Asymmetry of the site of choline incorporation into phosphatidylcholine of rat liver microsomes.

[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.     

Microsomal membranes contain phosphatidylcholine that equilibrates across the bilayer, and phosphatidylcholine that does not

Results of experiments using phosphatidylcholine transfer protein and phospholipase C as probes indicate that there are at least two pools of phosphatidylcholine in rat liver microsomes. One of these is preferentially labelled with [14C]choline and does not equilibrate across the bilayer. The second pool is labelled with [3H]glycerol and does equilibrate across the bilayer. Our observations also confirm that phosphatidylcholine exchange protein does not modify the distribution of phospholipids or cause randomization of the inner and outer leaflet pools of phosphatidylcholine when these are differentially labelled by [14C]choline.     

Asymmetric synthesis and transmembrane movement of phosphatidylethanolamine synthesised by base-exchange in rat liver endoplasmic reticulum

Using trinitrobenzenesulphonic acid (TNBS) as a probe we have observed that phosphatidylethanolamine (PE) formed by base-exchange is initially concentrated in the cytosolic leaflet of the membrane bilayer. At 2 min, the specific activity of the PE in this leaflet was 3-times that of the PE in the cisternal leaflet. After 30 min, the specific activities of the two pools of PE, determined with either phospholipase C or TNBS, were similar. Transbilayer movement of PE was slow at low temperature, prevented by EDTA and restored by the addition of calcium ions after EDTA treatment. Trypsin treatment of microsomes, under conditions in which the vesicles remained closed, inhibited the incorporation of ethanolamine into PE by 87%. The cytosolic location of the ethanolamine base-exchange enzyme is consistent with the initial concentration of newly synthesised PE at this site prior to its transmembrane movement to the cisternal leaflet.     

Topological Biosynthesis of Phosphatidylcholine in Brain Microsomes

The sidedness of the biosynthesis of phosphatidylcholine and its transbilayer movement in brain microsomes were investigated. Microsomes were labelled in vitro or in vivo either through Kennedy's pathway or by the base-exchange reaction. The vesicles were treated with phospholipase C under conditions where only the phospholipids present in the external leaflet were hydrolyzed. The incubation of microsomes with CDP-[14C]choline or [14C]choline showed that most of the newly synthesized phosphatidylcholine molecules were localized in the external leaflet. With time a few molecules were transferred into the inner leaflet. When phosphatidylcholine was labelled in vivo by intraventricular injection of [3H]choline the specific activities of the phosphatidylcholine in the outer leaflet were higher than those in the inner leaflet after short times of labelling but became similar after long times of labelling. The results suggest that in brain microsomes the synthesis of phosphatidylcholine through Kennedy's pathway or by the base-exchange reaction takes place on the external leaflet which corresponds to the cytoplasmic one in situ. The transfer of these molecules from the outer leaflet to the inner one is a slow process and the mechanisms that control the transbilayer movement of the phosphatidylcholine seem to be independent of those that control their biosynthesis.     

In vitro synthesis and transbilayer movement of phosphatidylethanolamine molecules labelled with different fatty acids in chick brain microsomes

The transbilayer fatty acid distribution of diacylglycerophosphoethanolamine and the translocation of newly synthesized phosphatidylethanolamine molecules labelled with different fatty acids has been investigated in chick brain microsomes using trinitrobenzensulfonic acid. The determination of the fatty acid composition of diacylglycerophosphoethanolamine in both the outer and the inner leaflet of the microsomal vesicles revealed a similar distribution indicating that both leaflets share the same molecular species. The in vitro incorporation of radioactive fatty acids (16:0, 18:1 and 20:4(n-6] into ethanolamine phospholipids, known to be catalyzed by the lyosphosphatidylethanolamine acyl transferase, showed that the radioactive diacylglycerophosphoethanolamine molecules appeared first in the outer leaflet and were thereafter transferred to the inner leaflet. The apparent rate of translocation of the newly synthesized ethanolamine phospholipid molecules was the highest for those labelled with 16:0 and the lowest for those labelled with 20:4(n-6). The results indicate that the active site of the acyl-CoA:lysophosphatidylethanolamine acyltransferases is located on the outer leaflet of the microsomal vesicles and that the different newly synthesized molecular species of diacylglycerophosphoethanolamine may be translocated from the outer to the inner leaflet at different rates.     

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium weichii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

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.     

