The mechanism of protein transport across specific intracellular membranes is a central issue in cell biology. This project focuses on the hydrophobic signal sequences which direct the transfer of secretory proteins across the endoplasmic reticulum (ER) membrane, the first step in the secretion of proteins from the cell. The importance of signal sequences has been well-established, but their fate, once removed from precursor proteins by the action of the ER membrane protein signal peptidase, is unknown. The objective of the experiments to be carried out is to investigate the proteolysis of different polypeptides containing functional signal sequences in an effort to define a proposed signal peptide hydrolase activity in Xenopus laevis oocytes. The intracellular fates of both engineered and unmodified cleaved signal peptides will be observed, and the properties of their degradation will be characterized. The results of these investigations will be used to develop a working assay for signal peptide hydrolases in Xenopus oocytes. Such an assay is essential to efforts to identify and purify the proteins which specifically metabolize cleaved signal sequences. These proteins may be important not only for clearance of signal sequences from cells, but also in the actual mechanism of translocation across the ER membrane. The technique which will be employed to follow the fate of cleaved signal peptides in Xenopus oocytes may provide a powerful tool for the dissection of discrete steps in the transfer of proteins across the ER membrane in living cells. %%% The transfer of proteins across the ER membrane as they are synthesized in eukaryotic cells is a critical first step in the trafficking of proteins to a variety of destinations, including lysosomes and the extracellular space (secretion). Advances in our understanding of this fundamental process are important not only for general understanding of how cells function, but also for biotechnology. This project addresses the question of the fate of the cleaved signal sequence, that is, the piece of the newly- synthesized protein which is required for transport across the ER membrane, but which is not part of the "finished" protein product and is therefore enzymatically cleaved from the rest of the protein very soon after the transfer is effected. This is an area of cell biology which has received very little attention, despite the high level of research activity on other aspects of the secretory process. There is a good likelihood that unforeseen new and potentially important insights will emerge from the work.