Depletion of phosphatidylcholine in yeast induces shortening and increased saturation of the lipid acyl chains: evidence for regulation of intrinsic membrane curvature in a eukaryote

To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to choline-free medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range.     

Yeast cells accumulate excess endogenous palmitate in phosphatidylcholine by acyl chain remodeling involving the phospholipase B Plb1p

In the yeast Saccharomyces cerevisiae, the molecular species profile of the major membrane glycerophospholipid phosphatidylcholine (PC) is determined by the molecular species-selectivity of the biosynthesis routes and by acyl chain remodeling. Overexpression of the glycerol-3-phosphate acyltransferase Sct1p was recently shown to induce a strong increase in the cellular content of palmitate (C16:0). Using stable isotope labeling and mass spectrometry, the present study shows that wild type yeast overexpressing Sct1p incorporates excess C16:0 into PC via the methylation of PE, the CDP-choline route, and post-synthetic acyl chain remodeling. Overexpression of Sct1p increased the extent of remodeling of PE-derived PC, providing a novel tool to perform mechanistic studies on PC acyl chain exchange. The exchange of acyl chains occurred at both the sn-1 and sn-2 positions of the glycerol backbone of PC, and required the phospholipase B Plb1p for optimal efficiency. Sct1p-catalyzed acyl chain exchange, the acyl-CoA binding protein Acb1p, the Plb1p homologue Plb2p, and the glycerophospholipid:triacylglycerol transacylase Lro1p were not required for PC remodeling. The results indicate that PC serves as a buffer for excess cellular C16:0.     

Metabolism of phosphatidylcholine and its implications for lipid acyl chain composition in Saccharomyces cerevisiae

Phosphatidylcholine (PC) is a very abundant membrane lipid in most eukaryotes including the model organism Saccharomyces cerevisiae. Consequently, the molecular species profile of PC, i.e. the ensemble of PC molecules with acyl chains differing in number of carbon atoms and double bonds, is important in determining the physical properties of eukaryotic membranes, and should be tightly regulated. In this review current insights in the contributions of biosynthesis, turnover, and remodeling by acyl chain exchange to the maintenance of PC homeostasis at the level of the molecular species in yeast are summarized. In addition, the phospholipid class-specific changes in membrane acyl chain composition induced by PC depletion are discussed, which identify PC as key player in a novel regulatory mechanism balancing the proportions of bilayer and non-bilayer lipids in yeast.     

酿酒酵母酚类抑制物耐受性脂质组学研究

通过脂质组学分析方法从细胞膜磷脂分布方面探究适应进化酿酒酵母酚酸耐受性机制。主要利用高效液相色谱-质谱(LC-MS)对酚酸胁迫下适应进化菌株和原始菌株脂质成分检测并进行统计学比较分析。检测出565种脂质代谢物,包含细胞膜磷脂185种。相比初始菌株,适应进化菌株细胞膜中磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)和磷脂酰肌醇(PI)类磷脂分子相对含量增加,含有长链(C32-C36)和双不饱和脂酰链的磷脂分子含量增加。统计学分析表明显著性差异磷脂分子主要为含有长链不饱和脂酰链的PC和PE类磷脂分子。推测适应进化菌株通过膜磷脂重塑提高细胞膜完整性,对酚类抑制物起到选择性屏障作用,从而保持细胞活性。     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Self-adaptive modification of red-cell membrane lipids in lecithin: Cholesterol acyltransferase deficiency. Lipid analysis and spin labeling

In a patient with lecithin: cholesterol acyltransferase deficiency, free cholesterol was markedly increased, and esterified cholesterol was diminished. In the patient's plasma, an increase in phosphatidylcholine (PC) and a decrease in sphingomyelin were observed. Concomitantly, an increase in a shorter acyl chain 16:0 was noted in PC, sphingomyelin and phosphatidylethanolamine (PE). In contrast to these results, longer chains such as 22:0 and 24:0 were decreased, especially in sphingomyelin. Unsaturated double bonds such as 18:1 was also increased in PC and PE. In the red-cell membrane lipids, the increase in free cholesterol was counteracted by an increase in PC and by a decrease in sphingomyelin and PE, reflecting changes in the patient's plasma lipids. Increased 16:0 (in PC) and decreased 18:0 and 24:0 were observed. The increased plasma free cholesterol due to metabolic defect (lecithin:cholesterol acyltransferase deficiency) led to decreased red-cell membrane fluidity. This effect appeared to be counteracted by changing phospholipid composition (increased PC and decreased sphingomyelin and PE), by increasing shorter chains (16:0), by decreasing longer chains (18:0 and 24:0) and by increasing unsaturated double bonds (18:2). These results can be interpreted as a self-adaptive modification of lecithin:cholesterol acyltransferase deficiency-induced red-cell membrane abnormalities, to maintain normal membrane fluidity. This speculation was supported by the ESR spin-label studies on the patient's membrane lipids. The normal order parameters in intact red cells and in total lipid liposomes were decreased if cholesterol-depleted membrane liposomes were prepared. Thus, the hardening effect of cholesterol appeared to be counteracted by the softening effects described above. Overall membrane fluidity in intact red cells of the lecithin:cholesterol acyltransferase-deficient patient was maintained normally, judged by order parameters in ESR spin-label studies.     

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates a variety of lysophospholipids including bis(monoacylglycero)phosphate

When the lysoglycerophospholipid (GPL) acyltransferase At1g78690 from Arabidopsis thaliana is over-expressed in Escherichiacoli a headgroup acylated GPL, acyl phosphatidylglycerol (PG), accumulates despite that in vitro this enzyme catalyzes the transfer of an acyl chain from acyl-CoA to the sn-2 position of 1-acyl phosphatidylethanolamine (PE) or 1-acyl PG to form the sn-1, sn-2, di acyl PE and PG respectively; it does not acylate PG to form acyl PG. To begin to understand why the overexpression of a lyso GPL acyltransferase leads to the accumulation of a headgroup acylated GPL in E. coli we investigated the headgroup specificity of At1g78690. Using membranes prepared from E. coli overexpressing At1g78690, we assessed the ability of At1g78690 to catalyze the transfer of acyl chains from acyl-coenzyme A to a variety of lyso GPL acyl acceptors including lyso-phosphatidic acid (PA), -phosphatidylcholine (PC), -phosphatidylserine (PC), -phosphatidylinositol (PI) and three stereoisoforms of bis(monoacylglycero)phosphate (BMP). The predicted products were formed when lyso PI and lyso PC were used as the acyl acceptor but not with lyso PC or lyso PA. In addition, At1g78690 robustly acylates two BMP isoforms with sn-2 and/or sn-2′ hydroxyls in the R-stereoconfiguration, but not the BMP isoform with the sn-2 and sn-2′ hydroxyls in the S-stereoconfiguration. This strongly suggests that At1g78690 is stereoselective for hydroxyls with R-stereochemistry. In addition, this robust acylation of BMPs by At1g78690, which yields acyl PG like molecules, may explain the mechanism by which At1g78690 so strikingly alters the lipid composition of E. coli.     

Lipid composition of Silybum marianum cell cultures treated with methyl jasmonate

Elicitation of cell cultures of Silybum marianum with methyl jasmonate (MeJA) increases the production and release of the secondary metabolite silymarin into the culture medium and this process seems to be dependent on phospholipase D activity and its product phosphatidic acid (PA). However, MeJA did not alter total membrane lipid content or overall fatty acid composition. A progressive increase in some galactolipids was observed with elicitation time. Phospholipids were mainly represented by phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) and phosphatidylinositol (PI). MeJA caused losses of PC species that contain two unsaturated acyl species, 36:5 and 36:6 and an increase in 36:2 species. A drop in the ratio of compounds with 18:3 in PI and PE was also observed. The presence of the lysophospholipids (LP) LPC (16:0, 18:3, 18:2, 18:1) and LPE (16:0, 18:3, 18:2, 18:1) and the high contents of PA, represented by the molecular species 34:3, 34:2 and 36:5 and 36:4, indicates high basal level of phospholipase activity in cultures and a high phospholipid turnover. MeJA treatment did not quantitatively alter these lipid classes.     

Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila

Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. Here we describe a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. We demonstrate that in Atg2 mutants (Atg2^?), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2^?. Significant differences in the lipid profiles of control and Atg2^? flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species.     

Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis

The effect of the phospholipid acyl chain carbon number on the activity of the branched-chain amino acid transport system of Lactococcus lactis has been investigated. Major fatty acids identified in a total lipid extract of L. lactis membranes are palmitic acid (16:0), oleic acid (18:1) and the cyclopropane-ring containing lactobacillic acid (19 delta). L. lactis membrane vesicles were fused with liposomes prepared from equimolar mixtures of synthetic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with cis mono-unsaturated acyl chains. The activity of the branched-chain amino acid carrier is determined by the bulk properties of the membrane (Driessen, A.J.M., Zheng, T., In 't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). PE acts as an activator and PC is ineffective. Counterflow and protonmotive-force driven transport of leucine is sensitive to changes in the acyl chain carbon number of both phospholipids and maximal with dioleoyl-PE/dioleoyl-PC. Above the gel to liquid-crystalline phase transition temperature of the lipid species, membrane fluidity decreased with increasing acyl chain carbon number. Our data suggest that the carbon number of the acyl chains of PE and PC determine to a large extent the activity of the transport system. This might be relevant for the interaction of PE with the transport protein. Variations in the acyl chain composition of PC exert a more general effect on transport activity. The acyl chain composition of phospholipids determines the membrane thickness (Lewis, B.A. and Engelman, D.M. (1983) J. Mol. Biol. 166, 211-217). We therefore propose that the degree of matching between the lipid-bilayer and the hydrophobic thickness of the branched-chain amino acid carrier is an important parameter in lipid-protein interactions.     