Electrophoretic patterns of protein synthesis and turnover in apical plasma membrane and outer bilayer of Schistosoma mansoni

Polypeptide fractions labelled with [¹⁴C]leucine and associated with fractioned inner plasma membrane and outer bilayer (envelope) from the apical double bilayer complex of the surface epithelium of the human blood fluke, Schistosoma mansoni, were analyzed by two-dimensional electrophoresis and fluorography. In contrast to the distribution of alkaline phosphatase, the polypeptide profiles of the two bilayer fractions were similar due to cross contamination between one membrane containing larger amounts of protein (inner) and the second bilayer having more heavily labelled proteins (outer bilayer). Convincing evidence for only two of 35 polypeptides could be provided for localization to the outer bilayer. These results suggest that the marker enzyme used for the inner bilayer, alkaline phosphatase, may not be homogeneously distributed in this membrane. In pulse-chase studies a correction factor for cross-contamination was derived. The rate to turnover of the polypeptide fractions was twice as fast for the outer compared to the inner membrane, this difference being consistent with the view that multilamellar bodies are the precursors of the apical double bilayer complex. Comparing the rates of surface renewal in adult and juvenile schistosomes leads to the suggestion that membrane turnover can be correlated with susceptibility to host immune effector mechanisms.     

The transverse distribution of phospholipids in the membranes of Golgi subfractions of rat hepatocytes.

The transverse distribution of phospholipids in the membranes of subfractions of the Golgi complex was investigated by using phospholipase C and 2,4,6-trinitrobenzenesulphonic acid as probes. In trans-enriched Golgi membranes, 26% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate or for hydrolysis by phospholipase C, and 72% of the phosphatidylcholine is hydrolysed by phospholipase C. In cis-enriched Golgi membranes, 45% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate and for hydrolysis by phospholipase C, and 95% of the phosphatidylcholine is hydrolysed by phospholipase C. Under the conditions used with either probe the contents of the Golgi vesicles labelled with either [3H]palmitic acid or [14C]leucine were retained. Galactosyltransferase activity of the membrane vesicles was partially inhibited by the experimental procedures used to investigate the transverse distribution of phospholipids. However, the residual activity was latent, suggesting that the vesicles remained closed. Trinitrobenzenesulphonic acid caused no detectable morphological change in either Golgi fraction. Phospholipase C treatment caused morphological changes, including fusion of vesicles and the appearance of 'signet-ring' profiles in some vesicles; however, the vesicles remained closed and the bilayer was retained. It appears, therefore, that neither probe causes major disruption of the Golgi vesicles nor gains access to the inner surface of the membrane bilayer. These observations suggest that phospholipids have a transverse asymmetry in Golgi membranes, that this distribution differs in trans and cis membranes, and that the phospholipid structure of Golgi membranes is inconsistent with a simple flow of membrane bilayer from endoplasmic reticulum to Golgi membranes to plasma membrane.     

Base-exchange reactions for the synthesis of phospholipids in nervous tissue: the incorporation of serine and ethanolamine into the phospholipids of isolated brain microsomes

Abstract— The calcium-dependent incorporation of l -[3-³H]serine and [1,2-¹⁴C]ethanol-amine into the phospholipid of isolated subcellular fractions from chick brain was studied and the properties of incorporation were examined. The microsomal fraction was found to possess the highest rate of incorporation and was able to convert under optimal conditions about 120 nmol of labelled serine and 220 nmol of ethanolamine/g of fresh brain microsomes/h. The requirement for Ca²⁺ ion appeared to be absolute. Mg²⁺ ion caused a gradual reduction in the existing enzymic activity, only when pre-incubated with microsomes and labelled bases before adding Ca²⁺ ion. The incorporation of serine and ethanolamine was actively inhibited by Hg²⁺, Co²⁺, Cu²⁺ and Mn²⁺ ions, and was abolished by ethylenediamine tetra-acetate treatment. Ethanolamine, but not choline, inositol or carnitine, competitively inhibited serine incorporation, while d -serine had slight effect. Conversely, l -serine inhibited competitively the incorporation of ethanolamine. The greater part of the incorporated serine (85 per cent) was localized in microsomal phosphatidylserine, while a small percentage was found in phosphatidylethanolamine. Similarly, 90 per cent of the incorporated ethanolamine was confined to phosphatidylethanolamine and a small percentage was found in the plasmalogen derivative. The mechanism of serine and ethanolamine incorporation was investigated. The results are compared with those published for similar mammalian and non-mammalian systems.     

Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes

The exchange of phosphatidylcholine between [³²P]phosphatidylcholine liposomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of [³²P]phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homogeneous pool of small single walled vesicles. Treatment of phosphatidyl [¹⁴C]choline liposomes with phospholipase D (phosphatidylcholine phosphatidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed could not be hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of Phosphatidylcholine from the inner to the outer layer of the liposome bilayer.     

