Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells.
ABSTRACT: Addition of oleic acid to Krebs II cells induced a rapid incorporation of [3H]choline into phosphatidylcholine, since 500 microM of the fatty acid stimulated choline incorporation by 5-fold over the control after 5 min of incubation. In fact, a noticeable increase in phosphatidylcholine labelling could be monitored immediately after 1 min of cell incubation with [3H]choline, at which time 50% of cytosolic cytidylyltransferase activity (EC 188.8.131.52), the regulatory enzyme of phosphatidylcholine synthesis, was translocated on to membranes. Non-esterified [3H]oleic acid content was also increased in the same range of time in the particulate fraction. Subcellular fractionation indicated that endoplasmic reticulum was the unique binding site for cytidylyltransferase even after 1 min of incubation. Also, [3H]oleic acid accumulated mainly in the same internal membrane. Addition of exogenous albumin to cells prelabelled with [3H]oleic acid induced the release of 50% of membrane-bound cytidylyltransferase activity within 1 min, together with a decrease in unesterified oleic acid in the same membrane. Although total depletion of oleic acid was obtained, total release of membrane-bound cytidylyltransferase was not. The remaining minor pool of membrane-bound cytidylyltransferase was not affected by cell incubation with dibutyryl cyclic AMP, suggesting that this pool was neither regulated by fatty acid nor modulated by cyclic-AMP-dependent protein phosphorylation. Addition of [3H]oleic acid directly to an homogenate led to a less specific accumulation of the fatty acid in the endoplasmic reticulum, but cytidylyltransferase remained exclusively associated with this membrane. We concluded that in vivo translocation of cytidylyltransferase provoked by oleic acid concerns one specific pool of the enzyme distinct from the enzyme firmly bound to endoplasmic reticulum, but other factor(s) than fatty acid seem to be required to explain the specificity of endoplasmic reticulum for cytidylyltransferase binding.
Project description:The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the 'de novo' pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the 'de novo' pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 184.108.40.206) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).
Project description:Phosphatidylcholine synthesis by rat type II pneumonocytes was altered either by depleting the cells of choline or by exposing the cells to extracellular lung surfactant. Effects of these experimental treatments on the activity of a regulatory enzyme, CTP:phosphocholine cytidylyltransferase, were investigated. Although choline depletion of type II pneumonocytes resulted in inhibition of phosphatidylcholine synthesis, cytidylyltransferase activity (measured in cell homogenates in either the absence or presence of added lipids) was greatly increased. Activation of cytidylyltransferase in choline-depleted cells was rapid and specific, and was quickly and completely reversed when choline-depleted cells were exposed to choline (but not ethanolamine). Choline-dependent changes in enzymic activity were apparently not a result of direct actions of choline on cytidylyltransferase and they were largely unaffected by cyclic AMP analogues, oleic acid, linoleic acid or cycloheximide. The Km value of cytidylyltransferase for CTP (but not phosphocholine) was lower in choline-depleted cells than in choline-repleted cells. Subcellular redistribution of cytidylyltransferase also was associated with activation of the enzyme in choline-depleted cells. When measured in the presence of added lipids, 66.5 +/- 5.0% of recovered cytidylyltransferase activity was particulate in choline-depleted cells but only 34.1 +/- 4.5% was particulate in choline-repleted cells. An increase in particulate cytidylyltransferase also occurred in type II pneumonocytes that were exposed to extracellular surfactant. This latter subcellular redistribution, however, was not accompanied by a change in cytidylyltransferase activity even though incorporation of [3H]choline into phosphatidylcholine was inhibited by approx. 50%. Subcellular redistribution of cytidylyltransferase, therefore, is associated with changes in enzymic activity under some conditions, but can also occur without a resultant alteration in enzymic activity.
