Shape changes of giant liposomes induced by an asymmetric transmembrane distribution of phospholipids.

The influence of a phospholipid transmembrane redistribution on the shape of nonspherical flaccid vesicles was investigated at a fixed temperature by optical microscopy. In a first series of experiments, a transmembrane pH gradient was imposed on egg phosphatidylcholine (EPC)-egg phosphatidylglycerol (EPG) (100:1) giant vesicles. The delta pH induced an asymmetric distribution of EPG. Simultaneously, discoid vesicles were transformed into tubular or a series of connected small vesicles. The fraction of phospholipid transfer necessary for a shape change from discoid to two connected vesicles was of the order of 0.1% of the total phospholipids. Additional lipid redistribution was accompanied by a sequence of shape changes. In a second series of experiments, lyso phosphatidylcholine (L-PC) was added to, or subtracted from, the external leaflet of giant EPC vesicles. The addition of L-PC induced a change from discoid to a two-vesicle state without further evolution, suggesting that lipid transfer and lipid addition are not equivalent. L-PC depletion from the outer leaflet generated stomatocyte-like vesicles. Whenever possible, we have determined whether the giant vesicles undergoing shape changes were unilamellar or multilamellar by measuring the elastic area compressibility modulus, K, by the micropipette assay (Kwok and Evans, 1981). Shape transformations triggered by phospholipid modification of the most external bilayer were indeed influenced by the presence of other underlying membranes that played a role comparable to that of a passive cytoskeleton layer. It appears that in real cells, invaginations of the plasma membrane or budding of organelles could be triggered by a phospholipid transfer from one leaflet to the other caused, for instance, by the aminophospholipid translocase which is present in eukaryotic membranes.     

Bilayer balance and regulation of red cell shape changes

Discocytic human red cells undergo discocyte-echinocyte and discocytestomatocyte transformations under the action of a wide variety of lipid-soluble anionic and cationic agents respectively. These shape transformations are explained by the bilayer couple hypothesis of Sheetz and Singer to be the result of preferential distribution of the anionic agents in the outer half of the bilayer and the cationic agents in the inner half of the bilayer. We demonstrate that echinocytogenic effects indeed occur when the naturally occurring phospholipid lysophosphatidylcholine (LPC) is localized in the outer half of the bilayer, and stomatocytogenic effects occur when LPC is in the inner half. However, in contrast to the bilayer couple hypothesis, our results show that simple equivalent membrane surface area expansion on each layer is insufficient to maintain the discocytic shape and there exists a differential concentration effect of LPC on the two halves of the bilayer.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Cyclopropane fatty acid synthase of Escherichia coli. Stabilization, purification, and interaction with phospholipid vesicles.

The cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes the methylenation of the unsaturated moieties of phospholipids in a phospholipid bilayer. The methylene donor is S-adenosyl-L-methionine. The enzyme is loosely associated with the inner membrane of the bacterium and binds to and is stabilized by phospholipid vesicles. The enzyme has been purified over 500-fold by flotation with phospholipid vesicles and appears to be a monomeric protein having a molecular weight of about 90 000. The enzyme binds only to vesicles of phospholipids which contain either unsaturated or cyclopropane fatty acid moieties. CFA synthase is active on phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, the major phospholipids of E. coli, and also has some activity on phosphatidylcholine. The enzyme is equally active on phospholipid vesicles in the ordered or the disordered states of the lipid phase transition. Studies with a reagent that reacts only with the phosphatidylethanolamine molecules of the outer leaflet of a phospholipid bilayer indicate that CFA synthase reacts with phosphatidylethanolamine molecules of both the outer and the inner leaflets of phospholipid vesicles.     

Phospholipid Flip-Flop and the Distribution of Surface Charges in Excitable Membranes

There is now good evidence that most of the lipids in a biological membrane are arranged in the form of a bilayer. Charged lipids in the membrane of an excitable cell are subject to a significant driving force, the gradient of the intramembrane potential, which will tend to redistribute the lipids between the two halves of the bilayer by a “phospholipid flip-flop” mechanism. We have calculated, by combining the Boltzmann relation from statistics and the Gouy equation from the theory of the diffuse double layer, the steady-state distribution of charged lipids in the bilayer. This distribution is completely determined, within the framework of the model, by three experimentally accessible variables; the percentage of charged lipid in the bilayer as a whole, the resting potential and the ionic strength. The known values for the percentage of anionic phospholipids in squid axons (10-15%), the membrane potential (50-100 mV) and ionic strength (0.5 M) imply that the charge density and double layer potential at the outer surface of the nerve will be substantially greater than the charge density and double layer potential at the inner surface, in agreement with the best available evidence from physiological measurements.     

