Phosphatidylethanolamine Methylation Pathway Research Articles

Phosphocholine cytidylyltransferase MoPct1 is crucial for vegetative growth, conidiation, and appressorium-mediated plant infection by Magnaporthe oryzae.

Phosphatidylcholine (PC) plays crucial biological roles in eukaryotic cells. In Saccharomyces cerevisiae, apart from phosphatidylethanolamine (PE) methylation pathway, PC is also synthesized via CDP-choline pathway. Phosphocholine cytidylyltransferase Pct1 is the rate-limiting enzyme to catalyze the conversion from phosphocholine to CDP-choline in this pathway. Here, we report the identification and functional characterization of an ortholog of the budding yeast PCT1 in Magnaporthe oryzae, named MoPCT1. Targeted gene deletion mutants of MoPCT1 were impaired in vegetative growth, conidiation, appressorium turgor accumulation and cell wall integrity. Also, the mutants were severely compromised in appressorium-mediated penetration, infectious growth and pathogenicity. Western blot analysis revealed that cell autophagy was activated by the deletion of MoPCT1 under nutrient-rich conditions. Moreover, we found several key genes in PE methylation pathway, such as MoCHO2, MoOPI3, and MoPSD2, were significantly up-regulated in the ΔMopct1 mutants, indicating that a pronounced compensation effect exists between the two PC biosynthesis pathways in M. oryzae. Interestingly, in the ΔMopct1 mutants, histone H3 was hypermethylated and expression levels of several methionine cycling-related genes were significantly up-regulated, suggesting that MoPCT1 is involved in histone H3 methylation and methionine metabolism. Taken together, we conclude that the phosphocholine cytidylyltransferase coding gene MoPCT1 plays important roles in vegetative growth, conidiation and appressorium-mediated plant infection by M. oryzae.

LPGAT1 controls the stearate/palmitate ratio of phosphatidylethanolamine and phosphatidylcholine in sn-1 specific remodeling

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.

Petit-High Pressure Carbon Dioxide stress increases synthesis of S-Adenosylmethionine and phosphatidylcholine in yeast Saccharomyces cerevisiae

Petit-High Pressure Carbon Dioxide (p-HPCD) is a promising nonthermal technology for foods pasteurization. Cluster analysis of gene expression profiles of Saccharomyces cerevisiae exposed to various stresses exhibited that gene expression profile for p-HPCD stress (0.5MPa, 25°C) was grouped into a cluster including profiles for Sodium Dodecyl Sulfate and Roundup herbicide. Both are detergents that can disorder membrane structurally and functionally, which suggests that cell membrane may be a target of p-HPCD stress to cause cell growth inhibition. Through metabolomic analysis, amount of S-Adenosylmethionine (AdoMet) that is used as methyl donor to participate in phosphatidylcholine synthesis via phosphatidylethanolamine (PE) methylation pathway, was increased after p-HPCD treatment for 2h. The key gene OPI3 encoding phospholipid methyltransferase that catalyzes the last two steps in PE methylation pathway was confirmed significantly induced by RT-PCR. Transcriptional expression of genes (MET13, MET16, MET10, MET17, MET6 and SAM2) related to AdoMet biosynthesis was also significantly induced. Choline as the PC precursor and ethanolamine as PE precursor in Kennedy pathway were also found increased under p-HPCD condition. We also found that amounts of most of amino acids involving protein synthesis were found decreased after p-HPCD treatment for 2h. Moreover, morphological changes on cell surface were observed by scanning electron microscope. In conclusion, the effects of p-HPCD stress on cell membrane appear to be a very likely cause of yeast growth inhibition and the enhancement of PC synthesis could contribute to maintain optimum structure and functions of cell membrane and improve cell resistance to inactivation.

Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis.

Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)-choline to diacylglycerol. Phosphocholine, the direct precursor to CDP-choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C.merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [(32) P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [(32) P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C.sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas.

