scholarly journals Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum.

1977 ◽  
Vol 72 (3) ◽  
pp. 530-551 ◽  
Author(s):  
G K Ojakian ◽  
G Kreibich ◽  
D D Sabatini
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Thin Section ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Lateral Displacement ◽  
Homogeneous Distribution ◽  
Antibody Treatment ◽  
Section Electron ◽  
Microsomal Membranes

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High-salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase-treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.

The Role of Membrane Proteins and Phospholipids in the Interaction of Ribosomes with Endoplasmic Reticulum Membranes

1975 ◽  
Vol 53 (9) ◽  
pp. 1039-1045 ◽  
Author(s):  
Serge Jothy ◽  
Jean-Louis Bilodeau ◽  
Henry Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Molecular Weights ◽  
Low Concentrations ◽  
Phospholipid Headgroups ◽  
Hydrolysis Of

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.

scholarly journals Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes.

1978 ◽  
Vol 78 (3) ◽  
pp. 874-893 ◽  
Author(s):  
E Rodriguez Boulan ◽  
G Kreibich ◽  
D D Sabatini
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Binding Sites ◽  
Microsomal Fraction ◽  
Lectin Binding ◽  
Membrane Glycoproteins ◽  
Con A ◽  
Microsomal Membranes ◽  
Carbohydrate Chains ◽  
Lectin Binding Sites

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.

scholarly journals Endoplasmic Reticulum Export of Glycosyltransferases Depends on Interaction of a Cytoplasmic Dibasic Motif with Sar1

2003 ◽  
Vol 14 (9) ◽  
pp. 3753-3766 ◽  
Author(s):  
Claudio G. Giraudo ◽  
Hugo J.F. Maccioni
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Type I ◽  
Type Ii ◽  
Transport Vesicles ◽  
Microsomal Membranes ◽  
Copii Vesicles ◽  
Er Export ◽  
Selective Process ◽  
Selection Of

Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p–Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.

scholarly journals Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽  
2018 ◽  
Vol 363 (6422) ◽  
pp. 84-87 ◽  
Author(s):  
Samuel Itskanov ◽  
Eunyong Park
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Binding Sites ◽  
Protein Translocation ◽  
Secretory Proteins ◽  
Conducting Channel ◽  
Cryo Electron Microscopy ◽  
Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

The protein translocation machinery of the endoplasmic reticulum

1982 ◽  
Vol 300 (1099) ◽  
pp. 225-228 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Rough Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Integral Membrane Proteins ◽  
Multiple Copies ◽  
And Function ◽  
Lysosomal Proteins

The rough endoplasmic reticulum (r.e.r.) has been postulated to possess a single translation-coupled translocation system (in multiple copies) that effects signal sequence-mediated translocation of all secretory and lysosomal proteins and integration of all integral membrane proteins whose port of entry is the rough endoplasmic reticulum (G. Blobel 1980 Proc. natn. Acad. Sci. U.S.A. 77, 1496—1500). Two proteins have been isolated that are components of the r.e.r. translocation system. Their properties and function in protein translocation across and integration into membranes are discussed.

scholarly journals Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

1970 ◽  
Vol 116 (4) ◽  
pp. 617-630 ◽  
Author(s):  
J. R. Tata
Keyword(s):  
Growth Hormone ◽  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Time Course ◽  
Seminal Vesicles ◽  
Membrane Phospholipid ◽  
Phospholipid Synthesis ◽  
Membrane Phospholipids ◽  
Short Period ◽  
Microsomal Membranes

1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [32P]orthophosphate, [14C]choline and [14C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with 32P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with 32P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis.

scholarly journals Ribosome binding sites visualized on freeze-fractured membranes of the rough endoplasmic reticulum.

1980 ◽  
Vol 85 (1) ◽  
pp. 147-152 ◽  
Author(s):  
T H Giddings ◽  
L A Staehelin
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Polypeptide Chain ◽  
Freeze Fracture ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Spiral Form ◽  
Micrasterias Denticulata ◽  
Polypeptide Chains

Freeze-fracture micrographs of cells of the green alga Micrasterias denticulata stabilized by ultrarapid freezing reveal imprints of polysomes on the rough endoplasmic reticulum membranes. The imprints appear as broad, spiral ridges on the P faces and as corresponding wide grooves on the E faces of the membranes. Distinct 110-A particles with a spacing of 270 +/- 45 A are associated with the P-face ridges. Where imprints of individual ribosomes can be discerned, it is seen that there is a 1:1 relationship between the ribosomes and the 110-A particles, and that the 110-A particles are located in a peripheral position with respect to the polysome spirals. We propose that the 110-A particles could be structural equivalents of ribosome-binding sites, consisting of a molecule each of ribophorins I and II and a nascent polypeptide chain. These observations suggest that the spiral form of polysomes could result from the forces generated by the extrusion of the growing polypeptide chains to one side of the polysome.

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