Phosphoethanolamine bases as intermediates in phosphatidylcholine synthesis by lemna

The pathway for synthesis of phosphatidylcholine, the dominant methyl-containing end product formed by Lemna paucicostata, has been investigated. Methyl groups originating in methionine are rapidly utilized by intact plants to methylate phosphoethanolamine successively to the mono-, di-, and tri-methyl (i.e. phosphocholine) phosphoethanolamine derivatives. With continued labeling, radioactivity initially builds up in these compounds, then passes on, accumulating chiefly in phosphatidylcholine (34% of the total radioactivity taken up by plants labeled to isotopic equilibrium with l-[(14)CH(3)]methionine), and in lesser amounts in soluble choline (6%). Radioactivity was detected in mono- and dimethyl derivatives of free ethanolamine or phosphatidylethanolamine only in trace amounts. Pulse-chase experiments with [(14)CH(3)]choline and [(3)H] ethanolamine confirmed that phosphoethanolamine is rapidly methylated and that phosphocholine is converted to phosphatidylcholine. Initial rates indicate that methylation of phosphoethanolamine predominates over methylation of either phosphatidylethanolamine or free ethanolamine at least 99:1. Although more studies are needed, it is suggested this pathway may well turn out to account for most phosphatidylcholine synthesis in higher plants. Phosphomethylethanolamine and phosphodimethylethanolamine are present in low quantities during steady-state growth (18% and 6%, respectively, of the amount of phosphocholine). Radioactivity was not detected in CDP-choline, probably due to the low steady-state concentration of this nucleotide.     

Modulation of phosphatidylethanolamine biosynthesis by exogenous ethanolamine and analogues in the hamster heart

In the hamster heart, exogenous ethanolamine is taken up by the heart and utilized for the biosynthesis of phosphatidylethanolamine. The role of the exogenous supply of ethanolamine on phosphatidylethanolamine biosynthesis was examined by perfusing hamster heart with various concentrations of labelled ethanolamine. Analysis of the radioactivity distributed in the ethanolamine-containing metabolites indicated that at low exogenous ethanolamine concentrations ( 0.1 M), the conversion of phosphoethanolamine to CDP-ethanolamine was rate-limiting for phosphatidylethanolamine biosynthesis. However, perfusion with higher concentrations of ethanolamine ( 0.4 M) resulted in the phosphorylation of ethanolamine becoming rate-limiting. Since the intracellular ethanolamine levels remained unchanged, the alterations in radioactivity distribution suggested that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis. The effects of ethanolamine analogues on ethanolamine uptake and subsequent conversion to phosphatidylethanolamine at physiological concentrations of exogenous ethanolamine were examined. Monomethylethanolamine was found to inhibit ethanolamine uptake, the conversion of ethanolamine to phosphoethanolamine and incorporation of radioactivity into phosphatidylethanolamine.The accumulation of radioactivity in the ethanolamine fraction by monomethylethanolamine, despite of the inhibition of ethanolamine uptake, further confirms the rate-limiting role of ethanolamine kinase in the biosynthesis of phosphatidylethanolamine. (Mol Cell Biochem116: 69–73, 1992)     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Characterization of the pathways for phosphatidylethanolamine biosynthesis in Chinese hamster ovary mutant and parental cell lines

A tritium suicide procedure was devised to facilitate the isolation of Chinese hamster ovary cell mutants defective in phosphatidylethanolamine biosynthesis. One mutant with a 20-50% reduction in [3H]ethanolamine incorporation was chosen for further analysis and was shown to have reduced activity of CTP: phosphoethanolamine cytidylyltransferase. Levels of phosphatidylethanolamine and rates of its biosynthesis were compared in the mutant and parent cell lines. Despite the reduced activity of the CDP-ethanolamine pathway in the mutant, levels of phosphatidylethanolamine were the same in mutant and parent cells. Rates of phosphatidylethanolamine synthesis de novo, as measured by incorporation of 32PO4 into phosphatidylethanolamine, were also the same in mutant and parent cells, as was the rate of incorporation of [3H]serine into both phosphatidylserine and phosphatidylethanolamine. After a long term labeling with [3H]serine, the specific radioactivity of phosphatidylserine was the same as that of phosphatidylethanolamine, and there was no difference in the specific radioactivities of the two lipids between mutant and parent cells. These results implicate decarboxylation of phosphatidylserine as the sole route for synthesis of phosphatidylethanolamine under normal culture conditions.     

Biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine route is an important pathway in isolated rat hepatocytes

In the present study pulse-label and pulse-chase experiments with isolated rat hepatocytes in suspension were designed to investigate the effects of the presence of either serine or ethanolamine in the medium on the rate of phosphatidylethanolamine synthesis via the CDPethanolamine pathway and by decarboxylation of phosphatidylserine. Addition of serine to the medium did not affect the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-label experiments showed that the incorporation of [3H]serine into phosphatidylserine decreased in the presence of ethanolamine with a corresponding decrease of the incorporation of label into the ethanolamine base moiety of phosphatidylethanolamine. However, the radioactivity in the diacylglycerol part of phosphatidylethanolamine was considerably higher in the presence of ethanolamine than in its absence. Pulse-chase experiments with labelled serine demonstrated that the conversion of phosphatidylserine to phosphatidylethanolamine was not affected by varying concentrations of ethanolamine. Our observations indicate that in the presence of ethanolamine the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway is enhanced relative to the synthesis by decarboxylation of phosphatidylserine.     

Conversion of ethanolamine, monomethylethanolamine and dimethylethanolamine to choline-containing compounds by neurons in culture and by the rat brain.

The incubation of neurons from chick embryos in primary culture with [3H]ethanolamine revealed the conversion of this base into monomethyl, dimethyl and choline derivatives, including the corresponding free bases. Labelling with [methyl-3H]monomethylethanolamine and [methyl-3H]dimethylethanolamine supported the conclusion that in chick neuron cultures, phosphoethanolamine appears to be the preferential substrate for methylation, rather than ethanolamine or phosphatidylethanolamine. The methylation of the latter two compounds, in particular that of phosphatidylethanolamine, was seemingly stopped at the level of their monomethyl derivatives. Fetal rat neurons in primary culture incubated with [3H]ethanolamine showed similar results to those observed with chick neurones. However, phosphoethanolamine and phosphatidylethanolamine and, to a lesser extent, free ethanolamine, appeared to be possible substrates for methylation reactions. The methylation of water-soluble ethanolamine compounds de novo was further confirmed by experiments performed in vivo by intraventricular injection of [3H]ethanolamine. Phosphocholine and the monomethyl and dimethyl derivatives of ethanolamine were detected in the brain 15 min after injection.     

Choline Synthesis in Spinach in Relation to Salt Stress

Choline metabolism was examined in spinach (Spinacia oleracea L.) plants growing under nonsaline and saline conditions. In spinach, choline is required for phosphatidylcholine synthesis and as a precursor for the compatible osmolyte glycine betaine (betaine). When control (nonsalinized) leaf discs were incubated for up to 2 h with [1,2-14C]ethanolamine, label appeared in the N-methylated derivatives of phosphoethanolamine including phosphomono-, phosphodi-, and phosphotri- (i.e. phosphocholine) methyl-ethanolamine, as well as in choline and betaine, whereas no radioactivity could be detected in the mono- and dimethylated derivatives of the free base ethanolamine. Leaf discs from salinized plants showed the same pattern of labeling, although the proportion of label that accumulated in betaine was almost 3-fold higher in the salinized leaf discs. Enzymes involved in choline metabolism were assayed in crude leaf extracts of plants. The activites of ethanolamine kinase and of the three S-adenosylmethionine:phospho-base N-methyltransferase enzymes responsible for N-methylating phosphoethanolamine to phosphocholine were all higher in extracts of plants salinized step-wise to 100, 200, or 300 mM NaCI compared with controls. In contrast, choline kinase, phosphocholine phosphatase, and cytidine 5[prime]-triphosphate: phosphocholine cytidylyltransferase activities showed little variation with salt stress. Thus, the increased diversion of choline to betaine in salt-stressed spinach appears to be mediated by the increased activity of several key enzymes involved in choline biosynthesis.     

