Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum.

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.

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Reversible Ca Gradients between the Subplasmalemma and Cytosol Differentially Activate Ca-dependent Cl Currents

The Journal of General Physiology ◽

10.1085/jgp.113.2.249 ◽

1999 ◽

Vol 113 (2) ◽

pp. 249-266 ◽

Cited By ~ 33

Author(s):

Khaled Machaca ◽

H. Criss Hartzell

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Confocal Microscopy ◽

Driving Force ◽

Xenopus Oocytes ◽

Biophysical Properties ◽

Temporal Features ◽

Cl Channels ◽

Spatio Temporal ◽

Stimulation Of

Xenopus oocytes express several different Ca-activated Cl currents that have different waveforms and biophysical properties. We compared the stimulation of Ca-activated Cl currents measured by two-microelectrode voltage clamp with the Ca transients measured in the same cell by confocal microscopy and Ca-sensitive fluorophores. The purpose was to determine how the amplitude and/or spatio-temporal features of the Ca signal might explain how these different Cl currents were activated by Ca. Because Ca release from stores was voltage independent, whereas Ca influx depended upon the electrochemical driving force, we were able to separately assess the contribution of Ca from these two sources. We were surprised to find that Ca signals measured with a cytosolic Ca-sensitive dye, dextran-conjugated Ca-green-1, correlated poorly with Cl currents. This suggested that Cl channels located at the plasma membrane and the Ca-sensitive dye located in the bulk cytosol were sensing different [Ca]. This was true despite Ca measurement in a confocal slice very close to the plasma membrane. In contrast, a membrane-targeted Ca-sensitive dye (Ca-green-C18) reported a Ca signal that correlated much more closely with the Cl currents. We hypothesize that very local, transient, reversible Ca gradients develop between the subplasmalemmal space and the bulk cytosol. [Ca] is higher near the plasma membrane when Ca is provided by Ca influx, whereas the gradient is reversed when Ca is released from stores, because Ca efflux across the plasma membrane is faster than diffusion of Ca from the bulk cytosol to the subplasmalemmal space. Because dissipation of the gradients is accelerated by inhibition of Ca sequestration into the endoplasmic reticulum with thapsigargin, we conclude that [Ca] in the bulk cytosol declines slowly partly due to futile recycling of Ca through the endoplasmic reticulum.

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The sarcoplasmic–endoplasmic reticulum Ca2+ ATPase 2b regulates the Ca2+ transients elicited by P2Y2 activation in PC Cl3 thyroid cells

Journal of Endocrinology ◽

10.1677/joe.1.06455 ◽

2006 ◽

Vol 190 (3) ◽

pp. 641-649 ◽

Cited By ~ 4

Author(s):

Luca Ulianich ◽

Maria Giovanna Elia ◽

Antonella Sonia Treglia ◽

Antonella Muscella ◽

Bruno Di Jeso ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Thyroid Cell ◽

Myristate Acetate ◽

Thyroid Cells ◽

Pkc Inhibitor ◽

Kinase C ◽

Thyroid Cell Line ◽

Rat Thyroid ◽

Stimulation Of

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2 purinoceptor activation provoked a transient increase of [Ca2+]i, followed by a decreasing sustained phase. The α and β1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca2+]i level and increased the peak of Ca2+ entry of the P2Y2-provoked Ca2+transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2+ and Ba2+ uptake were not changed by Gö 6976. Similarly, the Na+/Ca2+ exchanger was not implicated, since the rate of decrement to the basal [Ca2+]i level was equally decreased in physiological and Na+-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2+ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca2+]i level after P2Y2 stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2+ transients caused by P2Y2 stimulation.

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Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle

Biochemical Journal ◽

10.1042/bj2310737 ◽

1985 ◽

Vol 231 (3) ◽

pp. 737-742 ◽

Cited By ~ 7

Author(s):

J Verbist ◽

F Wuytack ◽

L Raeymaekers ◽

R Casteels

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Muscle ◽

Plasma Membrane ◽

Atpase Activity ◽

Membrane Fraction ◽

Transport Atpase ◽

Calmodulin Binding ◽

Dependent Atpase ◽

Inhibitory Antibodies ◽

Stimulation Of

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.

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Plasma membrane—endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis

EMBO Reports ◽

10.1038/embor.2013.36 ◽

2013 ◽

Vol 14 (5) ◽

pp. 434-440 ◽

Cited By ~ 74

Author(s):

Shabnam Tavassoli ◽

Jesse T Chao ◽

Barry P Young ◽

Ruud C Cox ◽

William A Prinz ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Contact Sites ◽

Phosphatidylcholine Synthesis

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Intracellular Ca2+Release Triggers Translocation of Membrane Marker FM1–43 from the Extracellular Leaflet of Plasma Membrane into Endoplasmic Reticulum in T Lymphocytes

Journal of Biological Chemistry ◽

10.1074/jbc.m501202200 ◽

2005 ◽

Vol 280 (16) ◽

pp. 16377-16382 ◽

Cited By ~ 15

Author(s):

Sepehr Dadsetan ◽

Vyacheslav Shishkin ◽

Alla F. Fomina

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

T Cell ◽

T Cell Receptor ◽

Membrane Trafficking ◽

Cell Receptor ◽

Multivesicular Bodies ◽

Close Proximity ◽

Intracellular Ca ◽

Stimulation Of

Stimulation of T cell receptor in lymphocytes enhances Ca2+signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1–43 to explore membrane trafficking upon mobilization of intracellular Ca2+in Jurkat T cells. We established that liberation of intracellular Ca2+with T cell receptor agonist phytohemagglutinin P or with Ca2+-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1–43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1–43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1–43-containing ER were free of dye. Intralumenal FM1–43 accumulation was observed even when Ca2+signaling in the cytosol was abolished by the removal of extracellular Ca2+. Our findings strongly suggest that release of intracellular Ca2+may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca2+elevation.