Project description:The mechanism by which phospholipase C (PLC) digestion of cultured cells mediates binding of CTP:phosphocholine cytidylyltransferase to cellular membranes was investigated. Incubation of choline-depleted rat hepatocytes with PLC caused a translocation of enzyme from cytosol to membranes concomitant with a decrease in the concentration of phosphatidylcholine with no effect on the concentration of other phospholipids. Removal of PLC and supplementation with choline restored the amount of phosphatidylcholine in the cells and translocated cytidylyltransferase to the cytosol. However, when phosphatidylcholine levels were decreased by incubation with phospholipase A2 (PLA2), there was no significant redistribution of cytidylyltransferase activity. With PLA2 the concentration of phosphatidylethanolamine, as well as of phosphatidylcholine, was significantly decreased. Since PLC, but not phospholipase A2, raised the cellular concentration of diacylglycerol, possibly diacylglycerol mediated the binding of cytidylyltransferase to membranes. This possibility was examined, but is unlikely, since addition of lysophosphatidylcholine to PLC-treated cells restored the concentration of phosphatidylcholine and released cytidylyltransferase into the cytosol, but did not lower diacylglycerol levels to normal values. Studies in vitro, incubations of cells with choline analogues and a survey of the literature suggested that the over-riding common factor in regulation of cytidylyltransferase binding to membranes may be the ratio of bilayer to non-bilayer lipids in that membrane.
Project description:Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.
Project description:Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS:AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.
Project description:When type II pneumonocytes from adult rats were maintained in a medium that lacked choline, the incorporation of [14C]glycerol into phosphatidylcholine was not greatly diminished during the period that the cells displayed characteristics of type II pneumonocytes. Cells that were maintained in choline-free medium that contained choline oxidase and catalase, however, became depleted of choline and subsequent synthesis of phosphatidylcholine by these cells was responsive to choline in the extracellular medium. Incorporation of [14C]glycerol into phosphatidylcholine by choline-depleted cells was stimulated maximally (approx. 6-fold) by extracellular choline at a concentration (0.05 mM) that also supported the greatest incorporation into phosphatidylglycerol. The incorporation of [14C]glycerol into other glycerophospholipids by choline-depleted cells was not increased by extracellular choline. When cells were incubated in the presence of [3H]cytidine, the choline-dependent stimulation of the synthesis of phosphatidylcholine and phosphatidylglycerol was accompanied by an increased recovery of [3H]CMP. This increased recovery of [3H]CMP reflected an increase in the intracellular amount of CMP from 48 +/- 9 to 76 +/- 16 pmol/10(6) cells. Choline-depleted cells that were exposed to [3H]choline contained [3H]CDP-choline as the principal water-soluble choline derivative. As the extracellular concentration of choline was increase, however, the amount of 3H in phosphocholine greatly exceeded that in all other water-soluble derivatives. Choline-depletion of cells resulted in an increase in the specific activity of CTP:phosphocholine cytidylyltransferase in cell homogenates (from 0.40 +/- 0.15 to 1.31 +/- 0.20 nmol X min-1 X mg of protein-1). These data are indicative that the biosynthesis of phosphatidylcholine is integrated with that of phosphatidylglycerol and are consistent with the proposed involvement of CMP in this integration. The choline-depleted type II pneumonocyte provides a new model for investigating the regulation of CTP:phosphocholine cytidylyltransferase activity.
Project description:It has been known for 40 years that oestrogens stimulate phospholipid metabolism in roosters. We have investigated in vivo the mechanism for this effect. Young roosters were injected daily with 1 mg of diethylstilboestrol for 1--3 days. At 4 h after the last injection, 30 microCi of [Me-3H]choline was injected into the portal vein. At periods up to 3 min the livers were freeze-clamped and choline and its metabolites were extracted and resolved by t.l.c. Hormone treatment in the first 2 days resulted in a 2-fold increase in phosphorylation of [Me-3H]choline and a decrease in the oxidation of [Me-3H]choline to [3H]betaine. The concentrations of phosphocholine in liver were increased 2-fold during the first 2 days concomitant with a 2-fold increase in the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, many of the above effects were reversed and the rate of phosphatidylcholine biosynthesis decreased to approx. 60% of the control value. The results suggest that the initial hormone treatments activate choline kinase within 4 h and, thereby, divert choline form oxidation to betaine. The resulting increased phosphocholine concentrations cause an increase in the activity of CTP:phosphocholine cytidylyltransferase, which results in a doubling of the rate of phosphatidylcholine biosynthesis. After 3 days of hormone treatment, the biosynthesis of phosphatidylcholine is decreased, most likely by an effect on the cytidylyltransferase reaction.
