The Structural Basis of Transit Peptide Interaction(s) with the Chloroplast Toc Receptors

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Plastids have evolved into semi-autonomous organelles whose metabolic function is heavily dependent upon the import of nuclear-encoded proteins that were initially encoded by ancestral cyanobacterial genomes. The majority of nuclear-encoded proteins that function within the plastid are synthesized as higher molecular weight precursors with an N-terminal extension known as a transit peptide, which mediates specific and efficient targeting of the precursor to plastids. Analysis of the Arabidopsis genome predicts that more than 3000 precursors are plastid-targeted. The structural and functional criteria that define these thousands of transit peptides are not well understood. Analysis of hundreds of transit peptides to date has failed to reveal significant conserved primary sequence, suggesting that a common secondary or tertiary structure may account for the specific targeting activity of transit peptides. In this study, site-directed molecular mutagenesis, NMR, isothermal titration calorimetry, and analytical ultracentrifugation will be used to directly determine the structural basis of peptide-receptor interactions in Arabidopsis. This project will provide a new level of mechanistic understanding of chloroplast protein import. Findings from the project will provide a starting point for additional structure/function analysis of other transit peptides and their interaction with other components of the chloroplast translocation apparatus. An applied aspect of this research may be the enhanced targeting of novel plastid precursor proteins to plastids, thereby improving both the utility and safety of many different agronomic plants and plant products.