Assay for the transbilayer distribution of glycolipids. Selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine- egg phosphatidylethanolamine;-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A(2) confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing <1 mol% radiolabeled glucosylceramide or short-chain C(6)-glucosylceramide, carboxy-TEMPO oxidized half the glucosylceramide. However, if surface C(6)-glucosylceramide was first depleted by bovine serum albumin (BSA) (extracting 49 +/- 1%), 94% of the remaining C(6)-glucosylceramide was resistant to oxidation. Carboxy-TEMPO oxidized glucosylceramide on the surface of LUVs without affecting inner leaflet glucosylceramide. At pH 9.5 and at 0 degrees C, the reaction reached completion by 20 min.     

Transverse asymmetry of phospholipids in subcellular membranes of rat liver

Subcellular membranes isolated from rat liver in a form impermeable to macromolecules were treated with phospholipase A₂ from Naga naja venom. The phosphatidylserine, phosphatidylethanolamine and about half of the phosphatidylcholine of microsomes, Golgi membranes, inner mitochondrial membranes, lysosomes and nuclear membranes were hydrolyzed. It is proposed that these phospholipids are localized in the outer surface of the membrane bilayer, which represents the cytoplasmic side in the living cell, while the remaining phosphatidylcholine and most of the phosphatidylinositol, sphingomyelin and cardiolipin may be assigned to the inner side of the bilayer.     

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

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

Asymmetric distribution of phospholipids in acetylcholine receptor-rich membranes from T. marmorata electric organ

1. The distribution of phospholipids between the two leaflets of the lipid bilayer in acetylcholine receptor (AChR)-rich membranes from T. marmorata has been examined with two complementary techniques: chemical derivatization with the membrane-impermeable reagent trinitrobenzenesulphonate (TNBS) and B.cereus phospholipase C hydrolysis. 2. AChR-membranes were reacted with TNBS at 0-4 and 37 degrees C and the accessibility of their aminophospholipids was compared to that of rod outer segment and erythrocyte membranes. The results indicate that more of the total ethanolamine glycerophospholipid (EGP) than of the total phosphatidylserine (PS) is located in the outer monolayer. 3. Nearly half the phospholipid content of AChR membranes is hydrolyzed by phospholipase C with a half-time of ca. 1.6 min at 25 degrees C. Consistent with the TNBS results, more of the total EGP than of the total PS is degraded. Beyond 3 min the reaction slows down, relatively smaller additional amounts of lipids are hydrolyzed, and all phospholipid classes are attacked to a similar extent, indicating that after half the lipid is removed all phospholipids become accessible to the enzyme. 4. The results indicate that the outer leaflet of the bilayer is richer in ethanolamine and choline glycerophospholipids, whereas phosphatidylinositol, most of the sphingomyelin, and ca 65% of the PS are located on the inner leaflet.     

Asymmetric distribution of phospholipids in membranes from Mycobacterium phlei

The distribution of phospholipids in the membranes of Mycobacterium phlei has been studied by the use of phospholipase C and trinitrobenzenesulfonic acid. In inverted membrane vesicles, whose external surface apparently corresponds topologically to the cytoplasmic surface of the membrane in intact cells, 80% of the phosphatidyl ethanolamine, 24% of diphosphatidyl glycerol, and 13% of phosphatidyl inositol are accessible to cleavage by phospholipase C. These results are in agreement with the finding that 70–75% of phosphatidyl ethanolamine in the membrane is accessible to chemical modification by trinitrobenzenesulfonic acid or dimethylsuberimidate at 4 °C. It can be inferred that in the inverted membrane the majority of phosphatidyl ethanolamine is present on the outer half of the lipid bilayer while inner half constitutes primarily other phospholipids namely phosphatidyl inositol and diphosphatidyl glycerol. Phospholipase C treatment of ETP membranes selectively impairs the active transport of Ca²⁺ without affecting the generation of a proton gradient, respiration, and coupled phosphorylation.     

Phosphoglycerides and phospholipase C in membrane fractions of Escherichia coli B.

The phospholipid composition and the phospholipase C activity of envelope fractions of Escherichia coli B were determined with special consideration of fractions containing sites at which an attachment of inner and outer membranes had been observed in the electron microscope (Int.M). Phosphoglycerides labeled with [14C]palmitic acid and [3H]serine were extracted from membrane fractions and identified by two-dimensional thin-layer chromatography. The amount of phosphatidylethanolamine was highest in the outer membrane, whereas the amounts of phosphatidylglycerol and cardiolipin were highest in the inner membrane. The Int.M fractions were observed to have concentrations of phospholipids intermediate to those of the inner and outer membranes. This result supports the assumption that a concentration gradient of inner membrane-outer membrane lipids might exist at the membrane contact sites. The highest phospholipase C activity was detected in the inner membrane and Int.M fractions. The presence of phospholipase C and other lipolytic enzymes in the Int.M fractions suggests a possible involvement of adhesion sites in lipid metabolism, adding a further set of activities to the function of these domains.     

Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes.     

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.