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

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

Lipid scrambling facilitates membrane vesiculation through decreasing membrane stiffness

Membrane blebbing plays important roles in cell migration, cytokinesis, apoptosis, as well as extracellular vesicle formation for intercellular communication. During budding of the plasma membrane (PM) to form extracellular vesicles, phosphatidylserine is often translocated from the PM inner leaflet to the outer leaflet, indicative of lipid scrambling that results in loss of native membrane asymmetry. Using micrometer-sized giant plasma membrane vesicles (GPMV) as a readout of PM vesiculation, we found that knocking out (KO) the lipid scramblase TMEM16F reduced lipid scrambling and dramatically inhibited GPMV formation, in line with previous observations. TMEM16F repletion could rescue GPMV formation, indicating that lipid scrambling is necessary for membrane budding. We hypothesized three mechanisms to explain this surprising observation, namely that lipid scrambling: (1) depletes PI(4,5)P2 from the inner leaflet leading to detachment of the PM from the actin cytoskeleton, (2) makes PM bilayers softer and easier to bend, (3) externalizes lipids that stabilize highly curved bud necks. Depletion of PI(4,5)P2 was indeed observed during vesiculation, however this occurred in both wild type and KO cells, implying that detachment of PM from actin cytoskeleton due to PIP2 depletion is not sufficient to make large vesicles. Next, through fluorescence lifetime imaging microscopy of a lipid packing reporter (Di4), we found that lipid scrambling leads to a decrease of PM packing, potentially making the membrane softer for bending and thus facilitating membrane budding. Finally, we observed that lipid scrambling resulted in externalization of phosphatidylethanolamine (PE). This PE was preferentially located at the highly curved vesicle necks, suggesting that the intrinsic curvature of PE stabilizes this highly curved membrane structure. Thus, we conclude that lipid scrambling changes membrane biophysical properties, potentially explaining mechanisms underlying vesicle-budding in physiological contexts including apoptotic cell removal, bone mineralization, fertilization, and blood coagulation

Myelin-Associated MAL and PLP Are Unusual among Multipass Transmembrane Proteins in Preferring Ordered Membrane Domains

Eukaryotic membranes can be partitioned into lipid-driven membrane microdomains called lipid rafts, which function to sort lipids and proteins in the plane of the membrane. As protein selectivity underlies all functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of raft affinity. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multipass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored. Here we used cell-derived giant plasma membrane vesicles (GPMVs) to systematically measure multipass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multipass TMPs, the large majority (92%) had minimal raft affinity. The only exceptions were two myelin-associated four-pass TMPs, myelin and lymphocyte protein (MAL), and proteo lipid protein (PLP). We characterized the potential mechanisms for their exceptional raft affinity and observed that PLP requires cholesterol and sphingolipids for optimal association with ordered membrane domains and that PLP and MAL appear to compete for cholesterol-mediated raft affinity. These observations suggest broad conclusions about the composition of ordered membrane domains in cells and point to previously unrecognized drivers of raft affinity for multipass transmembrane proteins

Myelin-Associated MAL and PLP Are Unusual among Multipass Transmembrane Proteins in Preferring Ordered Membrane Domains

Eukaryotic membranes can be partitioned into lipid-driven membrane microdomains called lipid rafts, which function to sort lipids and proteins in the plane of the membrane. As protein selectivity underlies all functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of raft affinity. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multipass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored. Here we used cell-derived giant plasma membrane vesicles (GPMVs) to systematically measure multipass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multipass TMPs, the large majority (92%) had minimal raft affinity. The only exceptions were two myelin-associated four-pass TMPs, myelin and lymphocyte protein (MAL), and proteo lipid protein (PLP). We characterized the potential mechanisms for their exceptional raft affinity and observed that PLP requires cholesterol and sphingolipids for optimal association with ordered membrane domains and that PLP and MAL appear to compete for cholesterol-mediated raft affinity. These observations suggest broad conclusions about the composition of ordered membrane domains in cells and point to previously unrecognized drivers of raft affinity for multipass transmembrane proteins

Partitioning to ordered membrane domains regulates the kinetics of secretory traffic

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery