IMPORT OF POLYPEPTIDES INTO CHLOROPLASTS

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The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase and the constituent (AB) polypeptides of the light-harvesting chlorophyll a/b protein complex are synthesized on cytoplasmic ribosomes as precursor polypeptides. These precursors (preSS and preAB) are characterized by an N-terminal extension known as the transit peptide. We have recently demonstrated that the transit peptide for preSS can mediate the posttranslational import into chloroplasts of a foreign polypeptide. This proposal is concerned with defining both how general this observation is and the sequences within the transit peptide that mediate the individual steps of import. For these studies we will use in vitro expression systems to generate the labeled polypeptides of interest. The polypeptides we propose to study include a variety of fusion polypeptides of distinguishing characteristics and mutant polypeptides in which we will have generated deletions within the transit peptide of either preSS or preAB. We will examine the import into isolated chloroplasts for individual polypeptides and for those that are defective we will determine whether the defect occurs with bindings, translocation or processing. By these procedures we expect to characterize domains within the transit peptides that mediate destinct import processes. We will define sequences within these domains by oligonucleotide-directed mutagenesis. The proposed studies will contribute to our understanding of the process of import of polypeptides into organelles and will aid the future manipulation of phenotypic characteristics of plants.