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

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

Science ◽

10.1126/science.aav6740 ◽

2018 ◽

Vol 363 (6422) ◽

pp. 84-87 ◽

Cited By ~ 32

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.

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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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Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex.

The Journal of Cell Biology ◽

10.1083/jcb.126.4.925 ◽

1994 ◽

Vol 126 (4) ◽

pp. 925-934 ◽

Cited By ~ 121

Author(s):

K U Kalies ◽

D Görlich ◽

T A Rapoport

Keyword(s):

Salt Concentration ◽

Binding Sites ◽

Protein Translocation ◽

Tight Binding ◽

Binding Activity ◽

Ribosome Binding ◽

Ribosome Binding Sites ◽

Low Salt ◽

Unspecific Binding ◽

Membrane Anchoring

The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p-complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane-anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.

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

The Journal of Cell Biology ◽

10.1083/jcb.85.1.147 ◽

1980 ◽

Vol 85 (1) ◽

pp. 147-152 ◽

Cited By ~ 15

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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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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