Cubic phases in phosphatidylcholine-cholesterol mixtures: cholesterol as membrane "fusogen"

X-ray diffraction reveals that mixtures of some unsaturated phosphatidylcholines (PCs) with cholesterol (Chol) readily form inverted bicontinuous cubic phases that are stable under physiological conditions. This effect was studied in most detail for dioleoyl PC/Chol mixtures with molar ratios of 1:1 and 3:7. Facile formation of Im3m and Pn3m phases with lattice constants of 30-50 nm and 25-30 nm, respectively, took place in phosphate-buffered saline, in sucrose solution, and in water near the temperature of the Lalpha-HII transition of the mixtures, as well as during cooling of the HII phase. Once formed, the cubic phases displayed an ability to supercool and replace the initial Lalpha phase over a broad range of physiological temperatures. Conversion into stable cubic phases was also observed for mixtures of Chol with dilinoleoyl PC but not for mixtures with palmitoyl-linoleoyl PC or palmitoyl-oleoyl PC, for which only transient cubic traces were recorded at elevated temperatures. A saturated, branched-chain PC, diphytanoyl PC, also displayed a cubic phase in mixture with Chol. Unlike the PEs, the membrane PCs are intrinsically nonfusogenic lipids: in excess water they only form lamellar phases and not any of the inverted phases on their own. Thus, the finding that Chol induces cubic phases in mixtures with unsaturated PCs may have important implications for its role in fusion. In ternary mixtures, saturated PCs and sphingomyelin are known to separate into liquid-ordered domains along with Chol. Our results thus suggest that unsaturated PCs, which are excluded from these domains, could form fusogenic domains with Chol. Such a dual role of Chol may explain the seemingly paradoxical ability of cell membranes to simultaneously form rigid, low-curvature raft-like patches while still being able to undergo facile membrane fusion.     

Activation of dolichyl-phospho-mannose synthase by phospholipids

Dolichyl-phospho-mannose synthase, or GDPmannose:dolichyl-phosphate mannosyltransferase (EC 2.4.1.83), was solubilized from rat liver microsomes with 1.0% Nonidet P-40 and the enzyme was further purified by column chromatography on DEAE-cellulose in the presence of 0.1% Nonidet P-40. The purified enzyme preparation (880-fold over microsomes) was unstable in the presence of detergent and had no activity in the presence of Nonidet P-40, Triton X-100, octyl beta-glucoside, or deoxycholate. Detergent-free enzyme was active in the presence of phosphatidylethanolamine (PtdEtn) and in the presence of phospholipid mixtures of PtdEtn and phosphatidylcholine (PtdCho) when the molar proportion of PtdCho was 70% or less. The enzyme was inactive in the presence of PtdCho alone. Unsaturated species of PtdEtn have a tendency to destabilize membrane bilayers [Cullis, P. R. & de Kruijff, B. (1978) Biochim. Biophys. Acta 507, 207-218] and we have shown that dolichol promotes the destabilizing effect of PtdEtn on membranes composed of PtdCho and PtdEtn [Jensen, J. W. & Schutzbach, J. S. (1984) Biochemistry 23, 115-1119]. These results suggest that dolichyl-P-mannose synthase is optimally active in a phospholipid matrix that contains some component phospholipids that prefer non-bilayer structural organization in isolation. Heat-inactivation and sedimentation experiments demonstrated that the synthase associated with PtdEtn in the presence of dolichyl-P. The PtdEtn-reconstituted enzyme catalyzed the reversible transfer of mannose from GDP-mannose to dolichyl-P. The Km for GDP-mannose was found to be 0.69 microM and the apparent Km for dolichyl-P was 0.3 microM. GMP, GDP, and GTP inhibited mannosyl transfer 50% at concentrations of 16 microM, 1.3 microM and 3 microM respectively.     

