Arabidopsis 2010: Towards a Comprehensive Arabidopsis Protein Interactome Map: Systems Biology of the Membrane Proteins and Signalosome

生物系统通过蛋白质与其他大分子（蛋白质、DNA、RNA、复合碳水化合物和脂质）以及小分子（包括代谢物和次级化合物）的相互作用来发挥功能。膜提供了一个表面，用于感知和转导来自相邻细胞和外部条件（如营养物质、病原体和不利的非生物条件的可用性）的信号。此外，膜控制分子的细胞和亚细胞进入和离开。为了更好地了解细胞-环境、细胞-细胞和细胞外基质与胞质溶胶相互作用的调控，本项目计划确定参考植物拟南芥中约6000种蛋白质的相互作用。它基本上包含所有的膜蛋白（5，000）（除了那些被预测定位于线粒体和质体的）和大量（1，000种）被预测参与信号传导或蛋白质修饰的蛋白质，如激酶、磷酸酶、钙调蛋白等。由于膜蛋白固有地难以与之一起工作，一种特殊的酵母双杂交系统，分裂泛素系统，用于确定膜蛋白相互作用。在该项目中，将使用分裂泛素系统对这大约6000种蛋白质的二元相互作用进行系统分析，最终测试超过2500万种相互作用。 这些数据将进行生物信息学和图论荟萃分析，以确定相互作用网络中的主要信号转导途径和蛋白质复合物（信号体）。关键的功能信号介质（枢纽），以及丰富的监管模式将被确定。将使用分离GFP系统验证相互作用的子集。此外，将对编码80种主要功能未知的蛋白质的基因的T-DNA插入敲除进行宏观表型表征，所述蛋白质包含用于维持预测途径和复合物的结构和/或功能的关键节点。一组较小的20个基因，预测将在保卫细胞或根表皮从以前的微阵列分析中表达，将获得详细的表征，他们在这些细胞类型中的功能，反映了特定的专业领域的PI和这些细胞的潜力，了解植物膜生物学的许多方面。数据将通过我们的项目网站（www.associomics.org）、TAIR以及IntAct、DIP和bioGRID等互动数据库公开。第一年年底将提供相互作用和克隆的初始数据集，项目期结束时将提供最终的相互作用和克隆集。在整个项目期间，将定期（例如每半年）向TAIR提交验证和文献整理信息。该项目将提供全长ORF的额外来源，并将为蛋白质的功能分配做出重大贡献;例如，我们数据集中37%的膜蛋白目前在GO“功能”和“过程”的一个或两个类别中被注释为未知。此外，由于这些数据将提供第一套完整的膜蛋白相互作用和信号蛋白质组在任何生物体，他们将提供一个社区资源的产生新的假说，追求生物技术的应用，并发展比较基因组学和蛋白质网络研究。为少数民族服务的学校有圣何塞州立大学、弗朗西斯科城市学院、弗朗西斯科州立大学，这些学校历史上都是非洲裔美国人大学，和斯沃斯莫尔学院将有针对性的本科生谁将选择和评估特定的信号模块在我们的膜相互作用组项目使用湿台，生物信息学和理论工具的来源。来自圣地亚哥普鲁斯学校和宾夕法尼亚州立大学附近的秃鹰地区高中的高中生，这些学校的经济困难学生比例很大，将被选中参与T-DNA插入突变体的鉴定和表型分析。我们的目标是确保真正参与研究项目将鼓励这些学生考虑科学事业。此外，该项目将培训参与跨学科研究的研究生和博士后，特别是系统生物学的概念和工具。

Biological systems function through interactions of proteins with other macromolecules (proteins, DNA, RNA, complex carbohydrates and lipids), as well as small molecules, including metabolites and secondary compounds. Membranes provide a surface for perceiving and transducing signals from adjacent cells and external conditions such as availability of nutrients, pathogens and adverse abiotic conditions. In addition, membranes control cellular and subcellular entry and exit of molecules. To understand the regulation of cell-environment, cell-cell, and extracellular matrix to cytosol interactions better, this project plans to determine the interactions of approximately 6000 proteins from the reference plant Arabidopsis. It comprises essentially all integral membrane proteins (5,000) (except those predicted to be localized to mitochondria and plastid) and a large number (1,000) of proteins predicted to be involved in signaling or protein modification such as kinases, phosphatases, calmodulins, etc. Since membrane proteins are inherently difficult to work with, a special yeast two-hybrid system, the split-ubiquitin system, was developed for determining membrane protein interactions. In this project, a systematic analysis of the binary interactions of these approximately 6000 proteins will be performed using the split ubiquitin system, culminating in testing more than 25 million interactions. The data will be subjected to bioinformatic and graph-theoretical meta-analyses to identify the main signal transduction pathways and protein complexes (signalosomes) in the interaction network. Key functional signal mediators (hubs) as well as abundant regulatory patterns will be identified. A subset of interactions will be verified using the split GFP system. In addition, T-DNA insertional knockouts of genes encoding 80 proteins of mainly unknown function and comprising critical nodes for maintaining the structure and/or function of the predicted pathways and complexes will be characterized for macroscopic phenotypes. A smaller set of 20 genes, predicted to be expressed in guard cells or the root epidermis from previous microarray analyses, will receive detailed characterization regarding their functions in those cell types, reflecting the specific expertise areas of the PIs and the potential of these cells for understanding many aspects of plant membrane biology. Data will be made public through our project website (www.associomics.org), TAIR, and interaction data repositories such as IntAct, DIP and bioGRID. An initial dataset of interactions and clones will be available at the end of the first year and final set of interactions and clones will be available at the end of the project period. Validation and literature-curated information will be submitted to TAIR on a regular basis (e.g. biannually) throughout the project period. The project will provide an additional source of full length ORFs and will contribute significantly to the assignment of functions to proteins; for example 37% of the membrane proteins in our dataset are currently annotated as unknowns in one or both of the categories of GO 'function' and 'process'. Moreover, since these data will provide the first complete set of membrane protein interactions among each other and the signaling proteome in any organism, they will provide a community resource for generation of novel hypotheses, pursuit of biotechnological applications, and development of comparative genomics and protein network studies.Broader Impacts: The minority-serving schools San Jose State University, City College of San Francisco, San Francisco State University, historically African-American universities, and Swarthmore College will be targeted as sources of undergraduates who will select and assess specific signaling modules within our membrane interactome project using wet bench, bioinformatic and theoretical tools. High school students from the Preuss School in San Diego and the Bald Eagle Area High School near Penn State, schools with a large proportion of economically disadvantaged students, will be selected to participate in identification and phenotypic analysis of T-DNA insertional mutants. Our goal is to ensure that genuine participation in a research project will encourage these students to consider careers in science. In addition, the project will train participating graduate students and postdocs in interdisciplinary research, specifically the concepts and tools of systems biology.