TARGETING AND ASSEMBLY OF THYLAKOID MEMBRANE PROTEINS

真核细胞含有各种膜和膜结合的 执行特殊功能的细胞器。蛋白质靶向和 运输系统是维持这些基本过程的基础， 隔间蛋白质靶向性的重要性 健康从疾病的存在是显而易见的，如I-cell和 高尿酸I型，其中酶定位于错误的细胞 隔间我们研究的长期目标是了解 参与各种蛋白质转运系统的分子机制。 这里提出的工作将调查一个奇怪的现象， 三个进化上相关但不同的系统针对亚种群 蛋白质的蛋白质的同一个膜，叶绿体的类囊体膜。 实验提出了检查的三个阶段的运输 这些系统的过程：靶向，初始膜插入， 链移位穿过双层。特定体外实验 将识别和剖析靶向序列的特定元件 使前蛋白进入通路。分析了拟议的 受体的这些信号的两个途径，一个叶绿体SecA同源 和叶绿体SRP同源物，将评估它们的结合特异性 以及结合发生的叶绿体位置。 其他实验 将试图确定叶绿体SRP的分子组成。 最后，对膜上事件的研究将表征初始的 跨双层插入和多肽链移位。在 特别是，实验旨在确定这三种途径是否 有不同的膜成分，如果它们在一个水平上合并， 普通孔这些后面的研究将采用体内实验， 减少一种可疑的气孔成分，叶绿体SecY 同源物，通过表达反义RNA。的遗传学研究 酵母内质网、细菌细胞质膜和植物类囊体膜 揭示了多蛋白靶向的体内本质 通向同一膜的通路。圆满完成 这里提出的实验将描述这一点的分子基础。 现象，并提供必要的实验基础 解决蛋白质靶向途径多样性的原因？

Eukaryotic cells contain various membranes and membrane-bounded organelles that perform specialized functions. Protein targeting and transport systems are the fundamental processes that maintain these compartments. The importance of faithful protein targeting to human health is evident from the existence of diseases, such as I-cell and hyperoxaluria type I, where enzymes are localized to the wrong cellular compartments. The long range goal of our research is to understand the molecular mechanisms involved in the various protein transport systems. The work proposed here will investigate a curious phenomenon, whereby three evolutionarily related, but distinct systems target sub-populations of proteins to the same membrane, the thylakoid membrane of chloroplasts. Experiments are proposed to examine the three phases of the transport process for these systems: targeting, initial membrane insertion, and chain translocation across the bilayer. Specific in vitro experiments will identify and dissect the specific elements of targeting sequences that commit a preprotein to pathway. An analysis of the proposed receptors of these signals for two pathways, a chloroplast SecA homologue and a chloroplast SRP homologue, will assess their binding specificity and the chloroplast location where binding occurs. Other experiments will attempt to determine the molecular makeup of the chloroplast SRP. Finally, studies of the events at the membrane will characterize initial insertion across the bilayer and polypeptide chain translocation. In particular, experiments are designed to determine if these three pathways have distinct membrane components of if they merge at the level of a common pore. These latter studies will employ in vivo experiments to reduce the amount of one suspected pore component, a chloroplast SecY homologue, by the expression of antisense RNA. Genetic studies of the yeast ER, bacterial cytoplasmic membrane, and plant thylakoid membrane have revealed the essential in vivo nature of multiple protein targeting pathways to the same membrane. The successful completion of the experiments proposed here will describe the molecular basis for this phenomenon and provide the foundation necessary for experimentally addressing the reason for multiplicity of protein targeting pathways?