The Regulation of Phosphatidylcholine Synthesis at the Subcellular Level in Krebs II Ascite Cells

Tercé, François; Record, Michel; Ribbes, Gérard; Chap, Hugues; Douste‐Blazy, Louis

doi:10.1007/978-3-642-73184-6_5

Cited by 12 publications

(14 citation statements)

References 16 publications

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“…The purity of the preparations was assessed by electron microscopy (negatively stained in 2% uranyl acetate on carbon grids, and examined in a JEOL 200 CTX microscope) and by typical clathrin detection on SDS-PAGE. Furthermore, potential contaminations by other subcellular compartments were detected by measurement of specific enzymatic markers such as alkaline phosphatase or 5′-nucleotidase for plasmic membranes, galactosyltransferase for Golgi, lactate dehydrogenase for cytosol, NADHdehydrogenase for total endoplasmic reticulum, N-acetyl--D-glucosaminidase for lysosomes, and monoamine oxidase for mitochondria (Tercé et al, 1988). Possible contamination by caveolae vesicles was assessed by Western blotting using anti-caveolin antibody (Transduction Laboratories).…”

Section: Methodsmentioning

confidence: 99%

High-Density Lipoprotein 3 Receptor-Dependent Endocytosis Pathway in a Human Hepatoma Cell Line (HepG₂)

et al. 1996

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The internalization of HLD3 into HepG2 cells at 37 degrees C was precisely measured, taking advantage of the previously observed rapid dissociation of HDL3 from its two binding sites [Barbaras, R., et al. (1994) Biochemistry 33, 2335-2340]. We observed a high level of HDL3 internalization (100 ng/mg of cell protein, corresponding to 45.5% of the total HDL3 associated to the cells at 37 degrees C) reaching a plateau at 15 min. Apolipoprotein A-I (the main HDL3 apolipoprotein) associated with dimyristoylphosphatidylcholine (DMPC) complexes was also internalized by HepG2 cells, at levels comparable to those obtained with HDL3 lipid-free apolipoprotein A-I, which can bind only to the HDL3 high-affinity binding site, and displayed a weak internalization (5 ng internalized/mg of cell protein compared to 250 ng/mg for apolipoprotein A-I complexed with DMPC). Clathrin-coated vesicle purification following HDL3 or LDL internalization at 37 degrees C showed radioactivity associated with these vesicles, and further content analysis evidenced the presence of radiolabeled apoA-I and apoB, respectively. Treatment of the cells either by saccharose hypertonic shock or by potassium depletion, in order to block clathrin-coated vesicle formation, completely inhibited HDL3 internalization, as also observed with LDL. Altogether, these observations clearly demonstrate that HDL3 internalization into HepG2 cells occurs through an endocytosis pathway involving an interaction between apolipoprotein A-I and a cell surface protein, leading to the formation of clathrin-coated vesicles.

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Section: Methodsmentioning

confidence: 99%

High-Density Lipoprotein 3 Receptor-Dependent Endocytosis Pathway in a Human Hepatoma Cell Line (HepG₂)

et al. 1996

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“…CTP: phosphocholine cytidylyltransferase activity was assayed as previously described [7,25]. The incubation mixture contained 20 mM-Tris/succinate, pH 7.8, 6 mM-MgCl2, 8 mM-CTP, 4 mMphospho[methyl-'4C]choline (0.5 Ci/mol) and up to 300 ,ug of protein. A sonicated suspension of total lipid extract from Krebs II cells was added to the assay for cytosolic enzyme at a final concentration of I mm lipid P as previously described [7].…”

