Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Phospholipid base exchange in human leukocyte membranes: quantitation and correlation with other phospholipid biosynthetic pathways

The biosynthesis of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) by base-exchange reactions, and of PC and PE by the CDP pathways, was assessed in the membrane phospholipids of human leukocytes (neutrophils, lymphocytes, T lymphocytes, non-T lymphocytes, and monocytes). Of the three base-exchange activities, ethanolamine exchange was the highest and choline exchange the lowest in each leukocyte membrane. In the CDP pathways, ethanolaminephosphotransferase (EPT) and cholinephosphotransferase (CPT) had comparable activities. Among subpopulations of leukocytes, T lymphocytes showed the highest levels of each enzyme activity, and neutrophils showed the least. In contrast to the enzymes of the CDP pathways, each base-exchange activity was directly proportional to the Ca2+ concentration, but markedly inhibited by Mg2+. Despite this Ca2+ dependence, the base-exchange activities were increased in a dose-dependent manner by calmodulin antagonists and, except for ethanolamine exchange, inhibited by the addition of calmodulin; EPT and CPT activities were only slightly inhibited by calmodulin antagonists and were unaffected by calmodulin. PE formation in both neutrophil and lymphocyte base-exchange reactions was enhanced in a dose-dependent manner by the presence of low concentrations of bioactive stimulants (zymosan, 0.05-0.2 mg/ml; Con A, 0.5-2 micrograms/ml), while EPT and CPT activities were not increased by these cell stimulants. Taken together, our data suggest that base-exchange activity, the biological significance of which has been hitherto unclear, may be related to cell activation; in contrast, the CDP pathways appear primarily to involve the constitutive biosynthesis of phospholipids. Our data further suggest that ethanolamine required for base-exchange reactions is a precursor of PE, N-transmethylation of which can serve as a source of cell activation, leading to production of arachidonic through PC by mediation of phospholipase A2 activity.     

Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects

Most of the phosphatidylethanolamine (PE) in mammalian cells is synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. Electron microscopy of Pisd-/- embryos revealed large numbers of aberrantly shaped mitochondria. In addition, fluorescence confocal microscopy of Pisd-/- embryonic fibroblasts showed fragmented mitochondria. PS decarboxylase activity and mRNA levels in Pisd+/- tissues were approximately one-half of those in wild-type mice. However, heterozygous mice appeared normal, exhibited normal vitality, and the phospholipid composition of livers, testes, brains, and of mitochondria isolated from livers, was the same as in wild-type littermates. The amount and activity of a key enzyme of the CDP-ethanolamine pathway for PE synthesis, CTP:phosphoethanolamine cytidylyltransferase, were increased by 35-40 and 100%, respectively, in tissues of Pisd+/- mice, as judged by immunoblotting; PE synthesis from [3H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDP-ethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality.     

Occurrence of phosphatidyl-d-serine in the rat cerebrum

Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-l-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-d-serine (d-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of d-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence of d-PS. Free d-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the occurrence of a divalent cation-dependent enzyme that synthesizes d-PS by a base-exchange reaction.     

The biosynthesis of phosphatidylserine and phosphatidylethanolamine from L-[3-14C]serine in isolated rat hepatocytes

Incorporation of L-[3-14C]serine into phosphatidylserine (PS) and phosphatidylethanolamine (PE) has been studied in isolated rat hepatocytes. Ethanolamine inhibited the incorporation, indicating competition with serine in the base-exchange reaction. Choline, monomethylethanolamine, dimethylethanolamine and dimethyl-3-aminopropan-1-ol had no such effect. The observed rate of PS biosynthesis corresponded to 7-17 nmol/min per liver at 0.55 mM L-serine. The results indicate that only a small fraction (1/25 to 1/70) of the PS pool equilibrates with the base-exchange enzyme, and that decarboxylation to PE occurs preferentially from this pool. The rate of PS synthesis and decarboxylation can therefore not be calculated by methods which assume random, homogeneous labelling of the total PS pool. The apparent rate of PS decarboxylation increased approx. 4-fold when L-serine increased from 0.5 to 2.25 mM, suggesting that decarboxylation of PS to PE might be regulated by the concentration of L-serine or by the amount of PS present in the hepatocyte cell membranes. Lauric, palmitic, stearic, oleic and linoleic acid decreased the rate of PS synthesis. At 0.5 mM, lauric and palmitic acid were most inhibitory. At 1.0 mM, linoleic acid was the least inhibitory fatty acid. The saturated hexaenoic and saturated tetraenoic species of PS contained 51 and 29%, respectively, of the incorporated L-[3-14C]serine. The combined monoene dienoic/diene dienoic fraction had the highest rate of synthesis judged by its relative specific activity. At 0.9 mM concentration, linoleic acid doubled the relative specific activity of the combined monoene dienoic/diene dienoic fraction of PS. Incorporation of L-[3-14C]serine into molecular species of PE resembled that into PS, both in the absence and presence of linoleic acid, suggesting that the phosphatidylserine decarboxylase (EC 4.1.1.65) has a low specificity towards the fatty acid composition of PS. The results indicate that biosynthesis of PS from L-serine occurs mainly by the base-exchange with only negligible contribution from direct incorporation of phosphatidic acid or diacylglycerol. Furthermore, the deacylation-reacylation pathway seem to contribute only little to the determination of the fatty acid composition of hepatocyte PS. Active PS turnover seems to be confined to a small fraction of the PS pool.     

