Protein folding and maturation in a cell-free system

1998 ◽  
Vol 76 (5) ◽  
pp. 867-873 ◽  
Author(s):  
Daniel N Hebert ◽  
Jian-Xin Zhang ◽  
Ari Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Effective Means ◽  
Cellular Systems ◽  
Temporal Association ◽  
Dependent Manner ◽  
Viral Glycoprotein ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Reduced cellular systems have provided important tools to study complex cellular processes. Here we describe the oxidation, oligomerization, and chaperone binding of the viral glycoprotein influenza hemagglutinin in a cell-free system. The cell-free system, comprised of rough endoplasmic reticulum derived microsomes and a reticulocyte lysate, supported the complete maturation of hemagglutinin from the earliest oxidative intermediate to the mature homo-oligomer. Hemagglutinin disulfide bond formation and oligomerization were found to occur in a time- and temperature-dependent manner. Hemagglutinin's temporal association with the molecular chaperones calnexin and calreticulin was similar to that observed for their association with elongating ribosome-attached nascent chains in live cells. Furthermore, a procedure is described that permits the translocation of protein into microsomes that are depleted of lumenal contents. This cell-free system, therefore, provided an effective means to study the biological maturation processes of a protein that traverses the secretory pathway.Key words: protein folding, endoplasmic reticulum, molecular chaperone.

scholarly journals Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

1991 ◽  
Vol 266 (7) ◽  
pp. 4322-4328 ◽  
Author(s):  
P Moreau ◽  
M Rodriguez ◽  
C Cassagne ◽  
D M Morré ◽  
D J Morré
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Ligand-dependent and -independent activation of the transcription factor γRF-1 in a cell-free system

1995 ◽  
Vol 310 (2) ◽  
pp. 461-467 ◽  
Author(s):  
C A Feghali ◽  
T M Wright
Keyword(s):  
Transcription Factor ◽  
Tyrosine Phosphatase ◽  
Cell Fractionation ◽  
Dependent Manner ◽  
Sodium Orthovanadate ◽  
Ifn Gamma ◽  
Free System ◽  
Cell Free System ◽  
A Cell

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.

scholarly journals Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Tomoyuki Iwasaki ◽  
Naoe Kaneko ◽  
Yuki Ito ◽  
Hiroyuki Takeda ◽  
Tatsuya Sawasaki ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Early Onset ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Blau Syndrome ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Early Onset Sarcoidosis

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

scholarly journals A Cell-Free System for Regulated Exocytosis in Pc12 Cells

2000 ◽  
Vol 148 (2) ◽  
pp. 317-324 ◽  
Author(s):  
Julia Avery ◽  
Darren J. Ellis ◽  
Thorsten Lang ◽  
Phillip Holroyd ◽  
Dietmar Riedel ◽  
...  
Keyword(s):  
Acridine Orange ◽  
Dependent Manner ◽  
Intact Cells ◽  
Free System ◽  
Regulated Exocytosis ◽  
Cell Free System ◽  
Force Microscopy ◽  
Atomic Force ◽  
A Cell

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca2+ at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca2+-dependent manner dur- ing 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimula- tion. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.

scholarly journals Yolk platelets impede nuclear expansion in Xenopus embryos

2021 ◽  
Author(s):  
Sora Shimogama ◽  
Yasuhiro Iwao ◽  
Yuki Hara
Keyword(s):  
Gene Expression ◽  
Endoplasmic Reticulum ◽  
Gene Expression Regulation ◽  
Early Embryogenesis ◽  
Dependent Manner ◽  
Nucleus Size ◽  
Free System ◽  
Dependent Cell ◽  
A Cell ◽  
Nuclear Expansion

ABSTRACTDuring metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. There was slower nuclear expansion during longer interphase durations in blastomeres from vegetal hemispheres than those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, there was slower nuclear expansion and DNA replication as compared to normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion in the yolk-rich vegetal blastomeres.

scholarly journals Temperature- and acceptor-specificity of cell-free vesicular transfer from transitional endoplasmic reticulum to the cis Golgi apparatus

1992 ◽  
Vol 288 (3) ◽  
pp. 969-976 ◽  
Author(s):  
S Dunkle ◽  
T Reust ◽  
D D Nowack ◽  
L Waits ◽  
M Paulik ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
Transitional Endoplasmic Reticulum ◽  
Acceptor Specificity ◽  
Membrane Compartment ◽  
A Cell

The temperature dependence and specificity of transfer of membrane constituents from donor transitional endoplasmic reticulum to the cis Golgi apparatus were investigated using a cell-free system from rat liver. The radiolabelled transitional endoplasmic reticulum donors were prepared from slices of rat liver prelabelled with [14C]leucine. The acceptor Golgi apparatus elements were unlabelled and immobilized on nitrocellulose. When Golgi apparatus stacks were separated by preparative free-flow electrophoresis into subfractions enriched in cisternae derived from the cis, medial and trans portions of the stack respectively, efficient specific transfer was observed only to cis elements. Trans elements were devoid of specific acceptor capacity. Similarly, when transfer was determined as a function of temperature, a transition was observed in transfer activity between 12 degrees C and 18 degrees C similar to that seen in vivo for formation of the so-called 16 degrees C cis Golgi-located membrane compartment. Transfer at temperatures below 16 degrees C and transfer to trans Golgi apparatus compartments at temperatures either above or below 16 degrees C was similar and unspecific. The unspecific transfer at low temperature was pH independent, whereas specific transfer was greatest at the physiological pH of 7, and was reduced to 10% and 18% of that occurring at pH 8 and pH 5.5 respectively. These findings show that the cell-free system derived from rat liver exhibits a high degree of fidelity to transfer in vivo, an efficiency approaching that observed in vivo, and a nearly absolute acceptor specificity for cis Golgi apparatus. The acceptor-, temperature- and pH-specificity of the cell-free transfer, as well as the saturation kinetics exhibited with respect to acceptor Golgi apparatus, support the concept of transition-vesicle-specific docking sites of finite number associated with cis Golgi apparatus cisternae.

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