Proton transport by a fraction of endoplasmic reticulum and Golgi membranes of corn roots: comparison with the plasma membrane and tonoplast H+ pumps

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

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SUBSURFACE CISTERNS AND THEIR RELATIONSHIP TO THE NEURONAL PLASMA MEMBRANE

The Journal of Cell Biology ◽

10.1083/jcb.13.3.405 ◽

1962 ◽

Vol 13 (3) ◽

pp. 405-421 ◽

Cited By ~ 287

Author(s):

Jack Rosenbluth

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Nerve Cell ◽

Plasma Membranes ◽

Granular Endoplasmic Reticulum ◽

Supporting Cells ◽

Golgi Membranes ◽

Subsurface Cisterns ◽

Central Nervous Systems ◽

Neuronal Plasma Membrane

Subsurface cisterns (SSC's) are large, flattened, membrane-limited vesicles which are very closely apposed to the inner aspect of the plasma membranes of nerve cell bodies and the proximal parts of their processes. They occur in a variety of vertebrate and invertebrate neurons of both the peripheral and central nervous systems, but not in the surrounding supporting cells. SSC's are sheet-like in configuration, having a luminal depth which may be less than 100 A and a breadth which may be as much as several microns. They are separated from the plasmalemma by a light zone of ∼50 to 80 A which sometimes contains a faint intermediate line. Flattened, agranular cisterns resembling SSC's, but structurally distinct from both typical granular endoplasmic reticulum (ER) and from Golgi membranes, also occur deep in the cytoplasm of neurons. It is suggested that membranes which are closely apposed may interact, resulting in alterations in their respective properties. The patches of neuronal plasmalemma associated with subsurface cisterns may, therefore, have special properties because of this association, resulting in a non-uniform neuronal surface. The possible significance of SSC's in relation to neuronal electrophysiology and metabolism is discussed.

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Fractionation of Golgi, Endoplasmic Reticulum, and Plasma Membrane from Cultured Cells in a Preformed Continuous Iodixanol Gradient

The Scientific World JOURNAL ◽

10.1100/tsw.2002.286 ◽

2002 ◽

Vol 2 ◽

pp. 1435-1439 ◽

Cited By ~ 4

Author(s):

John Graham

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Cultured Cells ◽

Density Range ◽

Cultured Cell ◽

Golgi Membranes ◽

Cell Homogenate ◽

Membrane Compartments

A continuous iodixanol gradient within the range 0-30% (w/v) iodixanol can resolve the major membrane compartments of the endoplasmic reticulum, Golgi membranes, and plasma membrane from a postnuclear supernatant prepared from a cultured cell homogenate. The precise density range of the gradient and the centrifugation conditions (100,000-200,000gfor 2-16 h) vary with the type of cell and the requirements of the separation. The strategy is widely used to study the processing of proteins within cells.

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Absence of cytochrome P-450 and presence of autolysosomal membrane antigens on the isolation membranes and autophagosomal membranes in rat hepatocytes.

Journal of Histochemistry & Cytochemistry ◽

10.1177/38.11.2212617 ◽

1990 ◽

Vol 38 (11) ◽

pp. 1571-1581 ◽

Cited By ~ 22

Author(s):

A Yamamoto ◽

R Masaki ◽

Y Fukui ◽

Y Tashiro

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Nuclear Envelope ◽

Protein A ◽

Rat Hepatocytes ◽

Immunogold Technique ◽

Microscopic Evidence ◽

Golgi Membranes ◽

Membrane Antigens ◽

Cytochrome P 450

We wished to determine if phenobarbital (PB)-inducible cytochrome P-450 [P-450(PB)] and autolysosomal membrane antigens could be localized immunocytochemically on the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by the post-embedding protein A-gold method. P-450(PB) was maximally induced by PB treatment; then formation of autophagosomes and accumulation of autolysosomes were induced by cessation of PB treatment and by injection of leupeptin, respectively. P-450(PB) was detected neither on the isolation membranes nor on the limiting membranes of autophagosomes and autolysosomes. Autolysosomal membrane antigens, which were localized by the immunogold technique exclusively in post-Golgi compartments such as lysosomes, endosomes, and plasma membrane but were not found in pre-Golgi compartments such as endoplasmic reticulum (ER) and nuclear envelope, were detected in large amounts on the isolation membranes. These results suggest that the isolation membranes originate not from ER membranes but from post-Golgi membranes. We also present direct immunoelectron microscopic evidence that P-450(PB) is indeed degraded in the autolysosomes: when rats were treated with leupeptin, P-450(PB) was detected not only within the autophagosomes but also within the autolysosomes, whereas without leupeptin treatment, P-450(PB) was detectable only within the autophagosomes.

