Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact.

Membrane association between mitochondria and the endoplasmic reticulum of the yeast Saccharomyces cerevisiae is probably a prerequisite for phospholipid translocation between these two organelles. This association was visualized by fluorescence microscopy and computer-aided three-dimensional reconstruction of electron micrographs from serial ultrathin sections of yeast cells. A mitochondria-associated membrane (MAM), which is a subfraction of the endoplasmic reticulum, was isolated and re-associated with mitochondria in vitro. In the reconstituted system, phosphatidylserine synthesized in MAM was imported into mitochondria independently of cytosolic factors, bivalent cations, ATP, and ongoing synthesis of phosphatidylserine. Proteolysis of mitochondrial surface proteins by treatment with proteinase K reduced the capacity to import phosphatidylserine. Phosphatidylethanolamine formed in mitochondria by decarboxylation of phosphatidylserine is exported to the endoplasmic reticulum where part of it is converted into phosphatidylcholine. In contrast with previous observations with permeabilized yeast cells [Achleitner, G., Zweytick, D., Trotter, P., Voelker, D. & Daum, G. (1995) J. Biol. Chem. 270, 29836-29842], export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy-independent in the reconstituted yeast system.     

Newly made phosphatidylserine and phosphatidylethanolamine are preferentially translocated between rat liver mitochondria and endoplasmic reticulum

The translocation of: (i) phosphatidylserine (PtdSer) from its site of synthesis on microsomal membranes to its site decarboxylation in mitochondrial membranes and (ii) phosphatidylethanolamine (PtdEtn) from the mitochondria to its site of methylation to phosphatidylcholine on microsomal membranes has been reconstituted in cell-free systems consisting of rat liver mitochondria and microsomes. Two types of systems have been reconstituted. In one, the translocation of newly made PtdSer or PtdEtn was examined by incubation of microsomes and mitochondria with [3-3H]serine. In the other, membranes were prelabeled with radioactive PtdSer or PtdEtn, and the transfer of these two lipids between mitochondria and microsomes was monitored. For the transfer of both PtdSer from microsomes to mitochondria and PtdEtn from mitochondria to microsomes, newly made phospholipids were translocated much more readily than pre-existing phospholipids. The data suggest that with respect to their translocation between these two organelles, the pools of newly synthesized PtdSer and PtdEtn were distinct from the pools of "older" phospholipids pre-existing in the membranes. Transfer of neither phospholipid in vitro depended on the presence of cytosolic proteins (i.e. soluble phospholipid transfer proteins) or on the hydrolysis of ATP, although there was some stimulation of PtdSer transfer by ATP and several other nucleoside mono-, di-, and triphosphates. The data are consistent with a collision-based mechanism in which the endoplasmic reticulum and mitochondria come into contact with one another, thereby effecting the transfer of phospholipids. The proposal that there is contact between the endoplasmic reticulum and mitochondria is supported by the recent isolation of a membrane fraction having many, but not all, of the properties of the endoplasmic reticulum, but which was isolated in association with mitochondria (Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256).     

Intracellular phospholipid movement and the role of phospholipid transfer proteins in animal cells

The mechanism of the intracellular movement of phospholipids from their site of synthesis in the endoplasmic reticulum to mitochondria and other cell membranes is a major unsolved problem of cell biology. Phospholipid transfer proteins of varying specificity found in the soluble supernatant fractions of many tissues catalyze the transfer of phospholipids from microsomes to mitochondria in vitro. They are postulated to play a similar role in vivo, but evidence for their function in living cells is lacking. We have now used an analogue of choline, N-propyl-N,N-dimethylethanolamine [PDME, (2-hydroxyethyl)dimethylpropylammonium hydroxide], to devise a test for the function of the transfer proteins in living cells. The rates of translocation of newly synthesized phosphatidylcholine and the analogue phosphatidyl-PDME in living cells were compared with the rates of transfer in vitro catalyzed by soluble transfer proteins extracted from the same cells. Labeled PDME, choline, and ethanolamine were found to be rapidly incorporated into the lipids of isolated rat hepatocytes and of baby hamster kidney (BHK-21) cells in culture. The translocation of newly synthesized phosphatidylcholine and phosphatidyl-PDME was very rapid in both types of cells with a half-time for equilibration of a few minutes, while the translocation of phosphatidylethanolamine was much slower, with a half-time 20-80 fold longer than those of the other two phospholipids. We then compared these relative rates of movement with the activities of the phospholipid transfer proteins of the respective cells. Partially purified phosphatidylcholine transfer protein from rat liver transfers phosphatidylcholine and phosphatidyl-PDME at identical rates but transfers phosphatidylethanolamine at a rate too low to be detected. This result is consistent with an essential function of this transfer protein in vivo. In contrast, partially purified phosphatidylcholine phospholipid transfer protein from BHK cells transfers phosphatidylcholine rapidly, while no transfer of phosphatidyl-PDME and phosphatidylethanolamine was detected. We further found that the specific phosphatidylcholine transfer protein of BHK cells accounts for nearly all of the transfer activity detected in the crude soluble fraction. The rapid translocation of phosphatidyl-PDME in vivo in BHK cells is therefore inconsistent with the postulate that soluble phospholipid transfer proteins are responsible for the rapid movement of phospholipids from microsomes to mitochondria in living cells.     

