Structural Insights into the Function and Auto-Regulation of Lipid Flippases

Biological membranes are composed of bilayers, typically with highly asymmetric distributions of lipids that are of key importance for biomembrane function and dynamics, such as in secretory pathways, lipid signaling, cell-cell interactions, and motility. The asymmetric distributions are actively maintained by so-called lipid flippases and floppases that also counteract the passive scrambling of lipid gradients. Dominating contributions in eukaryotes come from lipid flippases of the P4 subfamily of P-type ATPases, where the majority of lipid flippases form a binary complex with a member of the Cdc50 family. These transporters are involved in the inward active translocation, hence “flipping”, of phospholipids across the membrane bilayer. Central questions on P4 ATPase lipid flippases address i) their overall architecture and function in the take up and flipping of lipids in membranes, and ii) how they are able to accommodate and transport a much larger substrate than other P-type ATPases, which usually transport small cations. Additionally, some P4-ATPases are subjects to auto-inhibition mediated through extended cytosolic termini, a feature that is shared with other P-type ATPases, like the plasma membrane calcium ATPase. Here we present the first structures of a yeast lipid flippase, the phosphatidylserine (PS) specific flippase Drs2p/Cdc50p, with and without the auto-regulatory C-terminus, thus sampling inhibited and uninhibited conformations. Comparison of an autoinhibited and a truncated, PI4P activated state show re-arrangements of the cytosolic domains which transmit into flexible changes in the transmembrane domain. From these observations we propose a lipid substrate transport pathway. Our results provide a structural framework to understand lipid translocation between membrane leaflets and the auto-regulation of lipid flippases.