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.

Subcellular distribution and membrane association of Rho-related small GTP-binding proteins in kidney cortex

1995 ◽  
Vol 269 (2) ◽  
pp. F180-F189 ◽  
Author(s):  
D. Boivin ◽  
R. Beliveau
Keyword(s):  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Covalent Modification ◽  
Kidney Cortex ◽  
Gtp Binding Proteins ◽  
Gtp Binding

We have examined the subcellular distribution of Rho-related small GTP-binding proteins in the kidney. RhoA, CDC42, and Rac1 small GTP-binding proteins were found to be expressed at high levels in rat outer kidney cortex. Western blot analysis showed that these proteins were predominantly associated with brush-border and basolateral plasma membranes, with the exception of Rac1 which was localized predominantly in the mitochondria. RhoA and CDC42 were also found in the cytosol, and a small fraction was associated with cytoskeletal elements. A GDP-dissociation inhibitor specific for the Rho family (RhoGDI) was also identified and found to be located exclusively in the cytosol. Upon fractionation of kidney cytosol with anion-exchange chromatography, RhoA and CDC42 proteins eluted in two major well-resolved peaks that coeluted with the RhoGDI protein, suggesting that they form heterodimers. Association of RhoA and CDC42 with RhoGDI was further suggested by coelution of these proteins with RhoGDI at an estimated size of approximately 45 kDa after gel-filtration chromatography. However, a second peak of RhoA eluted as a 20-kDa protein, indicating that not all RhoA is complexed to RhoGDI. Addition of RhoA- and CDC42-enriched fractions to purified membranes from kidney cortex resulted in their translocation to the membranes and their carboxyl methylation. Both processes were stimulated by guanosine 5'-O-(3-thiotriphosphate). Methylation inhibitors had no effect on the translocation of RhoA to membranes, suggesting that this covalent modification is not required for association to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of GTP-binding proteins in Golgi-associated membrane vesicles from rat adipocytes

1992 ◽  
Vol 283 (3) ◽  
pp. 795-801 ◽  
Author(s):  
A Schürmann ◽  
W Rosenthal ◽  
G Schultz ◽  
H G Joost
Keyword(s):  
G Proteins ◽  
Glucose Transport ◽  
Subcellular Distribution ◽  
Sucrose Gradient ◽  
Binding Proteins ◽  
Glucose Transporters ◽  
Beta Subunits ◽  
Transport Activity ◽  
Gtp Binding Proteins ◽  
Gtp Binding

We have previously reported that guanine nucleotides inhibit glucose transport activity reconstituted from adipocyte membrane fractions. In order to further investigate the hypothetical involvement of guanine-nucleotide-binding proteins (GTP-binding proteins) in the regulation of insulin-sensitive glucose transport activity, we studied their subcellular distribution in adipocytes treated or not with insulin. Adipocytes were homogenized and fractionated to yield plasma membranes (PM) and a Golgi-enriched fraction of intracellular membranes (low-density microsomes, LDM). In these membrane fractions, total guanosine 5′-[gamma-[35S]thio]triphosphate ([35S]GTP[S]) binding, alpha- and beta-subunits of heterotrimeric G-proteins, proto-oncogenes Ha-ras and K-ras, and 23-28 kDa GTP-binding proteins were assayed. The levels of alpha s and alpha i (the alpha-subunits of Gs and Gi) were approx. 8-fold lower in LDM than in PM; beta-subunits, Ha-ras and K-ras were not detectable in LDM. Total GTP[S]-binding sites and 23-28 kDa GTP-binding proteins were present in LDM in approximately the same concentrations as in PM. Insulin gave rise to the characteristic translocation of glucose transporters, but failed to alter the subcellular distribution of any of the GTP-binding proteins. Fractionation of the LDM on a discontinuous sucrose gradient revealed that alpha s and alpha i, as detected with antiserum against a common peptide sequence (alpha common), and the bulk of the 23-28 kDa G-proteins sedimented at different sucrose densities. None of the GTP-binding proteins co-sedimented with glucose transporters. Furthermore, the inhibitory effect of GTP[S] on the reconstituted transport activity was lost in the peak fractions of glucose transporters partially purified on the sucrose gradient. These data indicate that LDM from adipocytes contain several GTP-binding proteins in discrete vesicle populations. However, the intracellular GTP-binding proteins are not tightly associated with the vesicles containing the glucose transporter.

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.

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