The phosphatidylinositol transfer protein in 3T3 mouse fibroblast cells is associated with the Golgi system

Snoek, Gerry T.; Wit, I. S. C. de; Mourik, J. H. G. van; Wirtz, K.W.A.

doi:10.1002/jcb.240490404

Cited by 39 publications

(30 citation statements)

References 35 publications

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“…Total cell lysates were obtained by lysis of the cells in 50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 g/ml leupeptin, 10 mM NaF. Subsequently, proteins were separated by SDS-polyacrylamide gel electrophoresis in 12% acrylamide and 0.37% bis-acrylamide [12] and transferred to a nitrocellulose membrane (Schleicher & Schuell BA 85) by semidry Western blotting in a Multiphor II Nova Blot electrophoretic transfer unit (Pharmacia, Uppsala, Sweden) at 1 mA/cm 2 for 1 h at room temperature. The nonspecific binding sites of the nitrocellulose membrane were blocked by incubating the membrane for 1 h in 2% nonfat dry milk (w/v) in 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl (TBS) at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Clofibrate-Induced Relocation of Phosphatidylcholine Transfer Protein to Mitochondria in Endothelial Cells

Brouwer

Westerman

Kleinnijenhuis

et al. 2002

Experimental Cell Research

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The phosphatidylcholine transfer protein (PC-TP) is a specific transporter of phosphatidylcholine (PC) between membranes. To get more insight into its physiological function, we have studied the localization of PC-TP by microinjection of fluorescently labeled PC-TP in foetal bovine heart endothelial (FBHE) cells and by expression of an enhanced yellow fluorescent protein-PC-TP fusion protein in FBHE cells, human umbilical vein endothelial cells, and HepG2 cells. Analysis by confocal laser scanning microscopy showed that PC-TP was evenly distributed throughout the cytosol with an apparently elevated level in nuclei. By measuring the fluorescence recovery after bleaching it was established that PC-TP is highly mobile throughout the cell, with its transport into the nucleus being hindered by the nuclear envelope. Given the proposed function of PC-TP in lipid metabolism, we have tested a number of compounds (phorbol ester, bombesin, A23187, thrombin, dibutyryl cyclic AMP, oleate, clofibrate, platelet-derived growth factor, epidermal growth factor, and hydrogen peroxide) for their ability to affect intracellular PC-TP distribution. Only clofibrate (100 M) was found to have an effect, with PC-TP moving to mitochondria within 5 min of stimulation. This relocation did not occur with PC-TP(S110A), lacking the putative protein kinase C (PKC)-dependent phosphorylation site, and was restricted to the primary endothelial cells. Relocation did not occur in HepG2 cells, possibly due to the fact that clofibrate does not induce PKC activation in these cells.

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Section: Methodsmentioning

confidence: 99%

Clofibrate-Induced Relocation of Phosphatidylcholine Transfer Protein to Mitochondria in Endothelial Cells

Brouwer

Westerman

Kleinnijenhuis

et al. 2002

Experimental Cell Research

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“…The anti-PI-TP antibodies were raised in rabbits against synthetic peptides representing the amino acid sequence of predicted epitopes in rat brain PI-TP␣ (39). Geneticin G418 and goat anti-rabbit IgG conjugated with alkaline phosphatase were obtained from Sigma.…”

Section: Methodsmentioning

confidence: 99%

“…Localization studies by indirect immunofluorescence and by microinjection of fluorescently labeled purified PI-TP␣ and PI-TP␤ into intact mammalian cells have shown that PI-TP␣ is mainly localized in the nucleus and in cluster-like structures in the cytosol and that PI-TP␤ is mainly associated with the Golgi membranes (3,4,39,40). However, upon stimulation of the cells by different growth factors (bombesin, PDGF) that stimulate the phospholipase C-dependent degradation of PtdIns(4,5)P 2 , accumulation of PI-TP␣ near the plasma membrane was not observed.…”

