Aminophospholipid Asymmetry in Murine Synaptosomal Plasma Membrane

The asymmetric distribution of aminophospholipids in isolated murine synaptosomal plasma membranes was determined by a chemical labeling procedure. Under nonpenetrating conditions, mouse brain synaptosomes were reacted with trinitrobenzenesulfonic acid (TNBS) to label outermonolayer aminophospholipids covalently. About 10-15% of the phosphatidylethanolamine and 20% of the phosphatidylserine were found to be in the outer monolayer of the synaptosomal plasma membrane. Furthermore, the fatty acyl group composition of the labeled phosphatidylethanolamine (outer monolayer) consisted of more saturated fatty acid than did the unlabeled phosphatidylethanolamine (inner monolayer). These results demonstrated an aminophospholipid asymmetry in synaptosomal plasma membranes which was independent of serum-lipoprotein exchange processes and also of phosphatidylethanolamine-methylatingenzymes.     

Regulation of aminophospholipid asymmetry in murine fibroblast plasma membranes by choline and ethanolamine analogues

The regulation of the asymmetric distribution of aminophospholipids in mammalian cell plasma membranes is not understood at this time. One approach to determine the nature of such regulatory mechanisms is to attempt alteration of the plasma membrane phospholipid composition. Choline analogues such as N,N'-dimethylethanolamine and N-monomethylethanolamine lowered the quantity of phosphatidylethanolamine in the plasma membrane of LM fibroblasts grown in defined medium without serum. Ethanolamine supplementation increased the phosphatidylethanolamine content while ethanolamine analogues such as 2-amino-2-methyl-1-propanol, 2-amino-1-butanol, 1-aminopropanol, and 3-aminopropanol did not alter the aminophospholipid content significantly. The transverse distribution of aminophospholipids in the plasma membrane was determined by use of a chemical labelling reagent trinitrobenzenesulfonic acid. The percent phosphatidylethanolamine trinitrophenylated by trinitrobenzenesulfonate in the outer plasma membrane monolayer of LM cells supplemented with choline analogues was not altered. In contrast, ethanolamine analogue supplementation increased the percentage of aminophospholipid in the outer monolayer 2--3-fold. Ethanolamine analogue-containing phospholipids were distributed asymmetrically across the plasma membrane with 85 to 91% being located in the inner monolayer of the plasma membrane, a distribution similar to that of phosphatidylethanolamine. The fatty acyl composition of aminophospholipids in the outer monolayer was in all cases more saturated than in the corresponding phospholipids of the inner monolayer. However, choline analogues and especially the ethanolamine analogues reduced this difference. Thus, base analogues of choline and ethanolamine may alter the aminophospholipid asymmetry, the surface charge, and the acyl chain asymmetry of LM cell plasma membranes.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane.

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography. The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography.The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

Surface distribution of the fatty acid side chains of phosphatidylethanolamine in mixed phospholipid vesicles

A method has been developed for the selective determination of the fatty acid side chain distribution associated with the amino containing phospholipids located in the inner and outer surfaces of membranes. Using sonicated phosphatidylethanolamine/phosphatidylcholine vesicles as a model, the analysis consists of selective labeling of the outer surface amino groups with the membrane impermeable reagent 2,4,6-trinitrobenzenesulfonic acid. Outer and inner surface phosphatidylethanolamine fractions are separated by thin-layer chromatography. Analysis of methyl esters derived from these two fractions, by gas-liquid chromatography, yields the fatty acid side chain distribution. Our results show that there is no mol fraction dependence of the incorporation of any specific fatty acid side chains of egg yolk phosphatidylethanolamine into the vesicle or any preferential distribution of these side chains in the inner or outer vesicle surface. The surface distribution of the egg yolk phosphatidylethanolamine molecules in these vesicles appears to be determined by the head group packing requirements and not the fatty acid side chain composition.     

Surface distribution of the fatty acid side chains of phosphatidylethanolamine in mixed phospholipid vesicles.

A method has been developed for the selective determination of the fatty acid side chain distribution associated with the amino containing phospholipids located in the inner and outer surfaces of membranes. Using sonicated phosphatidylethanolamine/phosphatidylcholine vesicles as a model, the analysis consists of selective labeling of the outer surface amino groups with the membrane impermeable reagent 2,4,6-trinitrobenzenesulfonic acid. Outer and inner surface phosphatidylethanolamine fractions are separated by thin-layer chromatography. Analysis of methyl esters derived from these two fractions, by gas-liquid chromatography, yields the fatty acid side chain distribution. Our results show that there is no mol fraction dependence of the incorporation of any specific fatty acid side chains of egg yolk phosphatidylethanolamine into the vesicle or any preferential distribution of these side chains in the inner or outer vesicle surface. The surface distribution of the egg yolk phosphatidylethanolamine molecules in these vesicles appears to be determined by the head group packing requirements and not the fatty acid side chain composition.     