Ethanol potentiates the uptake of [14C]Serine into phosphatidylserine by base-exchange reaction in NG 108-15 cells

Phospholipid base-exchange enzymes catalyze the incorporation of nitrogenous bases into phosphoglycerides by a calcium-dependent mechanism. In this study, we describe the effect of ethanol on the incorporation of radioactive serine, choline and ethanolamine into their respective phospholipids in a neuroblastoma x glioma hybrid cell line (NG 108-15). Long term ethanol exposure induced a potentiation of the incorporation of [¹⁴C]serine into phosphatidylserine. Moreover, the phosphorus content of PS was found to be increased after long-term ethanol exposure. No concomitant changes in the phosphorus content of other phospholipids were observed. The results indicate that in NG 108-15 cells, the incorporation of radiolabelled serine into PS is potentiated during chronic ethanol exposure.     

Ethanolamine Base-Exchange Reaction in Rat Brain Microsomal Subfractions

Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subfractions A, B, and C, corresponding to light smooth, heavy smooth, and rough microsomal membranes, respectively. The purity and the vesiculation of the membranes were checked biochemically. Subfraction C showed the highest ethanolamine base-exchange activity, both on phospholipid and protein bases. The other two subfractions had roughly similar activities. The kinetic behavior of the enzyme activity, although anomalous, was similar in the three subfractions. Treatment of the vesicles with Pronase or with mercury-dextran produced inactivation of the ethanolamine base-exchange reaction in the three subfractions. These findings suggest that the active site of base-exchange activity would be localized on the external leaflet of the vesicles. Treatment of the membranes with trinitrobenzenesulfonic acid (TNBS) has shown that the newly synthesized phosphatidylethanolamine (PE) belongs to a pool easily reacting with the probe, independent of the subfraction investigated. On the other hand, the distribution of the bulk membrane PE reacting with TNBS differs in the three subfractions examined. It is concluded that the newly synthesized PE and probably the active site of the enzyme are on the external leaflet of the membrane in all subfractions and that the ethanolamine base-exchange reaction has similar properties in all subfractions.     

The reaggregation of rat brain microsomal membranes after the treatment with octyl-beta-D-glucopyranoside. A study on ethanolamine base-exchange

The ethanolamine base-exchange activity of rat brain microsomes has been studied after treating the membranes with the non-ionic detergent n-octyl-beta-D-glucopyranoside. The detergent could solubilize membrane lipid and protein. The concentrations of the detergent and of membrane protein were both important for this effect. The presence of disaggregating concentrations of octylglucopyranoside in the base-exchange incubation mixture strongly inhibited the incorporation of radioactive ethanolamine into lipid; however, the removal of the detergent through dialytic procedures before assaying the base-exchange reaction restored the enzymic activity almost completely. As shown by exposing the membranes to trinitrobenzenesulfonic acid (TNBS), the phosphatidylethanolamine (PE) which was newly synthesized by base-exchange was also compartmented in the microsomal membrane. The treatment with the detergent after the base-exchange reaction abolished the compartmentation of the newly synthesized lipid. However, if microsomes were solubilized and the detergent was removed by dialysis before the assay of base-exchange, the reassembly of membranes occurred with a recovery of the compartmentation of the newly synthesized PE. The presence of Ca2+ in the dialytic medium was important for the preservation of base-exchange activity, probably affecting the reassembly of membrane components.     

A phosphatidylserine decarboxylase activity in root cells of oat (Avena sativa) is involved in altering membrane phospholipid composition during drought stress acclimation.