Role for two conserved intermembrane space proteins, Ups1p and Up2p, in intra-mitochondrial phospholipid trafficking

Mitochondrial membranes maintain a specific phospholipid composition. Most phospholipids are synthesized in the endoplasmic reticulum (ER) and transported to mitochondria, but cardiolipin and phosphatidylethanolamine are produced in mitochondria. In the yeast Saccharomyces cerevisiae, phospholipid exchange between the ER and mitochondria relies on the ER-mitochondria encounter structure (ERMES) complex, which physically connects the ER and mitochondrial outer membrane. However, the proteins and mechanisms involved in phospholipid transport within mitochondria remain elusive. Here, we investigated the role of the conserved intermembrane space proteins, Ups1p and Ups2p, and an inner membrane protein, Mdm31p, in phospholipid metabolism. Our data show that loss of the ERMES complex, Ups1p, and Mdm31p causes similar defects in mitochondrial phospholipid metabolism, mitochondrial morphology, and cell growth. Defects in cells lacking the ERMES complex or Ups1p are suppressed by Mdm31p overexpression as well as additional loss of Ups2p, which antagonizes Ups1p. Combined loss of the ERMES complex and Ups1p exacerbates phospholipid defects. Finally, pulse-chase experiments using [(14)C]serine revealed that Ups1p and Ups2p antagonistically regulate conversion of phosphatidylethanolamine to phosphatidylcholine. Our results suggest that Ups proteins and Mdm31p play important roles in phospholipid biosynthesis in mitochondria. Ups proteins may function in phospholipid trafficking between the outer and inner mitochondrial membranes.     

Use of 3-aminopropanol as an ethanolamine analogue in the study of phospholipid metabolism in Tetrahymena.

About 50% of the ethanolamine in phosphatidylethanolamine in Tetrahymena is replaced by 3-aminopropan-1-ol when the compound is added to the growth medium. The phosphatidylpropanolamine which is formed is not converted into the corresponding phosphatidylcholine analogue by methylation. There is an increase in phosphatidylcholine formed by the phosphotransferase pathway from free [3H]choline and a decrease in the phosphatidylcholine formed by the methylation pathway from [14C]methionine. The nature of the observed phospholipid alterations suggests that the regulation of phosphatidylcholine biosynthesis in Tetrahymena may be different from that found in higher eukaryotes.     

Phenobarbital-induced alterations in phosphatidylcholine and triglyceride synthesis in hepatic endoplasmic reticulum

Biosynthetic pathways of phosphatidylcholine and triglyceride were studied in proliferating hepatic endoplasmic reticulum of rats pretreated with phenobarbital. Phosphatidylcholine accounted for the major increment in membrane phospholipid. In vitro measurements of hepatic microsomal enzymes which catalyze phosphatidylcholine biosynthesis revealed a significant increase in specific activity of the enzyme governing phosphatidylcholine synthesis by sequential methylation of phosphatidylethanolamine. The specific activity of phosphorylcholine-glyceride transferase, which catalyzes phosphatidylcholine synthesis from d-1,2-diglyceride and CDP-choline, was not altered. Specific activity of diglyceride acyltransferase, which catalyzes triglyceride biosynthesis, was increased to a degree comparable to the increase in specific activity found in the phenobarbital-induced drug-metabolizing enzyme which oxidatively demethylates aminopyrine. In vivo incorporation of methyl-(3)H from l-methionine-methyl-(3)H into microsomal phosphatidylcholine was significantly increased, resulting in an increased methyl-(3)H to choline-1,2-(14)C incorporation ratio of more than three times that found in control animals. A comparable increase in this incorporation ratio was noted in serum phospholipids. The in vitro enzyme studies, in agreement with in vivo incorporation data, indicate that the increase in phosphatidylcholine content of phenobarbital-induced proliferating endoplasmic reticulum is related to increased activity of the pathway of phosphatidylcholine biosynthesis involving the sequential methylation of phosphatidylethanolamine.     

