素合成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，后三种酶在细胞质中。尽管所有的酶都是可溶的(当细胞或细胞器在没有洗涤剂的情况下被破坏时，它们会进入溶液中)，但生化研究表明，该途径是在原位高度组织的，作为一个功能单位，中间产物在酶和隔间之间输送。这些研究表明，这三种细胞质酶在线粒体膜上依次组织。研究还表明，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和它们各自的蛋白质一样，也定位在线粒体旁边。ASS基因的3‘非翻译区(3’UTR)可与位于肝线粒体外的蛋白质复合体特异性结合。该项目的具体目标是研究ASS和ASL mRNA定位的机制，以及维持蛋白质原位定位的大分子相互作用。本研究的目的有四个：1.通过构建融合了绿色荧光蛋白编码序列的各种mRNA序列的载体，将其导入体外培养的肝细胞，通过标准荧光显微镜和共聚焦显微镜确定绿色荧光蛋白在肝细胞中的定位，确定ASS和ASL基因中负责定位肝线粒体周围信息的序列(S)，以及3‘端非编码区蛋白结合序列的功能作用。鉴定ASSmRNA3‘非编码区结合复合体和/或多肽。该复合物和任何其他特异性结合的多肽将在RNA亲和层析柱上纯化，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以及基因的克隆和测序。为了确定尿素循环功能是否需要ASS和ASL mRNA的定位，靶向序列将在培养的肝细胞中过表达，与内源性mRNA竞争定位。在诱导内源性尿素循环酶的表达增加后，将检测这些细胞合成尿素的能力。鉴定可能与ASS和ASL蛋白相互作用的其他细胞成分，以维持后者的原位定位，并开始鉴定ASS和ASL的相互作用结构域。这种双杂交方法将被用来筛选与ASS或ASL相互作用的成分的肝脏cDNA文库。这些蛋白质将被克隆、测序、超量表达，并为目标2确定上述蛋白质的特征。我们将使用ASS和ASL选定区域的定点突变，以及分析突变对双杂交相互作用的影响来确定相互作用区域。可溶性酶系统的细胞内组织是细胞的重要和基本特征。其潜在机制的确定是代谢生物化学、细胞生物学、分化和信号转导等领域的一个重要研究课题，这些研究将增加我们对哺乳动物肝脏的主要功能-尿素合成的调控的认识和理解。这些研究还将提供与已知的与线粒体外膜相关的其他细胞质酶以及其他代谢途径的基本信息，这些代谢途径的功能可能依赖于特定的酶组织和定位。

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.