Formation Of eIF-2 Research Articles

Structure of the catalytic fragment of translation initiation factor 2B and identification of a critically important catalytic residue.

Eukaryotic initiation factor (eIF) 2B catalyzes the nucleotide activation of eIF2 to its active GTP-bound state. The exchange activity has been mapped to the C terminus of the eIF2Bepsilon subunit. We have determined the crystal structure of residues 544-704 from yeast eIF2Bepsilon at 2.3-A resolution, and this fragment is an all-helical protein built around the conserved aromatic acidic (AA) boxes also found in eIF4G and eIF5. The eight helices are organized in a manner similar to HEAT repeats. The molecule is highly asymmetric with respect to surface charge and conservation. One area in the N terminus is proposed to be directly involved in catalysis. In agreement with this hypothesis, mutation of glutamate 569 is shown to be lethal. An acidic belt and a second area in the C terminus containing residues from the AA boxes are important for binding to eIF2. Two mutations causing the fatal human genetic disease leukoencephalopathy with vanishing white matter are buried and appear to disrupt the structural integrity of the catalytic domain rather than interfering directly with catalysis or binding of eIF2.

M-RNA Binding-Properties of Wheat Germ Protein Synthesis Initiation Factor 2

The binding of protein synthesis initiation factor (eIF−) 2 to mRNA was measured by retention of the mRNA ·eIF-2 complexes on nitrocellulose filters. The binding of eIF-2 to mRNA was inhibited by GDP and GTP; the inhibition by GTP was enhanced by the presence of Met-tRNA i. In addition, the formation of eIF-2 ·GDP binary complex and eIF-2 ·GTPMet-tRNA i ternary complex was inhibited by mRNA. These data indicate that mRNA binds to a site on eIF-2 that is the same or overlaps the site to which guanine nucleotides bind eIF-2. This finding strongly suggests that inhibition of ternary complex formation by mRNA may be the result of competition between mRNA and GTP and not competition between mRNA and Met-tRNA i.

A protein complex of translational regulators of GCN4 mRNA is the guanine nucleotide-exchange factor for translation initiation factor 2 in yeast.

In Saccharomyces cerevisiae, phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2) by protein kinase GCN2 stimulates translation of GCN4 mRNA. In mammalian cells, phosphorylation of eIF-2 alpha inhibits the activity of eIF-2B, the GDP-GTP exchange factor for eIF-2. We present biochemical evidence that five translational regulators of GCN4 encoded by GCD1, GCD2, GCD6, GCD7, and GCN3 are components of a protein complex that stably interacts with eIF-2 and represents the yeast equivalent of eIF-2B. In vitro, this complex catalyzes guanine nucleotide exchange on eIF-2 and overcomes the inhibitory effect of GDP on formation of eIF-2.GTP.Met-initiator tRNA(Met) ternary complexes. This finding suggests that mutations in GCD-encoded subunits of the complex derepress GCN4 translation because they mimic eIF-2 alpha phosphorylation in decreasing eIF-2B activity. Our results indicate that translational control of GCN4 involves a reduction in eIF-2B function, a mechanism used in mammalian cells to regulate total protein synthesis in response to stress.

A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.

Mechanism of Stimulation of Globin Synthesis by Adenosine-3',5'-Monophosphate and Guanosine 5'-Triphosphate in a Rabbit Reticulocyte Lysate System

The inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2 alpha. High concentrations of cAMP (5-10 mM) and GTP (1-2 mM) stimulated the globin synthesis in hemin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide (NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined. As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2 alpha in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S]Met-tRNAf to the 40S ribosomal subunits, and promoted the dephosphorylation of eIF-2(alpha P), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H]GDP from the eIF-2(alpha P) X [3H]GDP complex and the formation of eIF-2(alpha P) X eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2 alpha and promoted both the release of GDP from eIF-2 and the formation of eIF-2(alpha P) X eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2 alpha. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2 alpha and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.

