A group of integral membrane proteins of the rat liver Golgi contains a conserved protein of 100 kDa

1994 ◽  
Vol 107 (12) ◽  
pp. 3425-3436
Author(s):  
J.G. Pryde
Keyword(s):  
Membrane Proteins ◽  
Rat Liver ◽  
Golgi Complex ◽  
Immunofluorescence Microscopy ◽  
Mammalian Species ◽  
Polyclonal Antiserum ◽  
Integral Membrane Proteins ◽  
Golgi Membrane ◽  
Wide Range ◽  
Triton X

Rat liver Golgi membranes were washed with KCl and urea, and a polyclonal antiserum that stained the Golgi complex by immunofluorescence microscopy was raised. A group of proteins of apparent molecular mass 500 kDa, 200 kDa and 100 kDa were identified by immunoblotting with the antiserum, and were enriched in the Golgi membrane fraction. These proteins were also localised to the Golgi by immunofluorescence microscopy with affinity-purified antibodies. They are integral membrane proteins, and protease digestion experiments suggested that they are not exposed on the cytoplasmic face of the Golgi membrane. Immunofluorescence microscopy showed that staining of the Golgi complex by antibodies to the 100 kDa Golgi protein can be demonstrated among a wide range of mammalian species. This conservation may point to an important structural or functional role for the molecule. When the 100 kDa protein was reduced with dithiothreitol it was no longer recognised by the anti-Golgi antiserum. During phase separation in Triton X-114 the 100 kDa protein partitioned into the aqueous phase, rather than into the detergent phase, suggesting that it has a large luminal domain of hydrophilic amino acids.

Evidence for a second class of membrane glycoprotein involved in cell adhesion

1989 ◽  
Vol 93 (4) ◽  
pp. 631-640
Author(s):  
W.E. Norris
Keyword(s):  
Extracellular Matrix ◽  
Biological Activity ◽  
Cell Adhesion ◽  
Membrane Proteins ◽  
Soluble Fraction ◽  
Biological Activities ◽  
Integral Membrane Proteins ◽  
Membrane Glycoprotein ◽  
Transmembrane Receptors ◽  
Triton X

It is believed that transmembrane relationships exist between the cytoskeleton and the extracellular matrix through integral membrane proteins, almost certainly glycoproteins, which would act as transmembrane receptors. Such receptors would include those involved in cell adhesion. I have been able to isolate a detergent-soluble fraction from chick embryo fibroblasts that is enriched in these integral membrane proteins by making use of their amphipathic character to phase-separate them in the detergent Triton X-114. Antisera raised to this fraction had biological activities interfering with cell adhesion and motility. A 45 X 10(3) Mr glycoprotein unique to this fraction appears to be responsible for this biological activity and is a candidate for a transmembrane receptor involved in cell adhesion.

scholarly journals Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina.

1988 ◽  
Vol 107 (6) ◽  
pp. 2029-2036 ◽  
Author(s):  
A Senior ◽  
L Gerace
Keyword(s):  
Membrane Proteins ◽  
Nuclear Membrane ◽  
Immunofluorescence Microscopy ◽  
Nuclear Lamina ◽  
Integral Membrane Proteins ◽  
Inner Nuclear Membrane ◽  
Attachment Sites ◽  
Nonionic Detergent ◽  
Pore Complexes ◽  
Integral Proteins

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.

scholarly journals Trypanosoma cruzi GP63 Proteins Undergo Stage-Specific Differential Posttranslational Modification and Are Important for Host Cell Infection

2009 ◽  
Vol 77 (5) ◽  
pp. 2193-2200 ◽  
Author(s):  
Manjusha M. Kulkarni ◽  
Cheryl L. Olson ◽  
David M. Engman ◽  
Bradford S. McGwire
Keyword(s):  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Host Cell ◽  
Posttranslational Modification ◽  
Immunofluorescence Microscopy ◽  
Polyclonal Antiserum ◽  
Cell Infection ◽  
Specific Expression ◽  
Metacyclic Trypomastigotes ◽  
Triton X

ABSTRACT The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.

scholarly journals Assembly and disassembly of the Golgi complex: two processes arranged in a cis-trans direction.

