Prokaryotic and eukaryotic secreted proteins are, in general, synthesized on membrane-bound polysomes with extra amino-terminal leader sequences which facilitate cotranslational insertion and transport across the cytoplasmic membrane. However, I have found that at least Subunit A of cholera toxin is synthesized in the cytoplasm as a precursor form on free, rather than membrane-bound polysomes, suggesting that cholera toxin is secreted by a different, post-translational mechanism. The goal of the proposed research is to continue to examine the mechanism of secretion cholera toxin by V. cholerae, difining the steps invloved in synthesis, processing, assemblyum, and release of holotoxin from the bacterial cell. Membrane fractions of V. cholerae will be examined for cholera toxin precursor processing activity(s) and we will attempt to reconstitute, in vitro, the secretion process for cholera toxin with prepared membrane vesicles. Transient assembly intermediates of holotoxin will be identified in various cell fractions by in vivo labeling, pulse chase experiments, and immunoprecipitation of cholera toxin-related polypeptides. Membrane perturbants and antibiotics will be utilized to specifically accumulate novel cholera toxin intermediate forms. Secretion mutants, defective in processing, assembly, or release of toxin, will be isolated and characterized. It is clear that the cholera enterotoxin molecule is primarily responsible for manifestations of cholera disease. Studies of secretion, processing, and assembly of cholera toxin will be pursued with the belief that an awareness of these processes at a molecular level is of fundamental importance for a clear understanding of the disease process and the posible development of an effective, safe, live oral vaccine for cholera.