Nuclear localization of initiation factor 2 in neuron primary cultures

1995 ◽  
Vol 53 ◽  
pp. 774-775
Author(s):  
F.J. Martinez Alonso ◽  
M.V. Toledo Lobo ◽  
S. Rodriguez Martínez ◽  
F.M. Muñoz Postigo ◽  
J.J. López-Fando Castro
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Control Function ◽  
Primary Cultures ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
40S Ribosomal Subunit ◽  
Initiation Factor 2 ◽  
Immunocytochemical Techniques ◽  
Embryo Brain

The dominant mechanism that controls protein synthesis is the phosphorylation/dephosphorylation of initiation and elongation factors, with a translational control function. Each phase of protein synthesis is promoted by some of these factors that transiently interact with ribosomes, mRNAs and aminoacyltRNAs. Eukaryotic initiation factor-2 (eIF2, 130 kD) is one of these proteins and it is composed of three subunits: alpha, beta and gamma. eIF2 forms a ternary complex (GTP-eIF2-Met tRNAi) that can then interact with the 40S ribosomal subunit which in turn binds mRNA and the 60S ribosomal subunit to form the 80S initiation complex. The relation between eIF2 and the ribosomes is then a well established aspect of protein synthesis, but there are no previous studies about the distribution of eIF2 within the cell.Using immunocytochemical techniques, we show the distribution of eIF2 within the cell found in primary cultures of rat embryo brain neurons, in which eIF2 and eIF2-kinases have been identified. Primary culture neuron cells were grown in D15 and N2 mediums for 8 days.

scholarly journals Effects of starvation, diabetes and acute insulin treatment on the regulation of polypeptide-chain initiation in rat skeletal muscle

1984 ◽  
Vol 223 (3) ◽  
pp. 687-696 ◽  
Author(s):  
C S Harmon ◽  
C G Proud ◽  
V M Pain
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Polypeptide Chain ◽  
Ribosomal Subunit ◽  
Diabetic Rats ◽  
Initiation Factor ◽  
Chain Initiation ◽  
Low Concentrations ◽  
40S Ribosomal Subunit

The rate of protein synthesis in skeletal muscle is greatly decreased in response to diabetes and starvation. Analysis of polyribosome profiles indicates that polypeptide-chain initiation is impaired under these conditions. To identify the step in initiation that is affected, we assayed the incorporation of [35S]methionyl-tRNAfMet into [35S]methionyl-tRNAfMet . 40S-ribosomal-subunit initiation complexes in cell-free extracts based on postmitochondrial supernatants prepared from gastrocnemius muscle. Extracts from either starved or diabetic rats were 30-40% less active in forming these complexes compared with those derived from fed or insulin-maintained controls respectively. This change could be reversed by treatment of either starved or diabetic rats with insulin in vivo 30 min before death. Formation of 40S initiation complexes by extracts from either fed or starved rats could be stimulated by the addition of exogenous purified initiation factor eIF-2, but extracts from starved or diabetic rats were more sensitive than controls to stimulation by low concentrations of the factor. These results provide evidence for the acute regulation by insulin of protein synthesis in skeletal muscle at the level of polypeptide-chain initiation, and suggest that in this tissue, as in certain other eukaryotic systems, control of initiation appears to be mediated by changes in the activity of initiation factor eIF-2.

scholarly journals Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 α-Subunit Kinase, PEK, Involved in Translational Control

1998 ◽  
Vol 18 (12) ◽  
pp. 7499-7509 ◽  
Author(s):  
Yuguang Shi ◽  
Krishna M. Vattem ◽  
Ruchira Sood ◽  
Jie An ◽  
Jingdong Liang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Pancreatic Islet ◽  
Translational Control ◽  
Islet Cells ◽  
Initiation Factor ◽  
Pancreatic Islet Cells ◽  
Α Subunit ◽  
Initiation Factor 2 ◽  
Identification And Characterization

ABSTRACT In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In mammals, the phosphorylation was shown to be carried out by eIF-2α kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2α kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2α kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced inEscherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2α on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In theSaccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2α kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2α. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2α kinase plays an important role in translational control from nematodes to mammals.

scholarly journals Fission Yeast Asc1 Stabilizes the Interaction between Eukaryotic Initiation Factor 3a and Rps0A/uS2 for Protein Synthesis

2019 ◽  
Vol 39 (19) ◽  
Author(s):  
Yi-Ting Wang ◽  
Yu-Chen Chien ◽  
Wan-Yi Hsiao ◽  
Chien-Chia Wang ◽  
Shao-Win Wang
Keyword(s):  
Protein Synthesis ◽  
Complex Formation ◽  
Fission Yeast ◽  
Ribosomal Subunit ◽  
Trna Synthetase ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Aminoacyl Trna Synthetase ◽  
Preinitiation Complex ◽  
40S Ribosomal Subunit

