PROJECT SUMMARY Regulated exocytosis – stimulus-dependent exocytic vesicle fusion – mediates a broad range of fundamental biological processes including nutrient homeostasis, cell-to-cell signaling, and elimination of transformed or virally infected cells. Imbalances in these exocytic pathways lead to major forms of human disease. The long- term goal of this project is to uncover the protein networks governing regulated exocytosis. A major model pathway we use is insulin-stimulated GLUT4 exocytosis, a physiologically important but poorly characterized regulated exocytic pathway. We also examine key aspects of other exocytic pathways, aiming to establish conserved and divergent mechanisms of regulated exocytosis. In our previous research, we carried out CRISPR genetic screens to systematically dissect insulin-stimulated GLUT4 exocytosis and other exocytic pathways, and identified a large number of new regulators. In this proposed research, we will carry out in-depth biochemical and genetic studies to characterize new exocytic regulators isolated in the screens. We will also investigate how exocytic vesicle fusion is coupled with other cellular processes such as clathrin-mediated endocytosis (CME) and intracellular signaling to achieve an appropriate exocytic response. In particular, we will examine how AP2 adaptor, a central player in CME, is assembled in the cytosol with the assistance of dedicated assembly chaperones. Besides these in-depth mechanistic studies, we will continue to identify and validate new regulators based on candidate genes isolated in our CRISPR screens. The mechanistic studies and CRISPR screens are fully complementary and will provide a comprehensive understanding of regulated exocytosis that neither approach alone could generate. Successful completion of this proposed research will fill major gaps in the knowledge of regulated exocytosis and will serve as a springboard for understanding the molecular basis of membrane trafficking in general. Ultimately, insights gleaned from this work will facilitate the development of new therapeutic strategies for diseases caused by dysregulated exocytosis.