scholarly journals The binding of ribosomal subunits to endoplasmic reticulum membranes

1972 ◽  
Vol 129 (3) ◽  
pp. 721-731 ◽  
Author(s):  
Francis S. Rolleston
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Messenger Rna ◽  
Mouse Liver ◽  
Peptide Bonds ◽  
Ribosomal Subunits ◽  
Membrane Bound

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60–80% of added 60S subunits and 10–15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane–60S subunit–40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.

A simple biochemical approach to quantitate rough endoplasmic reticulum

1995 ◽  
Vol 268 (2) ◽  
pp. C308-C316 ◽  
Author(s):  
A. K. Rajasekaran ◽  
S. A. Langhans-Rajasekaran ◽  
R. M. Gould ◽  
E. Rodriguez-Boulan ◽  
T. Morimoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Line ◽  
Rough Endoplasmic Reticulum ◽  
Cell Membranes ◽  
Fold Increase ◽  
Sucrose Density Gradient ◽  
Total Cell ◽  
Cell Fractionation ◽  
Cell Line Hela ◽  
Membrane Bound

In this report we demonstrate that the changes in size of the rough endoplasmic reticulum (RER) can be determined by quantifying the membrane-bound ribosomal population separated by cell fractionation and sucrose density gradient analysis. Total cell membranes, rather than microsomes, were used as the source of membrane-bound ribosomes to eliminate potential losses during the preparation of microsomes. Bound ribosomes were assayed after quantitative release and recovery from total cell membranes using puromycin in the presence of high-salt buffer. Using this analysis, we demonstrate a 4.2-fold increase in RER in estrogen-treated male Xenopus laevis liver. Furthermore, we show that the ratio of the distribution of free to membrane-bound ribosomes in a nonsecretory cell line (HeLa) was 3.3, while this ratio in a secretory cell line (AR42J) was 1.2, indicating that cells active in secretion contain more RER. We suggest that this biochemical technique provides a simpler assay to detect changes in the size of the RER.

Freeze-Fracture Replication of Rough Endoplasmic Reticulum of Mouse Liver Cells

1972 ◽  
Vol 11 (2) ◽  
pp. 477-489
Author(s):  
A. S. BREATHNACH ◽  
C. STOLINSKI ◽  
M. GROSS
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rough Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Mouse Liver ◽  
Freeze Fracture ◽  
The Other ◽  
Fracture Face ◽  
Mitochondrial Membranes ◽  
En Face

Fresh, chemically unfixed, glycerinated specimens of mouse liver were examined by the technique of freeze-fracture replication without sublimation (i.e. they were not ‘etched’). Where extensive areas of fractured lamellar membranes of the rough endoplasmic reticulum are revealed en face, 2 types of fracture face are distinguishable. One of these fracture faces (A) is directed towards the cytoplasm, and the other (B) towards the cisternal cavity. A characteristic mosaic, or patchwork pattern of flat areas circumscribed by particles, is evident on both faces, and more clearly so on face B, due to a greater number of more prominent particles. Similar mosaic patterns are revealed on convex faces of the nuclear membrane, and on concave fracture faces of mitochondrial membranes, but are not evident on fracture faces of the plasma membrane. Uncertainty in establishing the exact plane of fracture of membranes in this material, since glycerol is virtually non-sublimable, makes it difficult to assess the significance of these mosaic patterns. The fact that ribosomes are not identifiable on either face of fractured endoplasmic reticulum membranes, gives no certain indication of the plane of fracture.

scholarly journals Membrane-bound ribosomes of myeloma cells. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane-bound ribosomal particles.

1975 ◽  
Vol 67 (1) ◽  
pp. 16-24 ◽  
Author(s):  
B Mechler ◽  
P Vassalli
Keyword(s):  
Messenger Rna ◽  
Specific Radioactivity ◽  
Kinetic Studies ◽  
Chain Termination ◽  
High Turnover ◽  
Ribosomal Subunits ◽  
Myeloma Cells ◽  
High Specific Radioactivity ◽  
Membrane Bound ◽  
Kinetics Of

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.

scholarly journals Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes.

1975 ◽  
Vol 67 (1) ◽  
pp. 25-37 ◽  
Author(s):  
B Mechler ◽  
P Vassalli
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Membrane Fraction ◽  
Polypeptide Chain ◽  
Salt Treatment ◽  
Ribosomal Subunits ◽  
Myeloma Cells ◽  
Membrane Bound ◽  
Made In

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.

scholarly journals Co-translational excision of alpha-glucose and alpha-mannose in nascent vesicular stomatitis virus G protein.

