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.