Molecular Mechanism of Protein Transport into Chloroplasts

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A significant unresolved problem in cell biology is understanding how cytoplasmically synthesized proteins are targeted to the various compartments of eukaryotic cells. In plant cells, chloroplasts offer an excellent system for studying this problem. Most chloroplastic proteins are encoded in the nucleus and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported postranslationally into chloroplasts across the two envelope membranes and are subsequently directed to the proper location within the organelle. The long term goal of this project is to understand how the import process occurs. At the present time, the major barrier to gaining a more detailed understanding of protein transport into chloroplasts is a lack of information regarding the identity of transport intermediates. The basic strategy of the project will be to block protein transport at specific stages, thereby generating translocation intermediates containing precursors at discrete points in the transport process. Cross-linking reagents will be used to identify transport components that are in close physical proximity to the trapped intermediates. As putative components are identified, further work will be required to confirm their involvement in transport and to determine their function during the transport process. To accomplish these objectives, specific antibodies directed against each putative component will be prepared and cDNA clones encoding each component will be isolated and sequenced. These antibodies and CDNA clones will be valuable tools for investigating the functions of the putative components. %%% This work is significant for at least two reasons. First, it adds to the general understanding of a basic cellular process, i.e. intracellular protein trafficking. Second, it has practical significance for efforts to alter the metabolic pathways of chloroplasts using genetic engineering techniques, in which foreign proteins will need to be directed into chloroplasts.