Deficiency in phosphatidylserine decarboxylase activity in the psd1 psd2 psd3 triple mutant of Arabidopsis affects phosphatidylethanolamine accumulation in mitochondria

Phosphatidylserine (PS) decarboxylase is involved in the synthesis of the abundant phospholipid phosphatidylethanolamine (PE), particularly in mitochondria, in many organisms, including yeast (Saccharomyces cerevisiae) and animals. Arabidopsis (Arabidopsis thaliana) contains three genes with sequence similarity to PS decarboxylases, and the respective gene products were functionally characterized after heterologous expression in yeast and Escherichia coli. While the PSD1 protein localizes to mitochondria, PSD2 and PSD3 are found in the endomembrane system. To study the role of PSD genes in plant phospholipid metabolism, Arabidopsis insertional mutants for psd1, psd2, and psd3 were obtained. The single mutants were decreased in PS decarboxylase activity to various extents, but mutant plants showed no obvious growth or morphological phenotype. A triple mutant, psd1 psd2 psd3, was generated that was totally devoid of PS decarboxylase activity. While the phospholipid composition in whole leaves was unchanged, the PE content in isolated mitochondria of psd1 psd2 psd3 was decreased. Therefore, the predominant proportion of PE in Arabidopsis is synthesized by alternative pathways, but a significant amount of mitochondrial PE is derived from the PS decarboxylase reaction. These results imply that, similar to yeast and animal cells, a specific phospholipid transfer from the endoplasmic reticulum to mitochondria exists in plants.     

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.     

Continuous equilibration of phosphatidylcholine and its precursors between endoplasmic reticulum and mitochondria in yeast

In Saccharomyces cerevisiae phosphatidylcholine (PC) is synthesized in the ER and transported to mitochondria via an unknown mechanism. The transport of PC synthesized by the triple methylation of phosphatidylethanolamine was investigated by pulsing yeast spheroplasts with l-[methyl-3H]methionine, followed by a chase with unlabeled methionine and subcellular fractionation. During the pulse, increasing amounts of PC and its mono- and dimethylated precursors (PMME and PDME, respectively) appear in similar proportions in both microsomes and mitochondria, with the extent of incorporation in microsomes being twice that in mitochondria. During the chase, the [3H]-methyl label from the precursors accumulates into PC with similar kinetics in both organelles. The results demonstrate that transport of methylated phospholipids from ER to mitochondria is 1) coupled to synthesis, 2) not selective for PC, 3) at least as fast as the fastest step in the methylation of PE, and 4) bidirectional for PMME and PDME. The interorganellar equilibration of methylated phospholipids was reconstituted in vitro and did not depend on ongoing methylation, cytosolic factors, ATP, and energization of the mitochondria, although energization could accelerate the reaction. The exchange of methylated phospholipids was reduced after pretreating both microsomes and mitochondria with trypsin, indicating the involvement of membrane proteins from both organelles.     

Yeast mutant defective in phosphatidylcholine synthesis

The Saccharomyces cerevisiae opi3-3 mutant was shown to be defective in the synthesis of phosphatidylcholine via methylation of phosphatidylethanolamine. The opi3-3 mutant was isolated on the basis of an inositol excretion phenotype and was not auxotrophic for choline. Inositol, but not choline, stimulated growth of the mutant. The opi3-3 mutation was recessive and was genetically linked to the ino4 locus. When grown in the absence of exogenous choline, the opi3-3 mutant had a phospholipid composition consisting of 2 to 3% phosphatidylcholine compared with 40 to 50% in wild-type strains. In addition, the mutant accumulated elevated amounts of two intermediates, phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The incorporation of label from [methyl-14C]methionine into phosphatidylcholine was reduced 80 to 90% in the mutant compared with wild-type strains. However, label was recovered in the intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The mutant is believed to be defective in the third and possibly the second methylation reaction in the formation of phosphatidylcholine from phosphatidylethanolamine. The first methylation reaction appeared to be occurring at normal or even elevated levels. Based upon incorporation of choline into phosphatidylcholine, it is concluded that the opi3-3 mutant has no defect in the synthesis of phosphatidylcholine from exogenous choline. Furthermore, phosphatidylcholine represents over 25% of the phospholipid composition of the mutant when it is grown in the presence of exogenous choline.     

Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation.

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.     

Sterol carrier protein-2 localization in endoplasmic reticulum and role in phospholipid formation

Although sterol carrier protein-2 (SCP-2; also called nonspecific lipid transfer protein) binds fatty acids and fatty acyl-CoAs, its role in fatty acid metabolism is not fully understood. L-cell fibroblasts stably expressing SCP-2 were used to resolve the relationship between SCP-2 intracellular location and fatty acid transacylation in the endoplasmic reticulum. Indirect immunofluorescence double labeling and laser scanning confocal microscopy detected SCP-2 in peroxisomes > endoplasmic reticulum > mitochondria > lysosomes. SCP-2 enhanced incorporation of exogenous [(3)H]oleic acid into phospholipids and triacylglycerols of overexpressing cells 1.6- and 2.5-fold, respectively, stimulated microsomal incorporation of [1-(14)C]oleoyl-CoA into phosphatidic acid in vitro 13-fold, and exhibited higher specificity for unsaturated versus saturated fatty acyl-CoA. SCP-2 enhanced the rate-limiting step in microsomal phosphatidic acid biosynthesis mediated by glycerol-3-phosphate acyltransferase. SCP-2 also enhanced microsomal acyl-chain remodeling of phosphatidylethanolamine up to fivefold and phosphatidylserine twofold, depending on the specific fatty acyl-CoA, but had no effect on other phospholipid classes. In summary, these results were consistent with a role for SCP-2 in phospholipid synthesis in the endoplasmic reticulum.     

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.     

The stimulation by sodium fluoride of plasma-membrane Ca2+ inflow in isolated hepatocytes. Evidence that a GTP-binding regulatory protein is involved in the hormonal stimulation of Ca2+ inflow.

Bilirubin may be transported within intracellular membranes of the hepatocyte and may undergo membrane-membrane transfer to gain access to the conjugating enzyme UDP-glucuronyltransferase in the endoplasmic reticulum. We have demonstrated previously that the lipid composition of liposomal membranes incorporating bilirubin substrate influences the rate of transfer and glucuronidation of bilirubin by hepatic microsomes. To examine the mechanism(s) of substrate transfer, we incorporated radiolabelled bilirubin into small unilamellar model membranes of egg phosphatidylcholine or natural phospholipids in the proportions present in native hepatic microsomes. The rate at which bilirubin was transferred to rat liver microsomes and glucuronidated was then examined in the presence of various endogenous compounds that promote membrane fusion. For bilirubin substrate in membranes of egg phosphatidylcholine, the addition of Ca2+ (2 mM) increased the microsomal glucuronidation rate, whereas retinol enhanced microsomal conjugation rates for bilirubin in membranes of both lipid compositions. When the transfer of [3H]bilirubin from dual-labelled liposomes to microsomes was enhanced by Ca2+ or retinol, there was no associated increase in [14C]phospholipid transfer. Thus it appears likely that bilirubin is transferred to the endoplasmic reticulum by rapid cytosolic diffusion or membrane-membrane collisions, rather than by membrane fusion; this process may be modulated by changes in the lipid microenvironment of the substrate or the effective intracellular concentrations of Ca2+ or retinol. The observation that polymyxin B induced concomitant membrane-membrane transfer of [3H]bilirubin and [14C]phospholipid suggests that under certain circumstances membrane fusion or aggregation may promote the movement of lipophilic substrates in hepatocytes.     

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (method).

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.     

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (Method)

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.     

Myoinositol gets incorporated into numerous membrane glycoproteins of Saccharomyces cerevisiae; incorporation is dependent on phosphomannomutase (sec53).

We recently described a 125 kd membrane glycoprotein in Saccharomyces cerevisiae which is anchored in the lipid bilayer by an inositol-containing phospholipid. We now find that when S. cerevisiae cells are metabolically labeled with [3H]myoinositol, many glycoproteins become labeled more strongly than the 125 kd protein. Myoinositol is attached to these glycoproteins as part of a phospholipid moiety which resembles glycophospholipid anchors of other organisms. Labeling of proteins with [3H]myoinositol for short times and in secretion mutants blocked at various stages of the secretory pathway shows that these phospholipid moieties can be added to proteins in the endoplasmic reticulum and that these proteins are transported to the Golgi by the regular secretory pathway. sec53, a mutant which cannot produce GDP-mannose at 37 degrees C, does not incorporate myoinositol or palmitic acid into membrane glycoproteins at this temperature, suggesting that GDP-mannose is required for the biosynthesis of these phospholipid moieties. All other secretion and glycosylation mutants tested add phospholipid moieties to proteins normally.     

