Proc. Natl. Acad. Sci. USA 113, E4460–E4466 (2016)

Credit: PNAS

In eukaryotes, the asymmetry of the plasma membrane is critical to its proper function, and this asymmetry is maintained by phospholipid flippase enzymes that selectively transport phospholipids from the exofacial to the cytofacial leaflet of the membrane. In yeast, the P4-ATPase Dnf1 is a phosphatidylcholine (PC) flippase and does not transport sphingomyelin (SM), even though the two have the same choline headgroup and highly similar fatty acyl chains. To understand how Dnf1 exerts such selectivity for its phospholipid substrates, Roland et al. used a directed evolution library of Dnf1 variants in a screen with fluorescently labeled PC and SM to identify flippase sequences capable of selecting for SM instead of PC. A single amino acid change at the cytofacial side of the transmembrane domain enabled Dnf1 to select for the sphingosine backbone of SM over the glycerol backbone of PC. Homology modeling and additional mutagenesis further elucidated how this position, along with others nearby in the exit-gate region of the protein, discriminates between various phospholipid backbones as well as different headgroups. The insights into selectivity afforded by this study suggest how substrate phospholipids are coordinated by the exit gate and should enable the design of new substrate-specific flippases for future studies of membrane asymmetry.