In vitro Characterization of Uptake Mechanism of l-[methyl-3H]-methionine in Hepatocellular Carcinoma

Methionine (Met) could be a useful imaging biomarker for the diagnosis of hepatocellular carcinoma (HCC), as demonstrated by PET imaging with L-[methyl-(11)C]-Met. In HCC cells, protein synthesis mainly contributes to radiopharmaceutical uptake. In contrast, lipid synthesis via the phosphatidylethanolamine (PE) methylation pathway is the major metabolic route of L-[methyl-(11)C]-Met in normal hepatocytes, which contributes to the background contrast observed in PET images. However, the mechanisms of amino acid transport and the roles of the two key enzymes, methionine adenosyltransferase (MAT) and phosphatidylethanolamine N-methyltransferase (PEMT), are not yet completely understood. The aim of this study was to investigate the roles of the amino acid transporters and these two key enzymes in the uptake of L-[methyl-(11)C]-Met in HCC cells. A well-differentiated woodchuck HCC cell line, WCH17, was used for the study. The amino acid transporter of WCH17 cells was assayed to investigate the Met transport process in HCC. WCH17 cells were treated with 5 mM S-adenosylmethionine (SAM) for 8, 16, 24, and 48 h to downregulate MAT2A gene expression. Control or SAM-treated WCH17 cells were pulsed with L-[methyl-(3)H]-Met for 5 min and chased with cold media to mimic the rapid blood clearance of radiolabeled Met (pulse-chase experiment). In parallel, WCH17 cells were transfected with a mouse liver PEMT2 expression vector, and the pulse-chase experiment was performed to investigate the uptake of the radiolabeled Met in HCC cells. The water-soluble, protein, and lipid phases from the total uptake were subsequently extracted and measured, respectively. Met was transported into HCC cells via a facilitative transport process, which was characterized as system L and ASC-like, Na(+) dependent, and low affinity with partial energy dependence. The total uptake of L-[methyl-(3)H]-Met was decreased in HCC cells with SAM treatment. This reduction pattern followed that of MAT2A expression (the duration of SAM treatment). The incorporated (3)H was mostly distributed in the protein phase and, to a lesser degree, in the lipid phase via PE methylation pathway in HCC cells with SAM treatment. The downregulated MAT2A expression led to the decreased uptake in protein and water-soluble phases. In addition, an increased uptake in the lipid phase was observed in WCH17 cells transfected with PEMT2 expression vector. The amino acid transport processes may be responsible for the rapid accumulation of radiolabeled Met after the intravenous injection of tracers for the imaging of HCC. Upregulated MAT2A expression and impaired PEMT2 activities in HCC are associated with the specific metabolic pattern of L-[methyl-(11)C]-Met detected by PET.

