Principal Investigators:

Dr. Linda L. Walling, Department of Botany & Plant Sciences, University of CA, Riverside, CA
Dr. Ralph A. Bradshaw, Department of Physiology & Biophysics, University of CA, Irvine, CA
Dr. Ben Dunn, Department of Biochemistry & Molecular Biology, University of Florida, College of Medicine, Gainesville, FL

Intellectual Merit:

The N-terminus of a protein is often co- or post-translationally modified. These modifications may be required for activation or regulation of a protein's activity or may dictate protein half-life. We will determine the specificity and mechanisms of the Arabidopsis N-terminal modification enzymes (NTMEs) by focusing on the enzymes that: 1) remove one to three N-terminal amino acids; 2) modify the a-amino group; and 3) alter residue side chains. Virtually nothing is known about these processes in plants. There are 27 peptidases and 11 transferases that have likely roles in N-terminal modification of the Arabidopsis proteins. By understanding the program of expression, subcellular localization, and extended substrate specificity for each NTME, we will establish N-modification rules for the Arabidopsis proteome. Ultimately, we would like to predict the fate of a protein by the primary sequence of its N-terminus. We integrate state-of-the-art methods from plant genetics, cell biology and biochemistry to address the following questions: (1) Do NTMEs have enzymatic activities? (2) What are the specificities of each NTME, as determined by combinatorial peptide libraries? (3) When and where do NTME RNAs and proteins accumulate? (4) Do NTMEs act co-or post-translationally? and (5) What are the impacts of NTME T-DNA knock-out mutants?

Broader Impacts Criterion:

The NTME project will establish the biochemical rules to predict the fate of any protein in any subcellular compartment. This project will develop an accessible, interactive NTME web site, a collection of homozygous single and double NTME mutants, antisera for 38 NTMEs, and microarrays containing NTMEs, which will be readily available to the scientific community. This project also develops highly specialized combinatorial peptide libraries that can be accessed by the scientific community. This program provides training for graduate students and postdoctoral fellows in the newly emerging plant NTME field. In addition, there is a strong undergraduate training component. UC Riverside (UCR) has extraordinary diversity in its undergraduate population and it is easy to engage students in research during the academic year and summers. UCR undergraduates will receive plant biology training at UCR and, during their second year of training, students will have the opportunity to do an internship with collaborators at UC Irvine, University of Florida, Purdue, or Saskatoon (Canada). In addition, this 2010 program will exchange students/postdoctoral fellows with SUNY (Binghamton). SUNY visitors will make and use cDNA NTME/peptidase microarrays while at UCR. UCR investigators will engage in the proteomics approach to Arabidopsis endopeptidases while in Binghamton.