Chloroplast-localized N-terminal Protein Processing by Peptide Deformylase

Peptide deformylase (DEF), an essential co-translational protein-processing enzyme that catalyzes the removal of the N-formyl group from nascent polypeptides in prokaryotic organisms, has been identified in plant chloroplasts and mitochondria. Inhibitors of DEF are phytotoxic, exhibiting characteristics of broad-spectrum herbicides, but the mechanism by which DEF is essential for cell viability is unknown. Because chloroplast-translated proteins are relatively limited in number and many have been extensively characterized both functionally and physically, this organelle provides a unique opportunity to study the essential nature of protein processing by DEF. Understanding chloroplast-localized DEFs and their role in establishing the mature biologically active form of newly-translated chloroplast-proteins is important, and can be achieved by fulfilling the following objectives: 1) identifying amino acid residues instrumental in active-site metal ion coordination and catalysis utilizing high-resolution NMR analyses coupled with site-directed mutagenesis; 2) elucidating the functional significance of chloroplast N-terminal protein processing through comparative 2D-PAGE analysis of a T-DNA insertional Atdef2-knockout mutant; and 3) determining the authentic subcellular and suborganellar location of Arabidopsis DEFs using full length GFP fusions. Diverse chloroplast processes critical to plant survival rely on proteins produced in the chloroplast which interact directly with imported proteins. Thus, how these proteins are processed by peptide deformylase and the relationship with other protein processing events define a fundamental determinant of chloroplast function. The understanding gained by completing this research will validate peptide deformylase as a viable target for a new class of broad-spectrum herbicides without mammalian toxicity, and create predictive rules governing chloroplast protein processing that could substantially enhance the success of chloroplast-targeted foreign gene expression for enhancing crop productivity. Exposure of undergraduate, graduate, and post-doctoral scholars to these interdisciplinary studies and the multi-user facilities required will provide unparalleled, multifaceted research and learning experiences, including participation in the commercialization and technological transfer of ideas and discoveries.