1984 ◽  
Vol 98 (6) ◽  
pp. 2245-2249 ◽  
Author(s):  
P H Atkinson ◽  
J T Lee
Keyword(s):  
Endoplasmic Reticulum ◽  
High Resolution ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Rough Endoplasmic Reticulum ◽  
Hela Cells ◽  
Chain Elongation ◽  
Carbonyl Cyanide ◽  
Membrane Bound ◽  
Vesicular Stomatitis

Membrane bound polysomes were prepared from HeLa cells infected with vesicular stomatitis virus (VSV), after pulse labeling with [3H]mannose for various times from 15 to 90 min. Oligosaccharides on nascent chains were released from peptides by treatment with endoglycosidase H and sized by high resolution Biogel P4 chromatography. Processing on some nascent chains proceeded to the removal of all three types of alpha-linked glucose and one alpha-1,2-mannose from the Glc3Man9GlcNAc precursor showing that the enzymes responsible were not only active on nascent chains but were present in the rough endoplasmic reticulum (RER). Incubation of cells for various times in cycloheximide, where chain elongation had ceased, made no difference to the profile of oligosaccharides on the nascent chains, and trimming proceeded no further than Man8GlcNAc2Asn . Carbonyl cyanide m-chlorophenylhydrazone (CCCP), an energy inhibitor reportedly able to block the transfer of glycoproteins from the RER, increases the amount of Man8-oligosaccharides on the nascent chains and also the amount of Glc3Man9GlcNAc precursor. On completed G protein in the RER fraction from which membrane bound polysomes were prepared, processing occurred to Man6 - but not to Man5GlcNAc sized oligosaccharides in the CCCP-treated cells. By contrast, processing to Man5GlcNAc oligosaccharides was observed in unfractionated control cells.

scholarly journals Retention of mRNA on the endoplasmic reticulum membranes after in vivo disassembly of polysomes by an inhibitor of initiation.

1976 ◽  
Vol 71 (1) ◽  
pp. 307-313 ◽  
Author(s):  
M Adesnik ◽  
M Lande ◽  
T Martin ◽  
D D Sabatini
Keyword(s):  
Messenger Rna ◽  
Membrane Fraction ◽  
Human Fibroblasts ◽  
Ribosomal Subunits ◽  
Run Off ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Polypeptide Chains ◽  
Extensive Release

Membrane-bound ribosomes and messenger RNA remained associated with the microsomal membranes of human fibroblasts after cultures were treated with Verrucarin A, an inhibitor of initiation which led to extensive run-off of ribosomes from polysomal structures. When a membrane fraction from Verrucarin-treated cells containing such inactive ribosomes and mRNA was suspended in a medium of high salt concentration, extensive release of ribosomal subunits occurred without the need for puromycin. The mRNA nevertheless remained associated with the membranes. These results add support to the conclusion that, in human fibroblasts, mRNA is bound directly to ER membranes, independently of the ribosomes and nascent polypeptide chains.

scholarly journals The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro

1975 ◽  
Vol 146 (3) ◽  
pp. 513-526 ◽  
Author(s):  
T K Shires ◽  
C M McLaughlin ◽  
H C Pitot
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Large Subunit ◽  
Relative Concentration ◽  
Membrane Binding ◽  
Ribosomal Subunits ◽  
Derivatives Of ◽  
Membrane Binding Sites

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.

Analysis of Ca 2+ fluxes and Ca 2+ pools in pancreatic acini

1981 ◽  
Vol 296 (1080) ◽  
pp. 105-113 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Intact Cells ◽  
Pancreatic Acini ◽  
Mitochondrial Inhibitors ◽  
Intracellular Ca ◽  
Membrane Bound ◽  
Intracellular Storage ◽  
Carbamyl Choline

45 Ca 2+ movements have been analysed in dispersed acini prepared from rat pancreas in a quasi-steady state for 45 Ca 2+ . Carbamyl choline (carbachol; Cch) caused a quick 45 Ca 2+ release that was followed by a slower 45 Ca 2+ ‘reuptake’. Subsequent addition of atropine resulted in a further transient increase in cellular 45 Ca 2+ . The data suggest the presence of a Cch-sensitive ‘trigger’ pool, which could be refilled by the antagonist, and one or more intracellular ‘storage’ pools. Intracellular Ca 2+ sequestration was studied in isolated acini pretreated with saponin to disrupt their plasma membranes. In the presence of 45 Ca 2+ (1 µM), addition of ATP at 5 mM caused a rapid increase in 45 Ca 2+ uptake exceeding the control by fivefold. Maximal ATP-promoted Ca 2+ uptake was obtained at 10 µM Ca 2+ (half-maximal at 0.32 µM Ca 2+ ). In the presence of mitochondrial inhibitors it was 0.1 µM (half-maximal at 0.014 µM). 45 Ca 2+ release could still be induced by Cch but the subsequent reuptake was missing. The latter was restored by ATP and atropine caused further 45 Ca 2+ uptake. Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca 2+ , oxalate and ATP which were absent in intact cells or cells pretreated with A23187. The data suggest the presence of a plasma membrane-bound Cch-sensitive ‘trigger’ Ca 2+ pool and ATP-dependent Ca 2+ storage systems in mitochondria and rough endoplasmic reticulum of pancreatic acini. It is assumed that Ca 2+ is taken up into these pools after secretagogue-induced Ca 2+ release.

Synthesis of Albumin with Exogenous Mouse-Liver Messenger RNA in a Homologous Cell-Free System

1974 ◽  
Vol 52 (5) ◽  
pp. 429-432 ◽  
Author(s):  
A. J. Faber ◽  
S. H. Miall ◽  
T. Tamaoki
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Messenger Rna ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Free System ◽  
Cell Free System ◽  
Membrane Bound

RNA extracted from total and membrane-bound polysomes of mouse liver was capable of directing protein synthesis in a homologous cell-free system in the presence of a 0.5 M KCl ribosomal wash fraction. Analysis of the products by polyacrylamide gel electrophoresis and immunoprecipitation showed that newly formed albumin could account for up to 8% of the total protein synthesized.

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