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

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

Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.     

Plasma membrane—endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid‐synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)—ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N‐methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM–ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.     

Serum protein synthesis in mutant mice with abnormal hepatic endoplasmic reticulum.

Severe ultrastructural abnormalities of liver endoplasmic reticulum have been described in newborn mice homozygous for radiation-induced deletion alleles at the colour locus. The ultrastructural defects were accompanied by deficiencies of several enzymes and lowered serum protein levels. Studies on serum protein synthesis were undertaken to see if decreased rates of synthesis, especially of constituents thought to be synthesized on membrane-bound ribosomes, were the cause of the deficiencies. Although decreases or absence of several serum proteins were shown, radiopulse-immunoprecipitation studies of albumin and fibrinogen synthesis suggested that the decreased synthesis rates were a secondary defect. Serum glycoproteins were not altered more than other constituents in the mutant material.     

Cytidylyltransferase translocation onto endoplasmic reticulum and increased de novo synthesis without phosphatidylcholine accumulation in Krebs-II ascite cells.

Addition of oleic acid to Krebs-II cells stimulated by 9-fold [3H]choline incorporation into choline glycerophospholipids without affecting the selective incorporation of the precursor into diacyl subclass (90% of total [3H]choline glycerophospholipids). The total activity of cytidylyltransferase (E.C. 2.7.7.15), the regulatory enzyme of choline glycerophospholipid synthesis, was increased in the particulate fraction at the expense of cytosol. Free [3H]oleic acid was also associated with the particulate fraction. Subcellular fractionation of membranes on Percoll gradient, indicated that the endoplasmic reticulum, which contained 90% of total cell free oleic acid, was the unique target for the translocation of cytidylyltransferase. [3H]oleic acid was incorporated almost exclusively into phosphatidylcholine and corresponded to a synthesis of 9 nmol/h per 10(6) cells. Based on [3H]choline incorporation a net synthesis of 22 nmol/h per 10(6) cells was determined. However, oleic acid treatment did not change the total amount of phosphatidylcholine (45 nmol/10(6) cells) and other phospholipids; also no modification in the subcellular distribution of phospholipids was observed. It is concluded that the stimulation of the de novo synthesis of phosphatidylcholine which involves translocation of cytidylyltransferase onto the endoplasmic reticulum, is accompanied by a renewal of their polar head group. Also exogenous oleic acid induces an enhanced fatty acid turnover, highly specific for phosphatidylcholine. Therefore, Krebs-II cells exhibited a high degree of regulation of their phosphatidylcholine content, suggesting a parallel stimulation of both synthesis and degradation.     

Phosphatidylinositol synthesis at the endoplasmic reticulum

Phosphatidylinositol (PI) is a minor phospholipid with a characteristic fatty acid profile; it is highly enriched in stearic acid at the sn-1 position and arachidonic acid at the sn-2 position. PI is phosphorylated into seven specific derivatives, and individual species are involved in a vast array of cellular functions including signalling, membrane traffic, ion channel regulation and actin dynamics. De novo PI synthesis takes place at the endoplasmic reticulum where phosphatidic acid (PA) is converted to PI in two enzymatic steps. PA is also produced at the plasma membrane during phospholipase C signalling, where hydrolysis of phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) leads to the production of diacylglycerol which is rapidly phosphorylated to PA. This PA is transferred to the ER to be also recycled back to PI. For the synthesis of PI, CDP-diacylglycerol synthase (CDS) converts PA to the intermediate, CDP-DG, which is then used by PI synthase to make PI. The de novo synthesised PI undergoes remodelling to acquire its characteristic fatty acid profile, which is altered in p53-mutated cancer cells. In mammals, there are two CDS enzymes at the ER, CDS1 and CDS2. In this review, we summarise the de novo synthesis of PI at the ER and the enzymes involved in its subsequent remodelling to acquire its characteristic acyl chains. We discuss how CDS, the rate limiting enzymes in PI synthesis are regulated by different mechanisms. During phospholipase C signalling, the CDS1 enzyme is specifically upregulated by cFos via protein kinase C.     

Assembly of the endoplasmic reticulum phospholipid bilayer: the phosphatidylcholine transporter

Phospholipids are synthesized and concomitantly inserted on the cytoplasmic surface of the endoplasmic reticulum. Assembly of the phospholipid bilayer requires translocation to the lumenal monolayer. The hypothesis that rat liver microsomes contain a protein transporter, or "flippase," for phosphatidylcholine was tested by measuring the transport of sn-1,2,-dibutyroylphosphatidylcholine (diC4PC). This homolog retains the polar head group, the portion of the phospholipid unable to undergo spontaneous transmembrane movement in vesicles, and its water solubility permits application of standard transport methods. DiC4PC entered the lumenal compartment of microsomal vesicles. Transport was saturable and was dependent on time, amount of microsomes, and an intact permeability barrier. DiC4PC transport was inhibited by structural analogs (but not by sn-2,3-diC4PC) and by treatment of microsomes with proteases, N-ethylmaleimide, and trinitrobenzenesulfonic acid. These data suggest that the transmicrosomal movement of diC4PC is protein mediated. DiC4PC was not transported across PC vesicles or red cell membranes, where PC translocation is slow.     

