Inactivation of the gene for phospholipid N-methyltransferase in Sinorhizobium meliloti: phosphatidylcholine is required for normal growth

In phosphatidylcholine (PC)-containing prokaryotes, only the methylation pathway of PC biosynthesis was thought to occur. However, a second choline-dependent pathway for PC formation, the PC synthase (Pcs) pathway, exists in Sinorhizobium (Rhizobium) meliloti in which choline is condensed with CDP-diacylglyceride. Here, we characterize the methylation pathway of PC biosynthesis in S. meliloti. A mutant deficient in phospholipid N-methyltransferase (Pmt) was complemented with a S. meliloti gene bank and the complementing DNA was sequenced. A gene coding for a S-adenosylmethionine-dependent N-methyltransferase was identified as the sinorhizobial Pmt, which showed little similarity to the corresponding enzyme from Rhodobacter sphaeroides. Upon expression of the sinorhizobial Pmt, besides phosphatidylcholine, the methylated intermediates of the methylation pathway, monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine, are also formed. When Pmt-deficient mutants of S. meliloti are grown on minimal medium, they cannot form PC, and they grow significantly more slowly than the wild type. Growth of the Pmt-deficient mutant in the presence of choline allows for PC formation via the Pcs pathway and restores wild-type-like growth. Double knock-out mutants, deficient in Pmt and in Pcs, are unable to form PC and show reduced growth even in the presence of choline. These results suggest that PC is required for normal growth of S. meliloti.     

Phosphatidylethanolamine is not essential for growth of Sinorhizobium meliloti on complex culture media

In addition to phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylethanolamine (PE), Sinorhizobium meliloti also possesses phosphatidylcholine (PC) as a major membrane lipid. The biosynthesis of PC in S. meliloti can occur via two different routes, either via the phospholipid N-methylation pathway, in which PE is methylated three times in order to obtain PC, or via the phosphatidylcholine synthase (Pcs) pathway, in which choline is condensed with CDP-diacylglycerol to obtain PC directly. Therefore, for S. meliloti, PC biosynthesis can occur via PE as an intermediate or via a pathway that is independent of PE, offering the opportunity to uncouple PC biosynthesis from PE biosynthesis. In this study, we investigated the first step of PE biosynthesis in S. meliloti catalyzed by phosphatidylserine synthase (PssA). A sinorhizobial mutant lacking PE was complemented with an S. meliloti gene bank, and the complementing DNA was sequenced. The gene coding for the sinorhizobial phosphatidylserine synthase was identified, and it belongs to the type II phosphatidylserine synthases. Inactivation of the sinorhizobial pssA gene leads to the inability to form PE, and such a mutant shows a greater requirement for bivalent cations than the wild type. A sinorhizobial PssA-deficient mutant possesses only PG, CL, and PC as major membrane lipids after growth on complex medium, but it grows nearly as well as the wild type under such conditions. On minimal medium, however, the PE-deficient mutant shows a drastic growth phenotype that can only partly be rescued by choline supplementation. Therefore, although choline permits Pcs-dependent PC formation in the mutant, it does not restore wild-type-like growth in minimal medium, suggesting that it is not only the lack of PC that leads to this drastic growth phenotype.     

Plant-exuded choline is used for rhizobial membrane lipid biosynthesis by phosphatidylcholine synthase.

Phosphatidylcholine is a major lipid of eukaryotic membranes, but found in only few prokaryotes. Enzymatic methylation of phosphatidylethanolamine by phospholipid N-methyltransferase was thought to be the only biosynthetic pathway to yield phosphatidylcholine in bacteria. However, mutants of the microsymbiotic soil bacterium Sinorhizobium (Rhizobium) meliloti, defective in phospholipid N-methyltransferase, form phosphatidylcholine in wild type amounts when choline is provided in the growth medium. Here we describe a second bacterial pathway for phosphatidylcholine biosynthesis involving the novel enzymatic activity, phosphatidylcholine synthase, that forms phosphatidylcholine directly from choline and CDP-diacylglycerol in cell-free extracts of S. meliloti. We further demonstrate that roots of host plants of S. meliloti exude choline and that the amounts of exuded choline are sufficient to allow for maximal phosphatidylcholine biosynthesis in S. meliloti via the novel pathway.     

Novel pathway for phosphatidylcholine biosynthesis in bacteria associated with eukaryotes

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesised by either of two pathways, the CDP-choline pathway or the methylation pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of distantly related bacteria such as Rhizobacteria and Spirochetes. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a novel choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, a novel enzymatic activity, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. Surprisingly, genomes of some pathogens (Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) contain genes similar to the sinorhizobial gene for phosphatidylcholine synthase. We, therefore, suggest that the new PC synthase pathway is present in a number of bacteria displaying symbiotic or pathogenic associations with eukaryotes and that the eukaryotic host functions as the provider of choline for this pathway.     

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.     

Biosynthesis of phosphatidylcholine in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the methylation pathway or the CDP-choline pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of rather diverse bacteria and based on genomic data, we estimate that more than 10% of all bacteria possess PC. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. A number of symbiotic (Rhizobium leguminosarum, Mesorhizobium loti) and pathogenic (Agrobacterium tumefaciens, Brucella melitensis, Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) bacteria seem to possess the PC synthase pathway and we suggest that the respective eukaryotic host functions as the provider of choline for this pathway. Pathogens entering their hosts through epithelia (Streptococcus pneumoniae, Haemophilus influenzae) require phosphocholine substitutions on their cell surface components that are biosynthetically also derived from choline supplied by the host. However, the incorporation of choline in these latter cases proceeds via choline phosphate and CDP-choline as intermediates. The occurrence of two intermediates in prokaryotes usually found as intermediates in the eukaryotic CDP-choline pathway for PC biosynthesis raises the question whether some bacteria might form PC via a CDP-choline pathway.     

