Phospholipid asymmetry in human erythrocyte ghosts

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.     

Rapid kinetics of insertion and accessibility of spin-labeled phospholipid analogs in lipid membranes: a stopped-flow electron paramagnetic resonance approach.

Spin-labeled phospholipid analogs have been employed to probe the transbilayer distribution of endogenous phospholipids in various membrane systems. To determine the transmembrane distribution of the spin-labeled analogs, the analogs are usually inserted into the membrane of interest and subsequently the amount of analog in the outer membrane leaflet is determined either by chemical reduction with ascorbate or by back-exchange to bovine serum albumin (BSA). For accurate determination of the transbilayer distribution of analogs, both the kinetics of incorporation and those of accessibility of analogs to ascorbate or BSA have to be fast in comparison to their transbilayer movement. By means of stopped-flow electron paramagnetic resonance (EPR) spectroscopy, we have studied the kinetics of incorporation of the spin-labeled phosphatidylcholine (PC) analog 1-palmitoyl-2-(4-doxylpentanoyl)-sn-glycero-3-phosphocholine (SL-PC) and of its accessibility to chemical reduction and to back-exchange at room temperature. Incorporation of SL-PC into the outer leaflet of egg phosphatidylcholine (EPC) and red cell ghost membranes was essentially completed within 5 s. Ninety percent of the SL-PC molecules located in the outer membrane leaflet of those membranes were extracted by BSA within 15 s. All exterior-facing SL-PC molecules were reduced by ascorbate in a pseudo-first-order reaction within 60 s in EPC membranes and within 90 s in red cell ghost membranes. The rate of the reduction process could be enhanced by approximately 30-fold when 6-O-phenyl-ascorbic acid was used instead of ascorbate as the reducing agent. The results are discussed in light of assaying rapid transbilayer movement of spin-labeled analogs in biological membranes.     

Involvement of spectrin in the maintenance of phase-state asymmetry in the erythrocyte membrane

The fluorescent probe merocyanine 540 does not stain the plasma membrane of normal human or murine erythrocytes, nor of genetically abnormal human spherocytic erythrocytes. It does, however, stain erythrocyte membranes in several systems in which the underlying spectrin network is altered or missing. Because of the greater affinity of merocyanine 540 for fluid—phase lipid bilayers, these results suggest that the external leaflet of erythrocyte membranes becomes more disordered upon alteration or loss of the internal spectrin network. Analysis of the transbilayer arrangement of membrane phospholipids by digestion with phospholipase A2 suggests that lipid compositional asymmetry of the erythrocyte membrane is responsible for a phase-state asymmetry between the two lipid leaflets, and that spectrin is required to maintain this asymmetry and the gel-like state of the external leaflet.     

Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance

Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease.The loss of membrane phospholipid asymmetry in activated platelets seems to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of aminophospholipids to cytoskeletal proteins, and the involvement of a phospholipid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids.     

Transmembrane distribution of sterol in the human erythrocyte

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.     

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.     

Rapid loss and restoration of lipid asymmetry by different pathways in resealed erythrocyte ghosts.

The normal asymmetric distribution of phospholipids across the plasma membrane of erythrocytes can be abolished by lysing and resealing cells in the presence of Ca2+. In the present study, using flow cytometric analysis of the binding of merocyanine 540 to monitor transbilayer phospholipid distribution, Ca(2+)-induced loss of asymmetry is shown to be independent from the aminophospholipid translocase which catalyzes movement of normally internal phospholipids from the outer to the inner leaflet of the membrane. Loss of asymmetry is rapid, temperature-sensitive, and occurs in an uninterrupted, intact bilayer, rather than by diffusion of lipids through the hemolytic pore. Addition of ATP during lysis reverses loss of asymmetry, and this restoration can be blocked by inhibitors of the aminophospholipid translocase. These results suggest that the ATP-dependent translocase is essential for recovery of asymmetry, in turn suggesting that separate mechanisms mediate the loss and the recovery of lipid asymmetry in erythrocytes.     