Project description:The specificity of the phospholipid head-group for feedback regulation of CTP: phosphocholine cytidylyltransferase was examined in rat hepatocytes. In choline-deficient cells there is a 2-fold increase in binding of cytidylyltransferase to cellular membranes, compared with choline-supplemented cells. Supplementation of choline-deficient cells with choline, dimethylethanolamine, monomethylethanolamine or ethanolamine resulted in an increase in the concentration of the corresponding phospholipid. Release of cytidylyltransferase into cytosol was only observed in hepatocytes supplemented with choline or dimethylethanolamine. The apparent EC50 values (concn. giving half of maximal effect) for cytidylyltransferase translocation were similar for choline and dimethylethanolamine (25 and 27 microM respectively). The maximum amount of cytidylyltransferase released into cytosol with choline supplementation (1.13 m-units/mg membrane protein) was twice that (0.62) observed with dimethylethanolamine. Supplementation of choline-deficient hepatocytes with NN'-diethylethanolamine, N-ethylethanolamine or 3-aminopropanol also did not cause release of cytidylyltransferase from cellular membranes. The translocation of cytidylyltransferase appeared to be mediated by the concentration of phosphatidylcholine in the membranes and not the ratio of phosphatidylcholine to phosphatidylethanolamine. The results provide further evidence for feedback regulation of phosphatidylcholine biosynthesis by phosphatidylcholine.
Project description:1. The cholesterol content, proportions of different phospholipids and fatty acid components of phosphatidylcholine and phosphatidylethanolamine were studied in rat liver endoplasmic-reticulum membrane, after a single injection of 20-methylcholanthrene or injections of phenobarbitone for 5 days. 2. A marked decrease in the proportion of cholesterol occurred 5 days after injection of 20-methylcholanthrene or phenobarbitone. 3. The proportion of phosphatidylcholine was increased by injection of phenobarbitone and minor changes occurred in other phospholipids. 4. Phenobarbitone caused the proportion of linoleic acid in phosphatidylcholine and phosphatidylethanolamine to increase to 120-125% of the control and the proportion of oleic acid, arachidonic acid and docosahexaenoic acid to decrease. 5. 20-Methylcholanthrene caused an increase in the proportion of oleic acid in phosphatidylcholine and ethanolamine to 125-140% of the control, 1 day after injection. 6. The increased proportion of linoleic acid in phosphatidylcholine after phenobarbitone injection occurs simultaneously with the increase of cytochrome P-450 concentration, the rate of oxidative demethylation of aminopyrine and the rate of hydroxylation of biphenyl. It is therefore considered that distinct species of phosphatidylcholine or phosphatidylethanolamine containing linoleic acid in the beta position are essential in the endoplasmic-reticulum membrane for optimal activity of oxidative demethylation.
Project description:The antagonization of phorbol 12-myristate 13-acetate (PMA)-stimulated phosphatidylcholine (PtdCho) biosynthesis by the phospholipid analogue hexadecylphosphocholine (HePC) in MDCK cells was investigated and compared with the corresponding influence in HeLa cells. In both cell lines, PMA-stimulated PtdCho biosynthesis was antagonized by 50 microM HePC. However, subsequent experiments provided evidence that PMA enhances PtdCho biosynthesis by at least two mechanisms: (i) by stimulation of choline uptake and (ii) by translocation of CTP:choline phosphate cytidylyltransferase to membranes. In MDCK cells, 5 nM PMA caused a 4-fold increase in [methyl-3H]choline incorporation into PtdCho, which was paralleled by an approx. 2-fold stimulation of choline uptake. These data indicate that choline uptake might play an important role in the regulation of PtdCho biosynthesis in this cell line, especially since we could not detect any significant increase in membrane-bound cytidyltransferase activity in PMA-treated MDCK cells. In contrast, enhanced PtdCho biosynthesis in HeLa cells is achieved by a 2-fold increase in particulate cytidylyltransferase activity after PMA stimulation. Translocation of cytidylyltransferase from the cytosol to membranes is therefore important in HeLa cells. Nevertheless, in both cell lines, the main target of HePC seems to be the translocation process. In MDCK cells, addition of 50 microM HePC decreases membrane-bound cytidylyltransferase activity by about 45%, compared with control cells and PMA-treated cells. In HeLa cells, PMA-induced translocation of cytidylyltransferase to membranes is totally abolished by HePC.