Metabolism of Radiolabeled Methionine in Hepatocellular Carcinoma

Radiolabeled methionine (Met) promises to be useful in the positron emission tomography (PET) imaging of hepatocellular carcinoma (HCC). However, its metabolic routes in HCC have not yet been fully understood. In this study, the metabolic pathway(s) of radiolabeled Met in HCC were investigated. To simulate the rapid blood clearance of radiolabeled Met, pulse-chase experiments were conducted. L-[methyl-(3)H]-Met or L-[1-(14)C]-Met was pulsed over control or cycloheximide-treated WCH17 cells and rat hepatocytes for 5min and chased with cold media. The water-soluble, lipid-soluble, DNA, RNA, and protein phases were subsequently extracted and measured from the acid-precipitable and acid-soluble fractions of whole cells. The radioactive metabolites Met, S-adenosylmethionine (SAM), S-adenosylhomocysteine, Met sulfoxide, and Met sulfone were further separated by radio thin layer chromatography. (1) The uptake of L-[methyl-(3)H]-Met in both cell types was higher than that of L-[1-(14)C]-Met. In rat hepatocytes, the uptake of L-[methyl-(3)H]-Met was significantly higher than that of L-[1-(14)C]-Met, which may contribute to its physiologic accumulation in surrounding hepatic tissues seen in PET imaging of HCC using L-[methyl-(11)C]-Met. Compared to rat hepatocytes, WCH17 cells had significantly higher uptake of both radiotracers. (2) For L-[methyl-(3)H]-Met, the major intracellular uptake was found mostly in the protein phase and, to a lesser degree, in the phosphatidylethanolamine (PE) methylation pathway, which is fairly stabilized within the 55-min chase period (the main metabolites were SAM, Met, Met sulfoxide, and Met sulfone). In contrast, the uptake of Met in rat hepatocytes mainly points to phosphatidylcholine (PC) synthesis through the PE methylation pathway (the main metabolite was PC). (3) Both cell types incorporated L-[1-(14)C]-Met predominantly into protein synthesis. (4) Finally, when the protein synthesis pathway was inhibited, the incorporation of SAM derived from L-[methyl-(3)H]-Met to lipid class (PC was the main metabolite) occurred at a reduced rate in WCH17 cells, suggesting that the route may be impaired in HCC. This study demonstrated that different metabolic pathways of radiolabeled Met exist between HCC and surrounding hepatic tissue and contribute to the patterns of increased uptake of radiolabeled Met in HCC.

Imaging Lipid Synthesis in Hepatocellular Carcinoma with [Methyl-11C]Choline: Correlation with In Vivo Metabolic Studies

PET with [methyl-(11)C]-choline (11C-choline) can be useful for detecting well-differentiated hepatocellular carcinoma (HCC) that is not 18F-FDG-avid. This study was designed to examine the relationship between choline metabolism and choline tracer uptake in HCC for PET with 11C-choline. Dynamic PET scans of 11C-choline were acquired using the woodchuck models of HCC. After imaging, [methyl-(14)C]-choline was injected, and metabolites from both HCC tissue samples and the surrounding hepatic tissues were extracted and analyzed by radio-high-performance liquid chromatography. The enzymatic activities of choline kinase and choline-phosphate cytidylyltransferase were assayed for correlation with the imaging and metabolism data. PET with 11C-choline showed an HCC detection rate of 9 of 10. The tumor-to-liver ratio for the 9 detected HCCs was 1.89±0.55. Hematoxylin-eosin staining confirmed that all spots with high tracer uptake were well-differentiated HCCs. Variation of radioactivity distribution within HCCs indicated a heterogeneous uptake of choline. The activities of both choline kinase and choline-phosphate cytidylyltransferase were found to be significantly higher in HCC than in the surrounding hepatic tissues. The major metabolites of 11C-choline were phosphocholine in HCC and betaine and choline in the surrounding hepatic tissues at 12 min after injection; in HCC, phosphocholine rapidly converted to phosphatidylcholine at 30 min after injection. HCCs display enhanced uptake of radiolabeled choline despite a moderate degree of physiologic uptake in the surrounding hepatic tissues. Initially, increased radiolabeled choline uptake in HCCs is associated with the transport and phosphorylation of choline; as time passes, the increased uptake of radiolabeled choline reflects increased phosphatidylcholine synthesis derived from radiolabeled cytidine 5'-diphosphocholine (CDP-choline) in HCCs. In contrast, the surrounding hepatic tissues exhibit extensive oxidation of radiolabeled choline via the phosphatidylethanolamine methylation pathway, a major contributor to the observed physiologic uptake.