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The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases

Biochemical Journal ◽

10.1042/bj20021834 ◽

2003 ◽

Vol 371 (3) ◽

pp. 1013-1019 ◽

Cited By ~ 71

Author(s):

Dominique ARDAIL ◽

Iuliana POPA ◽

Jacques BODENNEC ◽

Pierre LOUISOT ◽

Daniel SCHMITT ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Marker Enzyme ◽

Sphingoid Base ◽

Cross Contamination ◽

Outer Leaflet ◽

Highly Active ◽

Golgi Membranes ◽

Marked Preference ◽

Intracellular Organelles

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed ‘mitochondria-associated membrane’ (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of GM3 ganglioside and small amounts of GD3. GM1 was also synthesized along with GM3 upon incubation of the fraction with exogenous unlabelled GM3, underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.

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The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture.

The Journal of Cell Biology ◽

10.1083/jcb.96.5.1197 ◽

1983 ◽

Vol 96 (5) ◽

pp. 1197-1207 ◽

Cited By ~ 13

Author(s):

C Tougard ◽

D Louvard ◽

R Picart ◽

A Tixier-Vidal

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Rough Endoplasmic Reticulum ◽

Primary Cultures ◽

Gh3 Cells ◽

Membrane Flow ◽

Golgi Membranes ◽

Membrane Components ◽

Golgi Zone

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.

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The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules

Molecular Biology of the Cell ◽

10.1091/mbc.e03-05-0325 ◽

2004 ◽

Vol 15 (2) ◽

pp. 447-455 ◽

Cited By ~ 85

Author(s):

Sylvie Le Gall ◽

Andrea Neuhof ◽

Tom Rapoport

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Low Temperature ◽

Small Molecules ◽

Endoplasmic Reticulum Membrane ◽

Membrane Permeation ◽

Permeabilized Cells ◽

Golgi Membranes ◽

Pass Through ◽

Er Membrane

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of ∼5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER.

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Differential distribution of a glucuronyltransferase, involved in glucuronoxylan synthesis, within the Golgi apparatus of pea (Pisum sativum var. Alaska)

Biochemical Journal ◽

10.1042/bj2770653 ◽

1991 ◽

Vol 277 (3) ◽

pp. 653-658 ◽

Cited By ~ 23

Author(s):

M C Hobbs ◽

M H P Delarge ◽

E A H Baydoun ◽

C T Brett

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Pisum Sativum ◽

Membrane Fraction ◽

Smooth Endoplasmic Reticulum ◽

Subcellular Location ◽

Sucrose Density Gradient ◽

Differential Distribution ◽

Golgi Stack ◽

Golgi Membranes

The subcellular location of a glucuronyltransferase (GT) involved in glucuronoxylan synthesis in pea (Pisum sativum) has been investigated. Most of the GT activity was found in the Golgi fraction, but activity was also detected in the plasma-membrane fraction. Separation of Golgi membranes on a shallow continuous sucrose density gradient resulted in three distinct subfractions, with GT activity being confined to Golgi membranes of a density similar to that of smooth endoplasmic reticulum. The differential distribution of GT within the Golgi stack indicates that glucuronoxylan synthesis occurs in specific cisternae and that there is functional compartmentalization of the Golgi with respect to hemicellulose biosynthesis.

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ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS

The Journal of Cell Biology ◽

10.1083/jcb.50.3.634 ◽

1971 ◽

Vol 50 (3) ◽

pp. 634-651 ◽

Cited By ~ 33

Author(s):

Francis J. Chlapowski ◽

R. Neal Band

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Specific Activity ◽

Food Vacuole ◽

Cell Fractionation ◽

Membrane Phospholipids ◽

Golgi Membranes ◽

Cytoplasmic Vesicles ◽

Food Vacuoles ◽

Phospholipid Pool

The membranes of Acanthamoeba palestinensis were studied by examination in fixed cells, and then by following the movements of glycerol-3H-labeled phospholipids by cell fractionation. Two previously undescribed structures were observed: collapsed cytoplasmic vesicles of cup shape, and plaques in food vacuole and plasma membrane similar in size to the collapsed vesicles. It appeared that the plaques formed by insertion of collapsed vesicles into membranes and/or that collapsed vesicles formed by pinching off of plaques. Fractions were isolated, enriched with nuclei, rough endoplasmic reticulum (RER), plasma membrane, Golgi-like membranes, and collapsed vesicles. The changes in specific activity of glycerol-3H-labeled phospholipids in these membranes during incorporation, turnover, and after pulse-labeling indicated an ordered sequence of appearances of newly synthesized phospholipids, first in nuclei and RER, then successively in Golgi membranes, collapsed vesicles, and finally, plasma membrane. In previous work we had found no large nonmembranous phospholipid pool in A. palestinensis. These observations are consistent with the hypothesis that membrane phospholipids are synthesized, perhaps as integral parts of membranes, in RER and nuclei. Subsequently, some of the newly synthesized phospholipids are transported to the Golgi complex to become integrated into the membranes of collapsed vesicles, which are precursors of the plasma membrane. Collapsed vesicles from the plasma membrane by inserting into it as plaques. When portions of the plasmalemma from food vacuoles, collapsed vesicles pinch off from their membranes and are recycled back to the cell surface.

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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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