Topological distribution of aminophospholipid fatty acids in trout intestinal brush-border membrane

The transbilayer distribution of aminophospholipids in trout intestinal brush-border membrane has been investigated using trinitrobenzene sulfonic acid (TNBS). In the middle intestine, phosphatidylethanolamine (PE) is symmetrically distributed between the two leaflets while 68% of the phosphatidylserine (PS) are located in the inner membrane leaflet. In the posterior intestine, 64% of the PE and 69% of the PS are located in the inner membrane leaflet. When asymmetrically distributed, the inner species of PE and PS have a higher content of 22:6(n − 3) than the outer ones. This asymmetric distribution of docosahexaenoic acid in trout intestinal brush-border membrane might be related to the rod-like shape of the microvillus membrane and to its metabolism to hydroxylated derivatives.     

Topological distribution of choline phospholipid fatty acids in trout intestinal brush-border membrane

The transbilayer distribution of choline phospholipids in trout intestinal brush-border membrane has been investigated using phospholipase C (from Clostridium welchii). In the middle intestine, 84% of phosphatidylcholine (PC) and 60% of sphingomyelin (SP) are located in the outer membrane leaflet. In the posterior intestine, 89% of PC and 52% of SP are located in the outer membrane leaflet. The externally located PC molecular species are (n - 3) fatty acid-rich in both parts of the intestine. While the sphingomyelin molecular species containing 24:1(n - 9) are exclusively located in the outer leaflet in the middle intestine, those containing 14:0 are more abundant in the same leaflet but in the posterior intestine. This strongly asymmetric distribution of both choline phospholipids may have numerous consequences on the brush-border membrane characteristics.     

Phospholipid asymmetry in cardiac sarcolemma. Analysis of intact cells and 'gas-dissected' membranes

The investigation focuses on the phospholipid composition of the sarcolemma of cultured neonatal rat heart cells and on the distribution of the phospholipid classes between the two monolayers of the sarcolemma. The plasma membranes are isolated by 'gas-dissection' technique and 38% of total cellular phospholipid is present in the sarcolemma with the composition: phosphatidylethanolamine (PE) 24.9%, phosphatidylcholine (PC) 52.0%, phosphatidylserine/phosphatidylinositol (PS/PI) 7.2%, sphingomyelin 13.5%. The cholesterol/phospholipid ratio of the sarcolemma is 0.5. The distribution of the phospholipids between inner and outer monolayer is defined with the use of two phospholipases A2, sphingomyelinase C or trinitrobenzene sulfonic acid as lipid membrane probes in whole cells. The probes have access to the entire sarcolemmal surface and do not produce detectable cell lysis. The phospholipid classes are asymmetrically distributed: (1) the negatively charged phospholipids, PS/PI are located exclusively in the inner or cytoplasmic leaflet; (2) 75% of PE is in the inner leaflet; (3) 93% of sphingomyelin is in the outer leaflet; (4) 43% of PC is in the outer leaflet. The predominance of PS/PI and PE at the cytoplasmic sarcolemmal surface is discussed with respect to phospholipid-ionic binding relations between phospholipids and exchange and transport of ions, and the response of the cardiac cell on ischemia-reperfusion.     

Differential modulation of human small intestinal brush-border membrane hemileaflet fluidity affects leucine aminopeptidase activity and transport of D-glucose and L-glutamate.

The fluidity of the exofacial (outer) and cytofacial (inner) leaflets of human proximal small intestinal brush-border membrane vesicles was studied by selective quenching by trinitrophenyl groups, steady-state fluorescence polarization, and differential polarized phase fluorometry techniques, utilizing the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Differences in the hemileaflet's phospholipid composition were also analyzed by trinitrophenylation of aminophospholipids and phospholipase A2 treatment of these preparations. The results of these studies demonstrated that the inner leaflet of these membranes was less fluid than its outer counterpart. Phosphatidylserine was located mainly in the inner hemileaflet, whereas phosphatidylethanolamine and phosphatidylcholine were more symmetrically distributed between the hemileaflets of this membrane. Moreover, in vitro addition of 2-[(2-methoxyethoxy)ethyl]-cis-8-(2-octylcyclopropyl)octanoate (final concentration, 7.5 microM) preferentially fluidized the cytofacial leaflet and concomitantly increased Na(+)-gradient-dependent D-glucose uptake, but decreased Na+, K+-dependent L-glutamic acid uptake in these membrane vesicles. In vitro addition of benzyl alcohol (final concentration, 25 mM) preferentially fluidized the exofacial leaflet and decreased leucine aminopeptidase activity in these preparations. These results, therefore, demonstrate that the hemileaflets of human small intestinal brush-border membranes have different phospholipid compositions and fluidities. Alterations of either the exofacial or cytofacial leaflet fluidity, moreover, modulate protein-mediated activities in a distinct manner.     

Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process.

The transbilayer movement of fluorescent and isotopically labeled analogs of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) from the outer to the inner leaflet (flip) and from the inner to the outer leaflet (flop) of human red blood cells (RBC) was examined. The inward movement of 1-oleoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole-aminocaproyl)- (C6-NBD-), 1-oleoyl-2-(N-(3-(3-[125I]iodo-4-hydroxyphenyl)propionyl)aminocaproyl)- (C6-125I-), or 1-oleoyl-2-(N-(3-3-[125I]iodo-4-azido-phenyl)propionyl)aminocaproyl- (C6-125I-N3-) analogs of PC and PE were relatively slow. In contrast, all analogs of PS and PE analogs containing aminododecanoic acid (C12 lipids) were rapidly transported to the cell's inner leaflet. Analysis of 125I-N3 lipids cross-linked to membrane proteins revealed labeling of 32-kDa Rh polypeptides that was dependent on the lipid's capacity to be transported to the inner leaflet but was independent of lipid species. To investigate whether lipids could also be transported from the inner to the outer leaflet, lipid probes residing exclusively in the inner leaflet were monitored for their appearance in the outer leaflet. Lipid movement could not be detected at 0 degrees C. At 37 degrees C, however, approximately 70% of the PC, 40% of the PE, and 15% of the PS redistributed to the cells outer leaflet, thereby attaining their normal asymmetric distribution. Continuous incubation in the presence of bovine serum albumin depleted the cells of the analogs (t1/2 approximately 1.5 h) in a manner that was independent of lipid species. Similar to the inward movement of aminophospholipids, the outward movement of PC, PE, and PS was ATP-dependent and could be blocked by oxidation of membrane sulfhydryls and by the histidine reagent bromophenacyl bromide. Evidence is presented which suggests that the outward movement of lipids is an intrinsic property of the cells unrelated to compensatory mechanisms due to an imbalance in lipid distribution.     

Phospholipid topology and flip-flop in intestinal brush-border membrane

The topological distribution of the two major phospholipids of brush-border membrane, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), has been investigated using brush-border membrane vesicles from rabbit small intestine. Bee venom phospholipase A2 and phosphatidylcholine exchange protein from bovine liver were used as membrane probes. It is shown that the brush-border membrane retains its integrity under conditions of phospholipase hydrolysis and intermembrane phospholipid exchange. Kinetic analysis of the data of phospholipase hydrolysis and phospholipid exchange at temperatures under 10 degrees C shows that both PC and PE occur in two pools: a minor (about 25%) more readily accessible pool and a major one (about 75%) less readily available. The rate of PC exchange between these two pools is relatively fast. The half-time derived under conditions of phospholipase hydrolysis is of the order of 20 min. Under conditions of phospholipid exchange the exchange rates may be even faster. The difference in exchange kinetics observed with the two methods of probing is probably due to changes in membrane properties such as the bilayer fluidity induced by the probing process itself. It is proposed that the two pools represent the transverse distribution of the phospholipids. The two major phospholipids of brush-border membranes, PC and PE, would be distributed mainly on the inner (cytoplasmic) side of the brush-border membrane. The phospholipid exchange between the brush-border vesicles and unilamellar phosphatidylcholine vesicles in the presence of phosphatidylcholine exchange protein reveals that significant quantities of phospholipid are taken up by brush-border membrane independently, i.e., in a separate process independent of the exchange protein-catalyzed phosphatidylcholine exchange.     