The Kap60-Kap95 Karyopherin Complex Directly Regulates Phosphatidylcholine Synthesis

Phosphatidylcholine is the major phospholipid in eukaryotic cells. There are two main pathways for the synthesis of phosphatidylcholine: the CDP-choline pathway present in all eukaryotes and the phosphatidylethanolamine methylation pathway present in mammalian hepatocytes and some single celled eukaryotes, including the yeast Saccharomyces cerevisiae. In S. cerevisiae, the rate-determining step in the synthesis of phosphatidylcholine via the CDP-choline pathway is catalyzed by Pct1. Pct1 converts phosphocholine and CTP to CDP-choline and pyrophosphate. In this study, we determined that Pct1 is in the nucleoplasm and at endoplasmic reticulum and nuclear membranes. Pct1 directly interacts with the alpha-importin Kap60 via a bipartite basic region in Pct1, and this region of Pct1 was required for its entry into the nucleus. Pct1 also interacted with the beta-importin Kap95 in cell extracts, implying a model whereby Pct1 interacts with Kap60 and Kap95 with this tripartite complex transiting the nuclear pore. Exclusion of Pct1 from the nucleus by elimination of its nuclear localization signal or by decreasing Kap60 function did not affect the level of phosphatidylcholine synthesis. Diminution of Kap95 function resulted in almost complete ablation of phosphatidylcholine synthesis under conditions where Pct1 was extranuclear. The beta-importin Kap95 is a direct regulator membrane synthesis.

Localization of the PE methylation pathway and SR-BI to the canalicular membrane: evidence for apical PC biosynthesis that may promote biliary excretion of phospholipid and cholesterol

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile.

Membrane lipids in plant-associated bacteria: their biosyntheses and possible functions.

Membrane lipids in most bacteria generally consist of the glycerophospholipids phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine (PE). A subset of bacteria also possesses the methylated derivatives of PE, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, and phosphatidylcholine (PC). In Sinorhizobium meliloti, which can form a nitrogen-fixing root nodule symbiosis with Medicago spp., PC can be formed by two entirely different biosynthetic pathways, either the PE methylation pathway or the recently discovered PC synthase pathway. In the latter pathway, one of the building blocks for PC formation, choline, is obtained from the eukaryotic host. Under phosphorus-limiting conditions of growth, S. meliloti replaces its membrane phospholipids by membrane-forming lipids that do not contain phosphorus; namely, the sulfolipid sulfoquinovosyl diacylglycerol, ornithine-derived lipids, and diacylglyceryl-N,N,N-trimethylhomoserine. Although none of these phosphorus-free lipids is essential for growth in culture media rich in phosphorus or for the symbiotic interaction with the legume host, they are expected to have major roles under free-living conditions in environments poor in accessible phosphorus. In contrast, sinorhizobial mutants deficient in PC show severe growth defects and are completely unable to form nodules on their host plants. Even bradyrhizobial mutants with reduced PC biosynthesis can form only root nodules displaying reduced rates of nitrogen fixation. Therefore, in the cases of these microsymbionts, the ability to form sufficient bacterial PC is crucial for a successful interplay with their host plants.

Cessation of Growth to Prevent Cell Death Due to Inhibition of Phosphatidylcholine Synthesis Is Impaired at 37 °C inSaccharomyces cerevisiae

Phosphatidylcholine is the most abundant phospholipid in eukaryotic cells, comprising 50% of total cellular phospholipid, and thus plays a major role in cellular and organellar biogenesis. In this study, we have used both nutritional deprivation as well as a conditional temperature sensitive allele of PCT1 (CTP:phosphocholine cytidylyltransferase) coupled with an inactivated phosphatidylethanolamine methylation pathway to determine how cells respond to inactivation of phosphatidylcholine synthesis. Metabolic studies determined that phosphatidylcholine biosynthesis decreased to negligible levels within 1 h upon shift to the nonpermissive temperature for the temperature-sensitive PCT1 allele. Phosphatidylcholine mass decreased to negligible levels upon removal of choline from the medium or growth at the nonpermissive temperature, with the levels of the other major phospholipids increasing slightly. Cell growth rate visibly slowed upon cessation of phosphatidylcholine synthesis. Cells remained viable for 7-8 h after phosphatidylcholine synthesis was prevented; however, at time points beyond 8 h, viability was significantly reduced but only if the cells had been previously grown at 37 degrees C and not 25 degrees C. The inhibition of phosphatidylcholine synthesis at 37 degrees C did not alter Golgi-derived vesicle transport to the vacuole as monitored by carboxypeptidase Y processing or to the plasma membrane as determined by invertase secretion. Immunofluorescence microscopy localized Pct1p to the nucleus and nuclear membrane. Pct1p activity is regulated by Sec14p, a cytoplasm/Golgi localized phosphatidylcholine/phosphatidylinositol binding protein that regulates Golgi-derived vesicle transport partially through its ligand-dependent regulation of PCT1 derived enzyme activity. Our nuclear localization of Pct1p indicates that the regulation of Pct1p by Sec14p is indirect.