1992 ◽  
Vol 116 (1) ◽  
pp. 69-83 ◽  
Author(s):  
J Alcalde ◽  
P Bonay ◽  
A Roa ◽  
S Vilaro ◽  
I V Sandoval
Keyword(s):  
Membrane Proteins ◽  
Golgi Complex ◽  
Rat Kidney ◽  
Brefeldin A ◽  
Golgi Membrane ◽  
Temperature Conditions ◽  
Selective Fusion ◽  
Normal Rat ◽  
Short Times

We have studied the disassembly and assembly of two morphologically and functionally distinct parts of the Golgi complex, the cis/middle and trans cisterna/trans network compartments. For this purpose we have followed the redistribution of three cis/middle- (GMPc-1, GMPc-2, MG 160) and two trans- (GMPt-1 and GMPt-2) Golgi membrane proteins during and after treatment of normal rat kidney (NRK) cells with brefeldin A (BFA). BFA induced complete disassembly of the cis/middle- and trans-Golgi complex and translocation of GMPc and GMPt to the ER. Cells treated for short times (3 min) with BFA showed extensive disorganization of both cis/middle- and trans-Golgi complexes. However, complete disorganization of the trans part required much longer incubations with the drug. Upon removal of BFA the Golgi complex was reassembled by a process consisting of three steps: (a) exist of cis/middle proteins from the ER and their accumulation into vesicular structures scattered throughout the cytoplasm; (b) gradual relocation and accumulation of the trans proteins in the vesicles containing the cis/middle proteins; and (c) assembly of the cisternae, and reconstruction of the Golgi complex within an area located in the vicinity of the centrosome from which the ER was excluded. Reconstruction of the cis/middle-Golgi complex occurred under temperature conditions inhibitory of the reorganization of the trans-Golgi complex, and was dependent on microtubules. Reconstruction of the trans-Golgi complex, disrupted with nocodazole after selective fusion of the cis/middle-Golgi complex with the ER, occurred after the release of cis/middle-Golgi proteins from the ER and the assembly of the cis/middle cisternae.

scholarly journals Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

2020 ◽  
Vol 12 (6) ◽  
pp. 1287-1302 ◽  
Author(s):  
Steven Lavington ◽  
Anthony Watts
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Drug Targets ◽  
Large Family ◽  
G Protein Coupled Receptors ◽  
Integral Membrane Proteins ◽  
Lipid Nanoparticle ◽  
Wide Range ◽  
Biophysical Studies ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function.

scholarly journals Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase.

1988 ◽  
Vol 107 (6) ◽  
pp. 2679-2688 ◽  
Author(s):  
W L Dentler
Keyword(s):  
Membrane Proteins ◽  
Atpase Activity ◽  
Tetrahymena Thermophila ◽  
High Molecular Mass ◽  
Integral Membrane Proteins ◽  
Matrix Proteins ◽  
Membrane Atpase ◽  
The Matrix ◽  
Triton X

Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.

Characterization of PrP binding proteins

1994 ◽  
Vol 343 (1306) ◽  
pp. 443-445 ◽  
Keyword(s):  
Membrane Proteins ◽  
Prion Protein ◽  
Binding Proteins ◽  
Mammalian Species ◽  
Integral Membrane Proteins ◽  
Infectious Particle ◽  
Spongiform Degeneration ◽  
The Brain ◽  
Total Membrane

Prions cause spongiform degeneration in various mammalian species. The scrapie prion protein (PrP Sc ) is part of the infectious particle and may mediate infection and spreading of the disease in the brain. It was therefore of interest to purify and analyse PrP ligands (Plis). Plis were identified on ligand blots using either intact PrP or peptides corresponding to the central portion of PrP. Here, characterization of a 110 and a 125 kDa Pli is reported. Both Plis were found in total membrane fractions and could be extracted with carbonate indicating that they are not integral membrane proteins. On sucrose gradients both PrP ligands sedimented with high density particles.

Extraction and identification of human platelet integral membrane proteins using triton X-114

1982 ◽  
Vol 27 (2) ◽  
pp. 221-224 ◽  
Author(s):  
Peter J. Newman ◽  
Michael A. Knipp ◽  
Richard A. Kahn
Keyword(s):  
Membrane Proteins ◽  
Human Platelet ◽  
Integral Membrane Proteins ◽  
Triton X
Sign in / Sign up

Export Citation Format

Share Document