Elucidation of the mechanism of export of protein in prokaryotic systems will provide information relevant to a phenomenon central to many biological process such as secretion and biogenesis of organelles. Furthermore, the recently developed recombinant DNA technology has made possible the production of medically important eukaryotic proteins in bacteria. Such production can be made most efficient if we understand the details of export in bacteria. Data derived from studies on export is currently organized in terms of either the signal hypothesis or the trigger hypothesis. We propose experiments designed to distinguish between the major features of these hypotheses. We shall determine at what point in synthesis nascent chains initially bind to the membrane, when and by what mechanism they are translocated and proteolytically processed and further characterize the involvement of proton motive force in export. We shall initiate investigations of export in a gram positive bacterium and compare the findings with what is known about export in gram negative bacteria. The proposed research involves techniques of molecular biology and biochemistry such as in vivo pulse-labeling, in vitro protein synthesis, membrane isolation and fractionation and analysis of proteolytic digests by high performance liquid chromatography. The work proposed should result in significant progress toward the understanding of the specific passage of protein through biological membranes.
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