scholarly journals Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

1978 ◽  
Vol 77 (2) ◽  
pp. 464-487 ◽  
Author(s):  
G Kreibich ◽  
BL Ulrich ◽  
DD Sabatini
Keyword(s):  
Membrane Proteins ◽  
Sodium Deoxycholate ◽  
Staining Intensity ◽  
High Salt ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Nonionic Detergent ◽  
Microsomal Membranes ◽  
Rnase Treatment ◽  
Low Ionic Strength

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin-KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

scholarly journals Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites

1978 ◽  
Vol 77 (2) ◽  
pp. 488-506 ◽  
Author(s):  
G Kreibich ◽  
CM Freienstein ◽  
BN Pereyra ◽  
BL Ulrich ◽  
DD Sabatini
Keyword(s):  
Binding Sites ◽  
Cross Linking ◽  
Ribosomal Subunits ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Close Proximity ◽  
Microsomal Membranes ◽  
Cross Links ◽  
Polypeptide Chains ◽  
Specific Membrane

Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS-acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These observations suggest that ribophorins are in close proximity to the bound ribosomes, as may be expected from components of the ribosome-binding sites.

scholarly journals Reconstitution into liposomes of membrane proteins involved in ribosome binding on rough endoplasmic reticulum. Ribosome-binding capacity

1981 ◽  
Vol 194 (3) ◽  
pp. 907-913 ◽  
Author(s):  
M Yamaguchi ◽  
M Sakai ◽  
T Horigome ◽  
S Omata ◽  
H Sugano
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Fraction ◽  
Proteolytic Enzyme ◽  
Binding Capacity ◽  
Binding Mechanism ◽  
Ribosome Binding ◽  
High Affinity ◽  
Microsomal Membranes

A membrane protein fraction having a high affinity for polyribosomes was isolated from microsomal membranes of rat liver and was incorporated into liposomes made from microsomal lipids to evaluate the polyribosome-binding capacity of the reconstituted liposomes, with the following results. (1) The polyribosome binding to the reconstituted liposomes depended on the amounts of polyribosomes added to the binding mixture. (2) Liposomes made from lipids alone did not bind any polyribosomes. (3) The polyribosome-binding capacity of the reconstituted liposomes was very sensitive to proteolytic enzyme and strongly inhibited by addition of 0.1 mM-aurintricarboxylic acid or by increasing KCl concentration. These results suggest that the binding mechanism of polyribosomes to the reconstituted liposomes is much like that for rough microsomal membrane stripped of endogenous polyribosomes.

Design of Ribosome Binding Sites in Streptomyces coelicolor

2017 ◽  
Vol 14 (4) ◽  
Author(s):  
Hong-Dou Luo ◽  
Yang Tao ◽  
Wen-Guang Wang ◽  
Tao Lin ◽  
Yue-Yue Wang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Streptomyces Coelicolor ◽  
Ribosome Binding ◽  
Ribosome Binding Sites

scholarly journals Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jinghui Xiong ◽  
Hefeng Chen ◽  
Ran Liu ◽  
Hao Yu ◽  
Min Zhuo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
Whole Cell ◽  
Cyclohexanone Monooxygenase ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Whole Cell Biocatalyst ◽  
Substrate Tolerance

Abstractε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites

1989 ◽  
Vol 21 (9) ◽  
pp. 987-996 ◽  
Author(s):  
Krassimir Alexciev ◽  
Anna Uscheva ◽  
Maja Pavlova ◽  
Libert Yavachev ◽  
Ivan Ivanov
Keyword(s):  
Binding Sites ◽  
Plasmid Vectors ◽  
Ribosome Binding ◽  
Ribosome Binding Sites

scholarly journals Evidence for Context-Dependent Complementarity of Non-Shine-Dalgarno Ribosome Binding Sites to Escherichia coli rRNA

2013 ◽  
Vol 8 (5) ◽  
pp. 958-966 ◽  
Author(s):  
Pamela A. Barendt ◽  
Najaf A. Shah ◽  
Gregory A. Barendt ◽  
Parth A. Kothari ◽  
Casim A. Sarkar
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Context Dependent

scholarly journals Multiple divergent picobirnaviruses with functional prokaryotic Shine-Dalgarno ribosome binding sites present in cloacal sample of a diarrheic chicken

Virology ◽  
2018 ◽  
Vol 525 ◽  
pp. 62-72 ◽  
Author(s):  
Ákos Boros ◽  
Beáta Polgár ◽  
Péter Pankovics ◽  
Hajnalka Fenyvesi ◽  
Péter Engelmann ◽  
...  
Keyword(s):  
Binding Sites ◽  
Ribosome Binding ◽  
Ribosome Binding Sites
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