Differential Utilization of the Ethanolamine Moiety of Phosphatidylethanolamine Derived from Serine and Ethanolamine during NGF-Induced Neuritogenesis of PC12 Cells

Neurite elongation involves the expansion of the plasma membrane and phospholipid synthesis. We investigated membrane phosphatidylethanolamine (PE) biosynthesis in PC12 cells during neurite outgrowth induced by nerve growth factor (NGF). When PE was prelabeled with [³H]ethanolamine and the radioactivity was chased by incubation with 1 mM unlabeled ethanolamine, the radioactivity of [³H]PE steadily declined and [³H]ethanolamine was released into the medium in NGF-treated cells during neurite outgrowth; in the absence of unlabeled ethanolamine, the radioactivity of [³H]PE remained relatively constant for at least 24 hr. In undifferentiated cells but not in NGF-treated cells, [³H]phosphoethanolamine accumulated in significant amounts during pulse labeling, and was converted partly to PE but largely released into the medium irrespective of incubation with unlabeled ethanolamine. The decline in the radioactivity of [³H]PE and release of [³H]ethanolamine following incubation with unlabeled ethanolamine were also observed in undifferentiated cells. Thus, the ethanolamine moiety of PE derived from ethanolamine is actively recycled in both differentiated and undifferentiated cells. When PE was derived from [³H]serine through phosphatidylserine (PS) decarboxylation, the decrease in radioactivity of [³H]PE and release of [³H]ethanolamine into the medium following incubation with unlabeled ethanolamine were observed only in NGF-treated cells, but not in undifferentiated cells, indicating that the ethanolamine moiety of PE derived from PS is actively recycled only in the cells undergoing NGF-induced neuritogenesis. Thus, in PC12 cells, the ethanolamine moiety of PE derived from PS is regulated differently from that of PE derived from ethanolamine.     

Synthesis of methylated ethanolamine moieties: regulation by choline in lemna

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[(3)H(3)C]methionine, l-[(14)CH(3)]methionine, or [1,2-(14)C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.     

Relationships Between Phosphatidylcholine, Phosphatidylethanolamine, and Sphingomyelin Metabolism in Cultured Oligodendrocytes

Abstract: In most cell types the major pathway of sphingomyelin synthesis is the direct transfer of the phosphocholine head group from phosphatidylcholine to ceramide catalyzed by the enzyme l -acylsphingosine:phosphatidylcholine phosphocholinetransferase (SM synthase; EC 2.7.8.-). Although this pathway has been demonstrated in brain tissue, its quantitative importance has been questioned. An alternative biosynthetic pathway for sphingomyelin synthesis in brain tissue has been proposed, viz., the direct transfer of phosphoethanolamine from phosphatidylethanolamine to ceramide, followed by methylation of the ethanolamine moiety to a choline group. We have evaluated various possible biosynthetic pathways of sphingomyelin synthesis in rat spinal cord oligodendrocytes, the myelin-forming cells of the CNS, by labeling cells in culture with radiolabeled choline, ethanolamine, or serine. Our results indicate that, in oligodendrocytes, most of the phosphocholine for the biosynthesis of sphingomyelin is provided by phosphatidylcholine, which is predominantly derived from de novo synthesis. No evidence was found for the operation of the alternative pathway via ceramide-phosphoethanolamine. Furthermore, our results indicate that a small pool of phosphatidylcholine is provided by methylation of phosphatidylethanolamine, which in turn is formed preferentially by decarboxylation of phosphatidylserine.     

Phosphatidylethanolamine is the donor of the ethanolamine residue linking a glycosylphosphatidylinositol anchor to protein.

Numerous cell surface glycoproteins from eukaryotic organisms including African trypanosomes and budding yeast (Saccharomyces cerevisiae), are anchored to the lipid bilayer by a glycophospholipid, glycosylphosphatidylinositol, covalently linked to the carboxyl terminus of the protein via a phosphoethanolamine bridge. In this paper we describe metabolic labeling experiments aimed at identifying the biosynthetic origin of the ethanolamine residue in the phosphoethanolamine bridge. Using yeast mutants generated by disruption of the ethanolaminephosphotransferase (EPT1) and cholinephosphotransferase (CPT1) genes, we report data consistent with the proposal that the ethanolamine residue is derived from phosphatidylethanolamine.     

