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

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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FUNCTIONAL AND STRUCTURAL CHARACTERISTICS OF ENDOPLASMIC RETICULUM PROTEINS ASSOCIATED WITH RIBOSOME BINDING SITES

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1980.tb47239.x ◽

1980 ◽

Vol 343 (1 Precursor Pro) ◽

pp. 17-37 ◽

Cited By ~ 16

Author(s):

Gert Kreibich ◽

Maria Czakó-Graham ◽

Ruth C. Grebenau ◽

David D. Sabatini

Keyword(s):

Endoplasmic Reticulum ◽

Binding Sites ◽

Structural Characteristics ◽

Ribosome Binding ◽

Ribosome Binding Sites ◽

Endoplasmic Reticulum Proteins

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

The Journal of Cell Biology ◽

10.1083/jcb.77.2.488 ◽

1978 ◽

Vol 77 (2) ◽

pp. 488-506 ◽

Cited By ~ 102

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.

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Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Biological Chemistry ◽

10.1515/bc.2000.126 ◽

2000 ◽

Vol 381 (9-10) ◽

pp. 1025-1028 ◽

Cited By ~ 23

Author(s):

Anke Prinz ◽

Enno Hartmann ◽

Kai-Uwe Kalies

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Binding Sites ◽

Mammalian Cells ◽

Protein Translocation ◽

Growth Conditions ◽

Ribosome Binding ◽

Ribosome Binding Sites ◽

Tight Association ◽

Biochemical Analyses

Abstract A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.

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Design of Ribosome Binding Sites in Streptomyces coelicolor

Current Proteomics ◽

10.2174/1570164614666170724120325 ◽

2017 ◽

Vol 14 (4) ◽

Cited By ~ 1

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

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

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00370-w ◽

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.

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