Plasma Membrane Packing Asymmetry Drives Transmembrane Protein Localization

Phospholipid asymmetry between the two plasma membrane leaflets has been discovered 40 years ago and since then, various physiological processes have been associated with asymmetric lipid distributions and changes to lipid asymmetry. Nevertheless, it remains unclear how lipid asymmetry affects the biophysical properties of individual leaflets and whether this putative biophysical asymmetry affects transmembrane proteins. To address these questions, we conducted a detailed analysis of asymmetric plasma membrane leaflet lipidomes and leaflet-specific biophysical properties. We further investigated whether distinct leaflets are maintained in intracellular organelles and how such intracellular membrane asymmetry may affect transmembrane protein localization and structure. Lipidomics revealed a striking disparity in lipid acyl chains, with the inner plasma membrane leaflet containing two-fold more acyl chain unsaturations than the outer leaflet. Consistent with the difference in unsaturation, we observed that the outer leaflet was highly packed, resembling a liquid ordered phase, whereas the inner leaflet was much more disordered. A bioinformatic analysis revealed that transmembrane domains (TMDs) of single-pass transmembrane proteins in the plasma membrane are broadly asymmetric in shape, with smaller accessible surface areas in the outer leaflet than the inner (i.e. thinner outside half of the TMD, fatter inside). We inferred that this shape asymmetry should facilitate insertion into the asymmetrically packed plasma membrane. We verified this hypothesis by creating TMD constructs with several shapes and discovered that proteins with a small accessible surface area in the outer leaflet preferred to localize at the plasma membrane regardless of their inner leaflet counterpart. This study delivers new insights into the structural organization of cell membranes and reveals that outer leaflet packing can drive protein localization to the plasma membrane