Co-ordinate regulation of ethanolamine kinase and phosphoethanolamine cytidylyltransferase in the biosynthesis of phosphatidylethanolamine in rat liver.

Essential-fatty acid deficiency produces a 52% increase in the rate of phosphatidyl-ethanolamine synthesis in rat liver as calculated from results obtained in vivo [Trewhella & Collins (1973) Biochem. Biophys. Acta 296, 34--50]. This flux change was used to test the possible regulatory roles of ethanolamine kinase and of phosphoethanolamine cytidylyltransferase, which are rate-limiting enzymes of the cytidine pathway for the synthesis of phosphatidylethanolamine [Infante (1977) Biochem. J. 167, 847--849]. The results show that essential-fatty acid deficiency produces 50% and 53% increases respectively in the specific activity of these enzymes, accounting for the increased rate of phosphatidylethanolamine synthesis produced by this dietary insufficiency. This evidence leads to the conclusion that ethanolamine kinase and phosphoethanolamine cytidylyl-transferase have co-ordinated regulatory roles in the flux control of the cytidine pathway, and its sphinganine 1-phosphate lyase branch reaction, for the synthesis of phosphatidylethanolamine.     

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[³H(G)]serine and [¹⁴C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of “correct” PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.     

Serine regulates phosphatidylethanolamine biosynthesis in the hamster heart.

The role of serine as a precursor and metabolic regulator for phosphatidylethanolamine biosynthesis in the hamster heart was investigated. Hearts were perfused with 50 microM [1-3H]ethanolamine in the presence or absence of serine for up to 60 min. Ethanolamine uptake was attenuated by 0.05-10 mM serine in a noncompetitive manner, and the incorporation of labeled ethanolamine into phosphatidylethanolamine was also inhibited by serine. Analysis of the ethanolamine-containing metabolites in the CDP-ethanolamine pathway revealed that the conversion of ethanolamine to phosphoethanolamine was reduced. The reduction was a result of an inhibition of ethanolamine kinase activity by an elevated pool of intracellular serine. Perfusion of the heart with 1 mM serine caused a 5-fold increase in intracellular serine pool. In order to examine the action of serine on other phosphatidylethanolamine metabolic pathways, hearts were perfused with [1-3H]glycerol in the presence and absence of serine. Serine did not cause any enhancement of phosphatidylethanolamine hydrolysis. The base-exchange reaction for phosphatidylserine formation or the decarboxylation of phosphatidylserine was not affected by serine perfusion. We conclude that circulating serine plays an important role in the modulation of phosphatidylethanolamine biosynthesis via the CDP-ethanolamine pathway in the hamster heart but does not affect the contribution of the decarboxylase pathway for phosphatidylethanolamine formation.     

Metabolism of phosphatidylethanolamine in the frog retina

The synthesis and the turnover of phosphatidylethanolamine in frog retinal rod outer segments and microsomes were studied by monitoring the incorporation of five radioactive precursors: 32PO4, 33PO4 [3H]glycerol, [3H]serine, and [3H]ethanolamine. 1. Labeled serine was actively incorporated into phosphatidylethanolamine. The kinetics of the labeling patterns in both microsomes and rod outer segments was consistent with formation via decarboxylation of phosphatidylserine. 2. Ethanolamine was found to be an ineffective precursor of phosphatidylethanolamine, suggesting that the major pathway for phosphatidylethanolamine synthesis in the retina is via the decarboxylation reaction. 3. An active methylation of phosphatidylethanolamine to phosphatidylcholine was observed in both retinal microsomes and rod outer segments. 4. The kinetics of labeling of phosphatidylethanolamine in the rod outer segments was different for the various isotopic precursors, and was found to depend on the relative turnover times of the precursor pools. Glycerol was the only precursor that gave a true pulse of radioactivity. 5. The specific activity of phosphatidylethanolamine derived from labeled glycerol declined exponentially, demonstrating that the labeled lipid was diffusely distributed throughout the rod outer segments. The half-life of phosphatidylethanolamine in the rod outer segments was determined to be 18 days. Comparison of this value to the turnover time of rod outer segment integral proteins revealed that rod outer segment lipid is renewed at a faster rate than protein.     