Fatty acids inhibit N-methylation of phosphatidylethanolamine in rat hepatocytes and liver microsomes

Supplementation of rat hepatocytes with various fatty acids in the culture medium reduced the conversion of [3H]phosphatidylethanolamine into phosphatidylcholine. Unsaturated fatty acids were the most effective inhibitors of phospholipid methylation. The inhibition of phosphatidylethanolamine methylation by oleate (2 mM) was reversed within 1 h after replacement with fatty acid-deficient medium. Fatty acids and their CoA derivatives (0.15-0.5 mM) produced 50% inhibition of phosphatidylethanolamine methyltransferase in rat liver microsomes. The first methylation reaction was the site of fatty acid inhibition, as methylation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was not reduced in the presence of oleate. The inhibition by oleate was reversed by inclusion of bovine serum albumin or by addition of phospholipid liposomes. Thus, while fatty acids stimulate phosphatidylcholine biosynthesis in hepatocytes via the CDP-choline pathway, the methylation pathway is inhibited.     

Metabolism and intracellular localization of a fluorescently labeled intermediate in lipid biosynthesis within cultured fibroblasts

In this paper we report on the uptake and distribution of an exogenously supplied fluorescent phosphatidic acid analogue by Chinese hamster fibroblasts. Under appropriate in vitro incubation conditions, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidic acid was rapidly and preferentially transferred from phospholipid vesicles to cells at 2 degrees C. However, unlike similar fluorescent derivatives of phosphatidylcholine and phosphatidylethanolamine that remain restricted to the plasma membrane under such incubation conditions (Struck, D. K., and R. E. Pagano. 1080. J. Biol. Chem. 255:5405--5410), most of the phosphatidic acid-derived fluorescence was localized at the nuclear membrane, endoplasmic reticulum, and mitochondria. This was shown by labeling cells with rhodamine- containing probes specific for mitochondria or endoplasmic reticulum, and comparing the patterns of intracellular NBD and rhodamine fluorescence. Extraction and analysis of the fluorescent lipids associated with the cells after treatment with vesicles at 2 degrees or 37 degrees C revealed that a large fraction of the fluorescent phosphatidic acid was converted to fluorescent diglyceride, phosphatidylcholine, and triglyceride. Our findings suggest that fluorescent phosphatidic acid may be useful in correlating biochemical studies of lipid metabolism in cultured cells and studies of the Intracellular localization of the metabolites by fluorescence microscopy. In addition, this compound provides a unique method for visualizing the endoplasmic reticulum in living cells.     

t-Butyl hydroperoxide alters fatty acid incorporation into erythrocyte membrane phospholipid

Because the ability of cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be an important determinant of the ability of cells to tolerate oxidative stress, incorporation of exogenous fatty acid into phospholipid by human erythrocytes has been examined following exposure of the cells to t-butyl hydroperoxide. Exposure of human erythrocytes to t-butyl hydroperoxide (0.5-1.0 mM) results in oxidation of glutathione, formation of malonyldialdehyde, and oxidation of hemoglobin to methemoglobin. Under these conditions, incorporation of exogenous [9,10-3H]oleic acid into phosphatidylethanolamine is enhanced while incorporation of [9,10-3H]oleic acid into phosphatidylcholine is decreased. These effects of t-butyl hydroperoxide on [9,10-3H]oleic acid incorporation are not affected by dissipating transmembrane gradients for calcium and potassium. When malonyldialdehyde production is inhibited by addition of ascorbic acid, t-butyl hydroperoxide still decreases [9,10-3H]oleic acid incorporation into phosphatidylcholine but no stimulation of [9,10-3H]oleic acid incorporation into phosphatidylethanolamine occurs. In cells pre-treated with NaNO2 to convert hemoglobin to methemoglobin, t-butyl hydroperoxide reduces [9,10-3H]oleic acid incorporation into phosphatidylcholine by erythrocytes but does not stimulate [9,10-3H]oleic acid incorporation into phosphatidylethanolamine. Under these conditions oxidation of erythrocyte glutathione and formation of malonyldialdehyde still occur. These results indicate that membrane phospholipid fatty acid turnover is altered under conditions where peroxidation of membrane phospholipid fatty acids occurs and suggest that the oxidation state of hemoglobin influences this response.