Section: The Addition Of Pi-tp␣ To a Total Lysate Of Myo-[mentioning

confidence: 99%

“…The homogenate was centrifuged for 10 min at 17,000 ϫ g, and the A 280 of the supernatant was used to calculate the protein content. 17.5 g of supernatant protein was loaded on an SDS-polyacrylmide gel, and gel electrophoresis was performed as described (39). The proteins were electrophoretically transferred to a nitrocellulose sheet in a Multiphor II Nova Blot electrophoretic transfer unit (Amersham Pharmacia Biotech) at room temperature applying 1 mA/cm 2 of gel for 2 h, and PI-TP␣ was detected as described (39).…”

Section: ϩmentioning

confidence: 99%

“…17.5 g of supernatant protein was loaded on an SDS-polyacrylmide gel, and gel electrophoresis was performed as described (39). The proteins were electrophoretically transferred to a nitrocellulose sheet in a Multiphor II Nova Blot electrophoretic transfer unit (Amersham Pharmacia Biotech) at room temperature applying 1 mA/cm 2 of gel for 2 h, and PI-TP␣ was detected as described (39). Quantification of the PI-TP␣ levels on an immunoblot was performed by scanning with a BioRad GS 700 imaging densitometer equipped with an integrating program, with known PI-TP␣ concentrations as a standard.…”

Section: ϩmentioning

confidence: 99%

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Overexpression of Phosphatidylinositol Transfer Protein α in NIH3T3 Cells Activates a Phospholipase A

Snoek¹,

Berrie²,

Geijtenbeek³

et al. 1999

Journal of Biological Chemistry

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In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein ␣ (PI-TP␣), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TP␣. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2-and 3-fold increase, respectively, in the level of PI-TP␣. Overexpression of PI-TP␣ resulted in a decrease in the duration of the cell cycle from 21 h for the wild type Upon equilibrium labeling of the cells with myo-[ 3 H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-bisphosphate were the same as those in control cells. The addition of PI-TP␣ to a total lysate of myo-[3 H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca 2؉ further increased this formation. Based on these observations, we propose that PI-TP␣ is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (plateletderived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.Phospholipid transfer proteins are proteins that are able to transfer phospholipids between membranes in vitro. A major phospholipid transfer protein in mammalian tissues is the phosphatidylinositol transfer protein (PI-TP) 1 (1). Recently, two isoforms of PI-TP have been identified (i.e. PI-TP␣ and PI-TP␤) that demonstrate differences in cellular localization and in specific lipid transfer activity (2-5).PI-TP␣ has been purified from both rat and bovine brain (6, 7). Cloning of the cDNA encoding rat brain PI-TP␣ showed that the protein consists of 271 amino acid residues (8). The subsequent isolation of the cDNAs encoding mouse and human PI-TP␣ revealed a high homology between the different mammalian PI-TPs (about 99% amino acid sequence identity) (9, 10). Furthermore, the cross-reactivity of the antibodies raised against bovine PI-TP␣ with a 35-kDa protein from other animals (e.g. rat, mouse, chicken, frog, and lizard) indicates an extensive conservation of the amino acid sequence between species (11). An exception is PI-TP from yeast (i.e. SEC14p) that has the same molecular weight as mammalian PI-TP and comparable phospholipid transfer activities yet shows no homology in the amino acid sequence (12)(13)(14).So far, very little is known about the precise cellular role of mammalian PI-TP␣. Since PI-TP␣ is able to transfer in vitro PtdIns between membrane...

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Phosphatidylinositol transfer proteins: a requirement in signal transduction and vesicle traffic

Cockcroft

1998

Bioessays

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The phosphatidylinositol transfer protein in 3T3 mouse fibroblast cells is associated with the Golgi system

Cited by 39 publications

References 35 publications

Clofibrate-Induced Relocation of Phosphatidylcholine Transfer Protein to Mitochondria in Endothelial Cells

Clofibrate-Induced Relocation of Phosphatidylcholine Transfer Protein to Mitochondria in Endothelial Cells

Overexpression of Phosphatidylinositol Transfer Protein α in NIH3T3 Cells Activates a Phospholipase A

Phosphatidylinositol transfer proteins: a requirement in signal transduction and vesicle traffic

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