During acclimation to drought stress, the lipid composition of oat root cell membranes is altered. The level of phosphatidylethanolamine (PE), a non-bilayer forming lipid, is increased relative to the bilayer-forming lipid phosphatidylcholine (PC). These changes are believed to increase stress tolerance by increasing the flexibility of the membranes. To elucidate if de novo lipid synthesis is involved in altering membrane lipid composition, oat plants, acclimated or non-acclimated, were incubated in vivo with radioactively labelled lipid precursors. The labelling pattern indicated that de novo synthesis, at least partly, is causing the alterations. In plants, phospholipids can be synthesized by the Kennedy pathway, with addition of activated head groups to diacylglycerol (DAG) or, alternatively, via the CDP-DAG pathway, where phospahtidylserine (PS) is decarboxylated to form PE. To reveal the importance of the respective pathways during acclimation, we studied the effect of a decarboxylase inhibitor and the relative incorporation of [(3)H]-serine and [(14)C]-ethanolamine in vivo. Activities of CTP:ethanolaminephosphate cytidyltransferase (EC 2.7.7.14), phosphatidylserine decarboxylase (EC 4.1.1.65) and phosphatidylserine synthase; CDP-DAG:L-serine o-phosphatidyltransferase (EC 2.7.8.8) were measured and additionally, the presence of a PS decarboxylase (PSD1) in oat was confirmed by immunoblotting. The results suggest that PE synthesis via the Kennedy pathway is downregulated during acclimation and that synthesis by PS decarboxylation, via the CDP-DAG pathway, is increased, mainly through an increased activity of PS synthase.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.     

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.     

质膜组分磷脂酰丝氨酸外翻的分子调控机制

磷脂酰丝氨酸(Phosphatidylserine, PS)是细胞质膜重要的磷脂成分之一,具有重要的生物学功能。在细胞凋亡及一些特殊的病理条件下,细胞内ATP供能不足,胞浆Ca²⁺浓度升高,引起PS发生外翻。PS外翻在不同类型细胞中具有不同的生物学功能,且外翻的程度与疾病发展程度密切相关,可作为癌症等多种疾病治疗的靶标。文章综述了细胞质膜中磷脂酰丝氨酸的重要生物学功能和意义、磷脂酰丝氨酸外翻的分子机制及在临床医学方面的应用,以期对未来的功能和临床应用研究提供参考。     

Differential reaction of cell membrane phospholipids and proteins with chemical probes.

The major aims of this study were to determine the degree of phospholipid asymmetry and the neighbor analysis of phospholipids in different types of cell membranes. For this study a penetrating probe (FDNB), a non-penetrating probe (TNBS) and a cross-linking probe (DFDNB) were used. The reaction of hemoglobin, membrane protein and membrane PE and PS of erythrocytes with DFNB and TNBS was studied over a concentration range of 0.5 to 10 mM probe. TNBS reacts to an extremely small extend with hemoglobin over the concentration range 0.4 to 4 mM whereas FDNB reacts with hemoglobin to a very large extent (50 fold more than TNBS). The reaction of membrane protein of intact erythrocytes reaches a sharp plateau at 1 mM TNBS whereas the reaction of membrane protein goes to a much larger extent with FDNB with no plateau seen up to 4 mM FDNB. This data shows that TNBS does not significantly penetrate into the cell under our conditions whereas FDNB does penetrate into the cell. The results show that there are four fold more reactive sites on proteins localized on the inner surface of the erythrocyte membrane as compared to the outer surface. TNBS at 0.5 to 2 mM concentration does not label membrane PS and labels membrane PE to a small extent. The reaction of PE with TNBS shows an initial plateau at 2 mM probe and a second slightly higher plateau between 4 to 10 mM probe. TNBS from 0.5-2.0 mM does not react with PS, but between 3 to 10 mM concentration, a very small amount of PS reacts with TNBS. Hence above 2 mM TNBS or FDNB a perturbation occurs in the membrane such that more PE and PS are exposed and react with these probes. These results demonstrate that essentially no PS is localized on the outer surface of the membrane and only 5% of the total membrane PE is localized on the outer surface of the erythrocyte membrane. TNBS and FDNB were reacted with yeast, E. coli, and Acholeplasma cells. With yeast cells, FDNB reacts to a much larger extent with PE than does TNBS, indicating that FDNB penetrates into the cell and labels more PE molecules. With E. coli, but not with erythrocytes or yeast cells, phospholipase A activity was very pronounced at pH 8.5 giving rise to a large amount of DNP-GPE from DNP-PE. A phosphodiesterase was also present which hydrolyized DNP-GPE to DNP-ethanolamine. The multilayered structure of the E. coli cell envelop did not permit a definitive interpretation of the results. It is clear, however, that TNBS and FDNB react to a different extent with PE in this cell. The Acholeplasma membrane had no detectable PE or PS but contains amino acid esters of phosphatidylglycerol. The reaction of these components with TNBS and FDNB indicate that these aminoacyl-PG are localized on both surfaces of the membrane, with 31% being on the outer surface and 69% on the inner surface...     