New reagents for phosphatidylserine recognition and detection of apoptosis

The phospholipid bilayer surrounding animal cells is made up of four principle phospholipid components, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). These four phospholipids are distributed between the two monolayers of the membrane in an asymmetrical fashion, with PC and SM largely populating the extracellular leaflet and PE and PS restricted primarily to the inner leaflet. Breakdown in this transmembrane phospholipid asymmetry is a hallmark of the early to middle stages of apoptosis. The consequent appearance of PS on the extracellular membrane leaflet is commonly monitored using dye-labeled Annexin V, a 36 kDa, Ca2+-dependent PS binding protein. Substitutes for Annexin V are described, including small molecules, nanoparticles, cationic liposomes, and other proteins that can recognize PS in a membrane surface. Particular attention is given to the use of these reagents for detecting apoptosis.     

The Curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content,and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm)radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, ~40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained ~20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets ofthe bilayer. The proportion oftotal PE residing in the outer leaflet was unaffected by changes in either the cholesterol orPE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions ofpalmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

The curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content, and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm) radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, approximately 40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained approximately 20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets of the bilayer. The proportion of total PE residing in the outer leaflet was unaffected by changes in either the cholesterol or PE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate- and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions of palmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

Aminophospholipid molecular species asymmetry in the human erythrocyte plasma membrane

The transbilayer distribution of the molecular species of aminophospholipids in human red blood cell plasma membrane has been investigated using a covalent labelling technique. Separation and quantitative analysis of the molecular species of phosphatidylethanolamine (PE) and phosphatidylserine (PS) was performed using high-performance liquid chromatography with UV detection of the trinitrophenyl derivatives obtained after reaction with trinitrobenzenesulfonic acid (TNBS). When the molecular species distribution obtained with intact cells was compared to that of the whole membrane, a molecular species asymmetry was evident. This phenomenon was most clearly evident when the reaction was performed at low temperatures (0 degrees C) and was obscured by the excessive labelling or probe permeation associated with higher temperatures or longer incubation times. The monoene species were enriched in the outer leaflet, they comprised about 30% of the PE species in this leaflet. The polyunsaturates were preferentially localized in the inner leaflet and this was true of the arachidonyl species in particular as they represented up to 35% of this pool. The w-3 polyunsaturated fatty acids displayed a preferential localization in the plasmalogen subclass in comparison to the diacyl fraction, i.e., they comprised about 58 of the former and 42% of the latter subclass of cellular PE w-3 species. Data concerning the separation, identification and quantification of PS molecular species in human erythrocytes is also presented. The internal localization of the polyunsaturated species as well as the compartmentalization of the w-3 and w-6 pools will have metabolic, structural and physical implications for membrane function.     

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell–cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.     

Phosphatidylserine decarboxylase: generation of asymmetric vesicles and determination of the transbilayer distribution of fluorescent phosphatidylserine in model membrane systems

Large unilamellar vesicles (LUV) that contained a fluorescent analog of phosphatidylserine (NBD-PS) were used in model systems to determine the feasibility of employing phosphatidylserine decarboxylase (PS-decarboxylase) to generate asymmetric vesicles and to determine the transbilayer distribution of PS. PS-decarboxylase prepared by sonication of Escherichia coli JA 200 pLC 8-47 was found to be stable in detergent-free buffers and catalyzed the conversion of NBD-PS to NBD-phosphatidylethanolamine (NBD-PE). PS-decarboxylase was capable of decarboxylating virtually all of the NBD-PS present in the outer leaflet of LUV containing a symmetric or asymmetric (outside only) distribution of NBD-PS, but not NBD-PS present in the inner leaflet of the vesicles. The ability of PS-decarboxylase to decarboxylate only NBD-PS located in the outer leaflet of the vesicles was independently verified by resonance energy transfer (between NBD-PS and (lissamine) rhodamine B-labeled phosphatidylethanolamine) and by derivatization with trinitrobenzenesulfonic acid (TNBS). These techniques revealed that the exchangeable pool (the fraction of NBD-PS on the outer leaflet) and the respective fraction of Tnp-(NBD-PS) formed were equivalent to the extent of PS-decarboxylase-mediated decarboxylation of NBD-PS to NBD-PE. These results show that PS-decarboxylase can be used to generate asymmetric vesicles (i.e., PS inside, PE outside) and determine the intrabilayer distribution of PS in model membranes.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A(2). We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A₂. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Inward relocation of exogenous phosphatidylserine triggered by IGF-1 in non-apoptotic C2C12 cells is concentration dependent

