scholarly journals Secretory protein translocation in a neurospora crassa in vitro system. Hydrolysis of a nucleoside triphosphate is required for posttranslational translocation.

1987 ◽  
Vol 262 (35) ◽  
pp. 17031-17037
Author(s):  
R Addison
Keyword(s):  
Neurospora Crassa ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Nucleoside Triphosphate ◽  
In Vitro System ◽  
Hydrolysis Of

scholarly journals Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.

1986 ◽  
Vol 102 (5) ◽  
pp. 1543-1550 ◽  
Author(s):  
M G Waters ◽  
G Blobel
Keyword(s):  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Translation System ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free System ◽  
In Vitro System ◽  
Alpha Factor ◽  
Microsomal Membranes

We describe an in vitro system with all components derived from the yeast Saccharomyces cerevisiae that can translocate a yeast secretory protein across microsomal membranes. In vitro transcribed prepro-alpha-factor mRNA served to program a membrane-depleted yeast translation system. Translocation and core glycosylation of prepro-alpha-factor were observed when yeast microsomal membranes were added during or after translation. A membrane potential is not required for translocation. However, ATP is required for translocation and nonhydrolyzable analogues of ATP cannot serve as a substitute. These findings suggest that ATP hydrolysis may supply the energy required for translocation of proteins across the endoplasmic reticulum.

scholarly journals Prepro-carboxypeptidase Y and a truncated form of pre-invertase, but not full-length pre-invertase, can be posttranslationally translocated across microsomal vesicle membranes from Saccharomyces cerevisiae.

1988 ◽  
Vol 106 (4) ◽  
pp. 1075-1081 ◽  
Author(s):  
W Hansen ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Full Length ◽  
Microsomal Membrane ◽  
Carboxypeptidase Y ◽  
Truncated Form ◽  
In Vitro System ◽  
Vesicle Membranes

We have determined that prepro-carboxypeptidase Y and a truncated form of pre-invertase can be translocated across the yeast microsomal membrane post-translationally in a homologous in vitro system. The yeast secretory protein prepro-alpha-factor which was previously shown to be an efficient posttranslational translocation substrate is therefore not unique in this regard, but rather the yeast ER protein translocation machinery is generally capable of accepting substrates from a ribosome-free, soluble pool. However, within our detection limits, full-length pre-invertase could not be translocated posttranslationally, but was translocated co-translationally. This indicates that not every fully synthesized pre-protein can use this pathway, presumably because normal or aberrant folding characteristics can interfere with translocation competence.

scholarly journals Effect of nucleoside triphosphate and magnesium ion concentration on the stability and function of rat liver polysomes in vitro

1968 ◽  
Vol 110 (4) ◽  
pp. 783-788 ◽  
Author(s):  
A. W. Pronczuk ◽  
B. S. Baliga ◽  
H. N. Munro
Keyword(s):  
Rat Liver ◽  
Ion Concentration ◽  
Nucleoside Triphosphate ◽  
Free Protein ◽  
A Cell ◽  
The Stability ◽  
And Function ◽  
Polysome Stability ◽  
Hydrolysis Of

The effects of different concentrations of ATP, GTP, UTP and CTP on polysome stability and function in a cell-free protein-synthesizing system prepared from rat liver were studied. Increasing the concentration of ATP in the incubation medium to 15mm resulted in progressive disaggregation of the polysomes; at ATP concentrations above 2mm their capacity to incorporate amino acids into peptide chains diminished. The same disaggregation phenomenon could be produced by incubating polysomes in a buffered medium containing 5mm-Mg2+ and increasing concentrations of ATP. Although the disaggregating action of ATP could be prevented by increasing Mg2+ concentration, the amino acid incorporation in the cell-free protein-synthesizing system remained impaired. The effects of different concentrations of GTP, UTP and CTP on polysome stability were similar to those of ATP. Increasing the concentrations of each nucleoside triphosphate also inhibited the hydrolysis of GTP in the cell-free protein-synthesizing system.

The role of molecular chaperones in protein transport into the endoplasmic reticulum

1993 ◽  
Vol 339 (1289) ◽  
pp. 335-341 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperones ◽  
Protein Transport ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Microsomal Membrane ◽  
Nucleoside Triphosphate ◽  
Secretory Proteins ◽  
Hsc 70 ◽  
Hydrolysis Of

In eukaryotic cells export of the vast majority of newly synthesized secretory proteins is initiated at the level of the membrane of the endoplasmic reticulum (microsomal membrane). The precursors of secretory proteins are not transported across the microsomal m em brane in their native state. Typically, signal peptides in the precursor proteins are involved in preserving the transport-competent state. Furthermore, there are two alternatively acting mechanisms involved in preserving transport competence in the cytosol. The first mechanism involves two ribonucleoparticles (ribosome and signal recognition particle) and their receptors on the microsomal surface and requires the hydrolysis of GTP. The second mechanism does not involve ribonucleoparticles and their receptors but depends on the hydrolysis of A TP and on cA-acting molecular chaperones, such as heat shock cognate protein 70 (hsc 70). In both mechanisms a translocase in the microsomal membrane mediates protein translocation. This translocase includes a signal peptide receptor on the cu-side of the microsomal membrane and a component that also depends on the hydrolysis of ATP. At least in certain cases, an additional nucleoside triphosphate-requiring step is involved which is related to the trans -acting molecular chaperone BiP.

scholarly journals A Cytosolic Reductase Pathway is Required for Complete N-Glycosylation of an STT3B-Dependent Acceptor Site

2021 ◽  
Author(s):  
Marcel van Lith ◽  
Marie Anne Pringle ◽  
Bethany Fleming ◽  
Giorgia Gaeta ◽  
Jisu Im ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protein Translocation ◽  
Site Occupancy ◽  
Secretory Protein ◽  
Glycosylation Site ◽  
Redox Conditions ◽  
Translation System ◽  
Acceptor Site ◽  
Post Translational Modification

AbstractN-linked glycosylation of proteins entering the secretory pathway is an essential post-translational modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide bond, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is dependent on the STT3A-containing oligosaccharide transferase. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

scholarly journals Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.

1994 ◽  
Vol 5 (4) ◽  
pp. 465-474 ◽  
Author(s):  
C Wachter ◽  
G Schatz ◽  
B S Glick
Keyword(s):  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Inner Membrane ◽  
In Vitro System ◽  
Mitochondrial Inner Membrane ◽  
Precursor Proteins ◽  
The Matrix

ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix is always needed for complete import of precursor proteins into this compartment, even when the precursors are presented to mitochondria in an unfolded conformation. In contrast, the requirement for external ATP is precursor-specific; depletion of external ATP strongly inhibits import of some precursors but has little or no effect with other precursors. A requirement for external ATP can often be overcome by denaturing the precursor with urea. We suggest that external ATP promotes the release of precursors from cytosolic chaperones, whereas matrix ATP drives protein translocation across the inner membrane.

In Vitro Assay of Platelet Adhesiveness with Washed and Tanned Human Red Cells

1968 ◽  
Vol 20 (03/04) ◽  
pp. 384-396 ◽  
Author(s):  
G Zbinden ◽  
S Tomlin
Keyword(s):  
Platelet Adhesion ◽  
Sodium Citrate ◽  
Blood Platelets ◽  
In Vitro Assay ◽  
Red Cells ◽  
Platelet Adhesiveness ◽  
Vitro Assay ◽  
In Vitro System ◽  
Human Red Cells

SummaryAn in vitro system is described in which adhesion of blood platelets to washed and tannic acid-treated red cells was assayed quantitatively by microscopic observation. ADP, epinephrine and TAME produced a reversible increase in platelet adhesiveness which was antagonized by AMP. With Evans blue, polyanetholsulfonate, phthalanilide NSC 38280, thrombin and heparin at concentrations above 1-4 u/ml the increase was irreversible. The ADP-induced increase in adhesiveness was inhibited by sodium citrate, EDTA, AMP, ATP and N-ethylmaleimide. EDTA, AMP and the SH-blocker N-ethylmaleimide also reduced spontaneous platelet adhesion to red cells. No significant effects were observed with adenosine, phenprocoumon, 5-HT, phthalanilide NSC 57155, various estrogens, progestogens and fatty acids, acetylsalicylic acid and similarly acting agents, hydroxylamine, glucose and KCN. The method may be useful for the screening of thrombogenic and antithrombotic properties of drugs.

The Mesothelial Cell as a Non-Thrombogenic Surface

1984 ◽  
Vol 52 (02) ◽  
pp. 102-104 ◽  
Author(s):  
L J Nicholson ◽  
J M F Clarke ◽  
R M Pittilo ◽  
S J Machin ◽  
N Woolf
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Blood Flow ◽  
Cell Surface ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Plastic Film ◽  
In Vitro System ◽  
Scanning Electron

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.

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