EXPORT OF PROTEINS IN ESCHERICHIA COLI

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The phenomenon of translocation of specific, newly synthesized polypeptides across or into biological membranes is common to eukaryotic secretion, organelle biogenesis and prokaryotic protein export. Although there are obvious differences among the systems, it seems likely that underlying principles are shared and similarities may exist in the molecular mechanisms; thus, as investigators attempt to elucidate the molecular mechanism of protein localization, it will be important to keep in mind the results obtained in each of the model systems. The research plan proposed here is aimed at elucidating the mechanism of export of protein in the bacterium, Escherichia coli. The three main objectives can be broadly stated as: 1) to delineate the pathway of export by defining the steps that exist in vivo and determining whether or not the leader peptide plays a role at each of these steps, 2) to characterize the cytosolic factor involved in export with respect to its function and the nature of interaction with the precursor, 3) to elucidate the role of conformation and folding of the precursors during the export process. A balance of in vivo and in vitro studies will be maintained during the course of the work. Thus, in vivo intermediates identified by pulse-labelling of cells in exponential growth phase will be analysed in vitro to determine their function in the pathway and the nature of interaction of the precursor with the apparatus. Studies designed to determine the rate of folding in intact cells will be combined with studies of folding using purified proteins and fluorescence spectroscopy. Attempts to purify the cytosolic factor will involve use of cross-linking reagents and standard column chromatography. The periplasmic protein maltose-binding protein was chosen for study not only because it is well characterized but also so that we can make use of the mutated strains which produce export- defective species of the protein that are available to us. The studies proposed here should complement those in other laboratories and contribute to the understanding of the fundamental process of protein localization.