Characterization of sulfate transport in the hepatic endoplasmic reticulum

The transport of sulfate ion across the endoplasmic reticulum membrane was investigated using rapid filtration and light scattering assays. We found a protein-mediated, bi-directional, low-affinity, and high-capacity, facilitative sulfate transport in rat liver microsomes, which could be inhibited by the prototypical anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. It was resistant to various phosphate transport inhibitors and was not influenced by high concentration of phosphate or pyrophosphate, which is contradictory to involvement of phosphate transporters. It was sensitive to S3483 that has been reported to inhibit the glucose 6-phosphate transporter (G6PT), but the weak competition between sulfate and glucose 6-phosphate did not confirm the participation of this transporter. Moreover, the comparison of the activity and S3483 sensitivity of sulfate transport in microsomes prepared from G6PT-overexpressing or wild type COS-7 cells did not show any significant difference. Our results indicate that sulfate fluxes in the endoplasmic reticulum are mediated by a novel, S3483-sensitive transport pathway(s).     

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

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

Assembly of the endoplasmic reticulum phospholipid bilayer. Transporters for phosphatidylcholine and metabolites

Phosphatidylcholine is synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported to the lumenal monolayer by a protein transporter, a phosphatidylcholine "flippase" (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). Since the endoplasmic reticulum contains enzymes involved in phosphatidylcholine turnover that have different locations within the organelle, transport systems may exist for phosphatidylcholine metabolites. To test the hypothesis that rat liver microsomes contain a lysophosphatidylcholine transporter, sn-1-monobutyroylphosphatidylcholine was employed. Since this homolog is highly water-soluble, transport of lysophosphatidylcholine could be measured using standard transport methods. sn-1-Monobutyroylphosphatidylcholine entered the lumenal compartment of microsomal vesicles. Transport was saturable and dependent on time and on amount of microsomes and required an intact permeability barrier. sn-1-Monobutyroylphosphatidylcholine transport was inhibited by treatment of microsomes with trypsin, N-ethylmaleimide, and trinitrobenzene-sulfonic acid. These findings suggest that sn-1-monobutyroylphosphatidylcholine transport is protein-mediated. sn-1-Monobutyroylphosphatidylcholine transported into microsomes was degraded to glycerophosphorylcholine. Glycerophosphorylcholine was also transported across the microsomal membrane. Glycerophosphorylcholine transport was also saturable and dependent on time, amount of microsomes, and an intact permeability barrier but was not inhibited by treatment with trypsin or the two protein modification agents. Thus, separate and distinct transport systems exist for phosphatidylcholine metabolites. Molecular events of phosphatidylcholine turnover in the endoplasmic reticulum are discussed.     

Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum

BACKGROUND: A long-standing problem in understanding the mechanism by which the phospholipid bilayer of biological membranes is assembled concerns how phospholipids flip back and forth between the two leaflets of the bilayer. This question is important because phospholipid biosynthetic enzymes typically face the cytosol and deposit newly synthesized phospholipids in the cytosolic leaflet of biogenic membranes such as the endoplasmic reticulum (ER). These lipids must be transported across the bilayer to populate the exoplasmic leaflet for membrane growth. Transport does not occur spontaneously and it is presumed that specific membrane proteins, flippases, are responsible for phospholipid flip-flop. No biogenic membrane flippases have been identified and there is controversy as to whether proteins are involved at all, whether any membrane protein is sufficient, or whether non-bilayer arrangements of lipids support flip-flop. RESULTS: To test the hypothesis that specific proteins facilitate phospholipid flip-flop in the ER, we reconstituted transport-active proteoliposomes from detergent-solubilized ER vesicles under conditions in which protein-free liposomes containing ER lipids were inactive. Transport was measured using a synthetic, water-soluble phosphatidylcholine and was found to be sensitive to proteolysis and associated with proteins or protein-containing complexes that sedimented operationally at 3.8S. Chromatographic analyses indicated the feasibility of identifying the transporter(s) by protein purification approaches, and raised the possibility that at least two different proteins are able to facilitate transport. Calculations based on a simple reconstitution scenario suggested that the transporters represent approximately 0.2% of ER membrane proteins. CONCLUSIONS: Our results clearly show that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane. These proteins are likely to be the flippases, which are required to translocate natural phosphatidylcholine and other phospholipids across the ER membrane. The methodology that we describe paves the way for identification of a flippase.     

Depletion of phosphatidylcholine affects endoplasmic reticulum morphology and protein traffic at the Golgi complex

The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their phosphatidylcholine (PC) level within 24 h when cultured at the nonpermissive temperature (40°C). This is due to a relative rapid breakdown of PC that is not compensated for by the inhibition of de novo PC synthesis. Despite this drastic decrease in cellular PC content, cells are viable and can proliferate by addition of lysophosphatidylcholine. By [³H]oleate labeling, we found that the FA moiety of the degraded PC is recovered in triacylglycerol. In accordance with this finding, an accumulation of lipid droplets is seen in MT58 cells. Analysis of PC-depleted MT58 cells by electron and fluorescence microscopy revealed a partial dilation of the rough endoplasmic reticulum, resulting in spherical structures on both sites of the nucleus, whereas the morphology of the plasma membrane, mitochondria, and Golgi complex was unaffected. In contrast to these morphological observations, protein transport from the ER remains intact. Surprisingly, protein transport at the level of the Golgi complex is impaired. Our data suggest that the transport processes at the Golgi complex are regulated by distal changes in lipid metabolism.     

Polyamine-dependent alterations in the structure of microfilaments,golgi apparatus,endoplasmic reticulum,and proteoglycan synthesis in BHK cells

The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, α-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6-NBD-ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a β-tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [³H]glucosamine and [³⁵S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine-deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation. J. Cell Biochem. 66:165-174, 1997. © 1997 Wiley-Liss, Inc.