scholarly journals Subcellular localization of inositol lipids in blood platelets as deduced from the use of labelled precursors

1987 ◽  
Vol 244 (3) ◽  
pp. 757-761 ◽  
Author(s):  
G Mauco ◽  
P Dajeans ◽  
H Chap ◽  
L Douste-Blazy
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Blood Platelets ◽  
Blood Platelet ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Inositol Lipids ◽  
Hormone Sensitive ◽  
Reported Data ◽  
Phosphatidylinositol 4

1. By rapid fractionation of blood platelet lysates on Percoll density gradients at alkaline pH (9.6), a very pure plasma-membrane fraction was obtained, as well as discrimination between endoplasmic reticulum and lysosomes. 2. Labelling of intact platelets with [32P]Pi followed by subcellular fractionation showed an exclusive localization of all inositol lipids in the plasma membrane. 3. Preincubation of whole platelets with myo-[3H]inositol in a buffer containing 1 mM-MnCl2 allowed incorporation of the label into PtdIns (phosphatidylinositol) of both plasma and endoplasmic-reticulum membrane, whereas [3H]PtdIns4P (phosphatidylinositol 4-phosphate) and [3H]PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) were exclusively found on the plasma membrane. 4. It is concluded that PtdIns4P and PtdIns(4,5)P2 are exclusively localized in the plasma membrane, whereas PtdIns is present in both plasma and endoplasmic-reticulum membranes. This could provide an explanation for previously reported data on hormone-sensitive and -insensitive inositol lipid pools.

scholarly journals Subcellular distribution of selenium in deficient mouse liver

1989 ◽  
Vol 258 (2) ◽  
pp. 541-545 ◽  
Author(s):  
R Reiter ◽  
R Otter ◽  
A Wendel
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Trace Element ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Fast Response ◽  
Cell Compartment ◽  
Deficient Mice

Selenium (Se)-deficient mice were labelled in vivo with single pulses of [75Se]selenite, and the intrahepatic distribution of the trace element was studied by subcellular fractionation. At 1 h after intraperitoneal injection of 3.3 or 10 micrograms of Se/kg body weight, 15% of the respective doses were found in the liver. Accumulation in the subcellular fractions followed the order: Golgi vesicular much greater than lysosomal greater than cytosolic = microsomal greater than mitochondrial, peroxisomal, nuclear and plasma-membrane fraction. At a dose of 3.3 micrograms/kg, more than 90% of the hepatic Se was protein-bound. When cross-contamination was accounted for, the following specific Se contents of the subcellular compartments were extrapolated: Golgi apparatus, 7.50 pmol/mg; cytosol, 0.90 pmol/mg; endoplasmic reticulum, 0.80 pmol/mg; mitochondria, 0.49 pmol/mg; nuclei, lysosomes, peroxisomes and plasma membrane, less than 0.4 pmol/mg. At 10 micrograms/kg, a roughly 2-3-fold increase in Se content of all fractions was found without major changes in the intrahepatic distribution pattern. An extraordinary rise in the cytosolic fraction was due to an apparently non-protein-bound Se pool. At 24 h after dosing, total hepatic Se had decreased to 6% of the initial dose and had become predominantly protein-bound. The 60% decrease in hepatic Se was reflected in a similar fall in the subcellular levels of the trace element. The Golgi apparatus still had the highest specific Se content, although accumulation was 5 times less than that after 1 h. The cytosolic pool accounted for 50% of the hepatic Se at both labelling times. After 1 h the Golgi apparatus was, with 19%, the second largest intrahepatic pool, followed by the endoplasmic reticulum with 16%. The high affinity and fast response of the Golgi apparatus to Se supplementation of deficient mice is interpreted in terms of a predominant function of this cell compartment in the processing and the export of Se-proteins from the liver.

scholarly journals TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (8) ◽  
pp. 2231-2247 ◽  
Author(s):  
Julia D. Romano ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Eukaryotic Proteins ◽  
Internal Site ◽  
Membrane Spanning ◽  
Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

Biochemical approaches to the study of cytosolic calcium regulation in nerve endings

1981 ◽  
Vol 296 (1080) ◽  
pp. 115-122 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Steady State ◽  
Nerve Ending ◽  
Cytosolic Calcium ◽  
Endoplasmic Reticulum Membrane ◽  
Mitochondrial Inner Membrane ◽  
Voltage Dependent ◽  
Minor Significance

The nerve ending cytosol is bounded by the plasma membrane, the mitochondrial inner membrane and the endoplasmic reticulum membrane, transport across each of which is capable, in theory, of regulating the cytosolic free Ca 2+ concentration. By parallel monitoring of mitochondrial and plasma membrane potentials, ATP levels, Na + gradients and intrasynaptosomal Ca 2+ distribution in preparations of isolated synaptosomes, we conclude the following: ( a ) mitochondria in situ represent a major Ca 2+ pool, regulating the upper steady-state limit of the cytosolic free Ca 2+ concentration by sequestering Ca 2+ reversibly; ( b ) this limit is responsive to the cytosolic Na + concentration, but is below the concentration required for significant exocytosis; ( c ) plasma membrane Ca 2+ transport can be resolved into a constant slow influx, a voltage-dependent and verapamil-sensitive influx and an ATP-dependent efflux, while Ca 2+ efflux driven by the sodium electrochemical potential cannot be detected; ( d ) Ca 2+ regulation by intrasynaptosomal endoplasmic reticulum appears to be of minor significance in the present preparation.

