This 2010 project will determine the specificity and mechanisms of N-terminal modifications of the Arabidopsis proteome. The N-terminal modification enzymes (NTMEs), which include 28 peptidases and 11 transferases, will be studied. NTMEs control important fundamental aspects of protein maturation, activation or regulation, degradation, and potentially of plant gene regulation. State-of-the-art methods from plant genetics, cell biology and both basic and high throughput biochemistry are integrated in this program to transition the NTME field from "activity by homology" to one grounded in solid biochemistry. The project will address the following questions: (1) Do Arabidopsis NTME proteins have enzymatic activities? (2) What are the specificities of NTME enzymes as determined by combinatorial peptide libraries? (3) When and where do NTME RNAs and proteins accumulate? (4) Do NTME proteins act co- or post-translationally? (5) What are the impacts of NTME T-DNA knock-out mutants? Collectively, these data will establish the specificity and location of each NTME and thereby establish a set of rules for the proteome, which will predict the fate of a protein by its subcellular localization and the primary sequence of its N-terminus.
Broader Impacts: This project will: (1) produce and distribute antisera to 39 NTME proteins, (2) develop methods to express and assay plant NTMEs, (3) synthesize novel combinatorial peptide libraries for determining the extended specificities of NTMEs, and (4) produce a collection of homozygous single and double mutants for NTMEs. The interactive NTME web site (www.cepceb.ucr.edu/members/walling/2010_NTMEs) will provide a list of the Arabidopsis NTME genes, NTME assays, links to the peptidase and transferase research communities, and mechanisms to access the resources developed by this project. The Arabidopsis Biological Resource Center (ABRC) will distribute mutants generated in this project. In addition to establishing the first biochemical rules to predict the fate of a protein in a subcellular compartment, this program provides a unique opportunity to train undergraduates, graduate students and postdoctoral fellows in a newly emerging field of N-terminal modifying enzymes in plants.
