Molecular Mechanism of Protein Transport into Chloroplasts

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Chloroplasts are the organelles within plant cells that are responsible for photosynthesis in addition to many other important aspects of metabolism. However, most of the proteins needed for the functions performed by chloroplasts are encoded in nuclear genes and imported into the organelle following their synthesis in the cytosol. While the basic outlines of the import process have been described, many aspects remain unknown. This project will investigate the details of precursor protein import into chloroplasts using a site-specific cross-linking strategy. Unnatural amino acids containing a photoreactive functional group will be incorporated into specific locations within precursor proteins and then allowed to interact with the transport apparatus of isolated intact chloroplasts. Following treatment with UV light to activate the cross-linking reagent, the products will be analyzed in an effort to map the set of interactions that occur as precursor proteins are transported across the chloroplastic envelope membranes. The expertise required to perform this specialized cross-linking strategy is not widely available, but is available at the University of Nagoya in Japan. Thus the project will be done in collaboration with the laboratory of Professor Endo to accomplish these experiments.