The plasma membrane is composed of two leaflets that are asymmetric with regard to their phospholipid composition with phosphatidylserine (PS) predominantly located within the inner leaflet whereas other phospholipids such as phosphatidylcholine (PC) are preferentially located in the outer leaflet. An intimate relationship between cellular physiology and the composition of the plasma membrane has been demonstrated, with for example apoptosis requiring PS exposure for macrophage recognition. In skeletal muscle development, differentiation also requires PS exposure in myoblasts to create cell-cell contact areas allowing the formation of multinucleate myotubes. Although it is clearly established that membrane composition/asymmetry plays an important role in cellular physiology, the role of cytokines in regulating this asymmetry is still unclear. When incubated with myoblasts, insulin-like growth factor I (IGF-1) has been shown to promote proliferation versus differentiation in a concentration dependent manner and therefore, may be a potential candidate regulating cell membrane asymmetry. We show, in non-apoptotic C2C12 cells, that relocation of an exogenous PS analogue, from the outer into the inner leaflet, is accelerated by IGF-1 in a concentration-dependent manner and that maintenance of membrane asymmetry triggered by IGF-1 is however independent of the PI3K inhibitor wortmannin.     

The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.     

Effects of docosahexaenoic and arachidonic acids on the synthesis and distribution of aminophospholipids during neuronal differentiation of PC12 cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.     

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells--under essentially non-lytic conditions--with phospholipases A2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.     

Disruption of the Lipid-Transporting LdMT-LdRos3 Complex in Leishmania donovani Affects Membrane Lipid Asymmetry but Not Host Cell Invasion

Maintenance and regulation of the asymmetric lipid distribution across eukaryotic plasma membranes is governed by the concerted action of specific membrane proteins controlling lipid movement across the bilayer. Here, we show that the miltefosine transporter (LdMT), a member of the P4-ATPase subfamily in Leishmania donovani, and the Cdc50-like protein LdRos3 form a stable complex that plays an essential role in maintaining phospholipid asymmetry in the parasite plasma membrane. Loss of either LdMT or LdRos3 abolishes ATP-dependent transport of NBD-labelled phosphatidylethanolamine (PE) and phosphatidylcholine from the outer to the inner plasma membrane leaflet and results in an increased cell surface exposure of endogenous PE. We also find that promastigotes of L. donovani lack any detectable amount of phosphatidylserine (PS) but retain their infectivity in THP-1-derived macrophages. Likewise, infectivity was unchanged for parasites without LdMT-LdRos3 complexes. We conclude that exposure of PS and PE to the exoplasmic leaflet is not crucial for the infectivity of L. donovani promastigotes.     

Ethanol specifically alters the synthesis, acylation and transbilayer movement of aminophospholipids in rat-liver microsomes

By experimenting with the aminoalcohols [3-3H]serine and [2-14C]ethanolamine we have been able to relate the effects of ethanol upon the biosynthesis of radioactive aminophospholipids (APL) in rat-liver microsomes and their distribution within the bilayer. The translocation of newly synthesized molecules of aminophospholipids labeled with different fatty acids was also investigated. The synthesis of phosphatidylserine (PS) and phosphatidylethanolamine (PE) by base-exchange reaction (BES) was inhibited in membranes exposed to ethanol in direct response to its concentration. In addition, 100 mM ethanol specifically inhibited the transport of newly synthesized PS to the inner leaflet, resulting in similar levels of PS in both leaflets of the bilayer. The inhibition of PE synthesis by ethanol caused a decrease in its distribution in both inner and outer leaflets. An in vitro study of the incorporation of radioactive palmitate and oleate into the PS and PE of microsomes incubated with ethanol showed a decrease in the radioactivity levels of PE, suggesting that ethanol was specifically inhibiting the corresponding acyltransferase. It specifically altered the transbilayer movement of newly acylated phospholipids, modifying the distribution of palmitoyl- and oleoyl-acylated PS and PE in both leaflets. These results demonstrate for the first time that ethanol interferes with both the synthesis and intramembrane transport of aminophospholipids in endoplasmic reticulum (ER) membranes. Bearing in mind that if a membrane is to function properly its structure must be in optimum condition; it is evident that the observed processes may be responsible to some degree for the pathophysiological effects of alcohol upon cells.