9722914 Schnell The mechanism by which cytoplasmically synthesized proteins are targeted and assembled into their proper subcellular compartment remains a major unsolved problem in cell biology. The cells of higher plants contain a unique organelle, the chloroplast, which offers an excellent model system for studying this problem. The majority of the protein components of the chloroplast are encoded in the nucleus, synthesized as precursors on cytoplasmic ribosomes, and posttranslationally imported into the organelle across the double membrane of the chloroplast envelope. The long-term goal of this research is to understand the mechanism of protein import into chloroplasts. In recent years, the signals responsible for targeting proteins to the chloroplast have been determined, and a number of components of the protein import apparatus at the chloroplast envelope have been identified. The major challenges to a more detailed understanding of the import process are determining the supramolecular structure and dynamics of the import apparatus and defining how the import components interact with precursor proteins during the import process. This proposal addresses both of these challenges. The first aim of this proposal is to investigate the structure and dynamics of membrane complexes that mediate protein import at the outer and inner envelope membranes. This strategy will use immunochemical and biochemical techniques to investigate the composition and regulation of import complexes within and between the two envelope membranes. These studies will indicate the supramolecular structure of the import apparatus and provide information on how the outer and inner membranes cooperate to facilitate transport of proteins across the envelope. The second aim of this proposal is to determine the sequence and specificity of the interaction of the import components with precursor proteins during the complete time course of protein import. This strategy will em ploy chemical cross-linking of precursor proteins to import components at intermediate stages in import ranging from initial binding of the precursor at the outer membrane to the completion of translocation at the inner membrane. The third aim is to characterize two newly discovered import components, IAP21 and IAP25, whose molecular properties have not been investigated. To this end, cDNA clones for each of the putative components will be isolated and sequenced, and antibodies specific for each component will be generated to localize the putative import components within the envelope. These reagents will provide the basic tools for functional studies aimed at determining the roles of each protein in envelope translocation. The sum of these studies will provide a considerable advancement in our goal of understanding the mechanism of protein import into chloroplasts. These studies are significant for several reasons. First, protein translocation plays a central role in chloroplast biogenesis. Therefore, definition of this process is essential to obtaining an understanding of the mechanism by which the photosynthetic and metabolic machinery of the organelle are targeted and assembled. Second, understanding the import process is important for applications directed at optimizing the metabolic and photosynthetic capabilities of genetically engineered plants by directing foreign or altered proteins to chloroplasts. Finally, the parameters governing the chloroplast protein import undoubtedly will contribute to the formulation of broader principles concerning the mechanism of protein targeting and membrane translocation in all cells. Chloroplasts are complex subcellular structures that support the photosynthetic metabolism of plants and algae. The interior of the chloroplasts contains many different proteins and a complex of membranes that mediate the photosynthetic process. Most of the proteins that support the photosynthetic metabolism are made in the cell outside of the chloroplast and are translocated into the chloroplast as precursors. Chloroplasts are enveloped by a double unit membrane that must be bridged in order to deposit proteins within its interior. This award will support detailed studies to examine the process by which proteins are translocated through the double membrane that bounds the chloroplast. The information gained from these studies will greatly advance the understanding of how the chloroplast is assembled. ***