Arachidonic Acid Incorporation and Redistribution in Human Neuroblastoma (SK-N-BE) Cell Phospholipids

The incorporation and redistribution of [1-14C]arachidonic acid in SK-N-BE human neuroblastoma cell phospholipids were investigated. By continuous labelling in serum-enriched medium, a rapid radioactivity incorporation into phosphatidylcholine (PtdCho), phosphatidylinositol, and phosphatidylserine was observed; initially, phosphatidylethanolamine (PtdEtn) was poorly labelled, but at later stages it displayed the highest level of arachidonic acid incorporation, in comparison with other phospholipid classes. Labelling of triacylglycerols was also observed. When cells were pulse-labelled with [1-14C]arachidonic acid and then reincubated in label-free medium, a decrease of the radioactivity in triacylglycerols was observed initially, paralleled by an increase of phospholipid labelling; thereafter, arachidonic acid redistribution was consistent with a net decrease of the radioactivity associated with PtdCho acid-stable forms (i.e., diacyl plus alkylacyl forms), concomitantly with a net labelling increase of both acid-stable PtdEtn and alkenylacyl-PtdEtn. Data indicate the following: (a) neuroblastoma cells incorporate arachidonic acid into phospholipids through complex kinetics involving transfer of the fatty acid from acid-stable PtdCho to both alkenylacyl-PtdEtn and acid-stable PtdEtn; and (b) triacylglycerols act as storage molecules for arachidonic acid which is subsequently incorporated into phospholipids. The possibility that arachidonic acid transfer to PtdEtn subclasses is driven by distinct mechanisms is discussed.     

Miscibility of ternary mixtures of phospholipids and cholesterol in monolayers, and application to bilayer systems

We investigate miscibility transitions of two different ternary lipid mixtures, DOPC/DPPC/Chol and POPC/PSM/Chol. In vesicles, both of these mixtures of an unsaturated lipid, a saturated lipid, and cholesterol form micron-scale domains of immiscible liquid phases for only a limited range of compositions. In contrast, in monolayers, both of these mixtures produce two distinct regions of immiscible liquid phases that span all compositions studied, the alpha-region at low cholesterol and the beta-region at high cholesterol. In other words, we find only limited overlap in miscibility phase behavior of monolayers and bilayers for the lipids studied. For vesicles at 25 degrees C, the miscibility phase boundary spans portions of both the monolayer alpha-region and beta-region. Within the monolayer beta-region, domains persist to high pressures, yet within the alpha-region, miscibility phase transition pressures always fall below 15 mN/m, far below the bilayer equivalent pressure of 32 mN/m. Approximately equivalent phase behavior is observed for monolayers of DOPC/DPPC/Chol and for monolayers of POPC/PSM/Chol. As expected, pressure-area isotherms of our ternary lipid mixtures yield smaller molecular area and compressibility for monolayers containing more saturated acyl chains and cholesterol. All monolayer experiments were conducted under argon. We show that exposure of unsaturated lipids to air causes monolayer surface pressures to decrease rapidly and miscibility transition pressures to increase rapidly.     

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.     

Phosphatidylcholine and N-methylated phospholipids are nonessential in Saccharomyces cerevisiae

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in numerous eukaryotes and is generally thought to be essential for membrane structure and cellular function. We designed a specific test of this idea by using genetic and biochemical manipulation of yeast. Yeast mutants (pem1 pem2Delta) lacking the phosphatidylethanolamine (PtdEtn) methyltransferase enzymes require choline for growth and cannot make N-methylated phospholipids. When these strains are grown on a glucose carbon source supplemented with 20 mm propanolamine (Prn), the PtdCho level declines precipitously to the limits of detection (<0.6%), and the hexagonal phase-forming, primary amine-containing lipids, PtdEtn and PtdPrn, constitute approximately 60% of the total phospholipid content of the cell. When the lipids were analyzed by mass spectrometry, there was no compensatory shift in unsaturation of the PtdEtn and PtdPrn toward more bilayer-forming species. Thus the majority of the cellular amino phospholipids remained hexagonal phase-forming. The pem1 pem2Delta cells will also grow without choline, in the presence of Prn, on nonfermentable carbon sources (requiring functional mitochondria) and accumulate nearly 70% of their phospholipid as hexagonal phase-forming types. These data provide compelling evidence that the functions of PtdCho and N-methylated lipids in membranes are nonessential in Saccharomyces cerevisiae.     

Calcium-induced phase separation phenomena in multicomponent unsaturated lipid mixtures

The ability of calcium to induce phase separation in multicomponent lipid mixtures containing various unsaturated species of acidic and neutral phospholipids has been investigated by 31P NMR, 3H NMR, and small-angle X-ray diffraction techniques. It is shown that, in unsaturated (dioleoyl-) phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) (1:1) and phosphatidic acid (PA)/phosphatidylcholine (PC) (1:1) mixtures, calcium is unable to induce lateral phase separation of the acidic and neutral lipids and that all the lipids adopt a hexagonal (HII) phase in the presence of calcium. In multicomponent mixtures containing one or more acidic species the presence of cholesterol either facilitates calcium-induced lamellar to hexagonal (HII) transitions for all the lipid components or, in systems already in a hexagonal (HII) phase, mitigates against calcium-induced lateral phase separations. Further, cholesterol is shown to exhibit no preferential interaction on the NMR time scale with either PC, PE, or phosphatidylserine (PS) when the lipids are in the liquid-crystal state. The ability of cholesterol to directly induce HII phase formation in PC/PE mixtures is also shown to be common to various other sterols including ergosterol, stigmasterol, coprostanol, epicoprostanol, and androstanol.     

Contribution of different biosynthetic pathways to species selectivity of aminoglycerophospholipids assembled into mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, three pathways lead to the formation of cellular phosphatidylethanolamine (PtdEtn), namely the mitochondrial conversion of phosphatidylserine (PtdSer) to PtdEtn catalyzed by phosphatidylserine decarboxylase 1 (Psd1p), the equivalent reaction catalyzed by phosphatidylserine decarboxylase 2 (Psd2p) in the Golgi, and the CDP-ethanolamine branch of the so-called Kennedy pathway which is located to the microsomal fraction. To investigate the contributions of these three pathways to the cellular pattern of PtdEtn species (fatty acid composition) we subjected lipids of wild-type and yeast mutant strains with distinct defects in the respective pathways to mass spectrometric analysis. We also analyzed species of PtdSer and phosphatidylcholine (PtdCho) of these strains because formation of the three aminoglycerophospholipids is linked through their biosynthetic route. We demonstrate that all three pathways involved in PtdEtn synthesis exhibit a preference for the formation of C34:2 and C32:2 species resulting in a high degree of unsaturation in total cellular PtdEtn. In PtdSer, the ratio of unsaturated to saturated fatty acids is much lower than in PtdEtn, suggesting a high species selectivity of PtdSer decarboxylases. Finally, PtdCho is characterized by its higher ratio of C16 to C18 fatty acids compared to PtdSer and PtdEtn. In contrast to biosynthetic steps, import of all three aminoglycerophospholipids into mitochondria of wild-type and mutant cells is not highly specific with respect to species transported. Thus, the species pattern of aminoglycerophospholipids in mitochondria is mainly the result of enzyme specificities, but not of translocation processes involved. Our results support a model that suggests equilibrium transport of aminoglycerophospholipids between mitochondria and microsomes based on membrane contact between the two compartments.     

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis by cytochalasin B in cultured glioma cells: potential regulation of biosynthesis by Ca(2+)-dependent mechanisms

The major route of phosphatidylcholine (PtdCho) biosynthesis in mammalian cells is the sequence: choline (Cho)----phosphocholine (PCho)----cytidinediphosphate choline (CDP-Cho)----PtdCho. Recently, we have found that intermediates of this pathway are not freely diffusible in cultured rat glioma (C6) cells but are channeled towards PtdCho biosynthesis (George et al. (1989). Biochim. Biophys. Acta. 1004, 283-291). Channeling of intermediates in other mammalian systems is thought to be mediated through adsorption of enzymes to membranes and cytoskeletal elements to form multienzyme complexes. In this study, agents which perturb the structure and function of cytoskeletal elements were tested for effects on phospholipid metabolism in glioma cells. The filament-disrupting agent cytochalasin B (CB), but not other cytochalasins or the microtubule depolymerizer colchicine inhibited PtdCho and phosphatidylethanolamine (PtdEtn) biosynthesis as judged by dose-dependent reduction of labeling from [3H]Cho and [14C]ethanolamine (Etn). 32Pi pulse-labeling indicated that CB selectively decreased PtdCho and PtdEtn biosynthesis without affecting synthesis of other phospholipids. Synthesis of water-soluble intermediates of PtdCho metabolism was unaffected but the conversion of phosphoethanolamine to CDP-ethanolamine was reduced by CB. Effects of CB on phospholipid biosynthesis were not due to inhibition of glucose uptake as shown by experiments with 2-deoxyglucose, glucose-starved cells and other cytochalasins. Experiments with Ca(2+)-EGTA buffers and digitonin-permeabilized cells, and the Ca(2+)-channel blocker verapamil suggest that effects of CB on PtdCho and PtdEtn biosynthesis are due to alteration of intracellular Ca2+. Taken together, these results suggest that CB acts at sites distinct from glucose transport and cellular microfilaments to specifically inhibit PtdCho and PtdEtn biosynthesis by mechanisms dependent on intracellular Ca2+.     

The influence of fermentation conditions and recycling on the phospholipid and fatty acid composition of the brewer’s yeast plasma membranes

Phospholipid (PL) and fatty acid (FA) compositions of the plasma membrane (PM), as well as the FA composition of the PM phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) in the pure culture (zero generation) and the first three recycled generations of the bottom-fermenting brewer’s yeast, have been determined. The PL composition differed markedly among the generations; in the zero generation, phosphatidylinositol (PtdIns) was the main PL, accounting for 27% of total PLs, followed by phosphatidic acid and PtdCho. In all recycled generations, the main PL was PtdCho with a marked increase in the first generation compared with the zero (32% and 20%, respectively), followed by PtdIns in the first and second generations. In the FA composition of the PM, 22 FAs were identified, ranging from C₁₀ to C₂₆. The compositions of the PM FAs, as well as those of PtdCho and PtdEtn, were characterised by a high preponderance of C₁₆ acids. Saturated FAs prevailed in the zero generation, whilst unsaturated prevailed in the first and second generation. Although the profiles of FAs in PtdCho and PtdEtn were similar, some marked differences were observed, pointing out to their specific functions in the regulation of membrane properties.     

Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDP-choline neuroprotection

We have previously demonstrated that cytidine 5'-diphosphocholine (CDP-choline or citicoline) attenuated arachidonic acid (ArAc) release and provided significant protection for the vulnerable hippocampal CA(1) neurons of the cornu ammonis after transient forebrain ischemia of gerbil. ArAc is released by the activation of phospholipases and the alteration of phosphatidylcholine (PtdCho) synthesis. Released ArAc is metabolized by cyclooxygenases/lipoxygenases to form eicosanoids and reactive oxygen species (ROS). ROS contribute to neurotoxicity through generation of lipid peroxides and the cytotoxic byproducts 4-hydroxynonenal and acrolein. ArAc can also stimulate sphingomyelinase to produce ceramide, a potent pro-apoptotic agent. In the present study, we examined the changes and effect of CDP-choline on ceramide and phospholipids including PtdCho, phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin, and cardiolipin (an exclusive inner mitochondrial membrane lipid essential for electron transport) following ischemia/1-day reperfusion. Our studies indicated significant decreases in total PtdCho, PtdIns, PtdSer, sphingomyelin, and cardiolipin and loss of ArAc from PtdEtn in gerbil hippocampus after 10-min forebrain ischemia/1-day reperfusion. CDP-choline (500 mg/kg i.p. immediately after ischemia and at 3-h reperfusion) significantly restored the PtdCho, sphingomyelin, and cardiolipin levels as well as the ArAc content of PtdCho and PtdEtn but did not affect PtdIns and PtdSer. These data suggest multiple beneficial effects of CDP-choline: (1) stabilizing the cell membrane by restoring PtdCho and sphingomyelin (prominent components of outer cell membrane), (2) attenuating the release of ArAc and limiting its oxidative metabolism, and (3) restoring cardiolipin levels.     

Mass‐spectrometric characterization of phospholipids and their primary peroxidation products in rat cortical neurons during staurosporine‐induced apoptosis

The molecular diversity of phospholipids is essential for their structural and signaling functions in cell membranes. In the current work, we present, the results of mass spectrometric characterization of individual molecular species in major classes of phospholipids – phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), sphingomyelin (CerPCho), and cardiolipin (Ptd₂Gro) – and their oxidation products during apoptosis induced in neurons by staurosporine (STS). The diversity of molecular species of phospholipids in rat cortical neurons followed the order Ptd₂Gro > PtdEtn >> PtdCho >> PtdSer > PtdIns > CerPCho. The number of polyunsaturated oxidizable species decreased in the order Ptd₂Gro >> PtdEtn > PtdCho > PtdSer > PtdIns > CerPCho. Thus a relatively minor class of phospholipids, Ptd₂Gro, was represented in cortical neurons by the greatest variety of both total and peroxidizable molecular species. Quantitative fluorescence HPLC analysis employed to assess the oxidation of different classes of phospholipids in neuronal cells during intrinsic apoptosis induced by STS revealed that three anionic phospholipids – Ptd₂Gro >> PtdSer > PtdIns – underwent robust oxidation. No significant oxidation in the most dominant phospholipid classes – PtdCho and PtdEtn – was detected. MS‐studies revealed the presence of hydroxy‐, hydroperoxy‐ as well as hydroxy‐/hydroperoxy‐species of Ptd₂Gro, PtdSer, and PtdIns. Experiments in model systems where total cortex Ptd₂Gro and PtdSer fractions were incubated in the presence of cytochrome c (cyt c) and H₂O₂, confirmed that molecular identities of the products formed were similar to the ones generated during STS‐induced neuronal apoptosis. The temporal sequence of biomarkers of STS‐induced apoptosis and phospholipid peroxidation combined with recently demonstrated redox catalytic properties of cyt c realized through its interactions with Ptd₂Gro and PtdSer suggest that cyt c acts as a catalyst of selective peroxidation of anionic phospholipids yielding Ptd₂Gro and PtdSer peroxidation products. These oxidation products participate in mitochondrial membrane permeability transition and in PtdSer externalization leading to recognition and uptake of apoptotic cells by professional phagocytes.     

Phospholipid-dependence of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase.

Hepatic UDP-glucuronyltransferase activity was resolved into two fractions, one exhibiting oestrone glucuronyltransferase activity and the other exhibiting p-nitrophenol glucuronyltransferase activity. Hydroxyapatite-column chromatography removed greater than 95% of the phospholipids from both preparations. The partially purified delipidated enzymes were essentially devoid of catalytic activity, but activities were restored by the addition of phospholipids or phosphatidylcholine mixtures containing various saturated and unsaturated fatty acids. Both oestrone and p-nitrophenol glucuronyl-transferase activities were reconstituted to similar degrees with the phosphatidylcholine mixtures. When purified phospholipids were tested, phosphatidylcholine and lysophosphatidylcholine were most effective in restoring activity, whereas phosphatidylethanolamine was the least effective. These results further suggest that oestrone and p-nitrophenol UDP-glucuronyltransferases are dependent on phospholipids for their activity.     

Calorimetric and spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylglycerol bilayer membranes

