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.
|Effective start/end date||9/1/06 → 8/31/11|
- National Science Foundation: $4,799,857.00