The membrane of the rough endoplasmic reticulum contains cytoplasmically exposed high affinity GTP-binding sites

1987 ◽  
Vol 148 (1) ◽  
pp. 478-484 ◽  
Author(s):  
Danièle Godelaine ◽  
Henri Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
High Affinity ◽  
Gtp Binding

scholarly journals Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum

1989 ◽  
Vol 262 (2) ◽  
pp. 497-503 ◽  
Author(s):  
J Lanoix ◽  
L Roy ◽  
J Paiement
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Types ◽  
Electrophoretic Analysis ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Dog Pancreas

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5′-[gamma-thio]triphosphate or guanosine 5′-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.

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 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.

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.

scholarly journals Electrostatics cause the molecular chaperone BiP to preferentially bind oligomerized states of a client protein

2021 ◽  
Author(s):  
Judy L.M. Kotler ◽  
Wei-Shao Wei ◽  
Erin E Deans ◽  
Timothy O. Street
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Binding Sites ◽  
Client Protein ◽  
High Affinity ◽  
Hsp70 Family ◽  
Differential Affinity ◽  
Peptide Region ◽  
Positively Charged ◽  
Micromolar Range

Hsp70-family chaperones bind short monomeric peptides with a weak characteristic affinity in the low micromolar range, but can also bind some aggregates, fibrils, and amyloids, with low nanomolar affinity. While this differential affinity enables Hsp70 to preferentially target potentially toxic aggregates, it is unknown how Hsp70s differentiate between monomeric and oligomeric states of a target protein. Here we examine the interaction of BiP (the Hsp70 paralog in the endoplasmic reticulum) with proIGF2, the pro-protein form of IGF2 that includes a long and mostly disordered E-peptide region that promotes proIGF2 oligomerization. We discover that electrostatic attraction enables the negatively charged BiP to bind positively charged E-peptide oligomers with low nanomolar affinity. We identify the specific BiP binding sites on proIGF2, and although some are positively charged, as monomers they bind BiP with characteristically low affinity in the micromolar range. We conclude that electrostatics enable BiP to preferentially recognize oligomeric states of proIGF2. Electrostatic targeting of Hsp70 to aggregates may be broadly applicable, as all the currently-documented cases in which Hsp70 binds aggregates with high-affinity involve clients that are expected to be positively charged.

The presence of epidermal growth factor binding sites in the intracellular organelles of term human placenta

1986 ◽  
Vol 84 (1) ◽  
pp. 19-40
Author(s):  
N. Ramani ◽  
N. Chegini ◽  
C.V. Rao ◽  
P.G. Woost ◽  
G.S. Schultz
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Rough Endoplasmic Reticulum ◽  
Human Placenta ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Smooth Endoplasmic Reticulum ◽  
Intracellular Organelles ◽  
Epidermal Growth

Highly purified lysosomes, rough and smooth endoplasmic reticulum, and Golgi apparatus, as well as microvillus plasma membranes, bound 125I-labelled epidermal growth factor ([125I]EGF) with similar affinity. Scatchard plots for all the organelles were curvilinear. The apparent number of available binding sites per mg protein of intracellular organelles was 27–71% of that found in microvillus plasma membranes. The bound and free [125I]EGF were not degraded by any of the organelles. Binding and dissociation of [125I]EGF in all organelles were dependent on the time and temperature of incubation. The specificity of [125I]EGF binding was similar in all organelles. The optimal pH for binding to lysosomes was 6.0, in contrast to 7.0 for all the other organelles. Exposure of different organelles to enzymes and protein-modifying reagents resulted in numerous binding differences between the intracellular organelles and microvillus plasma membranes. Covalent affinity labelling with [125I]EGF revealed two major proteins of 155 and 140(X10(3)) Mr in all the organelles. The 155 X 10(3) Mr protein was labelled predominantly in all organelles except rough endoplasmic reticulum, where both proteins were equally labelled. Addition of proteolytic inhibitors during isolation of organelles did not alter the pattern of [125I]EGF-labelled binding proteins found in the organelles. EGF also stimulated phosphorylation of the 155 and 140(X10(3)) Mr proteins in all the organelles. The 155 X 10(3) Mr protein was phosphorylated more than the 140 X 10(3) Mr protein in microvillus plasma membranes and smooth endoplasmic reticulum, whereas the 140 X 10(3) Mr protein was phosphorylated more than the 155 X 10(3) Mr protein in lysosomes and both proteins were equally phosphorylated in rough endoplasmic reticulum. Several organelles also contained minor [125I]EGF-binding proteins that did not show phosphorylation response and proteins that showed phosphorylation response but did not bind [125I]EGF. Thus, the present study demonstrates by a number of different criteria, that several intracellular organelles of term human placenta also contain EGF-binding and kinase activities.

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