大鼠心肌线粒体内、外膜磷脂动态结构的研究

我们以DPH为荧光探针.用毫微秒荧光分光光度计测定了大鼠心肌线粒体及线粒体内、外膜的动态微细结构;用HPLC分析了磷脂组成.实验结果提示.完整线粒体膜流动性主要反映了线粒体外膜的运动状态.线粒体内膜微粘度及磷脂分子摇动角大于外膜,扩散速率小于外膜.除去了蛋白质的线粒体内、外膜磷脂脂质体膜流动性无明显差异.提示线粒体内膜的高微粘度与膜中所含有的多量蛋白有关.     

Phospholipids,fatty acids and hemoglobin in rat erythrocytes under stress conditions (swimming at low temperature)

The phospholipid and fatty acid composition of rat erythrocytes was studied after stress exposure—swimming until drowning. This kind of stress was found to increase the content of phospholipids typical for the outer membrane layer (phosphatidylcholine by 13% and sphingomyelin by 23%). In contrast, the content of acid phospholipids, referring to the inner membrane layer, decreased (phosphatidylethanolamine by 16%, phosphatidylserine by 14% and monophosphoinositide by 23%). Our data indicate that under stress conditions the erythrocyte membrane undergoes certain structural changes, which appear to affect its functional properties. At the same time, the content of saturated and unsaturated fatty acids, as well as their “unsaturation index”, remain basically intact under the above stress conditions, probably, preserving functional properties of the erythrocyte membrane by compensating its impaired phospholipid structure. Based on the analysis of absorption spectra of lipid extracts, stress was established to induce a 2-fold spectrum enhancement in the heme-specific range of 390–410 nm. The appearance of heme in the extract indicates hemoglobin saponification induced by changes in pH of the erythrocyte internal environment. Indeed, during lipid extraction hemoglobin converts into a disordered state due to the effect not only of temperature and pH of the medium, but also of organic solvents, having a lower capacity to form hydrogen bonds than water. Probably, a small portion of phospholipids undergoes trans-esterification during their extraction from erythrocytes by the chloroform–methanol mixture.     

Membrane bilayer balance and erythrocyte shape: a quantitative assessment

When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and red cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorbed to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 +/- 1) X 10(6) molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% +/- 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% +/- 0.2%.     

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.     

The outer membrane of Proteus mirabilis. II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes.

1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.     

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.     

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.     

E.?coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

Phospholipid fatty acyl chain asymmetry in the membrane bilayer of isolated skeletal muscle sarcoplasmic reticulum

We previously showed [Herbette, L. G., Blasie, J. K., DeFoor, P., Fleischer, S., Bick, R. J., Van Winkle, W. B., Tate, C. A., & Entman, M. L. (1984) Arch. Biochem. Biophys. 234, 235-242; Herbette, L. G., DeFoor, P., Fleischer, S., Pascolini, D., Scarpa, A., & Blasie, J. K. (1985) Biochim. Biophys. Acta 817, 103-122] that the phospholipid head-group distribution in the membrane bilayer of isolated sarcoplasmic reticulum is asymmetric. From these studies, both the total number of phospholipid head groups and the total lipid, as well as the head-group species for these lipids, were found to be different for each monolayer of the membrane bilayer. In this paper, we demonstrate for the first time that there is significant asymmetry in the distribution of unsaturated fatty acids between the two monolayers; i.e., the outer monolayer of the sarcoplasmic reticulum contained more unsaturated and polyunsaturated chains when compared to the inner monolayer. X-ray diffraction measurements demonstrated that the time-averaged fatty acyl chain extension for the outer monolayer was approximately 20% less than for the inner monolayer. This is consistent with the concept that the greater degree of unsaturation in the outer monolayer may provide for a decreased average fatty acyl chain extension for that layer. This architecture for the bilayer may be related to both the "resting" state mass distribution of the calcium pump protein within the membrane bilayer and possible "conformational" states of the calcium pump protein during calcium transport by the sarcoplasmic reticulum.     

