Project Summary/Abstract Eukaryotic cells must maintain a specific protein and lipid composition of the plasma membrane and all of the internal membrane-bound organelles in order to function normally. Even though membrane biogenesis is crucial for life, mechanisms for establishing the composition and organization of membranes remain poorly understood. We study how membrane asymmetry is established, a fundamental feature of the eukaryotic cell plasma membrane defined by the enrichment of phosphatidylserine and phosphatidylethanolamine within the cytosolic leaflet, while sphingolipids and phosphatidylcholine are typically enriched in the extracellular leaflet of the bilayer. Regulated exposure of PS and PE on the extracellular leaflet contributes to cell signaling, cytokinesis, blood clotting, cell-cell fusion, apoptotic cell corpse removal and host-viral interactions. Membrane asymmetry is driven by type IV P-type ATPases (P4-ATPases), which are a large family of flippases that pump lipids from the extracellular leaflet to the cytosolic leaflet of the membrane bilayer. The P4-ATPase subfamily is highly conserved among eukaryotes and these transporters have been implicated in pathological conditions such as obesity-linked type 2 diabetes, cardiovascular disease, liver disease, hearing loss, immune deficiency, and severe neurological disease. In addition, P4-ATPases are critical components of the vesicle-mediated protein trafficking machinery within the Golgi complex and endosomal system. Through their role in protein trafficking, P4-ATPases help control the precise protein composition of the plasma membrane, Golgi complex endosomes and lysosomes. The proposed studies will determine how the P4-ATPases recognize and transport their lipid substrates to establish membrane asymmetry using structural, biochemical and molecular genetic approaches. These structure/function studies will include how P4-ATPase activity is regulated by post- translational modification and protein-protein interactions. We will also probe the cellular requirements for transport of specific substrate lipids, like glucosylceramide and phosphatidylserine, on cell morphogenesis, fungal pathogenesis, nutrient signaling, and protein trafficking. For the latter studies, we will probe how P4- ATPases help drive vesicle-mediated protein transport with a focus on carriers formed by COPI and retromer. Atypical roles for ubiquitination in the P4-ATPase- and COPI-dependent transport pathways will also be defined. In total, these studies should lead to a much better understanding of how P4-ATPases exert their essential function, and will be invaluable to our ability to understand and ultimately treat pathologies associated with P4-ATPase deficiency.