Phosphorus-free membrane lipids of Sinorhizobium meliloti are not required for the symbiosis with alfalfa but contribute to increased cell yields under phosphorus-limiting conditions of growth

The microsymbiont of alfalfa, Sinorhizobium meliloti, possesses phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, and phosphatidylcholine as major membrane phospholipids, when grown in the presence of sufficient accessible phosphorus sources. Under phosphate-limiting conditions of growth, S. meliloti replaces its phospholipids by membrane lipids that do not contain any phosphorus in their molecular structure and, in S. meliloti, these phosphorus-free membrane lipids are sulphoquinovosyl diacylglycerols (SL), ornithine-containing lipids (OL), and diacylglyceryl-N,N,N-trimethylhomoserines (DGTS). In earlier work, we demonstrated that neither SL nor OL are required for establishing a nitrogen-fixing root nodule symbiosis with alfalfa. We now report the identification of the two structural genes btaA and btaB from S. meliloti required for DGTS biosynthesis. When the sinorhizobial btaA and btaB genes are expressed in Escherichia coli, they cause the formation of DGTS in this latter organism. A btaA-deficient mutant of S. meliloti is unable to form DGTS but can form nitrogen-fixing root nodules on alfalfa, demonstrating that sinorhizobial DGTS is not required for establishing a successful symbiosis with the host plant. Even a triple mutant of S. meliloti, unable to form any of the phosphorus-free membrane lipids SL, OL, or DGTS is equally competitive for nodule occupancy as the wild type. Only under growth-limiting concentrations of phosphate in culture media did mutants that could form neither OL nor DGTS grow to lesser cell densities.     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.     

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.     

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.     

Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME.     

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.     

Regulation of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase by phospholipid precursors in Saccharomyces cerevisiae

Phosphatidylethanolamine methyltransferase (PEMT) and phospholipid methyltransferase (PLMT), which are encoded by the CHO2 and OPI3 genes, respectively, catalyze the three-step methylation of phosphatidylethanolamine to phosphatidylcholine in Saccharomyces cerevisiae. Regulation of PEMT and PLMT as well as CHO2 mRNA and OPI3 mRNA abundance was examined in S. cerevisiae cells supplemented with phospholipid precursors. The addition of choline to inositol-containing growth medium repressed the levels of CHO2 mRNA and OPI3 mRNA abundance in wild-type cells. The major effect on the levels of the CHO2 mRNA and OPI3 mRNA occurred in response to inositol. Regulation was also examined in cho2 and opi3 mutants, which are defective in PEMT and PLMT activities, respectively. These mutants can synthesize phosphatidylcholine when they are supplemented with choline by the CDP-choline-based pathway but they are not auxotrophic for choline. CHO2 mRNA and OPI3 mRNA were regulated by inositol plus choline in opi3 and cho2 mutants, respectively. However, there was no regulation in response to inositol when the mutants were not supplemented with choline. This analysis showed that the regulation of CHO2 mRNA and OPI3 mRNA abundance by inositol required phosphatidylcholine synthesis by the CDP-choline-based pathway. The regulation of CHO2 mRNA and OPI3 mRNA abundance generally correlated with the activities of PEMT and PLMT, respectively. CDP-diacylglycerol synthase and phosphatidylserine synthase, which are regulated by inositol in wild-type cells, were examined in the cho2 and opi3 mutants. Phosphatidylcholine synthesis was not required for the regulation of CDP-diacylglycerol synthase and phosphatidylserine synthase by inositol.     

Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding.     

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (< 20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.     

Brucella abortus synthesizes phosphatidylcholine from choline provided by the host

The Brucella cell envelope is characterized by the presence of phosphatidylcholine (PC), a common phospholipid in eukaryotes that is rare in prokaryotes. Studies on the composition of Brucella abortus 2308 phospholipids revealed that the synthesis of PC depends on the presence of choline in the culture medium, suggesting that the methylation biosynthetic pathway is not functional. Phospholipid composition of pmtA and pcs mutants indicated that in Brucella, PC synthesis occurs exclusively via the phosphatidylcholine synthase pathway. Transformation of Escherichia coli with an expression vector containing the B. abortus pcs homologue was sufficient for PC synthesis upon induction with IPTG (isopropyl-beta-d-thiogalactopyranoside), while no PC formation was detected when bacteria were transformed with a vector containing pmtA. These findings imply that Brucella depends on choline provided by the host cell to form PC. We could not detect any obvious associated phenotype in the PC-deficient strain under vegetative or intracellular growth conditions in macrophages. However, the pcs mutant strain displays a reproducible virulence defect in mice, which suggests that PC is necessary to sustain a chronic infection process.     

Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae.

chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. As expected, when chol mutants were starved for ethanolamine, the rates of synthesis of the phospholipids phosphatidylethanolamine and PC declined rapidly. Surprisingly, however, coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. The results obtained suggest that the slowing of PC biosynthesis in ethanolamine-starved chol cells leads to a coordinated decrease in the synthesis of all phospholipids. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed.     

Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes

Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. No formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 microM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylethanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatidylcholine biosyntheses.     

Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation

5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK.