N-Acylphosphatidylethanolamine accumulation in potato cells upon energy shortage caused by anoxia or respiratory inhibitors.

A minor phospholipid was isolated from potato (Solanum tuberosum L. cv Bintje) cells, chromatographically purified, and identified by electrospray ionization mass spectrometry as N-acylphosphatidylethanolamine (NAPE). The NAPE level was low in unstressed cells (13 +/- 4 nmol g fresh weight(-1)). According to acyl chain length, only 16/18/18 species (group II) and 18/18/18 species (group III) were present. NAPE increased up to 13-fold in anoxia-stressed cells, but only when free fatty acids (FFAs) started being released, after about 10 h of treatment. The level of groups II and III was increased by unspecific N-acylation of phosphatidylethanolamine, and new 16/16/18 species (group I) appeared via N-palmitoylation. NAPE also accumulated in aerated cells treated with NaN(3) plus salicylhydroxamate. N-acyl patterns of NAPE were dominated by 18:1, 18:2, and 16:0, but never reflected the FFA composition. Moreover, they did not change greatly after the treatments, in contrast with O-acyl patterns. Anoxia-induced NAPE accumulation is rooted in the metabolic homeostasis failure due to energy deprivation, but not in the absence of O(2), and is part of an oncotic death process. The acyl composition of basal and stress-induced NAPE suggests the existence of spatially distinct FFA and phosphatidylethanolamine pools. It reflects the specificity of NAPE synthase, the acyl composition, localization and availability of substrates, which are intrinsic cell properties, but has no predictive value as to the type of stress imposed. Whether NAPE has a physiological role depends on the cell being still alive and its compartmentation maintained during the stress period.

Impact of post-anoxia stress on membrane lipids of anoxia-pretreated potato cells. A re-appraisal.

The importance of lipid peroxidation and its contributing pathways (via reactive oxygen species and lipoxygenase) during post-anoxia was evaluated with respect to the biphasic behavior of membrane lipids under anoxia (A. Rawyler, D. Pavelic, C. Gianinazzi, J. Oberson, R. Brändle [1999] Plant Physiol 120: 293-300), using potato (Solanum tuberosum cv Bintje) cell cultures. When anoxic cells in the pre-lytic phase were re-oxygenated for 2 h, superoxide anion was not detectable, the hydrogen peroxide (H(2)O(2)) level remained small and similar to that of controls, and cell viability was preserved. Lipids were intact and no lipid hydroperoxides were detected. However, small amounts of lipid hydroperoxides accumulated upon feeding anoxic cells with H(2)O(2) and incubation for an additional 2 h under anoxia. When cells that entered the lytic phase of anoxia were re-oxygenated for 2 h, the H(2)O(2) and superoxide anion levels were essentially unchanged. However, cell respiration decreased, reflecting the extensive lipid hydrolysis that had already started under anoxia and continued during post-anoxia. Simultaneous with the massive release of free polyunsaturated fatty acids, small amounts of lipid hydroperoxides were formed, reaching 1% to 2% of total fatty acids. Catalase and superoxide dismutase activities were not greatly affected, whereas the amount and activity of lipoxygenase tended to increase during anoxia. Lipid peroxidation in potato cells is therefore low during post-anoxia. It is mainly due to lipoxygenase, whereas the contribution of reactive oxygen species is negligible. But above all, it is a late event that occurs only when irreversible damage is already caused by the anoxia-triggered lipid hydrolysis.

Analysis of the heat-shock response displayed by two Chaetomium species originating from different thermal environments.

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.

Membrane lipid integrity relies on a threshold of ATP production rate in potato cell cultures submitted to anoxia

In this paper we report on our study of the changes in biomass, lipid composition, and fermentation end products, as well as in the ATP level and synthesis rate in cultivated potato (Solanum tuberosum) cells submitted to anoxia stress. During the first phase of about 12 h, cells coped with the reduced energy supply brought about by fermentation and their membrane lipids remained intact. The second phase (12-24 h), during which the energy supply dropped down to 1% to 2% of its maximal theoretical normoxic value, was characterized by an extensive hydrolysis of membrane lipids to free fatty acids. This autolytic process was ascribed to the activation of a lipolytic acyl hydrolase. Cells were also treated under normoxia with inhibitors known to interfere with energy metabolism. Carbonyl-cyanide-4-trifluoromethoxyphenylhydrazone did not induce lipid hydrolysis, which was also the case when sodium azide or salicylhydroxamic acid were fed separately. However, the simultaneous use of sodium azide plus salicylhydroxamic acid or 2-deoxy-D-glucose plus iodoacetate with normoxic cells promoted a lipid hydrolysis pattern similar to that seen in anoxic cells. Therefore, a threshold exists in the rate of ATP synthesis (approximately 10 &mgr;mol g-1 fresh weight h-1), below which the integrity of the membranes in anoxic potato cells cannot be preserved.

Cyclodextrins: a new tool for the controlled lipid depletion of thylakoid membranes.

Cyclodextrins (CDs) have been used in controlled lipid depletion of thylakoid membranes avoiding the use of either detergents or lipolytic enzymes. Spinach thylakoid membranes were first treated with different CDs under various conditions. After removal of the CDs by washing, the amounts of mono-- and digalactosyldiacylglycerol (MGDG and DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), protein, pigment and plastoquinone remaining in the membranes were determined. The main results, obtained with alpha-CD and heptakis-(2,6-di-O-methyl)-beta-CD (DM-beta-CD), were as follows. (1)Acyl lipids were removed from thylakoid membranes by both CDs (DM-beta-CD being more efficient than alpha-CD; the extent of removal depended on both CD and chlorophyll concentrations. (2) alpha-CD presented a higher selectivity towards lip classes than did DM-beta-CD, but in both cases the removal order was SQDG > PG > MGDG > DGDG. (3) alpha-CD showed a preference for those lipids containing saturated 16-carbon acyl chains whereas DM-beta-CD was essentially insensitive to the fatty acid composition of the lipids. (4) The protein, chlorophyll and carotenoid contents of thylakoids were not affected by CD treatments. (5) Plastoquinones were removable but in small amounts only and with a low efficiency (DM-beta-CD > alpha-CD). (6) For all lipid classes, the extent of lipid removal was higher at 0 degrees than at 20 degrees C. (7) The presence of MgCl(2) reduced the removal of PG and SQDG but not affect galactolipid depletion levels. (8) Staple lipid depletion levels in thylakoid membranes were reached after 5-10 min of CD treatment at 0 degrees C. (9) Of the four CDs tested, only three (alpha-CD, beta-CD, and DM-beta-CD) promoted lipid depletion whereas one (hydroxypropyl-beta-CD) failed completely to do so. It is concluded that CD-mediated lipid removal provides a valuable and versatile tool to achieve controlled and specific lipid depletions in biological membranes. A few examples of the consequences of a CD-induced lipid depletion on fluorescence and electron transport properties of thylakoids are given to show the usefulness of CDs in the investigation of structure-function relationship in photosynthetic membranes.

(Galacto) lipid export from envelope to thylakoid membranes in intact chloroplasts. II. A general process with a key role for the envelope in the establishment of lipid asymmetry in thylakoid membranes.

The transfer of organelle of newly synthesized lipid molecules from inner envelope to thylakoid membranes, as well as their subsequent transbilayer distribution in these membranes, have been studied in intact chloroplasts isolated from young and mature spinach, young pea and mature lettuce leaves, using a recently developed methodology (Rawyler, A., Meylan, M. and Siegenthaler, P.A. (1992) Biochim. Biophys. Acta 1104, 331-341). Three radiolabelled precursors were used. UDP-[14C]galactose allowed to follow the fate of mono- and digalactosyldiacylglycerol (MGDG and DGDG) made from polyunsaturated, preexisting diacylglycerol (DAG), whereas [14C]acetate and [14C]glycerol 3-phosphate were used to follow the fate of MGDG and phosphatidylglycerol (PG), respectively, after de novo synthesis. MGDG, DGDG and PG molecules assembled at the envelope level were found to be exportable to thylakoids in amounts strictly proportional to the amounts synthesized, provided that the necessary substrates were not limiting. Lipid export was class-selective; under our conditions, as much as 50-80% of the MGDG, 87% of the PG and 20-30% of the DGDG synthesized were exported to thylakoids. However, within the MGDG class labelled from [14C]acetate, there was hardly any selectivity in the export of its various molecular species. For MGDG, the proportionality coefficient, which reflects the efficiency of the export process, was higher in chloroplasts from young than from mature leaves, and higher in spinach than in pea and lettuce. Temperature affected the efficiency of galactolipid export in a class-dependent way. MGDG synthesis and export had similar Q10 values of about 4 in young and 3 in mature spinach leaves, while the Q10 of DGDG export was higher than that of its synthesis. In most cases, the transmembrane distribution of labelled lipids in thylakoids was found to match closely the corresponding distribution of mass, regardless of plant age and species and of incubation time and temperature. In some cases however, small but significant differences occurred between the label and the mass transbilayer distributions of MGDG (labelled molecules more inwardly oriented), DGDG and PG (more outwardly oriented). We propose a general model in which the thylakoid lipid asymmetry is primarily preestablished in the chloroplast envelope by the topography of its lipid-synthesizing enzymes, together with the occurrence of relatively fast lateral diffusion and translocation rates of the newly synthesized lipids. Transient fusions between inner envelope and thylakoid membranes would allow lipid export by lateral diffusion and build the observed lipid asymmetry in the latter.

Galactolipid export from envelope to thylakoid membranes in intact chloroplasts. I. Characterization and involvement in thylakoid lipid asymmetry.

The galactolipid transfer from inner envelope to thylakoid membranes has been studied in intact spinach chloroplasts. Plastids, isolated from mature leaves, were dark-incubated in the presence of UDP-[14C]galactose. After various synthesis periods at 5 or 25 degrees C, intact plastids were reisolated and osmotically lysed. Thylakoid membranes were then prepared by a special procedure which removed greater than or equal to 99% of the envelope amount initially present. Under these conditions, purified thylakoids were found to contain radiolabelled MGDG and DGDG, indicating that galactolipids were exported from the inner envelope. The amounts exported were proportional to the amounts synthesized. About 55% of the MGDG and 25% of the DGDG synthesized in plastids were transferred to thylakoids, irrespectively of incubation time or temperature. The MGDG/DGDG radioactivity ratio was 7 in intact plastids and 18 in thylakoids, suggesting a preferential export of MGDG. Purified thylakoid membranes were then submitted to a lipolytic treatment designed to discriminate between the MGDG and DGDG pools belonging to the outer (stroma-facing) or to the inner monolayer. The radioactivity present in the lyso-products (corresponding to the outer pools) and in the residual parent lipids (corresponding to the inner pools) was measured. The labelled MGDG showed a transmembrane outside:inside distribution (mol%) of 50:50, which differed from the native (mass) MGDG asymmetry of 64:36. In contrast, the label and mass asymmetries of DGDG gave the same value of 15:85. These label distributions were affected neither by incubation time (from 5 to 90 min) nor by temperature (from 5 to 25 degrees C). We discuss the possibilities that transient fusions between the stroma-facing monolayers of the inner envelope and of the thylakoid membrane, and/or galactolipid transfer protein(s), together with lipid translocating activities in thylakoids, may account for the galactolipid export observed in mature spinach chloroplasts.

A single and continuous spectrophotometric assay for various lipolytic enzymes, using natural, non-labelled lipid substrates.

A rapid, continuous spectrophotometric assay for measuring the amount and activity of several lipolytic enzymes is described. It is based on the metachromatic properties of the cationic dye safranine, and makes use of the fact that an adequate combination of a lipolytic enzyme with one of its substrates leads to a change in the net negative charge at the lipid/water interface, which is monitored by the absorbance change of safranine. Utilizing this method, most lipolytic enzymes can be detected in very low amounts (milliunit or less) in about 1 min without employing radiolabelled lipids or synthetic lipid analogues. Over a wide range of enzyme concentrations, there is a good linearity between the initial hydrolysis rate (determination by the safranine method) and the amount of enzyme. The versatility of the assay is illustrated by examples showing how phospholipase A2, triacylglycerol hydrolase, phospholipase D or phospholipase C (either general or phosphatidylinositol-specific) activities can be detected, either separately or sequentially. Due to its high sensitivity, simplicity, and rapidity, this assay should find its main application in monitoring column effluents during the purification steps of lipolytic enzymes.

The transmembrane distribution of galactolipids in chloroplast thylakoids is universal in a wide variety of temperate climate plants.

The transmembrane distribution of monogalactosyldiacylglycerol and digalactosyldiacylglycerol was determined in chloroplast thylakoids from a range of temperate climate plants. These plants included dicotyledons, monocotyledons, C16:3 and C18:3 plants and herbicide-resistant species. In all the thylakoids examined monogalactosyldiacylglycerol was enriched in the outer leaflet (53-65%) while digalactosyldiacylglycerol was highly enriched in the inner leaflet (78-90%). The non-bilayer forming monogalactosyldiacylglycerol represented 55-81% of the total acyl lipids of the outer monolayer. The relative acyl lipid composition of both leaflets of the thylakoid membrane indicates that the lamellar structure is strongly favored in the inner monolayer, whereas the outer one presents a metastable character which allows the probable coexistence of both lamellar and non-lamellar phases. The consequence of this asymmetry for the stability and function of the thylakoid membrane is discussed.