The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7–45

The effects of incubation of yeast spheroplasts at elevated temperature (40°C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells ( wt A364A) and a temperature-sensitive mutant ( ts 7–45) derived from it. With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: (1) the ribosomal subunit-polysome pattern; (2) the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; (3) the translation of poly(U) in postpolysomal extracts; (4) the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; (5) the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and (6) the formation of eIF-2·GTP·Met-tRNA f ternary complex in the cytosol. With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations. Preincubation of temperature-sensitive spheroplasts at 40°C resulted in: (1) a further decrease in the level of 40 S subunits; (2) disaggregation of polysomes; (3) loss of ability to translate natural mRNA but not poly(U); (4) decreased ability to form 40 S preinitiation intermediates; and (5) production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly. The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.

Protein synthesis in brine shrimp embryos and rabbit reticulocytes. The effect of Mg2+ on binary (eukaryotic initiation factor 2 X GDP) and ternary (eukaryotic initiation factor 2 X GTP X met-tRNAf) complex formation.

We have prepared eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes and Artemia embryos and studied the effect of Mg2+ on binary (eIF-2 X GDP) and ternary (eIF-2 X GTP X Met-tRNAf) complex formation. Under conditions where Mg2+ inhibits Met-tRNAf binding to reticulocyte eIF-2, ternary complex formation with Artemia eIF-2 is not inhibited. Similarly, the formation of eIF-2 X GDP with Artemia eIF-2 is stimulated by Mg2+, whereas the corresponding reticulocyte binary complex is strongly inhibited. In the presence of 1 mM Mg2+, the isolated Artemia eIF-2 X GDP complex is stable in the absence of any added nucleotide, but readily exchanges bound GDP for free GTP. However, the reticulocyte eIF-2 X GDP complex is significantly more stable in the presence of GTP, and nucleotide exchange is dependent upon the addition of a factor isolated from either the postribosomal supernatant or the high salt wash of rabbit reticulocyte ribosomes. This factor also stimulates Met-tRNAf binding to both Artemia and reticulocyte eIF-2.

A ribosomal protein mediates eIF-2 phosphorylation by interferon-induced kinase.

We have previously reported that treatment of mouse L cells with interferon (IFN) induces a reduction of initiation factor (eIF-2) activity (ternary complex formation of eIF-2 with Met-tRNAf and GTP)1, and that the reduction of eIF-2 activity involves the phosphorylation of eIF-2 by an IFN-induced protein kinase2. In addition, we considered that the reduction of eIF-2 activity induced by the kinase (cyclic AMP-independent protein kinase) may be accompanied by the phosphorylation of some intermediary factor(s)2. This possibility was supported by early reports that at least two ribosomal proteins (of molecular weights (MWs) 35,000–38,000 and 64,000–67,000) of mouse L cells3–5 and cellular proteins of Ehrlich ascites tumour cells6 are highly phosphorylated by IFN treatment. Recently, we and others have purified IFN-induced protein kinases from different cells treated with IFN2,3,7–9. These purified kinases show several similarities. The reports suggested that the IFN-induced kinase may be responsible for the phosphorylation of the ribosomal proteins (MWs 35,000–38,000 and 65,000–67,000) and that the phosphorylation of these proteins by the kinase may be associated with the reduced eIF-2 activity in IFN-treated cells2–4,8,9. A similar inhibitory mechanism of eIF-2 activity has been demonstrated in rabbit reticulocyte lysates10–15. The present study was undertaken to purify a ribosomal protein [MW 65,000 (65K)] from mouse L cells and to examine the effect of the protein on eIF-2 activity, and also on the binding of the ternary complex to the 40S ribosomal subunit. We found that the eIF-2 activity was greatly reduced when both eIF-2 and the ribosomal protein were incubated with IFN-induced kinase in vitro. Available evidence suggests that the inhibitory mechanism of eIF-2 activity induced by the kinase in vitro may involve phosphorylation of the ribosomal proteins [a small subunit (MW 38,000) of eIF-2 and 65K ribosomal protein] by the IFN-induced kinase.