Inhibition of phosphatidylcholine synthesis via the phosphatidylethanolamine methylation pathway impairs incorporation of bulk lipids into VLDL in cultured rat hepatocytes

Inhibition of phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway was shown to decrease the secretion of VLDL from primary rat hepatocytes (Nishimaki-Mogami et al. 1996. Biochim. Biophys. Acta. 1304: 21–31). To understand further the role of PE methylation, we determined the effect of bezafibrate, an inhibitor of PE methylation, on VLDL assembly within the microsomal lumen. Bezafibrate was shown to decrease VLDL (triacylglycerol) secretion only when cellular PE methylation was active in the presence of methionine. Pulse-chase experiments showed that bezafibrate treatment did not impair the movement of [35S]apolipoprotein (apo)B-48 from microsomal membranes into the lumen. However, bezafibrate treatment resulted in reduced VLDL-[35S]apoB-48 and increased [35S]apoB-48-containing particles in the HDL density range (HDL-[35S]apoB-48) within the lumen. Inhibition of PE methylation by bezafibrate or 3-deazaadenosine after the completion of HDL-[35S]apoB-48 assembly effectively decreased VLDL-[35S]apoB-48 secretion with a concomitant increase in HDL-[35S]apoB-48 secretion.These findings suggest that inhibition of PC synthesis via the PE methylation pathway impairs the stage of bulk triacylglycerol incorporation during the assembly of VLDL.

NBD-labeled phosphatidylcholine enters the yeast vacuole via the pre-vacuolar compartment.

At low temperature, the short-chain fluorescent-labeled phospholipids, 1-myristoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminocaproyl]-phosphatidylcholine (M-C6-NBD-PC) and its phosphatidylethanolamine analog, M-C6-NBD-PE, are internalized by flip across the plasma membrane of S. cerevisiae and show similar enrichment in intracellular membranes including the mitochondria and nuclear envelope/ER. At higher temperatures (24-37 degrees C), or if low temperature internalization is followed by warming, M-C6-NBD-PC, but not M-C6-NBD-PE, is trafficked to the lumen of the vacuole. Sorting of M-C6-NBD-PC to the vacuole is blocked by energy-depletion and by null mutations in the VPS4 and VPS28 genes required for vesicular traffic from the pre-vacuolar compartment (PVC) to the vacuole. This sorting is not blocked by a temperature-sensitive mutation in SEC12, which inhibits ER to Golgi transport, a null mutation in VPS8, which inhibits Golgi to PVC transport, or temperature-sensitive and null mutations in END4, which inhibit endocytosis from the plasma membrane. Monomethylation or dimethylation of the primary amine head-group of M-C6-NBD-PE is sufficient for sorting to the yeast vacuole in both wild-type yeast and in strains defective in the phosphatidylethanolamine methylation pathway. These data indicate that methylation of M-C6-NBD-PE produces the crucial structural component required to sort these phospholipid analogues to the vacuole via the PVC.