scholarly journals Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase IIIα

2008 ◽  
Vol 19 (2) ◽  
pp. 711-721 ◽  
Author(s):  
Andras Balla ◽  
Yeun Ju Kim ◽  
Peter Varnai ◽  
Zsofia Szentpetery ◽  
Zachary Knight ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Small Interfering Rna ◽  
Type Ii ◽  
Measurable Effect ◽  
Down Regulation ◽  
Type Iii ◽  
Hormone Sensitive ◽  
Phosphatidylinositol 4 ◽  
Interfering Rna

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca2+ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2 levels in 32P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2 resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.

scholarly journals Synaptotagmins Maintain Diacylglycerol Homeostasis at Endoplasmic Reticulum-Plasma Membrane Contact Sites during Abiotic Stress

2020 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P. Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Phosphatidylinositol 4 ◽  
Polar Glycerolipids

SUMMARYEndoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

Coated Pits and Vesicles Transfer Plasma Components to Platelet Granules

1989 ◽  
Vol 62 (02) ◽  
pp. 718-722 ◽  
Author(s):  
O Behnke
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Blood Platelets ◽  
Fluid Phase ◽  
Membrane System ◽  
Blood Platelet ◽  
Ambient Fluid ◽  
Coated Pits ◽  
Initial Fixation ◽  
Mammalian Blood

SummaryElectron microscopy of mammalian blood platelets after contrast enhancement with tannic acid after an initial fixation in glutaraldehyde and osmium reveals numerous coated pits (c.p.) and vesicles (c.v.), indicating a process of receptor-mediated endocytosis. The c.p. may be located at any site of the plasma membrane or the canalicular, surface connected membrane system. C.v. fuse with platelet granules without losing their coat. Evidence for a continuous transfer of ambient fluid to granules via c. p. and c.v. was obtained by the use of fluid-phase markers.It is proposed that the endocytic process may play a role in blood platelet activation.

scholarly journals Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells

1996 ◽  
Vol 320 (3) ◽  
pp. 885-890 ◽  
Author(s):  
Christina DECKER ◽  
Maria Jesus MIRO OBRADORS ◽  
Daniel J. SILLENCE ◽  
David ALLAN
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Phospholipase D ◽  
Phorbol Ester ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Site Of Action

The localization of phorbol ester-sensitive phospholipase D (PLD) in baby hamster kidney cells has been investigated by determining the subcellular distribution of the phosphatidylbutanol produced when the cells are incubated with phorbol 12-myristate 13-acetate and n-butanol. Results derived by isolation of plasma membrane vesicles from intact cells or by subcellular fractionation on a sucrose density gradient suggest the PLD is specific for phosphatidylcholine and its primary site of action is not the plasma membrane but the endoplasmic reticulum.

scholarly journals Insulin secretory granule biogenesis. Co-ordinate regulation of the biosynthesis of the majority of constituent proteins

1991 ◽  
Vol 274 (1) ◽  
pp. 73-78 ◽  
Author(s):  
P C Guest ◽  
E M Bailyes ◽  
N G Rutherford ◽  
J C Hutton
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Granules ◽  
Signal Recognition Particle ◽  
Electrophoretic Analysis ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Translational Initiation ◽  
Insulin Secretory Granule ◽  
Fold Stimulation

Two-dimensional gel-electrophoretic analysis combined with fluorography and densitometric quantification was used to examine the effects of glucose on the biosynthesis of rat pancreatic islet proteins. An increase in the medium glucose concentration from 2.8 to 16.7 mM produced a 10-20 fold stimulation in the synthesis of 10 out of 260 detected islet proteins, as judged by incorporation of [35S]methionine during a 20 min incubation. The synthetic rates of the majority of the remaining proteins were stimulated by 2-4-fold. Greater resolution achieved by pulse-chase labelling and subcellular fractionation showed that, of 32 major proteins localized to insulin secretory granules, the biosynthesis of 25 were stimulated 15-30-fold by glucose. By contrast, only eight of 160 proteins in the soluble fraction showed a response of similar magnitude. It is concluded that there is a major and co-ordinated activation of the biosyntheses of proteins destined for secretory granules, which most likely occurs at the level of translational initiation and signal-recognition-particle-mediated translocation into the endoplasmic reticulum lumen. However, it is clear that not all granule proteins, or the majority of proteins translocated across the endoplasmic reticulum membrane, are affected in an equivalent manner. In addition, the synthesis of a small number of cytosolic proteins may be increased markedly by insulinotropic stimuli.

scholarly journals Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface

1997 ◽  
Vol 138 (2) ◽  
pp. 323-330 ◽  
Author(s):  
Harry F.G. Heijnen ◽  
Viola Oorschot ◽  
Jan J. Sixma ◽  
Jan W. Slot ◽  
David E. James
Keyword(s):  
Plasma Membrane ◽  
Energy Metabolism ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Shape Change ◽  
Blood Platelets ◽  
Blood Platelet ◽  
Clot Retraction ◽  
Isolated Cells

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (∼15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.

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