The precursors to small peptide hormones are excellent models to study the molecular signals that regulate intracellular trafficking, proteolytic processing and secretion. We have recently expressed recombinant proteins that consist of fusions between two prohormones yeast pro alpha factor and vertebrate preprosomatostain. One of these constructs becomes tranisently arrested in the membrane of the yeast endoplasmic reticulum; this novel phenotype is completely attributable to a minor aminoterminal sequence alteration. To pinpoint the structural requirements for membrane translocation, site-directed mutagenesis will be used to predictively alter the aminoterminal sequence of these hybrid precursors, followed by expression in vivo and in vitro in yeast. To analyze the conformational changes that accompany membrane translocation, the membrane topography of these molecules will be mapped by physical means, and their potential interaction with as-yet unidentified endoplasmic reticulum membrane proteins will be assessed by footprinting and crosslinking. A yeast-derived cell-free protein synthesis system will be used to dissect the translocation process in vitro, and to test whether multiple pathways exist for uptake into the endoplasmic reticulum. The goal of these experiments is to define sorting and processing domains within peptide hormone precursors, and to identify and purify the cellular machinery that mediates different stages in the biogenesis of bioactive peptides.