Disruption of Choline Methyl Group Donation for Phosphatidylethanolamine Methylation in Hepatocarcinoma Cells

Despite being widely hypothesized, the actual contribution of choline as a methyl source for phosphatidylethanolamine (PE) methylation has never been demonstrated, mainly due to the inability of conventional methods to distinguish the products from that of the CDP-choline pathway. Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway. When hepatocytes were labeled with d(9)-choline containing three deuterium atoms on each of the three methyl groups, d(3)-PC and d(6)-PC were detected, indicating that newly synthesized PC contained one or more individually mobilized methyl groups from d(9)-choline. The synthesis of d(3)-PC and d(6)-PC was sensitive to the general methylation inhibitor 3-deazaadenosine and were specific products of PE methylation using choline as a one-carbon donor. While the contribution to the CDP-choline pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to a previously identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.

Molecular Distinction of Phosphatidylcholine Synthesis between the CDP-Choline Pathway and Phosphatidylethanolamine Methylation Pathway

In addition to the CDP-choline pathway for phosphatidylcholine (PC) synthesis, the liver has a unique phosphatidylethanolamine (PE) methyltransferase activity for PC synthesis via three methylations of the ethanolamine moiety of PE. Previous studies indicate that the two pathways are functionally different and not interchangeable even though PC is the common product of both pathways. This study was designed to test the hypothesis that these two pathways produce different profiles of PC species. The PC species from these two pathways were labeled with specific stable isotope precursors, D9-choline and D4-ethanolamine, and analyzed by electrospray tandem mass spectrometry. Our studies revealed a profound distinction in PC profiles between the CDP-choline pathway and the PE methylation pathway. PC molecules produced from the CDP-choline pathway were mainly comprised of medium chain, saturated (e.g. 16:0/18:0) species. On the other hand, PC molecules from the PE methylation pathway were much more diverse and were comprised of significantly more long chain, polyunsaturated (e.g. 18:0/20:4) species. PC species from the methylation pathway contained a higher percentage of arachidonate and were more diverse than those from the CDP-choline pathway. This profound distinction of PC profiles may contribute to the different functions of these two pathways in the liver.

Bezafibrate and clofibric acid are novel inhibitors of phosphatidylcholine synthesis via the methylation of phosphatidylethanolamine

The effects of bezafibrate and clofibric acid, fibrate hypolipidemic agents, on phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway were studied. In cultured rat hepatocytes, bezafibrate and clofibric acid added to the medium rapidly and markedly reduced the conversion of ethanolamine-labeled PE to PC (IC 50 30 and 150 μM, respectively). Furthermore, the methylation of PE derived from serine was also blocked by bezafibrate, as was the secretion of PC derived from either serine or ethanolamine. The microsomal activity of PE N-methyltransferase was inhibited by these agents. Perfluorooctanoic acid but not DCQVA, though both are potent peroxisome proliferators comparable to fibrates, produced this inhibition. The inhibitory effects produced by these agents were diminished by dithiothreitol (DTT) added to the assay or alkaline pH assay condition. Inhibition by oleic acid was also attenuated under these conditions, suggesting a common mechanism of inhibition. However, unlike fatty acids, fibrates did not have rapid stimulatory effects on the CDP-choline pathway in hepatocytes. These results suggest that fibrates may mimic fatty acids in regulating PC synthesis from the PE methylation pathway but not the CDP-choline pathway.

The role of phosphatidylethanolamine methylation in the secretion of very low density lipoproteins by cultured rat hepatocytes: rapid inhibition of phosphatidylethanolamine methylation by bezafibrate increases the density of apolipoprotein B48-containing lipoproteins