Section: Enzyme Assaymentioning

confidence: 99%

“…A translocation process between cytosol and membranes has been pointed out to regulate the enzyme activity, following activation of cells by different stimuli [4][5][6]. Thus we previously demonstrated that, upon stimulation of phosphatidylcholine metabolism in Krebs II cells treated by an exogenous phospholipase C acting on plasma membrane, cytidylyltransferase was translocated specifically on to the endoplasmic reticulum and not to the plasma membrane [7,8]. A variety of processes have been described to try to explain the activation and translocation of cytidylyltransferase, including phosphorylation/dephosphorylation [9,10], a fatty acid effect [11][12][13][14][15], diacylglycerol action [4,[7][8], hydrophobic interactions [13] or, more recently, regulation by the membrane phosphatidylcholine content [16].…”

Section: Introductionmentioning

confidence: 99%

“…Thus we previously demonstrated that, upon stimulation of phosphatidylcholine metabolism in Krebs II cells treated by an exogenous phospholipase C acting on plasma membrane, cytidylyltransferase was translocated specifically on to the endoplasmic reticulum and not to the plasma membrane [7,8]. A variety of processes have been described to try to explain the activation and translocation of cytidylyltransferase, including phosphorylation/dephosphorylation [9,10], a fatty acid effect [11][12][13][14][15], diacylglycerol action [4,[7][8], hydrophobic interactions [13] or, more recently, regulation by the membrane phosphatidylcholine content [16]. Among all the stimuli, oleic acid was found to be the most potent activator of phosphatidylcholine synthesis, and we recently demonstrated that Krebs II cells stimulated by oleic acid increase phosphatidylcholine synthesis through translocation of both oleic acid and cytidylyltransferase to the endoplasmic reticulum, without increase in total cell phosphatidylcholine mass [17].…”

Section: Introductionmentioning

confidence: 99%

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Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells

Tercé

Record

Ribbes

et al. 1992

Biochemical Journal

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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 2.7.7.15), 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.

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“…The only marker found to coincide with acetyltransferase was the radioactivity of [ 3H]uridine previously incorporated into intact cells and precipitable with trichloroacetic acid. As discussed in a very recent work [25], this corresponds to RNA and allows identification of a specific subfraction of endoplasmic reticulum that is particularly rich in ribosomes ("heavyrough'' endoplasmic reticulum). A similar subfraction was previously observed in the same cell by Pryme et al [26], who found it to be specific for tumor cells.…”

Section: Subcellular Distribution Of Acetyltransferase and Alkylacyl-mentioning

confidence: 99%

Subcellular localization of phospholipids and enzymes involved in PAF‐acether metabolism

Record

Ribbes

Tercé

et al. 1989

J of Cellular Biochemistry

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The biosynthesis of platelet-activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187. A similar study was undertaken on the tumor strain Krebs-II cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl-GPC, the source of PAF-precursor alkyl-lyso-GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A2 in providing lyso-PAF to the acetyltransferase to form PAF-acether.

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The Regulation of Phosphatidylcholine Synthesis at the Subcellular Level in Krebs II Ascite Cells

Cited by 12 publications

References 16 publications

High-Density Lipoprotein 3 Receptor-Dependent Endocytosis Pathway in a Human Hepatoma Cell Line (HepG₂)

High-Density Lipoprotein 3 Receptor-Dependent Endocytosis Pathway in a Human Hepatoma Cell Line (HepG₂)

Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells

Subcellular localization of phospholipids and enzymes involved in PAF‐acether metabolism

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The Regulation of Phosphatidylcholine Synthesis at the Subcellular Level in Krebs II Ascite Cells

Cited by 12 publications

References 16 publications

High-Density Lipoprotein 3 Receptor-Dependent Endocytosis Pathway in a Human Hepatoma Cell Line (HepG2)

High-Density Lipoprotein 3 Receptor-Dependent Endocytosis Pathway in a Human Hepatoma Cell Line (HepG2)

Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells

Subcellular localization of phospholipids and enzymes involved in PAF‐acether metabolism

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Product

Resources

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High-Density Lipoprotein 3 Receptor-Dependent Endocytosis Pathway in a Human Hepatoma Cell Line (HepG₂)

High-Density Lipoprotein 3 Receptor-Dependent Endocytosis Pathway in a Human Hepatoma Cell Line (HepG₂)