Hexadecylphosphocholine inhibits phosphatidylcholine synthesis via both the methylation of phosphatidylethanolamine and CDP-choline pathways in HepG2 cells

We reported in a recent publication that hexadecylphosphocholine (HePC), a lysophospholipid analogue, reduces cell proliferation in HepG2 cells and at the same time inhibits the biosynthesis of phosphatidylcholine (PC) via CDP-choline by acting upon CTP:phosphocholine cytidylyltransferase (CT). We describe here the results of our study into the influence of HePC on other biosynthetic pathways of glycerolipids. HePC clearly decreased the incorporation of the exogenous precursor [1,2,3-3H]glycerol into PC and phosphatidylserine (PS) whilst increasing that of the neutral lipids diacylglycerol (DAG) and triacylglycerol (TAG). Interestingly, the uptake of L-[3-3H]serine into PS and other phospholipids remained unchanged by HePC and neither was the activity of either PS synthase or PS decarboxylase altered, demonstrating that the biosynthesis of PS is unaffected by HePC. We also analyzed the water-soluble intermediates and final product of the CDP-ethanolamine pathway and found that HePC caused an increase in the incorporation of [1,2-14C]ethanolamine into CDP-ethanolamine and phosphatidylethanolamine (PE) and a decrease in ethanolamine phosphate, which might be interpreted in terms of a stimulation of CTP:phosphoethanolamine cytidylyltransferase activity. Since PE can be methylated to give PC, we studied this process further and observed that HePC decreased the synthesis of PC from PE by inhibiting the PE N-methyltransferase activity. These results constitute the first experimental evidence that the inhibition of the synthesis of PC via CDP-choline by HePC is not counterbalanced by any increase in its formation via methylation. On the contrary, in the presence of HePC both pathways seem to contribute jointly to a decrease in the overall synthesis of PC in HepG2 cells.     

Calcium-dependent incorporation of choline into phosphatidylcholine (PC) by base-exchange in rat brain membranes occurs preferentially with phospholipid substrates containing docosahexaenoic acid (22:6(n-3))

The fatty acid composition of phosphatidylcholine (PC) formed by base-exchange was examined in rat brain membranes in vitro. The free choline incorporated into subspecies of PC by this phospholipase-D type activity can be distinguished from that which might enter indirectly via the last enzyme of the de novo pathway for phospholipid biosynthesis, cholinephosphotransferase, by its ionic requirements. Choline base-exchange in lysed synaptosomes is optimal when assayed at extracellular (mM) calcium concentrations and is blocked by magnesium. As much as 40% of the choline incorporated by base-exchange into rat brain membranes was recovered in subspecies of PC, representing no more than 10% of the total PC pool, which contained docosahexaenoic acid (22:6(n-3)). Docosahexaenoic acid is enriched in electrically-excitable membranes and its content in phospholipids of rat and human brain change during early development and increase with age.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

pH-dependent Ca2+ interaction with phospholipids related to phosphatidylethanolamine N-methylation

The interactions of PE and its N-methylated derivatives (PME, PDE AND PC) WITH Ca2+ were examined. PE and the intermediate phospholipids of PE N-methylation (PME and PDE) interacted with Ca2+ in a pH-dependent and reversible manner. When these phospholipids were present in the heptane phase, Ca2+ in the aqueous phase was translocated into the heptane phase at alkaline pH but not at acidic pH. PDE was also effective for the translocation even at around neutral pH, while PC hardly translocated Ca2+ at pH 6.0-9.2. The amounts of Ca2+ interacting with these phospholipids were in the following order: PDE is greater than PME is greater than PE is much greater than PC. P1, phosphatidic acid and PS interacted with Ca2+ in the whole pH range examined. The Ca2+ interactions with P1 and phosphatidic acid were independent of pH, while PS interacted with more Ca2+ at alkaline pH. These phospholipids interacted with Ca2+ most strongly among the cations studied. Liposomes containing PDE also bound the highest amounts Ca2+ among PE and its N-methylated derivatives. Furthermore, mammalian cultured cell membranes, which contain increased amounts of PDE by in vivo modification with N,N'-dimethylethanolamine, bound more Ca2+ than those prepared from choline-treated control cells.     

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs

Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with 32Pi and L-[U-14C]serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells.     

The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are major phospholipids in mammalian membranes. In liver, PC is synthesized via the choline pathway or by methylation of PE via phosphatidylethanolamine N-methyltransferase (PEMT). Pemt(-/-) mice fed a choline-deficient (CD) diet develop rapid steatohepatitis leading to liver failure. Steatosis is observed in CD mice that lack both PEMT and multiple drug-resistant protein 2 (MDR2), required for PC secretion into bile. We demonstrate that liver failure in CD-Pemt(-/-) mice is due to loss of membrane integrity caused by a decreased PC/PE ratio. The CD-Mdr2(-/-)/Pemt(-/-) mice escape liver failure by maintaining a normal PC/PE ratio. Manipulation of PC/PE levels suggests that this ratio is a key regulator of cell membrane integrity and plays a role in the progression of steatosis into steatohepatitis. The results have clinical implications as patients with nonalcoholic steatohepatitis have a decreased ratio of PC to PE compared to control livers.