Revisiting flippase specificity: Drs2-Cdc50 transports multiple anionic lipid substrates

Purup, A. B.; Funke, P.; Montigny, C.; Di Cicco, A.; Dieudonne, T.; Frosig, M. M.; Iwasaki, Y.; levy, D.; Guenther Pomorski, T.; Lopez Marques, R. L.; Lyons, J. A.; Lenoir, G.

doi:10.1101/2025.08.22.671827

bioRxiv · 10.1101/2025.08.22.671827

Revisiting flippase specificity: Drs2-Cdc50 transports multiple anionic lipid substrates

Purup, A. B.·Funke, P.·Montigny, C.·Di Cicco, A.·Dieudonne, T.·Frosig, M. M.·Iwasaki, Y.·levy, D.·Guenther Pomorski, T.·Lopez Marques, R. L.·Lyons, J. A.·Lenoir, G.

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Abstract

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.

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Purup, A. B., Funke, P., Montigny, C., Di Cicco, A., Dieudonne, T., Frosig, M. M., Iwasaki, Y., levy, D., Guenther Pomorski, T., Lopez Marques, R. L., Lyons, J. A., Lenoir, G.. 2025-08-27. Revisiting flippase specificity: Drs2-Cdc50 transports multiple anionic lipid substrates. https://doi.org/10.1101/2025.08.22.671827

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