Cholesterol affects spectrin-phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B_max) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in Δπ change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The Δπ change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a “raft-like” mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.     

Enhanced phospholipase B activity and alteration of phospholipids and neutral lipids in Saccharomyces cerevisiae exposed to N-nitrosonornicotine

A tobacco-specific nitrosamine (TSNA), N-nitrosonornicotine (NNN), is a potent carcinogen present in cigarette smoke, and chronic exposure to it can lead to pulmonary cancer. NNN causes changes in phospholipid metabolism and the mechanism is yet to be elucidated. Exposure of Saccharomyces cerevisiae to 50 μM NNN leads to a substantial decrease in phosphatidylserine (PS) by 63%, phosphatidylcholine (PC) by 42% and phosphatidylethanolamine (PE) by 36% with a concomitant increase in lysophospholipids (LPL) by 25%. The alteration in phospholipid content was dependent on increasing NNN concentration. Reduced phospholipids were accompanied with increased neutral lipid content. Here we report for the first time that NNN exposure, significantly increases phospholipase B (PLB) activity and the preferred substrate is PC, a major phospholipid responsible for a series of metabolic functions. Furthermore, NNN also promotes the alteration of fatty acid (FA) composition; it increases the long chain fatty acid (C18 series) in phospholipids specifically phosphatidylethanolamine (PE) and PS; while on the contrary it increases short chain fatty acids in cardiolipin (CL). NNN mediated degradation of phospholipids is associated with enhanced PLB activity and alteration of phospholipid composition is accompanied with acyl chain remodelling. Understanding the altered phospholipid metabolism produced by NNN exposure is a worthwhile pursuit because it will help to understand the toxicity of tobacco smoke.     

Checks and balances in membrane phospholipid class and acyl chain homeostasis,the yeast perspective

Glycerophospholipids are the most abundant membrane lipid constituents in most eukaryotic cells. As a consequence, phospholipid class and acyl chain homeostasis are crucial for maintaining optimal physical properties of membranes that in turn are crucial for membrane function. The topic of this review is our current understanding of membrane phospholipid homeostasis in the reference eukaryote Saccharomyces cerevisiae. After introducing the physical parameters of the membrane that are kept in optimal range, the properties of the major membrane phospholipids and their contributions to membrane structure and dynamics are summarized. Phospholipid metabolism and known mechanisms of regulation are discussed, including potential sensors for monitoring membrane physical properties. Special attention is paid to processes that maintain the phospholipid class specific molecular species profiles, and to the interplay between phospholipid class and acyl chain composition when yeast membrane lipid homeostasis is challenged. Based on the reviewed studies, molecular species selectivity of the lipid metabolic enzymes, and mass action in acyl-CoA metabolism are put forward as important intrinsic contributors to membrane lipid homeostasis.     

Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria

Heavy isotope-labeled ethanolamine and serine as well as exogenous PE and PS species were used to study trafficking of phosphatidylethanolamine (PE) and -serine (PS) molecular species between the endoplasmic reticulum (ER) and mitochondria in HeLa cells. Import of both endogenous and exogenous PS to IMM was a relatively slow process (T1/2 = several hours), but depended on the acyl chains. In particular, the 38:4 and 38:5 species were imported more efficiently compared to the other PS species. Knock-down of Mitofusin 2 or Mitostatin had no detectable effect on PS import to mitochondria, suggesting that the ER–mitochondria contacts regulated by these proteins are not essential. Knock-down of PS synthase 1 inhibited PS decarboxylation, suggesting that import of PS to mitochondria is coupled to its synthesis. Also the export of PE from IMM to microsomes is a relatively slow process, but again depends markedly on the acyl chain structure. Most notably, the polyunsaturated 38:4 and 38:5 PE species were less efficiently exported, which together with rapid import of the PS precursors most probably explains their enrichment in IMM. PE synthesized via the CDP-ethanolamine was also imported to IMM, but most of the PE in this membrane derives from imported PS. In contrast to PS, all PC species made in Golgi/ER translocated similarly and rapidly to IMM. In conclusion, selective translocation of PS species and PS-derived PE species between ER and mitochondria plays a major role in phospholipid homeostasis of these organelles.     

Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine.