Membrane phospholipid asymmetry as a factor in erythrocyte-endothelial cell interactions

The tendency of human erythrocytes to adhere to vascular endothelial cells was assessed as a function of the transbilayer distribution of the phospholipids of the erythrocyte membrane, using erythrocyte ghosts in which transbilayer lipid arrangement was manipulated by varying the conditions under which the ghosts were prepared. By two different assays, ghosts with symmetric lipid bilayers adhered strongly to monolayers of cultured endothelial cells, whereas ghosts with normal asymmetric membranes, like normal erythrocytes, did not. These results provide direct evidence that changes in phospholipid asymmetry can alter the tendency of erythrocytes to adhere to endothelial cells, and therefore imply that transbilayer phospholipid arrangement may influence the behavior of erythrocytes in the circulatory system and may contribute to the formation of microvascular occlusions.     

The mechanism of internalization of platelet-activating factor in activated human neutrophils. Enhanced transbilayer movement across the plasma membrane.

Recent studies suggest that cellular internalization of platelet-activating factor (PAF), a potent ether phospholipid mediator of inflammation, is modulated by, as yet undefined cellular mechanisms. Using an albumin extraction method, the internalization of PAF and several PAF analogues was studied in the resting and stimulated human neutrophil. Our data demonstrate that internalization of these analogues is largely dependent on the state of cellular activation and that the process is not specific for certain unique structural features of the PAF molecule including the 1-position ether linkage, 2-position acetyl substitution, or choline polar head group. Furthermore, the internalization process was shown not to be dependent on the PAF receptor, metabolism of the molecule, or the process of endocytosis. Data are presented to suggest that the route of internalization of PAF is enhanced transbilayer movement (flipping) across the plasma membrane occurring as a result of changes in membrane physical properties accompanying cellular activation. It is proposed that in addition to enhanced internalization of PAF, modulation of PAF biosynthesis and net release from the stimulated neutrophil may be consequences of enhanced transbilayer movement of PAF across the activated plasma membrane.     

Echinocytosis and microvesiculation of human erythrocytes induced by insertion of merocyanine 540 into the outer membrane leaflet

Echinocytosis and release of microvesicles from human erythrocytes treated with the impermeant fluorescent dye merocyanine 540 (MC540) has been correlated with the extent of dye binding to intact cells and ghosts. At 20 degrees C binding appeared to saturate at about 9.3.10(6) molecules per cell (3.6 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 2.7%. Stage 3 echinocytes were formed upon binding of (3-4).10(6) molecules of MC540/cell (about 1.3 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 1.0%. Negligible release of microvesicles was observed with MC540 at 20 degrees C. Binding of MC540 to permeable ghosts was approximately twice that to cells suggesting that there was no selective binding to the unsaturated (more fluid) phospholipids which are concentrated in the inner lipid leaflet of the membrane. At 37 degrees C apparent maximal binding of MC540 was about 3.2 mol/100 mol phospholipid and correlated with the maximal release of microvesicles from the cells as measured by release of phospholipid and acetylcholinesterase. These results are discussed in relation to the bilayer couple hypothesis of Sheetz and Singer (Proc. Natl. Acad. Sci. USA 71 (1974) 4457-4461).     

Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus.