ABSTRACT Aminoacyl-tRNA synthetase cofactors play important roles in coordinating aminoacylation and translation. In this study, we describe an additional function of the fission yeast aminoacyl-tRNA synthetase cofactor 1 (Asc1) in translation. We found that Asc1 directly binds and stabilizes the interaction between small ribosomal protein Rps0A/uS2 and eukaryotic initiation factor 3a (eIF3a). In the absence of Asc1, the interaction between eIF3a and Rps0A/uS2 was compromised. The interaction between Rps0A/uS2 and eIF3a mediated the 40S ribosomal subunit binding of eIF3 in 43S preinitiation complex formation to stimulate translation initiation. Keeping with this idea, in an asc1 mutant, the association of mRNA with the 40S ribosomal subunit was defective and protein synthesis was compromised. To show that Asc1 is directly involved in translation, we demonstrate that the addition of recombinant Asc1 is able to rescue the translation defect of the asc1 mutant in a cell-free system. Furthermore, this function of Asc1 is likely to be evolutionarily conserved, as a similar interaction with eIF3a and Rps0A/uS2 could be identified in the budding yeast Saccharomyces cerevisiae and human aminoacyl-tRNA synthetase cofactors. Together, these results identify a function of aminoacyl-tRNA synthetase cofactors in translation preinitiation complex formation, which adds significantly to the expanded functions associated with aminoacyl-tRNA synthetases and their cofactors.

scholarly journals Translation Initiation Factor 2γ Mutant Alters Start Codon Selection Independent of Met-tRNA Binding

2008 ◽  
Vol 28 (22) ◽  
pp. 6877-6888 ◽  
Author(s):  
Pankaj V. Alone ◽  
Chune Cao ◽  
Thomas E. Dever
Keyword(s):  
Binding Affinity ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Start Codon ◽  
Codon Recognition ◽  
40S Ribosomal Subunit ◽  
Initiation Factor 2

ABSTRACT Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNAi Met and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNAi Met to the 40S ribosomal subunit, and previous studies identified Sui− mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNAi Met binding. Consistently, an eIF2γ-N135D GTP-binding domain mutation impairs Met-tRNAi Met binding and causes a Sui− phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNAi Met binding affinity and cell growth; however, only A208V suppresses the Sui− phenotype associated with the eIF2γ-N135D mutation. An eIF2γ-A219T mutation impairs Met-tRNAi Met binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui− phenotype associated with the eIF2γ-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui− phenotype of the eIF2γ mutants. We propose that structural alterations in eIF2γ subtly alter the conformation of Met-tRNAi Met on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNAi Met binding affinity.

scholarly journals Interaction between membrane functions and protein synthesis in reticulocytes. An elongation-stage inhibitor of protein synthesis extracted from the reticulocyte membrane

1979 ◽  
Vol 180 (2) ◽  
pp. 379-387 ◽  
Author(s):  
D H Wreschner ◽  
M Herzberg
Keyword(s):  
Protein Synthesis ◽  
Cell Membrane ◽  
High Speed ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Membrane Component ◽  
Initiation Complex ◽  
Initiation Factor 2 ◽  
Reticulocyte Lysate ◽  
Inhibitor Of Protein Synthesis

A component of the reticulocyte cell membrane was found to inhibit protein synthesis severely in a reticulocyte lysate system. An investigation into the mode of action of the membrane inhibitor revealed the following facts. (1) The binding of the tertiary initiation complex (methionyl-tRNAfMet-Initiation Factor 2-GTP) to the 40S ribosomal subunit was unaffected by the membrane inhibitor. (2) The membrane component did not interfere with the binding of the 40S initiation complex to the AUG initiation codon and subsequent attachment of the 60S ribosomal subunit. (3) Elongation of the peptide chain, as assayed by peptidyl-puromycin formation, was markedly affected by the membrane inhibitor. Surprisingly, the membrane component caused a considerable increase in peptidyl-puromycin formation. (4) Reticulocyte ribosomes that had been reisolated by high-speed centrifugation, after preincubation with the membrane component, were found to be highly defective when assayed in a cell-free protein-synthesizing system. These results indicated that an extract of the reticulocyte cell membrane inhibited protein synthesis by interacting with the ribosome and thus interfered with the correct functions of the elongation stage of protein synthesis. The implications of this conclusion are discussed in the light of data showing that a highly purified preparation of the membrane inhibitor also displayed an endonucleolytic activity highly specific for 28S RNA.

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