Phospholipid Transverse Diffusion in Synaptosomes: Evidence for the Involvement of the Aminophospholipid Translocase

We have studied in Torpedo marmorata electric organ synaptosomes the equilibration kinetics of spin-labeled phospholipid analogues initially incorporated into the outer plasma membrane monolayer. As assayed by evoked releases of both ATP and acetylcholine, the nerve endings were closed vesicles containing an energy source. The aminophospholipids (phosphatidylethanolamine and phosphatidylserine) were translocated toward the inner membrane leaflet faster and to a higher extent than their choline-containing counterparts (phosphatidylcholine and sphingomyelin). This difference was abolished by incubation of synaptosomal membranes with N-ethylmaleimide, suggesting that the accumulation of aminophospholipids in the inner layer was driven by a protein. This phenomenon is comparable with what was described in plasma membranes of other eucaryotic cells (erythrocyte, lymphocyte, platelet, fibroblast), and thus we would suggest that an aminophospholipid translocase, capable of moving the aminophospholipids from the outer to the inner layer at the expense of ATP, is also present in the synaptosomal plasma membrane.     

Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme

When human erythrocytes are incubated with spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, with a short beta chain (C5) bearing a doxyl group at the fourth carbon position, the labeled lipids incorporate readily in the outer monolayer. The incorporation is followed in fresh erythrocytes by a selective inward diffusion of the amino derivatives. This observation led us to postulate the existence of a selective ATP-dependent system that would flip aminophospholipids from the outer to the inner monolayer [Seigneuret, M., & Devaux, P. F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3751-3755]. This study further examines the nature of this selective transport and demonstrates that it is mediated by a specific membrane protein. By measurement of the initial rate of transverse diffusion of spin-labeled lipids incorporated at various concentrations in the membrane outer leaflet of packed erythrocytes, apparent Km values were determined for the phosphatidylserine and phosphatidylethanolamine analogues. A ratio of approximately equal to 1/9.4 [corrected] was obtained (KmPS/KmPE). Using spin-labels bearing either a 14N or a 15N isotope, we have carried out competition experiments allowing us to measure simultaneously the transport of two different phospholipids. By this procedure, we show that phosphatidylserine and phosphatidylethanolamine compete for the same transport site but that phosphatidylserine has a higher affinity, in agreement with a lower apparent Km. On the other hand, the slow diffusion of the phosphatidylcholine or sphingomyelin analogues has no influence on the transport of phosphatidylserine or phosphatidylethanolamine. Experiments carried out in ghosts loaded with ATP enabled us to determine the activation energies for phosphatidylserine and phosphatidylcholine transverse diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid composition and phospholipid asymmetry of ram spermatozoa plasma membranes

The phospholipid composition of ram spermatozoa plasma membranes has been investigated. An exclusively high participation of the choline- and ethanolamine-plasmalogens in the phosphatidylcholine and phosphatidylethanolamine fractions has been established. Phosphatidylcholine of ram spermatozoa plasma membranes contains a great amount of polyunsaturated fatty acids. The phospholipid distribution in spermatozoa plasma membrane was investigated. It was established that the choline containing phospholipids are situated mainly in the outer membrane lipid monolayer, whereas diphosphatidylglycerol and phosphatidylserine are localized predominantly in the inner monolayer. The rest of the phospholipids are evenly distributed among the two monolayers. Ram spermal plasma membranes exhibit high phospholipase A2 activity.     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the     

Transmembrane redistribution of phospholipids of the human red cell membrane during hypotonic hemolysis.

The transmembrane distribution of spin-labeled phospholipids was measured in human erythrocytes before and after hypotonic hemolysis by electron paramagnetic resonance. With a first series of partially water soluble probes a complete randomization of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin analogues was achieved when cells were resealed in the absence of Mg-ATP or when the aminophospholipid translocase was inhibited by vanadate or calcium. If the ghosts were resealed with Mg-ATP inside, the transmembrane asymmetry of the aminophospholipids was reestablished. With long chain insoluble spin-labeled lipids complete randomization was obtained with the phosphatidylcholine analogue but even in the presence of vanadate only a small percentage (approx. 15%) of the spin-labeled phosphatidylserine flopped to the outer monolayer and comparable percentage of the spin-labeled sphingomyelin flipped to the inner monolayer, indicating a hierarchy in the phospholipid redistribution for these water insoluble lipids during hemolysis. The mechanism by which a selective randomization takes place is not known. It may involve phosphatidylserine-protein interactions in the inner leaflet and sphingomyelin-cholesterol or sphingomyelin-sphingomyelin interaction in the outer leaflet.     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus.

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the identity of the fatty acyl chains can uniquely specify or determine which side of the membrane individual phosphatidylethanolamine molecules come to occupy. Although fatty acyl chain asymmetry and phosphatidylethanolamine asymmetry are correlated in VSV, no simple rules can be discerned which uniquely relate the two paramaters.     

