ABSTRACT Vesicle trafficking is an essential process in all eukaryotic cells. Conserved protein complexes known as membrane tethering factors are required for vesicle fusion, and multiple models have been proposed for their mechanism of action: recruitment and tethering of vesicles at the target membrane, removal of the vesicle coat, and/or chaperoning the assembly of membrane fusogenic SNARE proteins. The simplest and best- characterized tethering factor, Dsl1, has been proposed to perform all of these functions. I intend to test this hypothesis by structurally characterizing Dsl1 in complex with SNAREs and vesicle coat subunits. I will also develop a novel assay to visualize the tethering of coated vesicles in the cell. These results will allow me to better define the molecular mechanism of Dsl1 action and will lead to a more thorough understanding of how tethering factors, coats, and SNAREs work together to regulate vesicle fusion. My proposed work will also form the basis for a better understanding of coat and tethering factor dysfunction, which has been implicated in both heritable diseases, such as congenital autoimmune disorders and spinal muscular atrophy, and sporadic diseases including Alzheimer's and Parkinson's disease.