Two phospholipid exchange proteins and two phospholipases C have been employed to determine the phospholipid composition of the outer surface of the membrane of influenza virus. These four protein probes have defined the same accessible and inaccessible pool for each viral phospholipid. Phospholipids which are exchangeable or hydrolyzable are located on the outer surface, whereas the inaccessible pool is located at the inner surface of the viral bilayer. The two pools are unequal in size, with ca. 30% of the total phospholipid accessible to the four proteins, and ca. 70% inaccessible. The membrane is thus highly asymmetric with regard to the amount of phospholipid on each side of the membrane. There is also a marked asymmetry of phospholipid composition. Phosphatidylcholine and phosphatidylinositol are enriched in the outer surface, and sphingomyelim is enriched in the inner surface, whereas phosphatidylethanolamine and phosphatidylserine are present in similar proportions in each surface. This distribution is qualitatively different from that previously reported for the human erythrocyte. The close agreement between results obtained with excahnge proteins and phospholipases C demonstrates that the hydrolytic action of these enzymes does not alter phospholipid asymmetry. The nonperturbing nature of the exchange proteins has permitted the rate of transmembrane movement of phospholipids (flip-flop) in the intact virion to be studied. This process could not be detected after 2 days at 37 degrees C. It was estimated that the half-time for flip-flop is indeterminately in excess of 30 days for sphingomyelin and 10 days for phosphatidylcholine at 37 degrees C. These extremely long times provide a simple explanation for the maintenance of transbilayer asymmetry in influenza virions and possibly, other membranes. Since the viral membrane is acquired by budding through the host cell plasma membrane, the transbilayer distribution of phospholipids observed in the virions presumably reflects a similar asymmetric distribution of phospholipids in the host cell surface membrane. Because animal cells in culture do not incorporate extracellular phospholipid, our results demonstrate that individual cells have the capacity to generate asymmetric membranes.     

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts.

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg(2+)-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable Kd of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 microM free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.     

Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue

Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by [14C]AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric ghosts, suggesting that its relationship with the bilayer is normal in these lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes.     

Light-induced reorganization of phospholipids in rod disc membranes

The transbilayer redistribution of spin-labeled phospholipid analogues (SL-PL) with choline, serine, and ethanolamine head groups (PC, PS, and PE, respectively) was studied on intact disc vesicles of bovine rod outer segment membranes in the dark and after illumination. Redistribution was measured by the extraction of spin-labeled lipid analogues from the outer leaflet of membrane using the bovine serum albumin back-exchange assay. In the dark, PS was distributed asymmetrically, favoring the outer leaflet, whereas PC and PE showed small if any asymmetry. Green illumination for 1 min caused lipid head group-specific reorganization of SL-PL. Extraction of SL-PS by bovine serum albumin showed a fast transient (<10 min) enhancement, which was further augmented by a peptide stabilizing the active metarhodopsin II conformation. The data suggest a direct release of 1 molecule of bound PS per rhodopsin into the outer leaflet and subsequent redistribution between the two leaflets. SL-PE and SL-PC showed more complex kinetics, in both cases consistent with a prolonged period of reduced extraction (2 phospholipids per rhodopsin in each case). The different phases of SL-PL reorganization after illumination may be related to the formation and decay of the active rhodopsin species and to their subsequent regeneration process.     

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.Abbreviations PBS phosphate buffered saline - DFP diisopropyl fluorophosphate - ESR electron spin resonance - RBC red blood cells - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SM sphingomyelin - (0,2)PC 1-palmitoyl-2(4doxylpentanoyl)-PC - (0,2)PE 1-palmitoyl-2(4-doxylpentanoyl)-PE - (0,2) PS 1-palmitoyl-2(4-doxylpentanoyl)-PS     

Sphingomyelinase activity causes transbilayer lipid translocation in model and cell membranes