Phospholipid turnover in soybean tissue cultures

The degradation rates of phospholipids in soybean (Glycine max L. Merrill) suspension cultures were studied by pulse-chase experiments. The only chloroform-soluble product of incorporation of radioactive choline was phosphatidylcholine, the bulk of which had a half-life of 36 hours. Ethanolamine was incorporated primarily into phosphatidylethanolamine, phosphatidylcholine at an intermediate level, and phosphatidylmonomethylethanolamine to a small extent. The phosphatidylethanolamine decayed in a triphasic fashion with half-lives of 12, 34, and 136 hours. Phosphatidylcholine in this case increased in radioactivity up to day 4 and thereafter declined with a 92-hour half-life. The radioactivity rose slightly to day 4 in phosphatidylmonomethylethanolamine after an initial rapid decline. When serine was used as a substrate, half-lives similar to those obtained with ethanolamine were obtained. Phosphatidylcholine contained the greatest amount of label, however, with phosphatidylethanolamine containing slightly less, and phosphatidylserine contained the least. Data also are presented for glycerol and acetate phospholipid product degradation.     

Phosphatidylethanolamine is the donor of the terminal phosphoethanolamine group in trypanosome glycosylphosphatidylinositols.

A variety of eukaryotic cell surface proteins, including the variant surface glycoproteins of African trypanosomes, rely on a covalently attached lipid, glycosylphosphatidylinositol (GPI), for membrane attachment. GPI anchors are synthesized in the endoplasmic reticulum by stepwise glycosylation of phosphatidylinositol (via UDP-GlcNAc and dolichol-P-mannose) followed by the addition of phosphoethanolamine. The experiments described in this paper are aimed at identifying the biosynthetic origin of the terminal phosphoethanolamine group. We show that trypanosome GPIs can be labelled via CDP-[3H]ethanolamine or [beta-32P]CDP-ethanolamine in a cell-free system, indicating that phosphoethanolamine is acquired en bloc. In pulse-chase experiments with CDP-[3H]ethanolamine we show that the GPI phosphoethanolamine is not derived directly from CDP-ethanolamine, but instead from a relatively stable metabolite, such as phosphatidylethanolamine (PE), generated from CDP-ethanolamine in the cell-free system. To test the possibility that PE is the immediate donor of the GPI phosphoethanolamine moiety, we describe metabolic labelling experiments with [3H]serine and show that GPIs can be labelled in the absence of detectable radiolabelled CDP-ethanolamine, presumably via [3H]PE generated from [3H]phosphatidylserine (PS). The data support the proposal that the terminal phosphoethanolamine group in trypanosome GPIs is derived from PE.     

Enzymes of phosphatidylcholine synthesis in lemna, soybean, and carrot

Cell-free extracts from Lemna and suspension cultured carrot (Daucus Carota L.) catalyze S-adenosylmethionine-dependent N-methylations of phosphoethanolamine, phosphomethylethanolamine, and phosphodimethylethanolamine; extracts of suspension cultured soybean (Glycine max), of phosphoethanolamine only. Material pelleted from each tissue between 15,000 and 100,000g catalyzes S-adenosylmethionine-dependent N-methylations of phosphatidylmethylethanolamine and phosphatidyl-dimethylethanolamine, but not phosphatidylethanolamine. Extracts from each tissue catalyze CTP-dependent cytidylyltransfers to each of the three methylated phosphoethanolamine derivatives, forming the corresponding CDP derivatives. Some of the properties of the activities investigated are reported. On the basis of in vivo labeling experiments, we have proposed (AH Datko, SH Mudd 1988 Plant Physiol 88: 854-861) differing pathways for phosphatidylcholine synthesis in which, after a common committing step, N-methylation of phosphoethanolamine, subsequent methylations occur in Lemna almost exclusively at the phospho-base level; in soybean, at the phosphatidyl-base level; and in carrot, at both levels. Thus, among the activities investigated, at least those required for the operation of the proposed pathways have been positively demonstrated. The extent to which the present results explain the differences between these pathways is discussed, and a speculation offered as to how these differences may have arisen phylogenetically.     

Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.