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Calcium influx in mammalian eggs

Reproduction ◽

10.1530/rep-12-0496 ◽

2013 ◽

Vol 145 (4) ◽

pp. R97-R105 ◽

Cited By ~ 16

Author(s):

Chunmin Wang ◽

Zoltan Machaty

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Assisted Reproductive Technologies ◽

Calcium Influx ◽

Critical Role ◽

Reproductive Technologies ◽

Signaling Cascades ◽

Mammalian Eggs ◽

Stimulation Of

Calcium (Ca2+) signals are involved in the regulation of oocyte maturation and play a critical role during fertilization. In the egg, Ca2+is stored in the lumen of the endoplasmic reticulum and a signal is generated when the stored Ca2+is released through specialized channels in the membrane of the endoplasmic reticulum to elevate the free Ca2+concentration in the cytoplasm. Extracellular Ca2+is also important, indicated by the fact that the mobilization of luminal Ca2+is typically followed by Ca2+entry across the plasma membrane. The transmembrane Ca2+flux replenishes the endoplasmic reticulum, and thus, it is essential to sustain prolonged Ca2+signals. It also seems to be responsible for the stimulation of important signaling cascades required for complete egg activation. Characterization of the pathway that mediates Ca2+entry implies that its major components include STIM1, a protein that senses the filling status of the stores, and ORAI1, a channel protein located in the plasma membrane. Defining the mechanism and functions of Ca2+entry will not only lead to a better understanding of egg physiology but may also help improving the efficiency of a number of assisted reproductive technologies.

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Dietary orotic acid accentuates the hepatic response to phenobarbital in rats

Biochemistry and Cell Biology ◽

10.1139/o90-171 ◽

1990 ◽

Vol 68 (9) ◽

pp. 1146-1149 ◽

Cited By ~ 1

Author(s):

D. S. Milton Haines ◽

Margaret Wu ◽

Sonya D. Tokmakjian

Keyword(s):

Endoplasmic Reticulum ◽

Total Protein ◽

Orotic Acid ◽

Smooth Endoplasmic Reticulum ◽

Limiting Factor ◽

Semisynthetic Diet ◽

Structural Framework ◽

Cytochrome P 450 ◽

Phosphatidylcholine Synthesis ◽

Stimulation Of

In rats treated with phenobarbital for 3 days and simultaneously fed a semisynthetic diet containing 1.0% orotic acid, the extent of the increases in liver microsomal phosphatidylcholine, phosphatidylethanolamine, total RNA, total protein, and cytochrome P-450 were significantly greater than they were in rats treated identically with phenobarbital but without dietary orotic acid. This is attributed primarily to the stimulation of hepatic phosphatidylcholine synthesis by dietary orotic acid. In the absence of phenobarbital, orotic acid was shown to cause some increase in liver smooth endoplasmic reticulum components, but not cytochrome P-450. Orotic acid also decreased the activity of microsomal phosphatidylethanolamine N-methyltransferase, which may have contributed to the increase in the microsomal content of phosphatidylethanolamine. The hypothesis is advanced that phospholipid availability is a limiting factor in the hepatic response to phenobarbital. When more phospholipid is available to provide the structural framework for biogenesis of endoplasmic reticulum, all of the hepatic actions of phenobarbital, including induction of cytochrome P-450, are amplified.Key words: phosphatidylcholine, phosphatidylethanolamine, cytochrome P-450, phenobarbital, orotic acid, endoplasmic reticulum.

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Triple Fixation For Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100196 ◽

1968 ◽

Vol 26 ◽

pp. 90-91 ◽

Cited By ~ 1

Author(s):

M. A. Hayat

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Electron Beam ◽

Plasma Membrane ◽

Myelin Sheath ◽

Good Deal ◽

Granular Structure ◽

Oxidizing Agent ◽

Fixation Technique ◽

Large Clusters

Potassium permanganate has been successfully employed to study membranous structures such as endoplasmic reticulum, Golgi, plastids, plasma membrane and myelin sheath. Since KMnO4 is a strong oxidizing agent, deposition of manganese or its oxides account for some of the observed contrast in the lipoprotein membranes, but a good deal of it is due to the removal of background proteins either by dehydration agents or by volatalization under the electron beam. Tissues fixed with KMnO4 exhibit somewhat granular structure because of the deposition of large clusters of stain molecules. The gross arrangement of membranes can also be modified. Since the aim of a good fixation technique is to preserve satisfactorily the cell as a whole and not the best preservation of only a small part of it, a combination of a mixture of glutaraldehyde and acrolein to obtain general preservation and KMnO4 to enhance contrast was employed to fix plant embryos, green algae and fungi.

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Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

The Plant Cell ◽

10.1093/plcell/koab122 ◽

2021 ◽

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 ◽

Fluorescent Protein ◽

Mechanical Damage ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana 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 wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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