This research seeks to determine the mechanism by which proteins are exported from the cytoplasm of E. coli. Exported periplasmic proteins, which include two leucine binding proteins and Beta-lactamase, will be used as model systems. In vitro studies of processing and translocation of these proteins will be carried out using cell-free synthesis systems in conjunction with bacterial membrane vesicles. In vivo experiments will be performed in whole cells and speroplasts of E. coli. The energy requirements and manner of energy use for export events will be examined in vitro and in vivo using ionophoric uncouplers of protonmotive force and transmembrane potential. The export properties of different fractions and cell extracts from export mutant strains will be investigated in vitro to identify the cellular location and manner of action of different components of export machinery. Crosslinkers will be used to identify components that interact with exported proteins along the export pathway. Group-specific chemical probes and proteases will be used to determine the topography of exported proteins with respect to the cell membrane at different stages in export. Sequence regions that are critical for export will be identified in exported protein sequences by site-directed modification and creation of internal deletions within the genes. Studies of bacterial protein export should lead to better understanding of protein dynamics in membranes and utilization of membrane energy. Fundamental information about bacterial protein export may eventually be helpful in production of recombinant DNA gene products for pharmaceutical purposes.