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)-deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane-associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well-known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn-requiring rat mammary cell line 64-24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12,13-dibutyrate (PDBu) under conditions where cells have either normal or PE-deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (approximately fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE-deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP-choline pathway of PC synthesis indicated that the regulatory enzyme, CTP:phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE-deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation.     

Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli

Phosphatidylethanolamine is the major membrane phospholipid of Escherichia coli, and two experimental approaches were used to investigate the metabolic activity of the fatty acids occupying the 1-position of this phospholipid. [3H]Acetate pulse-chase experiments with logarithmically growing cells indicated that 3-5% of the acyl groups were removed from the phosphatidylethanolamine pool/generation. The reacylation aspect of the turnover cycle was demonstrated by the incorporation of fatty acids into the 1-position of pre-existing phosphatidylethanolamine when de novo phospholipid biosynthesis was inhibited using the plsB acyltransferase mutant. 2- Acylglycerophosphoethanolamine would be the intermediate in a 1-position turnover cycle, and this lysophospholipid was identified as a membrane component that could re-esterified by a membrane-bound acyltransferase. The acyltransferase either utilized acyl-acyl carrier protein directly as an acyl donor or activated fatty acids for acyl transfer in the presence of ATP and Mg2+. Acyl-acyl carrier protein was also indicated as an intermediate in the latter reacylation reaction by the complete inhibition of phosphatidylethanolamine formation from fatty acids by acyl carrier protein-specific antibodies and by the observation that the inhibition of the acyltransferase by LiCl was reversed by the addition of acyl carrier protein. Coenzyme A thioesters were not substrates for this acyltransferase. These results suggest the existence of a metabolic cycle for the utilization of 1-position acyl moieties of phosphatidylethanolamine followed by the resynthesis of this membrane phospholipid from 2- acylglycerophosphoethanolamine by an acyl carrier protein-dependent 1-position acyltransferase.     

Electrospray ionization mass spectrometry and exogenous heavy isotope-labeled lipid species provide detailed information on aminophospholipid acyl chain remodeling

Mammalian cells maintain the phospholipid compositions of their different membranes remarkably constant. Beside de novo synthesis, degradation, and intracellular trafficking, acyl chain remodeling plays an important role in phospholipid homeostasis. However, many key details of this process remain unresolved, largely because of limitations of existing methodologies. Here we describe a novel approach that allows one to study metabolism of individual phospholipid species in unprecedented detail. Forty different phosphatidylethanolamine (PE) or -serine (PS) species with a deuterium-labeled head group were synthesized and introduced to BHK21 or HeLa cells using cyclodextrin-mediated transfer. Their metabolism was then monitored in detail by electrospray ionization mass spectrometry. Atypical PE and PS species were rapidly remodeled at both sn1 and sn2 position, yielding a molecular species profile similar to that the endogenous PE and PS. In contrast, remodeling of exogenous species identical or similar to major endogenous ones was more limited and much slower. Major differences in remodeling pathways and kinetics were observed between species within a class, as well as between corresponding PE and PS species. These data along with those obtained with pharmacological inhibitors strongly suggest that multiple lipid class-specific A-type phospholipases and acyl transferases are involved in aminophospholipid remodeling. In conclusion, the approach described here provides highly detailed information on remodeling of exogenously added (amino)glycerophospholipids and should thus be very helpful when elucidating the proteins and processes maintaining molecular species homeostasis.     

AtPGAP1 functions as a GPI inositol-deacylase required for efficient transport of GPI-anchored proteins

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) play an important role in a variety of plant biological processes including growth, stress response, morphogenesis, signaling, and cell wall biosynthesis. The GPI anchor contains a lipid-linked glycan backbone that is synthesized in the endoplasmic reticulum (ER) where it is subsequently transferred to the C-terminus of proteins containing a GPI signal peptide by a GPI transamidase. Once the GPI anchor is attached to the protein, the glycan and lipid moieties are remodeled. In mammals and yeast, this remodeling is required for GPI-APs to be included in Coat Protein II-coated vesicles for their ER export and subsequent transport to the cell surface. The first reaction of lipid remodeling is the removal of the acyl chain from the inositol group by Bst1p (yeast) and Post-GPI Attachment to Proteins Inositol Deacylase 1 (PGAP1, mammals). In this work, we have used a loss-of-function approach to study the role of PGAP1/Bst1 like genes in plants. We have found that Arabidopsis (Arabidopsis thaliana) PGAP1 localizes to the ER and likely functions as the GPI inositol-deacylase that cleaves the acyl chain from the inositol ring of the GPI anchor. In addition, we show that PGAP1 function is required for efficient ER export and transport to the cell surface of GPI-APs.

The inositol deacylase AtPGAP1 mediates the first step of glycosylphosphatidylinositol (GPI) anchor-lipid remodeling and is required for efficient transport of GPI-anchored proteins     

Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells

Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.     

Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics

Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species.