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.     

The dependence of lipid asymmetry upon polar headgroup structure

The effect of lipid headgroup structure upon the stability of lipid asymmetry was investigated. Using methyl-β-cyclodextrin -induced lipid exchange, sphingomyelin (SM) was introduced into the outer leaflets of lipid vesicles composed of phosphatidylglycerol, phosphatidylserine (PS), phosphatidylinositol, or cardiolipin, in mixtures of all of these lipids with phosphatidylethanolamine (PE), and in a phosphatidylcholine/phosphatidic acid mixture. Efficient SM exchange (>85% of that expected for complete replacement of the outer leaflet) was obtained for every lipid composition studied. Vesicles containing PE mixed with anionic lipids showed nearly complete asymmetry which did not decay after 1 day of incubation. However, vesicles containing anionic lipids without PE generally only exhibited partial asymmetry, which further decayed after 1 day of incubation. Vesicles containing the anionic lipid PS were an exception, showing nearly complete and stable asymmetry. It is likely that the combination of multiple charged groups on PE and PS inhibit transverse diffusion of these lipids across membranes relative to those lipids that only have one anionic group. Possible explanations of this behavior are discussed. The asymmetry properties of PE and PS may explain some of their functions in plasma membranes.     

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.     

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.     

The penetrating properties of the tumor homing peptide LyP‐1 in model lipid membranes

Cell‐penetrating peptides (CPPs) have the property to cross the plasma membrane and enhance its permeability. CPPs were successfully used to deliver numerous cargoes such as drugs, proteins, nucleic acids, imaging and radiotherapeutic agents, gold and magnetic nanoparticles, or liposomes inside cells. Although CPPs were intensively investigated over the past 20 years, the exact molecular mechanisms of translocation across membranes are still controversial and vary from passive to active mechanisms. LyP‐1 is a cyclic 9‐amino‐acids homing peptide that specifically binds to p32 receptors overexpressed in tumor cells. tLyP‐1 peptide is the linear truncated form of LyP‐1 and recognizes neuropilin (NRP) receptors expressed in glioma tumor tissue. Here, we investigate the interaction of the cyclic LyP‐1 peptide and linear truncated tLyP‐1 peptide with model plasma membrane in order to understand their passive, energy‐independent mechanism of uptake. The experiments reveal that internalization of tLyP‐1 peptides depends on membrane lipid composition. Inclusion of negatively charged phosphatidylserine (PS) or cone‐shaped phosphatidylethanolamine (PE) lipids in the composition of giant unilamellar vesicles facilitates the membrane adsorption and direct penetration but without inducing pore formation in membranes. In contrast, cyclic LyP‐1 peptide mostly accumulates on the membrane, with very low internalization, regardless of the lipid composition. Thus, the linear tLyP‐1 peptide has enhanced penetrating properties compared with the cyclic LyP‐1 peptide. Development of a mutant peptide containing higher number of arginine amino acids and preserving the homing properties of tLyP‐1 may be a solution for new permeable peptides that facilitate the internalization in cells and further the endosomal escape as well.     

Phosphatidylserine Asymmetry Promotes the Membrane Insertion of a Transmembrane Helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. Here, we studied the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.     

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.     

Ionic control of the metastable inner leaflet of the plasma membrane: Fusions natural and artefactual

The phospholipids of the inner and outer leaflets of the plasma membrane face chemically very different environments, and are specialized to serve different needs. While lipids of the outer leaflet are inherently stable in a lamellar (bilayer) phase, the main lipid of the inner layer, phosphatidylethanolamine (PE), does not form a lamellar phase unless evenly mixed with phosphatidylserine (PS⁻). This mixture can be readily perturbed by factors that include an influx of Ca²⁺ that chelates the negatively charged PS⁻, thereby destabilizing PE. The implications of this metastability of the inner leaflet for vesicular trafficking, and experimentally for the isolation of detergent-resistant membrane domains (DRMs) at physiological temperature, are considered.     

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.     

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab′ fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.     

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.     

Insulin effect on the phospholipid organization and some enzyme activities of rat liver membrane fractions

1. The influence of insulin on rat liver membrane lipid composition, fluidity, some enzyme activities and asymmetry of microsomal phospholipids were investigated. 2. The total phospholipids and cholesterol were increased in microsomes and reduced in plasma membranes from insulin-treated rats. 3. Of all the investigated enzymes participating in the lipid metabolism, only the neutral sphingomyelinase activity was observed to be enhanced, whereas the ceramide-phosphatidylethanolamine (PE) synthetase and phospholipase A2 activities remained unchanged. 4. Insulin administration caused translocation of phosphatidylserine (PS) and PE to the outer leaflet and of phosphatidylinositol (PI) to the inner leaflet of microsomal membranes.     

Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.     

Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP₂), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP₂, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.     

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.     