Mitochondrial membrane contact sites of yeast. Characterization of lipid components and possible involvement in intramitochondrial translocation of phospholipids

Submitochondrial membrane fractions from yeast that are enriched in inner and outer membrane contact sites were analyzed with respect to their lipid composition. Characteristic features were the significantly reduced content of phosphatidylinositol, the decreased amount of phosphatidylcholine, and the enrichment in phosphatidylethanolamine and cardiolipin. Coisolation of phosphatidylserine synthase with the outer membrane portion and enrichment of phosphatidylserine decarboxylase in the inner membrane portion of isolated contact sites provided the basis for a metabolic assay to study phosphatidylserine transfer from the outer to the inner mitochondrial membrane via contact sites. The efficient conversion to [3H]phosphatidylethanolamine of [3H]phosphatidylserine synthesized from [3H]serine in situ supports the notion that mitochondrial membrane contact sites are zones of intramitochondrial translocation of phosphatidylserine.     

Alterations in erythrocyte membrane lipid and fatty acid composition in Chediak-Higashi syndrome

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease characterized by the presence of abnormally large cytoplasmic organelles in all body granule producing cells. The molecular mechanism for this disease is still unknown. Functional disorders in membrane-related processes have been reported. Erythrocyte membranes from four CHS patients and 15 relatives including obligatory heterozygous were studied to examine potential alterations in the lipid and fatty acid profile of erythrocyte membranes associated with this syndrome. Plasma concentrations of cholesterol, triglycerides, phospholipids, and apolipoproteins AI and B100, and the lipid components of very low-, intermediate-, low- and high-density lipoproteins were also determined. CHS erythrocyte membranes were found to be enriched with lipids in relation to protein and to show: (1) an increase in cholesterol and choline-containing phospholipids (sphingomyelin and phosphatidylcholine) that predominate in the outer monolayer, which is higher than the increase in phosphatidylserine and phosphatidylethanolamine, that are chiefly limited to the inner monolayer in normal red blood cells; (2) a relative palmitic acid and saturated fatty acid increase and arachidonic acid and unsaturated fatty acid decrease, this resulting in a lower unsaturation index than controls. Changes in CHS erythrocyte membrane lipids seem to be unrelated to serum lipid disorders as plasma lipid and apolipoprotein concentrations were apparently in the normal range, with the exception of a modest hypertriglyceridemia in patients and relatives and a decreased concentration of HDL cholesterol in patients. These findings indicate that CHS erythrocyte membranes contain an abnormal lipid matrix with which membrane proteins are defectively associated. The anomalous CHS membrane composition can be explained on the postulated effects of the CHS1/Lyst gene.     

Phospholipid and fatty acid changes produced by cholecalciferol on intestinal mitochondrial membranes

Cholecalciferol administration to vitamin D-deficient chicks produces, 24 h after treatment, a specific increase of the phosphatidylcholine content in the intestinal mitochondrial inner membrane plus matrix fraction without changes in its proportion in the outer membrane. The ratio of unsaturated/saturated fatty acids in the outer membrane phosphatidylcholine was increased by that treatment. The inner membrane plus matrix presents a decrease of 16:1 in phosphatidylethanolamine and 18:0 in the phosphatidylcholine fraction. Cardiolipin shows the largest change in the ratio of unsaturated/saturated fatty acids predominantly by an increase in the linoleic acid. The present data suggest that phosphatidylcholine and fatty acids modifications in both mitochondrial subfractions caused by vitamin D3 might have some role in the intestinal mitochondrial Ca transport.     

Control of the transmembrane phospholipid distribution in eukaryotic cells by aminophospholipid translocase

The aminophospholipid translocase is a plasma membrane Mg2(+)-ATPase which selectively pumps the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the outer to the inner monolayer in eukaryotic cells and is predominantly responsible for the asymmetric phospholipid distribution of the plasma membrane. Similar ATP-dependent transport of phospholipid takes place in some organelles such as chromaffin granules. On the other hand, the phospholipid flippase of rat liver endoplasmic reticulum does not require ATP and has a low lipid specificity. The biological implications of these phospholipid flippases are discussed.     

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.     

Regulation of hepatic HMG-CoA reductase in vivo by reversible phosphorylation

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.     

Phospholipid outside-inside translocation in lymphocyte plasma membranes is a protein-mediated phenomenon

We have measured the transbilayer diffusion of spin-labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in pig lymphocyte plasma membrane. At 4 degrees C and 37 degrees C the aminophospholipids are rapidly transported from the outer to the inner leaflet of the membrane, whereas the choline-containing phospholipids experience a slower diffusion. This selectivity is abolished after cell treatment by SH-group reagents indicating that the aminophospholipid translocation is protein-dependent and must be driven by a system analogous to the one existing in the human red cell membrane. The fact that the selectivity exists at low temperature, that it does not depend on cytoskeleton integrity and that there is a competition between the two aminophospholipids show that this translocation is not purely an endocytic process.     

Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs.

The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane.