scholarly journals Legionella pneumophila Replication Vacuole Formation Involves Rapid Recruitment of Proteins of the Early Secretory System

2004 ◽  
Vol 72 (5) ◽  
pp. 3048-3053 ◽  
Author(s):  
Isabelle Derré ◽  
Ralph R. Isberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Legionella Pneumophila ◽  
Recruitment Process ◽  
Free System ◽  
Vacuole Formation ◽  
Potential Host ◽  
Bacterial Uptake ◽  
A Cell ◽  
Secretory System ◽  
Vacuole Biogenesis

ABSTRACT Legionella pneumophila vacuole biogenesis was analyzed by using a cell-free system. We show that calnexin, Sec22b, and Rab1 are recruited to the vacuole very shortly after bacterial uptake, and we have identified Rab1 as a potential host factor involved in the endoplasmic reticulum recruitment process.

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 Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine

1985 ◽  
Vol 232 (2) ◽  
pp. 485-491 ◽  
Author(s):  
R Hopewell ◽  
P Martin-Sanz ◽  
A Martin ◽  
J Saxton ◽  
D N Brindley
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Unsaturated Fatty Acids ◽  
Free System ◽  
Phosphatidate Phosphohydrolase ◽  
Percoll Gradients ◽  
Microsomal Membranes ◽  
A Cell ◽  
Nadh Cytochrome

The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, α-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes.

Cell-free assembly of rough and smooth endoplasmic reticulum

1996 ◽  
Vol 109 (6) ◽  
pp. 1415-1425 ◽  
Author(s):  
C. Lavoie ◽  
J. Lanoix ◽  
F.W. Kan ◽  
J. Paiement
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Freeze Fracture ◽  
Tubule Formation ◽  
Free System ◽  
Phosphatase Cytochemistry ◽  
A Cell ◽  
Freeze Fracture Electron Microscopy ◽  
Membrane Networks ◽  
Gamma S

Smooth endoplasmic reticulum assembly was studied in a cell-free system using thin-section and freeze-fracture electron microscopy. Incubation of rat hepatocyte rough and smooth microsomes in the presence of ATP, GTP, cytosol (Xenopus egg) and an ATP-regenerating system led to assembly of membrane networks comprising a central core of interconnecting smooth tubules continuous with peripherally located rough membrane cisternae. Glucose-6-phosphatase cytochemistry confirmed the endoplasmic reticulum origin of the reconstituted membranes. When both ATP and GTP were omitted from the incubation medium, or when GTP was replaced by a variety of nucleotide analogues, including GTP gamma S, membrane aggregates contained only unfused microsomes. The presence of GTP alone stimulated assembly of rough membrane cisternae but had no effect on smooth membranes. Smooth tubule formation occurred independent of cytosol and an ATP-regenerating system, but did require GTP and ATP. Omission of ATP, or replacement of this nucleotide with a variety of analogues, including ATP gamma S, prevented tubule formation but did not affect the assembly of the rough membrane cisternae. Morphometric studies revealed sequential formation of rough membrane cisternae (0-60 minutes) followed by appearance of interconnecting smooth tubules (> 60 minutes). The amount of rough membrane cisternae per membrane network diminished with time after 60 minutes; that of smooth tubules increased. Thus GTP is required for reconstitution of rough membrane cisternae, both GTP and ATP are required for smooth tubule formation, and assembly of smooth tubules occurs as an outgrowth (i.e. via tubulation) from rough membranes.

scholarly journals Reconstitution of transport of vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell-free system.

1987 ◽  
Vol 104 (3) ◽  
pp. 749-760 ◽  
Author(s):  
W E Balch ◽  
K R Wagner ◽  
D S Keller
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Free System ◽  
A Cell ◽  
Vesicular Stomatitis ◽  
High Mannose

Transport of the vesicular stomatitis virus-encoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (man GlcNAc2) ER form of G protein to the man5GlcNAc5 form by the cis Golgi enzyme alpha-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the man5GlcNAc2 form without the appearance of the intermediate man GlcNAc2 oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi alpha-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supernatant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro.

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 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.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

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