The role of phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway in the secretion of very low density lipoproteins (VLDL) by cultured rat hepatocytes has been investigated by determining the effects of inhibitors. We have shown that bezafibrate and clofibric acid, known hypolipidemic agents, are potent inhibitors of PE methylation (see accompanying paper by Nishimaki-Mogami et al. (1996) Biochim. Biophys. Acta 1304). In hepatocytes incubated with ethanolamine, which maintained cellular PE levels and PE methylation activity, bezafibrate (200 μM) decreased the secretion of triacylglycerol (TG), PC, apolipoproteins B48, and E in VLDL by 50–75%. In contrast, bezafibrate at this concentration had marginal effect on VLDL secretion (83–115% of control) by hepatocytes that had been cultured in the absence of ethanolamine. In these cells PE levels and PE methylation activity had decreased by approx. 40%. VLDL secretion was decreased at concentrations similar to those required to inhibit PE methylation, and was accompanied by an increase in cellular TG levels. The same ethanolamine-dependent effects were produced by clofibric acid and also by 3-deazaadenosine (DZA), an inhibitor of cellular methylation reactions. These results indicate that PC synthesis via the PE methylation pathway plays a significant role in VLDL secretion in rat hepatocytes if the pathway is maintained at levels comparable to those in vivo. The reductions of PE methylation by bezafibrate and DZA did not affect the total amount of apolipoprotein B48 secreted into the medium. The decrease in apolipoprotein B48 in VLDL caused by bezafibrate was accompanied by an increase in apolipoprotein B48 in the HDL density range. In contrast, the amount of apolipoprotein B100 in VLDL and density determined by sequential flotation were unaffected. These findings indicate that rapid reduction of PC synthesis via PE methylation does not affect the secretion of apolipoprotein B48- or B100-containing lipoprotein particles, but does impair the lipidation of particles containing apolipoprotein B48, but not apolipoprotein B100.

Identification of the Upstream Activation Sequences Responsible for the Expression and Regulation of the <italic>PEM1</italic> and <italic>PEM2</italic> Genes Encoding the Enzymes of the Phosphatidylethanolamine Methylation Pathway in <italic>Saccharomyces cerevisiae</italic><xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

The yeast phosphatidylethanolamine methylation pathway is encoded by two structural genes, PEM1 and PEM2. The abundance of their transcripts was coordinately repressed by myo-inositol and choline. The most upstream transcriptional start sites for PEM1 and PEM2 were mapped at positions -142 and -42 relative to their first ATG codons, respectively. Promoter deletion analysis defined the 5' boundary of the regulatory region of PEM1 between -336 and -332 and that of PEM2 between -177 and -158. The 38-bp sequence between -336 and -299 from PEM1 and the 48-bp sequence between -177 and -130 from PEM2 conferred regulated transcription upon an upstream-activation-sequence-deficient test gene, CYC1-lacZ. Comparison of these two regions revealed the presence of a common octameric sequence, 5-CATRTGAA-3', which occurred twice in the 38-bp PEM1 regulatory region and once, followed by the 5'-AAACCCACACATG-3' GRFI site, in the 48-bp PEM2 regulatory region. When synthesized chemically and placed in front of CYC1-lacZ, a single copy of CATATGAA directed a rather low level of gene expression, but multiple copies produced high-level expression. In both cases, gene expression was sensitive to myo-inositol and choline. The synthesized GRFI site directed considerable but constitute lacZ expression. When used in conjunction with CATATGAA, synergistic, regulated gene expression was obtained. Hence CATRTGAA was concluded to play an important role in the myo-inositol-choline regulation of PEM1 and PEM2. Binding proteins to these sequences were demonstrated by electrophoretic mobility shift assay. Protein binding to CATRTGAA was not competitive with binding to the GRFI sequence, and vice versa. CATRTGAA was also found in the upstream regions of other genes encoding phospholipid-synthesizing enzymes, such as choline kinase, phosphatidylserine synthase, and myo-inositol-1-phosphate synthase, known to be repressed by myo-inositol and choline.

Characterization of the methyltransferases in the yeast phosphatidylethanolamine methylation pathway by selective gene disruption.

