9630459 Keegstra A significant unsolved problem in cell biology is the question of 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, synthesized in the cytoplasm as higher molecular weight precursors, and imported posttranslationally across the two envelope membranes. The long term goal of this research is to provide a detailed molecular understanding of how this import process occurs. Significant process has been made in recent years in identifying and characterizing the targeting signals contained within precursor proteins, and in identifying and characterizing the molecular components of the transport apparatus. The goal of this project is to understand how precursor proteins interact with the components of the transport apparatus to achieve proper targeting and translocation. To this end, three specific objectives will be pursued. The first is to investigate the supramolecular structure of transport components present in the outer membrane of the chloroplast envelope and determine how their arrangement is altered by precursors. The second is to investigate the formation of translocation contact sites that bring together transport components present in the outer envelope membrane, the inner envelope membrane and in the chloroplastic stromal space. Both of these objectives will be pursued using chemical cross-linking strategies to stabilize translocation complexes, followed by sensitive immunological and biochemical techniques to determine the composition of each complex. The third objective will be to investigate the topology of two components of the transport apparatus present in the outer envelope membrane. These two proteins were chosen for detailed study because they are thought to be important in the transport apparatus and because they appear to have unusual properties for membrane p roteins. Understanding how these proteins are situated in the envelope membrane should eventually lead to new insights into how they mediate the transport of precursor proteins across the envelope membranes. %%% Understanding protein import into chloroplasts is important for at least two reasons. First, it adds to our understanding of a basic cellular process, i.e., intracellular protein trafficking. Second, many economically important functions within plants occur in the chloroplasts, and therefore an increased understanding of protein transport into chloroplasts will have practical significance for efforts to employ genetic engineering to alter the metabolic processes inside chloroplasts. ***