Phospholipid localization in the plasma membrane of Friend erythroleukemic cells and mouse erythrocytes.

The distribution of phospholipids over outer and inner layers of the plasma membranes of Friend erythroleukemic cells (Friend cells) and mature mouse erythrocytes has been determined. The various techniques which have been applied to establish the phospholipid localization include the following: phospholipase A2, phospholipase C, and sphingomyelinase C treatment, fluorescamine labeling of phosphatidylethanolamine, and a phosphatidylcholine transfer protein mediated exchange procedure. The data obtained with these different techniques were found to be in good agreement with each other. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were found to be distributed symmetrically over both layers of the plasma membrane of Friend cells. In contrast, sphingomyelin was found to be enriched in the outer layer of the membrane (80-85%), and phosphatidylserine appeared to be present mainly in the inner layer (80-90%). From these results, it was calculated that the outer and inner layers accounted for 46% and 54%, respectively, of the total phospholipid complement of that membrane. Analogous studies on the plasma membrane of mature mouse erythrocytes showed that the transbilayer distribution of the total phospholipid mass appeared to be the same as in the plasma membrane of the Friend cell, namely, 46% and 54% in outer and inner layers, respectively. The outer layer of this membrane contains 57% of the phosphatidylcholine, 20% of the phosphatidylethanolamine, 85% of the sphingomyelin, and 42% of the phosphatidylinositol, and none of the phosphatidylserine was present.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of fluorescamine as a permeant probe to localize phosphatidylethanolamine in intact friend erythroleukaemic cells.

Intact Friend erythroleukaemic cells (Friend cells) were incubated at 0-4 degrees C with increasing amounts of fluorescamine. Phospholipids were extracted and the amounts of phosphatidylethanolamine and of its fluorescamine derivative were determined. (1). The plasma membrane of intact Friend cells appeared to be permeable to fluorescamine in a concentration-dependent way. (2). Three pools of phosphatidylethanolamine could be detected as the fluorescamine concentration was raised. The two first pools were ascribed to the outer monolayer (16-17% of the total cellular phosphatidylethanolamine) and inner (17-18%) monolayer of the plasma membrane, respectively, indicating an essentially symmetrical distribution of this phospholipid. The third pool of phosphatidylethanolamine (66%) corresponds to the contribution of intracellular membranes. (3). These data were used in turn, to calculate the relative amount of each phospholipid class present in the plasma membrane. The results are in perfect agreement with those obtained by an independent method involving the use of sphingomyelinase C (Rawyler, A., Roelofsen, B., Op den Kamp, J.A.F. and Van Deenen, L.L.M. (1983) Biochim. Biophys. Acta 730, 130-138). The present method is discussed in terms of its applicability for the localization of phosphatidylethanolamine in eukaryotic cells.

Isolation and characterization of plasma membranes from Friend erythroleukaemic cells. A study with sphingomyelinase C.

Plasma membranes have been prepared from Friend erythroleukaemic cells using a Dounce homogenization technique followed by differential and sucrose gradient centrifugations. (I) A plasma membrane fraction was obtained which showed a 20- to 30-fold enrichment in 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and in 32P-labeled (poly)phosphoinositides. About 1% of the total protein, 6-7% of phospholipid, 8-9% of cholesterol and 12-15% of each of the above markers were recovered in the plasma membrane fraction with an average yield of 15-20%. The plasma membrane was characterized by a high cholesterol to phospholipid molar ratio (0.626), a 2-fold enrichment in sphingomyelin and in phosphatidylserine as compared to the whole cell and by the complete absence of diphosphatidylglycerol. (2) When compared to the phospholipid composition of the mature mouse erythrocyte membrane, the plasma membrane of the Friend cell only differs by a higher phosphatidylcholine and a lower phosphatidylethanolamine content, whereas the levels of sphingomyelin and phosphatidylinositol plus phosphatidylserine are similar. (3) Friend cells were treated with sphingomyelinase C (S. aureus) under non-lytic conditions and subsequently submitted to subcellular fractionation. The results showed that the plasma membrane accounted for 38.5% of the total phospholipid, 64.1% of the total cholesterol and about 4.4% of the total protein content of Friend cells. (4) Sphingomyelin appeared to be asymmetrically distributed in the plasma membrane of Friend cells, with about 85% of this phospholipid being present in the outer monolayer.