PEM1 and PEM2 are structural genes for the yeast phosphatidylethanolamine methylation pathway which mediates the three-step methylation of phosphatidylethanolamine to phosphatidylcholine. Selective disruption of each locus in the yeast genome was performed using the in-vitro-inactivated gene with insertion of yeast LEU2 or HIS3. Complementation test and spore analysis indicated that the disruptants were allelic with our previous mutants that were isolated by chemical mutagenesis and used for the cloning of PEM1 and PEM2. The methyltransferase activities of the disruptants were assayed using their membrane fractions. When the PEM1 locus was disrupted, the activity for the first methylation was greatly decreased but was still detectable, while the activities for the second and third methylations were well retained. The remaining three activities exhibited nearly identical pH optima and apparent Km values for S-adenosyl-L-methionine. The disruptant incorporated radioactivity from L-[methyl-14C]Met into phosphatidylcholine at a low but measurable rate and required choline for optimal growth. When choline was omitted from the culture medium, the phosphatidylcholine content of the cells significantly decreased, but was restored by the addition of N-monomethylethanolamine or choline. When the PEM2 locus was disrupted, the activities for the second and third methylations were totally lost, but that for the first methylation remained. This activity could be distinguished from those remaining in the pem1 disruptant by its different pH optimum and apparent Km for S-adenosyl-L-methionine. When incubated with [methyl-14C]Met, the pem2 disruptant accumulated the radioactivity in phosphatidylmonomethylethanolamine. This disruptant also required choline for optimal growth. In the absence of choline, it accumulated phosphatidylmonomethylethanolamine with a concomitant decrease in phosphatidylcholine and phosphatidylethanolamine. When both loci were disrupted, all phospholipid-methylating activities were lost and cells absolutely required choline for growth. The flux through the pathway became negligible. Thus, the PEM1-encoded methyltransferase was strictly specific to the first step while the PEM2-encoded methyltransferase exhibited a somewhat broader specificity with a preference for the second and third steps of the pathway. These two enzymes accounted for all the activities in the yeast phosphatidylethanolamine methylation pathway.

Yeast phosphatidylethanolamine methylation pathway. Cloning and characterization of two distinct methyltransferase genes.

The structural genes (PEM1 and PEM2) encoding the enzymes involved in the yeast phosphatidylethanolamine (PE) methylation pathway were cloned by means of genetic complementation using yeast mutants. The cloned genes were expressed in a yeast mutant that was completely deficient in the PE methylation pathway. The membrane fraction obtained from the transformants carrying PEM1 only catalyzed the conversion of PE to phosphatidyl-N-monomethylethanolamine (PMME), the first step of the methylation pathway. Therefore, the enzyme encoded by PEM1 was designated as PE methyltransferase. In contrast, the membrane fraction from the transformants carrying PEM2 catalyzed the synthesis of phosphatidylcholine (PC) from PE, indicating that it contains all of the three methylation activities. PMME and phosphatidyl-N,N-dimethylethanolamine were found to be utilized more preferentially than PE. Because of its rather broad substrate specificity, the enzyme encoded by PEM2 is designated as phospholipid methyltransferase. The results of phospholipid composition analysis showed that the PEM1 transformant accumulated PMME whereas the PEM2 transformant contained a decreased amount of PC. Both genes were required for maintenance of the PC content of the yeast at a normal level. The results of nucleotide sequence analysis demonstrated that the coding frames of the PEM1 and PEM2 genes were capable of encoding 869- and 206-amino acid residues with calculated molecular weights of 101,202 and 23,150, respectively. The sizes of the PEM1 and PEM2 transcripts detected in the exponentially growing wild-type yeast were consistent with those of the deduced translation products. PE methyltransferase exhibits internal sequence homology as well as homology with phospholipid methyltransferase, suggesting that these two methyltransferase genes evolved through gene duplication. Furthermore, there was significant sequence homology between PE methyltransferase and bovine phenylethanolamine N-methyltransferase, and between phospholipid methyltransferase and Escherichia coli S-adenosylmethionine-6-N',N'-adenosyl (rRNA) dimethyltransferase.