Most chloroplast proteins are encoded in the nucleus, synthesized in the cytoplasm as higher molecular weight precursors and imported into chloroplasts posttranslationally. Our long term goal is to understand how this import process occurs. Protein import will be studied in an in vitro reconstituted assay. In this assay, radiolabeled precursor proteins are first synthesized by in vitro transcription and translation of cloned precursor genes and then incubated with isolated intact chloroplasts. The present studies will focus on two particular aspects of the import process. The first is the mechanism whereby precursor proteins get translocated across the two envelope membranes. Efforts will be made to identify translocation intermediates in order to gain a more complete description of the stages in the import process. Efforts will also be made to identify proteins of the chloroplast envelope membranes that are involved in precursor import. The second aspect of the proposed studies will be to investigate the pathway and mechanisms whereby cytoplasmically synthesized proteins are transported to the thylakoid lumen. This process is particularly interesting because these proteins cross three membranes between their site of synthesis and their final location.%%% The insights gained from this work will be important in two respects. First, they will add to basic knowledge regarding the targeting of cytoplasmically synthesized proteins to their proper locations in eukaryotic cells. Second, these results will have practical application during efforts to alter the metabolism of chloroplasts by genetic engineering.***//