Reorientation rates and asymmetry of distribution of lysophospholipids between the inner and outer leaflet of the erythrocyte membrane

Labelled lysophospholipids were inserted into the outer layer of the erythrocyte membrane and their reorientation (flip) to the inner layer quantified by following the increase of the fraction of lysophospholipids not extractable by albumin. Flip rate constants were calculated from the kinetics of equilibration of the lysophospholipids between two compartments, the outer and the inner leaf of the bilayer, in the early phase of the flip kinetics where correction for non-enzymatic hydrolysis and acylation could be omitted. The distribution of a lysophospholipid finally attained reflects its affinity for the two layers. Whereas lysophosphatidylcholine has a slight preference for the outer layer of the membrane, lysophosphatidylserine spontaneously concentrates in the inner layer up to a ratio of 4:1. This asymmetry mimics the distribution of phosphatidylserine in the native membrane. Flip rates depend on membrane lipid compositions. They are enhanced by cholesterol depletion. Comparison of various mammalian species demonstrates that erythrocytes with a higher phosphatidylcholine/sphingomyelin ratio and high content of polyunsaturated fatty acids (mouse and rat) have a high transbilayer mobility, in contrast to erythrocytes with a low phosphatidylcholine/sphingomyelin ratio and a low content of polyunsaturated fatty acids (ox). Molecular properties of lysophospholipids influence their transbilayer mobility. Flip rates of lysophospholipids are enhanced not only by unsaturation of their fatty acid, but also by a negative net charge on the headgroup. This indicates that the strongly asymmetric distribution of phosphatidylserine in the native erythrocyte membrane, which is maintained for the lifespan of the cell, does not result from a lack of transbilayer mobility.     

Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium.

The reversibility and specificity of phospholipid translocation between the inner and outer membrane of Salmonella typhimurium has been investigated by incorporating exogenous lipids from phospholipid vesicles into the outer membrane of intact cells. Translocation of newly incorporated phospholipids to the inner membrane was demonstrated by decarboxylation of vesicle-derived phosphatidylserine and by recovery of vesicle constituents in both inner and outer membrane fractions. All Salmonella phospholipids tested, as well as phosphatidylcholine and cholesteryl oleate were effectively translocated to the inner membrane. However, no translocation of vesicle-derived lipopolysaccharide or an incomplete biosynthetic precursor of lipid A could be detected. Translocation of phospholipids and cholesteryl ester was rapid and extensive, and appeared to lead to equilibration of the lipids between the two membranes. The mechanism of intermembrane translocation has not been established, but the results are suggestive of diffusional flow across zones of adhesion between the inner and outer membranes.     

Recovery of human erythrocytes from the echinocyte shape induced by added choline-phospholipids is dependent on the acyl chain length

Addition of an amphiphilic lipid, such as phosphatidylcholine (PC) species with two identical saturated chains or lysophosphatidylcholine (lysoPC) species with one saturated acyl chain of various lengths, into a suspension of intact human erythrocytes resulted in lipid incorporation into the erythrocytes membrane to produce echinocytes (crenated cells). The altered shape gradually reverted on incubation at 37 degrees C until the cells reassumed their normal disc shape. The rate of such recovery of shape increased with decreasing acyl chain length for both PC with C8-C12 acyl chains and lysoPC with a C14-C18 acyl chain, and was strongly influenced by incubation temperature. The identical rate of recovery of shape was observed for cells with normal, decreased or increased ATP content, implying that the metabolic state of the cell had no influence on the recovery process. Recovery of shape is therefore considered to be caused by translocation of the incorporated lipid molecules from the outer to the inner leaflet of the membrane lipid bilayer and the rate of recovery increases with decreasing hydrophobicity of the lipid.     

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts

Reliable protocols were established for investigating asymmetric distributions of 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-caproyl (C6NBD) phospholipids in the plasma membrane of boar sperm cells under physiological conditions. A method based on fluorescence resonance energy transfer was used to ensure that incorporation of the fluorescent phospholipids into the sperm proceeded via monomeric transfer. The total amount of incorporated phospholipid fluorescence and the proportion of translocated phospholipid fluorescence were determined by flow cytometric analysis before, and after, dithionite destruction of outer leaflet fluorescence. Catabolism of incorporated fluorescent phospholipids was blocked with phenylmethylsulfonyl fluoride. Membrane-damaged cells were detected with impermeant DNA stains, thereby enabling their exclusion from subsequent analyses of the flow cytometric data, whence it could be demonstrated that the labeled phospholipids were incorporated only via the outer plasma membrane leaflet in living sperm cells. Phospholipid uptake and internalization was followed at 38 degrees C. After 1 hr of labeling, about 96% of the incorporated C6NBD-phosphatidylserine, 80% of C6NBD-phosphatidylethanolamine, 18% of C6NBD-phosphatidylcholine, and 4% of C6NBD-sphingomyelin were found to have moved across the plasma membrane bilayer to the interior of the spermatozoa. These inward movements of fluorescent phospholipids were ATP-dependent and could be blocked with sulfhydryl reagents. Movements from the inner to the outer leaflet of the sperm plasma membrane were minimal for intact fluorescent phospholipids, but were rapid and ATP-independent for fluorescent lipid metabolites. The described method enables, for the first time, assessment of changes in lipid asymmetry under fertilizing conditions.     

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.