Previous work focussed on analysis of protein transport into chloroplasts using the small subunit of ribulose-1,5-bisphosphate carboxylase, pSSU, as the model. The effects of deletion mutations in the pSSU transit peptide (TP) which is essential for protein import were studied. Based on those results, mutants will now be generated with changes in the carboxyterminal and central regions of the TP. Site-directed mutagenesis will be used to alter a small number of specific residues previously identified as crucial to protein transport in those regions. Since functional residues in the aminoterminal region are less well defined, mutants in this region will be created by random chemical mutagenesis. Mutants will be analyzed for membrane binding, processing steps, and ATP requirements for transport. Preproteins, which are either located in the stroma or in the thylakoid membrane, will be isolated. They will be used to target and isolate different membrane receptor classes. Synthetic peptides representing functional regions of TP will be used for the isolation of membrane receptor components, which will be characterized. The long-term goal is to generate a mechanistic model of the transport process. The complexity of eucaryotic cells makes it necessary to target proteins to specific compartments which separate metabolic pathways. The routes to various major subcellular (and extracellular) compartments differ in specific ways. Even among different types of cells, the sorting may differ. For example, while green plants sort proteins destined for the two distinctly different double-membrane- enclosed intracellular compartments, mitochondria and for chloroplasts, precisely distinctly, yeast cells (which lack chloroplasts) will use the "chloroplast import signal" to target proteins into mitochondria. The mechanism(s) whereby information encoded in the amino acid sequence of specific proteins is "translated" into specific translocation into appropriate compartments is a fundamental problem in cell biology, which is addressed directly in this proposal.