Ceramide is known to induce structural rearrangements in membrane bilayers, including the formation of ceramide-rich and -poor domains and the efflux of aqueous solutes. This report describes a novel effect of ceramide, namely the induction of transbilayer lipid movements. This effect was demonstrated in both model (large unilamellar vesicles) and cell (erythrocyte ghost) membranes in which ceramide generation took place in situ through the action of an externally added sphingomyelinase. Two different novel assays were developed to detect transbilayer lipid movement. One of the assays required the preparation of vesicles containing a ganglioside only in the outer monolayer and entrapped neuraminidase. Sphingomyelinase activity induced ganglioside hydrolysis under conditions in which no neuraminidase was released from the vesicles. The second assay involved the preparation of liposomes or erythrocyte ghosts labeled with a fluorescent energy donor in their inner leaflets. Sphingomyelin hydrolysis was accompanied by fluorescence energy transfer to an impermeable acceptor in the outer aqueous medium. Ceramide-induced transbilayer lipid movement is explained in terms of another well known property of ceramide, namely the facilitation of lamellar to non-lamellar lipid-phase transitions. Thus, sphingomyelinase generates ceramide on one side of the membrane; ceramide then induces the transient formation of non-lamellar structural intermediates, which cause the loss of lipid asymmetry in the bilayer, i.e. the transbilayer movement of ceramide together with other lipids. As direct targets for ceramide tend to be intracellular, these observations may be relevant to the mechanism of transmembrane signaling by means of the sphingomyelin pathway.     

Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.     

Polyamine regulation of plasma membrane phospholipid flip-flop during apoptosis.

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.     

Transbilayer distributions of red cell membrane phospholipids in unilamellar vesicles

The phospholipid organization in unilamellar vesicles comprised of various purified phospholipid components of monkey erythrocyte membrane was ascertained using phospholipase A2 and trinitrobenzenesulfonic acid as external membrane probes. The vesicles were formed by sonication or detergent dialysis and fractionated by centrifugation or gel permeation chromatography. Experiments were done to confirm that the phospholipase A2 treatments did not cause lysis or induce fusion of the vesicles. This enzyme hydrolysed only the glycerophospholipids in the outer surface of the vesicles. The amounts of the external phospholipids determined by this enzymatic method were verified using the chemical probe, trinitrobenzenesulfonic acid. The choline-containing phospholipids and phosphatidylethanolamine localized randomly in the two surfaces of sonicated vesicles (outer diameter, about 30 nm), whereas phosphatidylserine preferentially distributed in the inner monolayer. This phosphatidylserine asymmetry virtually disappeared in detergent dialysed vesicles (outer diameter, about 45 nm). Furthermore, inclusion of cholesterol in both the types of vesicles resulted in more random glycerophospholipid distributions across the plane of vesicles bilayer, presumably due to the cholesterol-induced increases in the size of vesicles. These results demonstrate that the transbilayer distribution of erythrocyte membrane phospholipids in unilamellar vesicles are controlled mainly by the surface curvature rather than by interlipid interactions, and therefore suggest that phospholipid-phospholipid and phospholipid-cholesterol interactions should not play any significant role in determining the membrane phospholipid asymmetry in red cells. It is proposed that this asymmetry primarily originates from differential bindings of phospholipids with membrane proteins in the two leaflets of the membrane bilayer.     

Transbilayer asymmetry of phospholipids in the plasma membrane regulates exocytotic release in mast cells

To investigate the role of the asymmetric distribution of phospholipids of the plasma membrane in exocytosis, we examined the effects of disruption of this asymmetrical distribution of lipids on exocytotic release from mast cells (RBL-2H3). Lipid scramblase, which is activated by divalent cations and catalyzes the transbilayer movement of phospholipids, was overexpressed in mast cells. Exogenous lipid scramblase was expressed in the plasma membrane and the cytoplasm. Activation of scramblase by divalent cations disrupted the asymmetrical distribution of phospholipids in the plasma membrane. Exocytotic release induced by calcium ionophore and phorbol ester was significantly inhibited in the cells transfected with wild-type scramblase. This inhibition was observed with time lag of about 5 min. Furthermore, when the asymmetric distribution of lipids was disrupted before induction of exocytosis, the inhibition of exocytotic release was obvious from the beginning without time lag. These results suggest that the asymmetric distribution of phospholipids in the plasma membrane plays an essential role in fusion between secretory granules and the plasma membrane. This finding also demonstrates that the transbilayer asymmetry of phospholipids regulates exocytosis and gives a new insight into the significance of lipid asymmetry in the plasma membrane.