We have examined the effects of cholesterol (Chol) on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylglycerols (PGs) by high-sensitivity differential scanning calorimetry and Fourier transform infrared and ³¹P NMR spectroscopy. We find that the incorporation of increasing quantities of Chol alters the temperature and progressively reduces the enthalpy and cooperativity of the gel-to-liquid-crystalline phase transition of the host PG bilayer. With dimyristoyl-PG:Chol mixtures, cooperative chain-melting phase transitions are completely or almost completely abolished at Chol concentrations near 50 mol%, whereas with the dipalmitoyl- and distearoyl-PG:Chol mixtures, cooperative hydrocarbon chain-melting phase transitions are still discernable at Chol concentrations near 50 mol%. We are also unable to detect the presence of significant populations of separate domains of the anhydrous or monohydrate forms of Chol in our binary mixtures, in contrast to previous reports. We ascribe the previously reported large scale formation of Chol crystallites to the fractional crystallization of the Chol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. We further show that the direction and magnitude of the change in the phase transition temperature induced by Chol addition is dependent on the hydrocarbon chain length of the PG studied. This finding agrees with our previous results with phosphatidylcholine bilayers, where we found that Chol increases or decreases the phase transition temperature in a hydrophobic mismatch-dependent manner (Biochemistry 1993, 32:516-522), but is in contrast to our previous results for phosphatidylethanolamine (Biochim. Biophys. Acta 1999, 1416:119-234) and phosphatidylserine (Biophys. J. 2000, 79:2056-2065) bilayers, where no such hydrophobic mismatch-dependent effects were observed. We also show that the addition of Chol facilitates the formation of the lamellar crystalline phase in PG bilayers, as it does in phosphatidylethanolamine and phosphatidylserine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of Chol. Moreover, the formation of the lamellar crystalline phase in PG bilayers at lower temperatures excludes Chol, resulting in an apparent Chol immiscibility in gel-state PG bilayers. We suggest that the magnitude of the effect of Chol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipids dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.     

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides.

Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.     

Contribution of different pathways to the supply of phosphatidylethanolamine and phosphatidylcholine to mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast, three biosynthetic pathways lead to the formation of phosphatidylethanolamine (PtdEtn): (i) decarboxylation of phosphatidylserine (PtdSer) by phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria; (ii) decarboxylation of PtdSer by Psd2p in a Golgi/vacuolar compartment; and (iii) the CDP-ethanolamine (CDP-Etn) branch of the Kennedy pathway. The major phospholipid of the yeast, phosphatidylcholine (PtdCho), is formed either by methylation of PtdEtn or via the CDP-choline branch of the Kennedy pathway. To study the contribution of these pathways to the supply of PtdEtn and PtdCho to mitochondrial membranes, labeling experiments in vivo with [(3)H]serine and [(14)C]ethanolamine, or with [(3)H]serine and [(14)C]choline, respectively, and subsequent cell fractionation were performed with psd1Delta and psd2Delta mutants. As shown by comparison of the labeling patterns of the different strains, the major source of cellular and mitochondrial PtdEtn is Psd1p. PtdEtn formed by Psd2p or the CDP-Etn pathway, however, can be imported into mitochondria, although with moderate efficiency. In contrast to mitochondria, microsomal PtdEtn is mainly derived from the CDP-Etn pathway. PtdEtn formed by Psd2p is the preferred substrate for PtdCho synthesis. PtdCho derived from the different pathways appears to be supplied to subcellular membranes from a single PtdCho pool. Thus, the different pathways of PtdEtn biosynthesis play different roles in the assembly of PtdEtn into cellular membranes.     

New perspectives on the regulation of intermembrane glycerophospholipid traffic

In eukaryotes, phosphatidylserine (PtdSer) can serve as a precursor of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho), which are the major cellular phospholipids. PtdSer synthesis originates in the endoplasmic reticulum (ER) and its subdomain named the mitochondria-associated membrane (MAM). PtdSer is transported to the mitochondria in mammalian cells and yeast, and decarboxylated by PtdSer decarboxylase 1 (Psd1p) to form PtdEtn. A second decarboxylase, Psd2p, is also found in yeast in the Golgi-vacuole. PtdEtn produced by Psd1p and Psd2p can be transported to the ER, where it is methylated to form PtdCho. Organelle-specific metabolism of the aminoglycerophospholipids is a powerful tool for experimentally following lipid traffic that is now enabling identification of new proteins involved in the regulation of this process. Genetic and biochemical experiments demonstrate that transport of PtdSer between the MAM and mitochondria is regulated by protein ubiquitination, which affects events at both membranes. Similar analyses of PtdSer transport to the locus of Psd2p now indicate that a membrane-bound phosphatidylinositol transfer protein and the C2 domain of Psd2p are both required on the acceptor membrane for efficient transport of PtdSer. Collectively, these recent findings indicate that novel multiprotein assemblies on both donor and acceptor membranes participate in interorganelle phospholipid transport.     

