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.

scholarly journals GTP-binding proteins may stimulate insulin biosynthesis in rat pancreatic islets by enhancing the signal-recognition-particle-dependent translocation of the insulin mRNA poly-/mono-some complex to the endoplasmic reticulum

1991 ◽  
Vol 275 (1) ◽  
pp. 23-28 ◽  
Author(s):  
N Welsh ◽  
C Oberg ◽  
M Welsh
Keyword(s):  
Endoplasmic Reticulum ◽  
Binding Proteins ◽  
Specific Binding ◽  
Signal Recognition Particle ◽  
Insulin Biosynthesis ◽  
Signal Recognition ◽  
Insulin Synthesis ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Insulin Mrna

We aimed to elucidate the putative role of GTP-binding proteins in the regulation of insulin biosynthesis. For this purpose, freshly isolated rat islets were incubated in the presence of liposomes containing GDP, guanosine 5′-[beta-thio]diphosphate (GDP[S]), GTP, guanosine 5′-[gamma-thio]triphosphate (GTP[S]), guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG), guanosine 5′[beta gamma-imido]triphosphate (p[NH]ppG) and ATP, and the effects of the liposomal delivery of these substances on rates of biosynthesis of insulin and total protein were determined. Insulin biosynthesis during a 1 h incubation at 1.67 mM-glucose was stimulated by ATP- and GTP[S]-containing liposomes as compared with control liposomes. At 16.7 mM-glucose, only the GTP[S]-containing liposomes stimulated insulin biosynthesis. No inhibition of islet protein and insulin synthesis was observed with GDP-, GDP[S]-, p[CH2]ppG- and p[NH]ppG-containing liposomes. By determining the subcellular distribution of insulin mRNA, it was found that the mRNA content associated with microsomes was increased and that associated with the cytosolic mono-/poly-somes decreased when the islets were incubated with GTP[S]-containing liposomes, resulting in an approximate doubling of the ratio of microsomal to polysomal-associated insulin mRNA. ATP-containing liposomes produced no effects on the association of insulin mRNA with microsomes. By using photoaffinity labelling and immunoprecipitation techniques, specific binding of GTP[35S] to the alpha-subunit of the signal-recognition particle (SRP) receptor in islet homogenates containing physiological concentrations of GTP and GDP was demonstrated. These findings suggest that the GTP-binding subunit(s) of the SRP receptor, and possibly also of other GTP-binding proteins involved in this process, may regulate insulin biosynthesis by stimulating the translocation of insulin mRNA to the endoplasmic reticulum and by increasing preproinsulin-peptide translocation into the lumen of the reticulum.

scholarly journals Biosynthesis of rat liver pI-6.1 esterase, a carboxylesterase of the cisternal space of the endoplasmic reticulum

1987 ◽  
Vol 248 (2) ◽  
pp. 545-550 ◽  
Author(s):  
M Robbi ◽  
H Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Secretory Proteins ◽  
Structural Element ◽  
Oligosaccharide Moiety ◽  
Reticulocyte Lysate ◽  
Dog Pancreas ◽  
Processed Products

Biosynthesis of the rat liver microsomal esterase with pI 6.1 was investigated in cell-free systems and in cultured hepatocytes, by using a rabbit antiserum. Protein synthesis directed by total rat liver RNA in wheatgerm extract or reticulocyte lysate generated a single immunoprecipitable product, also found with the RNA extracted from bound, but not from free, polysomes. When dog pancreas microsomal fractions were included, reticulocyte lysates gave two processed products, a prominent one slightly larger, and another slightly smaller, than the precursor, both resistant to exogenous proteinases and, hence, segregated within vesicles. The processing was co-translational; it consisted of the removal of a peptide fragment and, for the large component, the addition of a single oligosaccharide chain. Indeed, this component bound to concanavalin A-Sepharose and gave the small one (approximately 2000 Mr loss) by cleavage with endo-beta-N-acetylglucosaminidase H (endo-H). A single labelled peptide was precipitated from hepatocytes incubated with [35S]methionine. Its apparent Mr was decreased by approximately 2000 after treatment with endo-H; it was then identical with that of an unglycosylated form produced in hepatocytes poisoned with tunicamycin. Even in that case, immunoreactive peptides were not detected in the culture medium. Whether synthesized in reticulocyte lysate or in hepatocytes, the glycosylated forms migrated in SDS/polyacrylamide-gel electrophoresis as the purified enzyme labelled with [3H]di-isopropyl fluorophosphate. Thus, although pI-6.1 esterase is not secreted, its biosynthesis is, as yet, indistinguishable from that of secretory proteins. Its oligosaccharide moiety is apparently not the structural element that retains it in the endoplasmic reticulum.

