A.B. Purup, P. Funke, C. Montigny, A. Di Cicco, T. Dieudonné, M.M. Frøsig, Y. Iwasaki, D. Lévy, T. Günther Pomorski, R.L. López-Marqués, J.A. Lyons, and G. Lenoir (2026).
Revisiting flippase specificity: Drs2-Cdc50 transports multiple anionic lipid substrates.
bioRxiv 2025.08.22.671827
doi: 10.1101/2025.08.22.671827
P4-ATPase lipid flippases maintain transbilayer lipid asymmetry in eukaryotic membranes, which is essential for many cellular processes. In yeast, the Drs2-Cdc50 flippase complex was previously shown to specifically transport phosphatidylserine (PS) from the exoplasmic to the cytosolic leaflet of the trans-Golgi network (TGN), thereby controlling vesicular trafficking in the secretory and endocytic pathways. Using an improved proteoliposome-based lipid flippase assay, we now show that the Drs2-Cdc50 complex transports multiple anionic glycerophospholipids, including PS, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. In vivo cell-based lipid uptake assays further support the transport of these lipids. To understand the basis of this substrate promiscuity, we analyzed cryo-EM structures of the complex with occluded lipids. These structures revealed that the water network surrounding the lipid headgroup plays a critical role in enabling Drs2-Cdc50 to recognize different lipids. These data unveil an unexpected broad specificity of the Drs2-Cdc50 complex for anionic lipids, which may significantly impact their transbilayer distribution in the yeast TGN.