The traffic patterns established by transport vesicles and other membrane carriers are of fundamental importance for protein localization, modification, and function within eukaryotic cells. Vesicle docking and fusion requires, in addition to soluble NSF attachment protein receptors (SNAREs), multisubunit tethering complexes (MTCs). This proposal focuses on three MTCs, conserved from yeast to mammals: the Dsl1 complex, the conserved oligomeric Golgi (COG) complex, and the homotypic fusion and vacuole protein sorting (HOPS) complex. The Dsl1 complex functions in COPI vesicle transport from the Golgi apparatus to the endoplasmic reticulum (ER), a pathway essential for the recycling of the anterograde transport machinery and the retrieval of ER-resident proteins. The COG complex functions in retrograde transport within the Golgi. As a result, COG is essential for normal Golgi structure and function, and defects in COG give rise to congenital disorders of glycosylation. Finally, membrane fusion at late endosomes and lysosomes/vacuoles depends on the HOPS complex. All six subunits of human HOPS are among the seven host proteins recently discovered to be required for Marburg and Ebola virus entry. We hypothesize that MTCs, through interactions with Rabs, SNAREs, Sec1/Munc18 proteins, vesicle coat proteins, and phospholipids, function to orchestrate the docking and fusion of transport vesicles. Achieving a deeper mechanistic understanding of MTC function depends critically on elucidating their structures and determining how they interact with other elements of the trafficking machinery. To this end, we propose three specific aims. In Aim 1, we will characterize functional interactions between the Dsl1 complex and other trafficking factors using x-ray crystallography and single particle electron microscopy (EM). In addition, we will capitalize on our complete structure of the Dsl1 complex by designing mutants to use in proteomic and synthetic genetic screens for additional Dsl1-interacting partners. In Aim 2, we will use single-particle EM to complete our mapping of the eight different subunits into the overall structure of the COG complex, complemented by x-ray crystallographic studies of interacting elements within COG sub- assemblies. Furthermore, we will determine COG-SNARE complex structures in order to elucidate how COG guides SNARE assembly. Finally, in Aim 3 we propose an entirely new project, structural studies of the HOPS complex and its interaction with SNAREs. We will determine structures of key HOPS subunits and sub- assemblies, which can then serve as blueprints for in vivo and in vitro functional studies. In addition, as in the first two Aims, w will use x-ray crystallography to study complexes with SNAREs. Because the HOPS complex is unrelated to the Dsl1 and COG complexes, this work should reveal both class-specific differences and common principles among MTCs, thereby deepening our mechanistic understanding of these fascinating components of the intracellular trafficking machinery.