Water permeability of asymmetric planar lipid bilayers: leaflets of different composition offer independent and additive resistances to permeation

To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:30176-30185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/P(AB) = 1/P(A) + 1/P(B) (Eq. l), where P(AB) is the permeability of a bilayer composed of leaflets A and B, P(A) is the permeability of leaflet A, and P(B) is the permeability of leaflet B. Using for the MDCK leaflet-specific liposomes gives an estimated value for the osmotic water permeability (P(f)) of 4.6 x 10(-4) cm/s (at 25 degrees C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:1711-1718). At 36 degrees C, P(f) was 3.44 +/- 0.35 x 10(-3) cm/s for symmetrical inner leaflet membranes and 3.40 +/- 0.34 x 10(-4) cm/s for symmetrical exofacial membranes. From, the estimated permeability of an asymmetric membrane is 6.2 x 10(-4) cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 +/- 0.7 x 10(-4) cm/s, which is within 10% of the calculated value. Direct experimental measurement of P(f) for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of.     

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.     

ATP-dependent transport of phosphatidylserine analogues in human erythrocytes

The plasma membrane of most cells contains a number of lipid transporters that catalyze the ATP-dependent movement of phospholipids across the membrane and assist in the maintenance of lipid asymmetry. The most well-characterized of these transporters is the erythrocyte aminophospholipid flippase, which selectively transports phosphatidylserine (PS) from the outer to the inner monolayer. Previous work has demonstrated that PS and to a lesser extent phosphatidylethanolamine (PE) are substrates for the flippase and that other phospholipids move across the membrane only by passive flip-flop. The present study re-evaluates these results. The incorporation and transbilayer movement of a number of short-chain (dilauroyl) phospholipid analogues in human erythrocytes was measured by observing lipid-induced changes in cell morphology, and the effect of an ATPase inhibitor (vanadate) and a sulfyhdryl reagent (N-ethylmaleimide) was determined. Incubation of cells with these lipids causes the rapid formation of echinocytes, because of the accumulation of the lipid in the outer monolayer. While dilauroylphosphatidylcholine-treated cells retained this shape, cells treated with sn-1,2-DLP-l-S, sn-1,2-DLP-d-S, or N-methyl-DLPS rapidly changed morphology to stomatocytes, which is consistent with the transport and accumulation of the lipid in the inner monolayer. A similar, although slower, stomatocytic shape change was induced by sn-2,3-DLP-l-S. Other lipids that were tested (dilauroylphosphatidylhydroxypropionate, dilauroylphosphatidylhomoserine, DLPS-methyl ester, or sn-2,3-DLP-d-S) reverted to discocytes only. In all cases, pretreatment with vanadate or N-ethylmaleimide inhibited the conversion of echinocytes to discocytes or stomatocytes. This is the first report of a protein- and energy-dependent pathway for the inwardly directed transbilayer movement of lipids other than PS and PE in the erythrocyte membrane and suggests that the flippase has broader specificity for substrates or that other lipid transporters are present.     

The transmembrane distribution of galactolipids in chloroplast thylakoids is universal in a wide variety of temperate climate plants

The transmembrane distribution of monogalactosyldiacylglycerol and digalactosyldiacylglycerol was determined in chloroplast thylakoids from a range of temperate climate plants. These plants included dicotyledons, monocotyledons, C_16:3 and C_18:3 plants and herbicide-resistant species. In all the thylakoids examined monogalactosyldiacylglycerol was enriched in the outer leaflet (53–65%) while digalactosyldiacylglycerol was highly enriched in the inner leaflet (78–90%). The non-bilayer forming monogalactosyldiacylglycerol represented 55–81% of the total acyl lipids of the outer monolayer. The relative acyl lipid composition of both leaflets of the thylakoid membrane indicates that the lamellar structure is strongly favored in the inner monolayer, whereas the outer one presents a metastable character which allows the probable coexistence of both lamellar and non-lamellar phases. The consequence of this asymmetry for the stability and function of the thylakoid membrane is discussed.     

Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids

Normal quescent cells maintain membrane lipid asymmetry by ATP-dependent membrane lipid transporters, which shuttle different phospholipids from one leaflet to the other against their respective concentration gradients. When cells are challenged, membrane lipid asymmetry can be perturbed resulting in exposure of phosphatidylserine [PS] at the outer cell surface. Translocation of PS from the inner to outer membrane leaflet of activated blood platelets and platelet-derived microvesicles provides a catalytic surface for interacting coagulation factors. This process is dramatically impaired in Scott syndrome, a rare congenital bleeding disorder, underscoring the indispensible role of PS in hemostasis. This also testifies to a defect of a protein-catalyzed scrambling of membrane phospholipids. The Scott phenotype is not restricted to platelets, but can be demonstrated in other blood cells as well. The functional aberrations observed in Scott syndrome have increased our understanding of transmembrane lipid movements, and may help to identify the molecular elements that promote the collapse of phospholipid asymmetry during cell activation and apoptosis.