(poly) Phosphoinositide phosphorylation is a marker for plasma membrane in Friend erythroleukaemic cells.

Upon subcellular fractionation of (murine) Friend erythroleukaemic cells (FELCs), purified plasma membranes were identified by their high enrichment in specific marker enzymes and typical plasma membrane lipids. When FELCs were incubated for short periods with 32Pi before cell fractionation, the lipid-bound radioactivity was almost exclusively present in phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI), and its distribution closely matched that of the plasma membrane markers. In addition, purified plasma membranes actively incorporated 32P from [gamma-32P]ATP into polyphosphoinositides, and the specific activities of the involved kinases were again mostly enriched in the plasma membrane fraction.

Transmembrane distribution of phospholipids and their involvement in electron transport, as revealed by phospholipids A2 treatment of spinach thylakoids.

Thylakoid membranes were treated with either pancreatic or snake venom phospholipase A2, and the residual phospholipid content of these membranes was determined and compared to the rates of Photosystem II and/or Photosystem I electron transports. The hydrolysis curves of both phosphatidylglycerol and phosphatidylcholine displayed a first, rapid phase which was almost temperature-insensitive, followed by a second, slower phase which depended strongly on the temperature. When pancreatic phospholipase A2 had access either to the outer face or to both faces of the thylakoid membrane, either only part of or all the phospholipids, respectively, could be hydrolysed. These results were interpreted as indicating an asymmetric distribution of phospholipids across the thylakoid membrane, phosphatidylglycerol and phosphatidylcholine being preferentially located in the outer and the inner layer, respectively. When acting on uncoupled thylakoid membranes, phospholipase A2 exerted an inhibitory effect on Photosystem II activity and a stimulatory effect on Photosystem I activity. The involvement of phosphatidylcholine and of phosphatidylglycerol in electron transport activities of Photosystem II and of Photosystem I are discussed with special reference to the role of the external and internal pools of these phospholipids.

Role of lipids in functions of photosynthetic membranes revealed by treatment with lipolytic acyl hydrolase.

1. Thylakoid membranes and subchloroplast particles I and II enriched in photosystem I and photosystem II, respectively, were treated with a potato lipolytic acyl hydrolase. 2. In the thylakoid membrane fraction, this treatment inhibited electron flows involving both photosystems and the associated photophosphorylations. However, electron flows involving either photosystem I or photosystem II were still preserved. The treatment of thylakoid membranes by lipolytic acyl hydrolase brought about a temporal convergence of different events such as maximal activity of reduced dichloroindophenol-supported electron flows, complete inhibition of photophosphorylations and electron transport activities through photosystem II + I, onset of the decay N,N,N',N-tetramethyl-rho-phenylenediamine-supported activity of photosystem activity and of the stimulation of photosystem activity (from reduced dichloroindophenol to NADP+ by exogenous plastocyanin. 3. Lipolytic acyl hydrolase catalyzed a limited hydrolysis of each lipid but in a stepwise manner, the galactolipids being attacked before the ionic lipids. The extent of the hydrolysis was not more than 50% for each lipid. Most of the hydrolytic process occurred before any significant change in photochemical activities could be observed. 4. In subchloroplast particles I, a treatment by lipolytic acyl hydrolase did not greatly affect the electron transport whilst lipid hydrolysis was almost complete. 5. In subchloroplast particles II, neither the electron flow activities nor lipid content were significantly altered by lipolytic acyl hydrolase. 6. The sites of lipolytic acyl hydrolase action appeared to be localized between plastoquinones and P700. It is suggested that it is not possible to establish a quantitative and/or temporal correlation between the extent of lipid hydrolysis and the inhibition of photochemical activities. 7. The profile of the hydrolysis of lipids in thylakoid membranes suggests that ionic lipids are less accessible to lipolytic acyl hydrolase than galactolipids.

Rapid analysis of membrane lipids using a combination of thin-layer chromatography and scanning of photographic negatives.

A simple method is proposed for the analysis of the distribution and changes in membrane lipids subjected to different treatments (lipolytic, aging, etc.). This technique involves only one thin-layer chromatographic step followed by a scanning of the photographic negative of the charred thin layer. This method is time saving, inexpensive and does not require the technical skill usually demanded in lipidology. The precision of this method is compared with that obtained with the classical TLC-GC method: its variability is roughly twice that of the TLC-GC method.