A fundamental feature of cellular plasma membranes (PMs) is an asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being approximately twofold more unsaturated than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asymmetric structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.

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All fluorescence microscopy was performed at the Center for Advanced Microscopy, Department of Integrative Biology & Pharmacology at McGovern Medical School, UTHealth. We thank N. Waxham for his generous sharing of the microinjection system. We acknowledge K. Simons, T. Steck, Y. Lange and G. Feigenson for their critical feedback on this manuscript. Funding for this work was provided by the NIH/National Institute of General Medical Sciences (GM114282, GM124072, GM120351 and GM134949), the Volkswagen Foundation (grant no. 93091) and the Human Frontiers Science Program (RGP0059/2019). E.S. is funded by Newton-Katip Ҫelebi Institutional Links grant no. 352333122. Anton2 computer time was provided by the National Resource for Biomedical Supercomputing (NRBSC), the Pittsburgh Supercomputing Center (PSC) and the Biomedical Technology Research Center for Multiscale Modeling of Biological Systems through grant no. P41GM103712-S1 from the National Institutes of Health.

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Asymmetric lipidomes.

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Lorent, J.H., Levental, K.R., Ganesan, L. et al. Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape. Nat Chem Biol 16, 644–652 (2020). https://doi.org/10.1038/s41589-020-0529-6

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