| Summary: | The plasma membrane of eukaryotic cells displays a distinctive asymmetry of the component phospholipids. Type-IV P-type ATPases (P4-ATPases) flip specific phospholipids across the membrane bilayer to establish and maintain this membrane asymmetry. P4-ATPases are closely related to ion-transporting P-type ATPases that use a single site within the center of the transmembrane domain to select and transport their substrates. This single binding site is spatially restrictive and will not accommodate ions of the same valence but different ionic radius. Fitting a phospholipid into this constrictive single binding site to is difficult to imagine and thus, P4-ATPase phospholipid recognition is referred to as the giant substrate problem. This work describes the characterization of the mechanism used by P4-ATPases to select and transport their phospholipid substrates. Surprisingly, P4-ATPases solve the giant substrate problem by using two gates at the protein-lipid interface to select and transport phospholipid, one at each face of the lipid bilayer.
A second defining feature of eukaryotes is membrane-bound organelles that serve a multitude of functions. Defined compartments present a challenge for coordinating separate steps in biochemical pathways carried out in distinct organelles. In addition to the role in membrane asymmetry, P4-ATPases play critical roles in communication between the organelles through vesicular-mediated protein transport. Using P4-ATPase variants with altered phospholipid specificity, we identified a role for phosphatidylserine flip in specific transport pathways. Here, we report phosphatidylserine flip facilitates traffic between the trans-Golgi network and the early endosomes, but not in other trafficking pathways within the Golgi-endosomal system. This dissertation has propelled the P-type ATPase and protein trafficking fields forward through a description of a novel P-type ATPase transport mechanism and a physiological role for the phospholipid flip in specific trafficking pathways.
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