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

2003 ◽  
Vol 371 (3) ◽  
pp. 1013-1019 ◽  
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.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

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.

scholarly journals Analytical subcellular fractionation of needle-biopsy specimens from human liver

1978 ◽  
Vol 174 (2) ◽  
pp. 435-446 ◽  
Author(s):  
T J Peters ◽  
C A Seymour
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Needle Biopsy ◽  
Human Liver ◽  
Resolving Power ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Acid Hydrolases ◽  
Biopsy Specimens

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.

Formation of chlamydospores in Gilbertella persicaria

1981 ◽  
Vol 59 (5) ◽  
pp. 908-928 ◽  
Author(s):  
Martha J. Powell ◽  
Charles E. Bracker ◽  
David J. Sternshein
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Light And Electron Microscopy ◽  
Multivesicular Bodies ◽  
Marker Enzyme ◽  
Transparent Layer ◽  
Thiamine Pyrophosphatase ◽  
Gilbertella Persicaria

The cytological events involved in the transformation of vegetative hyphae of the zygomycete Gilbertella persicaria (Eddy) Hesseltine into chlamydospores were studied with light and electron microscopy. Thirty hours after sporangiospores were inoculated into YPG broth, swellings appeared along the aseptate hyphae. Later, septa, traversed by plasmodesmata, delimited each end of the hyphal swellings and compartmentalized these hyphal regions as they differentiated into chlamydospores. Nonswollen regions adjacent to chlamydospores remained as isthmuses. Two additional wall layers appeared within the vegetative wall of the developing chlamydospores. An alveolate, electron-dense wall formed first, and then an electron-transparent layer containing concentrically oriented fibers formed between this layer and the plasma membrane. Rather than a mere condensation of cytoplasm, development and maturation of the multinucleate chlamydospores involved extensive cytoplasmic changes such as an increase in reserve products, lipid and glycogen, an increase and then disappearance of vacuoles, and the breakdown of many mitochondria. Underlying the plasma membrane during chlamydospore wall formation were endoplasmic reticulum, multivesicular bodies, vesicles with fibrillar contents, vesicles with electron-transparent contents, and cisternal rings containing the Golgi apparatus marker enzyme, thiamine pyrophosphatase. Acid phosphatase activity was localized cytochemically in a cisterna which enclosed mitochondria and in vacuoles which contained membrane fragments. Tightly packed membrane whorls and single membrane bounded sacs with finely granular matrices surrounding vacuoles were unique during chlamydospore development. Microbodies were rare in the mature chlamydospore, but endoplasmic reticulum was closely associated with lipid globules. As chlamydospores developed, the cytoplasm in the isthmus became highly vacuolated, lipid globules were closely associated with vacuoles, mitochondria were broken down in vacuoles, unusual membrane configurations appeared, and eventually the membranes degenerated. Unlike chlamydospores, walls of the isthmus did not thicken, but irregularly shaped appositions containing numerous channels formed at intervals on the inside of these walls. The pattern of cytoplasmic transformations during chlamydospore development is similar to events leading to the formation of zygospores and sporangiospores.

scholarly journals Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

2018 ◽  
Vol 92 (8) ◽  
Author(s):  
Maija K. Pietilä ◽  
Martijn J. van Hemert ◽  
Tero Ahola
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rna Viruses ◽  
Viral Rna ◽  
Cellular Membranes ◽  
Positive Strand ◽  
Membrane Markers ◽  
Highly Active ◽  
Replicase Proteins ◽  
Positive Strand Rna

ABSTRACTPositive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs.IMPORTANCESimilar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories, but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA andin vitroreplication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes were enriched with the replication complexes, demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have developed here a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

scholarly journals SUBSURFACE CISTERNS AND THEIR RELATIONSHIP TO THE NEURONAL PLASMA MEMBRANE

1962 ◽  
Vol 13 (3) ◽  
pp. 405-421 ◽  
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.

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

2002 ◽  
Vol 2 ◽  
pp. 1435-1439 ◽  
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.

scholarly journals Integrated stereological and biochemical studies on hepatocytic membranes. III. Relative surface of endoplasmic reticulum membranes in microsomal fractions estimated on freeze-fracture preparations.

1978 ◽  
Vol 78 (2) ◽  
pp. 289-308 ◽  
Author(s):  
G A Losa ◽  
E R Weibel ◽  
R P Bolender
Keyword(s):  
Endoplasmic Reticulum ◽  
Particle Density ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Biochemical Data ◽  
Numerical Density ◽  
Marker Enzyme ◽  
Freeze Fracturing ◽  
Outer Leaflet

New methods are required for identifying membranes in subcellular fractions with respect to their origin, if such preparations are to be evaluated morphometrically. One method is freeze-fracturing which reveals intramembrane particles whose size, pattern, and numerical density differ for various membrane types. The question is examined whether the differences in numerical particle density per square micrometer of membrane (alpha) can be used to differentiate membrane vesicles found in microsomal fractions from liver cells with respect to their origin in the hepatocytes. It is found that the range of alpha for the protoplasmic face (PF) of endoplasmic reticulum (ER) membrane (1,900 less than alpha less than 3,250) is intermediate between those for plasma and mitochondrial membranes. Since PF(ER) should appear in the outer leaflet of microsomal vesicles, alpha was estimated on concave profiles of freeze-fracture preparations; the numerical frequency distribution of vesicles with respect to alpha was trimodal, with a major peak around 2,900/micrometer2 and 66% of the vesicles in the range determined for PF(ER). Using a new stereological method, it was calculated that 63% of the membrane surface in these microsomal fractions was of ER origin by this criterion. On the same preparations, an attempt was made to label the ER-derived membranes cytochemically for glucose-6-phosphatase. A line intersection count revealed 62% of the membrane surface to be of ER origin on the basis of marker enzyme labeling. These findings indicate a smaller part of ER membranes in microsomal fractions than would be predicted from biochemical data (77%). The possible reasons for such discrepancies are discussed; shifts in particle densities due to the preparation procedure could lead to an underestimate by freeze-fracturing, whereas the prediction from biochemical data could be overestimates if marker enzymes were not homogeneously distributed.

scholarly journals ABCA1 causes an asymmetric cholesterol distribution to regulate intracellular cholesterol homeostasis

2021 ◽  
Author(s):  
Fumihiko Ogasawara ◽  
Kazumitsu Ueda
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cholesterol Level ◽  
Cholesterol Homeostasis ◽  
Contact Site ◽  
Cell Plasma Membrane ◽  
Cellular Cholesterol ◽  
Outer Leaflet ◽  
Cell Plasma ◽  
Intracellular Cholesterol

AbstractCholesterol is a major and essential component of the mammalian cell plasma membrane (PM) and the loss of cholesterol homeostasis leads to various pathologies. Cellular cholesterol uptake and synthesis are regulated by a cholesterol sensor in the endoplasmic reticulum (ER). However, it remains unclear how the PM cholesterol level is sensed. Here we show that the sensing depends on ATP-binding cassette A1 (ABCA1) and Aster-A, which cooperatively maintain the asymmetric transbilayer cholesterol distribution in the PM. ABCA1 translocates (flops) cholesterol from the inner to the outer leaflet of the PM to maintain a low inner cholesterol level. When the inner cholesterol level exceeds a threshold, Aster-A is recruited to the PM-ER contact site to transfer cholesterol to the ER. These results show unknown synergy between ABCA1 and Aster-A in intracellular cholesterol homeostasis.

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