A Biochemical Investigation of Mechanisms of Peripheral Phosphoinositide-Binding Proteins

The interaction of peripheral proteins with cellular lipids is essential to cellular function and homeostasis. Several conserved protein families dock at cellular membranes in order to target catalytic domains to membrane-localized substrates, to metabolize lipids, and to spatially re-organize or deform membranes. This thesis is an investigation of the biophysical and biochemical mechanisms of phosphoinositide-binding proteins at cellular membranes. Beginning with an investigation of the PI(4)P-binding protein FAPP1 and its role in Golgi vesicle formation and trafficking, it then progresses to an investigation of a family of lipid-binding C2 domains present in mammalian ubiquitin ligases, and of the Ebola virus lipid-binding protein VP40. The C2 domains, present in the Nedd4 family of ubiquitin ligases, are shown to have novel properties as both phosphoinositide-binding and charge sensing domains. As these Nedd4 ubiquitin ligases have been shown to regulate important cellular processes such as membrane receptor endocytosis, growth factor signaling, epithelial to mesenchymal transition in cancer cells, and viral egress, this work provides a platform on which to understand how these proteins regulate cellular substrates via their localization to membranes. The study of Ebola VP40 provides greater detail on the anionic lipid-targeting mechanism of the Ebola virus protein via phospholipids, revealing it to have specific affinity for phosphoinosities at the plasma membrane in live cells. In addition, a novel ELISA-based assay to assess virus-like particle egress from VP40-transfected cells is detailed. This data provides a better understanding of how the Ebola matrix protein targets membranes in virus replication. Overall, this work provides the lipid research community with a greater understanding of specific protein-lipid interactions and also with new insights on the diversity of lipid-binding mechanisms in the C2 domain family.