Thermodynamics of lipid interactions in complex bilayers

The mutual interactions between lipids in bilayers are reviewed, including mixtures of phospholipids, and mixtures of phospholipids and cholesterol (Chol). Binary mixtures and ternary mixtures are considered, with special emphasis on membranes containing Chol, an ordered phospholipid, and a disordered phospholipid. Typically the ordered phospholipid is a sphingomyelin (SM) or a long-chain saturated phosphatidylcholine (PC), both of which have high phase transitions temperatures; the disordered phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC). The unlike nearest-neighbor interaction free energies (ω_AB) between lipids (including Chol), obtained by an variety of unrelated methods, are typically in the range of 0-400 cal/mol in absolute value. Most are positive, meaning that the interaction is unfavorable, but some are negative, meaning it is favorable. It is of special interest that favorable interactions occur mainly between ordered phospholipids and Chol. The interpretation of domain formation in complex mixtures of Chol and phospholipids in terms of phase separation or condensed complexes is discussed in the light of the values of lipid mutual interactions.     

Substrate-specific forms of human platelet phospholipase A2

Purification of human platelet phospholipase A2 (PLA2) from a particulate fraction by ion-exchange chromatography at 4 degrees C yielded a single peak of enzyme activity, which catalyzed the hydrolysis of arachidonic acid from the 2-position of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). The activity toward PtdCho and that toward PtdEtn differed in stability during storage, pH optimum, Ca2+ requirement, and affinity for the substrate; however, each activity preferred phospholipid with arachidonate at the 2-position. The two activities appeared to be eluted as an aggregate in a single peak from the ion-exchange column. When the column was run at 22 degrees C, an additional PLA2 activity peak specific for PtdEtn was resolved from the original PLA2 peak. But when the particulate fraction was briefly sonicated in 0.1% octylglucoside before chromatography at 22 degrees C, a different PLA2 activity peak, specific for PtdCho, was obtained. Resolution of the two specific forms of PLA2 under different conditions probably resulted from selective solubilization of the aggregate. The specific PLA2 activities thus isolated were very labile, whereas those in the aggregate were relatively stable. These findings suggest that human platelets contain at least two substrate-specific forms of PLA2, one for PtdCho and another for PtdEtn.     

Molecular cloning of canine bullous pemphigoid antigen 2 cDNA and immunomapping of NC16A domain by canine bullous pemphigoid autoantibodies

The aminoglycerophospholipids of eukaryotic cells, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), can be synthesized by multiple pathways. The PtdSer pathway encompasses the synthesis of PtdSer, its decarboxylation to PtdEtn and subsequent methylation reactions to form PtdCho. The Kennedy pathways consist of the synthesis of PtdEtn and PtdCho from Etn and Cho precursors via CDP-Etn and CDP-Cho intermediates. The reactions along the PtdSer pathway are spatially segregated with PtdSer synthesis occurring in the endoplasmic reticulum or mitochondria-associated membrane (MAM), PtdEtn formation occurring in the mitochondria and Golgi/vacuole compartments and PtdCho formation occurring in the endoplasmic reticulum or MAM. The organelle-specific metabolism of the different lipids in the PtdSer pathway has provided a convenient biochemical means for defining events in the interorganelle transport of the aminoglycerophospholipids in intact cells, isolated organelles and permeabilized cells. Studies with both mammalian cells and yeast demonstrate many significant similarities in lipid transport processes between the two systems. Genetic experiments in yeast now provide the tools to create new strains with mutations along the PtdSer pathway that can be conditionally rescued by the Kennedy pathway reactions. The genetic studies in yeast indicate that it is now possible to begin to define genes that participate in the interorganelle transport of the aminoglycerophospholipids.