scholarly journals Distribution of polypeptides binding guanosine 5′-[γ-[35S]thio]triphosphate and anti-(ras protein) antibodies in liver subcellular fractions. Evidence for endosome-specific components

1990 ◽  
Vol 271 (1) ◽  
pp. 179-183 ◽  
Author(s):  
N Ali ◽  
W H Evans
Keyword(s):  
Rat Liver ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Subcellular Fractions ◽  
Relative Distribution ◽  
Ras Proteins ◽  
Ras Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The subcellular distribution in rat liver of polypeptides binding guanosine 5′-[gamma-[35S]thio]triphosphate [( 35S]GTP[S]) and seven antibodies against ras oncoproteins was evaluated. Multiple low-Mr (21,000-28,000) GTP-binding proteins were detected, but their relative distribution among the membrane fractions varied. A more specific compartmentation of polypeptides which bind antibodies generated against ras proteins was evident, with an Mr-28,000 polypeptide and a probable Mr-56,000 dimer, identified by six of the antibodies tested, being confined mainly to endosomes. An Mr-23,000 polypeptide was detected by some of the antibodies in all of the membrane fractions, but especially in the plasma membranes.

scholarly journals GTP-binding proteins in rat liver nuclear envelopes.

1990 ◽  
Vol 87 (18) ◽  
pp. 7080-7084 ◽  
Author(s):  
J B Rubins ◽  
J O Benditt ◽  
B F Dickey ◽  
N Riedel
Keyword(s):  
Rat Liver ◽  
Binding Proteins ◽  
Gtp Binding Proteins ◽  
Gtp Binding

scholarly journals Detection of 23-27 kDa GTP-binding proteins in platelets and other cells

1987 ◽  
Vol 245 (2) ◽  
pp. 617-620 ◽  
Author(s):  
R P Bhullar ◽  
R J Haslam
Keyword(s):  
Binding Proteins ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Soluble Form ◽  
Rat Tissues ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Membrane Bound ◽  
Red Cell Membranes

Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5′-[gamma-thio]triphosphate, but not by GMP or adenosine 5′-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).

scholarly journals Rat liver GTP-binding proteins mediate changes in mitochondrial membrane potential and organelle fusion

1999 ◽  
Vol 276 (3) ◽  
pp. C611-C620 ◽  
Author(s):  
Jorge Daniel Cortese
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Binding Proteins ◽  
Mitochondrial Fusion ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The variety of mitochondrial morphology in healthy and diseased cells can be explained by regulated mitochondrial fusion. Previously, a mitochondrial outer membrane fraction containing fusogenic, aluminum fluoride (AlF4)-sensitive GTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese, Exp. Cell Res. 240: 122–133, 1998). Quantitative confocal microscopy now reveals that mtg transiently increases mitochondrial membrane potential (ΔΨ) when added to permeabilized rat hepatocytes (15%), rat fibroblasts (19%), and rabbit myocytes (10%). This large mtg-induced ΔΨ increment is blocked by fusogenic GTPase-specific modulators such as guanosine 5′- O-(3-thiotriphosphate), excess GTP (>100 μM), and AlF4, suggesting a linkage between ΔΨ and mitochondrial fusion. Accordingly, stereometric analysis shows that decreasing ΔΨ or ATP synthesis with respiratory inhibitors limits mtg- and AlF4-induced mitochondrial fusion. Also, a specific G protein inhibitor ( Bordetella pertussis toxin) hyperpolarizes mitochondria and leads to a loss of AlF4-dependent mitochondrial fusion. These results place mtg-induced ΔΨ changes upstream of AlF4-induced mitochondrial fusion, suggesting that GTPases exert ΔΨ-dependent control of the fusion process. Mammalian mitochondrial morphology thus can be modulated by cellular energetics.

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