Control of Membrane Asymmetry by P4-ATPases

Abstract

The plasma membrane of eukaryotic cells is an asymmetric structure with the cytosolic leaflet highly enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE), whereas these lipids are rare in the extracellular leaflet. This asymmetric organization is generated by phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family that pump specific phospholipids (e.g. PS and PE) unidirectionally to the cytosolic leaflet. Cells typically express several P4-ATPases that localize to the Golgi, plasma membrane and endosomal system, and differ in substrate specificity. We discovered a crucial role for P4-ATPases in budding protein transport vesicles from the trans-Golgi network (TGN) and early endosomal membranes. Current studies are aimed at understanding how P4-ATPases from Saccharomyces cerevisiae recognize and flip specific phospholipid species to generate membrane asymmetry, and why this plays an important role in budding certain types of transport vesicles. We have succeeded in mapping a number of residues that determine the phospholipid substrate specificity of the P4-ATPases Drs2 and Dnf1 to the first four transmembrane segments. These studies imply that the P4-ATPases use a non-canonical mechanism to recognize and transport substrate relative to closely related Ca++-ATPase. In addition, we can mutationally “tune” the substrate specificity of Drs2 and Dnf1 and use these mutants to probe the cellular requirements for specific P4-ATPase substrates. Neo1 is an essential P4-ATPase in yeast that is closely related to ATP9A and 9B in mammalian cells. Recent studies imply that Neo1 is the primary PE flippase in yeast and a mutation in Drs2 that enhances its recognition of PE can functionally replace Neo1 in vivo.