Postribosomal complexes containing eukaryotic initiation factor eIF-2

Eukaryotic initiation factors are found in the postribosomal supernatant as well as bound to the 40S ribosomal subunits. We have analyzed the factor activities from the supernatant by means of zonal centrifugation followed by Sepharose-heparin affinity chromatography. They exist both as free factors, sedimenting in a broad range from 4 to 7S, and complexed with other protein(s) with a sedimentation value of 16–20S. This complexed fraction contains besides eIF-2 another activity which exhibits a profound stimulation on amino acid incorporation in crude lysates and appears to counteract the heme-regulated inhibitor.Abbreviations eIF-2, eIF-3, eIF-4A and eIF-4B are eukaryotic initiation factors, see FEBS Letters 76, 1-10 (1977).     

The effect of serum deprivation on the initiation of protein synthesis in mouse neuroblastoma cells

Growth of mouse neuroblastoma cells becomes stationary when cultured in serum-free medium. Within 60 h, the protein-synthesizing capacity of the cells declines to 25% as compared to that of exponentially growing cells. The transitional activity of the crude ribosomal salt washes from serum-deprived and control cells was compared in in vitro protein-synthesizing pH 5 systems. It appears that the ribosomal salt wash from serum-deprived cells has significantly (70%) lost its ability to support the translation of neuroblastoma poly(A)+ RNA. This activity of the ribosomal wash from serum-deprived cells can be restored to control level with rabbit reticulocyte initiation factor eIF-4B only. The ability of the ribosomal wash from serum-deprived cells to support the translation of encephalomyocarditis virus (EMC) and Semliki Forest virus (SFV) 42 S mRNA was tested. We found that EMC-mRNA is efficiently translated with the ribosomal salt wash from serum-deprived cells, whereas on the other hand the translation of SFV 42 S mRNA is severely impaired. Therefore, we conclude that in serum-deprived neuroblastoma cells protein synthesis is regulated in both a quantitative and a qualitative way. Modulation of the activity of initiation factor of protein synthesis eIF-4B is at least partly responsible for the observed (selective) blockade of protein synthesis in serum-deprived cells.     

Shutoff on Neuroblastoma Cell Protein Synthesis by Semliki Forest Virus: Loss of Ability of Crude Initiation Factors to Recognize Early Semliki Forest Virus and Host mRNA's

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing “pH5” system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (±80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5′ terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M_r, 80,000) and cap-binding protein (M_r, 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.     

Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity.

Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption. Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate. This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control. Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells. When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2. However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control. When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed. Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.     

Association of cap-binding protein with eucaryotic initiation factor 3 in initiation factor preparations from uninfected and poliovirus-infected HeLa cells.

Extracts from poliovirus-infected HeLa cells are unable to translate vesicular stomatitis virus or cellular mRNAs in vitro, probably reflecting the poliovirus-induced inhibition of host cell protein synthesis which occurs in vivo. Crude initiation factors from uninfected HeLa cells are able to restore translation of vesicular stomatitis virus mRNA in infected cell lysates. This restoring activity separates into the 0 to 40% ammonium sulfate fractional precipitate of ribosomal salt wash. Restoring activity is completely lacking in the analogous fractions prepared from poliovirus-infected cells. The 0 to 40% ammonium sulfate precipitates from both uninfected and infected cells contain eucaryotic initiation factor 3 (eIF-3), eIf-4B, and the cap-binding protein (CBP), which is detected by means of a cross-linking assay, as well as other proteins. The association of eIF-3 and cap binding protein was examined. The 0 to 40% ammonium sulfate precipitate of ribosomal salt wash from uninfected and infected cells was sedimented in sucrose gradients. Each fraction was examined for the presence of eIF-3 antigens by an antibody blot technique and for the presence of the CBP by cross-linking to cap-labeled mRNAs. From uninfected cells, a major proportion of the CBP cosedimented with eIF-3; however, none of the CBP from infected cells sedimented with eIF-3. The results suggest that the association of the CBP with eIF-3 into a functional complex may have been disrupted during the course of poliovirus infection.     

Identification of a protein kinase activity in rabbit reticulocytes that phosphorylates the mRNA cap binding protein

The 25 kDa mRNA cap binding protein can be purified in a partially phosphorylated state and the extent of its phosphorylation appears to be regulated during heat shock and mitosis in mammalian cells. We demonstrated that a nonabundant serine protein kinase activity exists in rabbit reticulocytes that phosphorylates the 25 kDa cap binding protein in both the free (eIF-4E) and complexed (eIF-4F) state. This kinase was not inhibited by the cAMP-dependent protein kinase inhibitory peptide IAAGRTGRRNAIHDILVAA, did not phosphorylate S6 ribosomal protein, did not phosphorylate p220 of eIF-4F as protein kinase C does and no other substrates for this kinase were apparent in reticulocyte ribosomal salt wash. The molecular identity of this kinase, the specific site(s) of eIF-4E that it phosphorylates and its in vivo regulatory role remain to be studied.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

The role of mammalian initiation factor eIF-4D and its hypusine modification in translation

Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.     

Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae

A 24 000-dalton protein [yeast eukaryotic initiation factor 4E (eIF-4E)] was purified from yeast Saccharomyces cerevisiae postribosomal supernatant by m7GDP-agarose affinity chromatography. The protein behaves very similarly to mammalian protein synthesis initiation factor eIF-4E with respect to binding to and elution from m7GDP-agarose columns and cross-linking to oxidized reovirus mRNA cap structures. Yeast eIF-4E is required for translation as shown by the strong and specific inhibition of cell-free translation in a yeast extract by a monoclonal antibody directed against yeast eIF-4E.     

Protein synthesis in brine shrimp embryos. Dormant and developing embryos of Artemia salina contain equivalent amounts of chain initiation factors 2.

Dormant and developing embryos of Artemia salina contain equivalent amounts of eIF-2, the eukaryotic initiation factor which forms a ternary complex with GTP and Met-tRNAf. The factor was purified from 0.5 M NH4Cl ribosomal washes by (NH4)2SO4 fractionation, followed by chromatography on heparin-Sepharose, DEAE-cellulose, hydroxyapatite and phosphocellulose. Purified preparations from dormant and developing embryos have similar specific activities and nucleotide requirements. The mobility of both proteins in dodecylsulfate gel electrophoresis is indistinguishable, and each contains three major polypeptide chains of molecular weight 52 000, 45 000 and 42 000. Both proteins are also immunologically identical, and each stimulates amino acid incorporation in a cell-free system of protein synthesis. The binding of [35S]Met-tRNAf to 40-S ribosomal subunits is catalyzed by eIF-2 isolated from dormant or developing embryos and is dependent upon GPT and AUG. Binding of [35S]Met-tRNAf to 40-S ribosomal subunits, and ternary complex formation with eIF-2, GTP, and [35S]Met-tRNAf is stimulated 2--3-fold by a factor present in the 0.5 M NH4Cl ribosomal wash and which elutes from DEAE-cellulose at 50 mM KCl. This protein does not exhibit GTP-dependent binding of [35S]Met-tRNAf. Binding of GDP and GTP was investigated with purified eIF-2 from developing embryos. The factor forms a binary complex with GDP or GTP, and eIF-2-bound [3H]GDP exchanges very slowly with free nucleotides. Our results suggest that eIF-2 does not limit resumption of embryo development following encystment, nor does it limit mRNA translation in extracts from dormant embryos.     

Subcellular localization of a lesion in protein synthesis in rabbit reticulocytes incubated at elevated temperatures.

When rabbit reticulocytes are incubated at 43-45 degrees C their rate of protein synthesis rapidly decreases, compared to a contol 37 degrees C incubation. Lysates prepared from cells incubated at this supra-optimal temperature have an equally decreased capacity for endogenous, but not poly(uridylic acid)-directed, protein synthesis. Subcellular fractionation traced the lesion to the crude ribosomal pellet, 0.5 M KCl ribosomal wash and postribosomal supernatant of the temperature-shocked cells. Preparation of purified ribosomal subparticles showed, however, that they were as active as the control in protein synthesis. In this paper we present evidence that the decreased activity of the heated lysate, 0.5 mM KCl wash and postribosomal supernatant is due to an inhibitor and can be overcome by the addition of 0.5 M KCl or supernatant from control cells. The results are discussed in terms of the inactivation of a component, essential for initiation of endogenous protein synthesis, which is probably partitioned between ribosomes and supernatant. We also suggest that the decreased protein synthetic activity of the heated cells may be related to their decreased synthesis of haem.     

Euglena gracilis chloroplast initiation factor 2. Identification and initial characterization

The chloroplast protein synthesis factor responsible for the binding of fMet-tRNAMeti to chloroplast 30 S ribosomal subunits (IF-2chl) has been identified in whole cell extracts of Euglena gracilis. The IF-2chl activity is present in considerably higher amounts in extracts of light-grown cells than in extracts of dark-grown cells. About 90% of this activity is found in the postribosomal supernatant of the cell. Chromatography on phosphocellulose results in the partial purification of IF-2chl and separates the chloroplast factor from the cytoplasmic factor eIF-2A. The binding of fMet-tRNAMeti to chloroplast 30 S subunits is message-dependent as observed for prokaryotic systems. In addition, GTP stimulates the IF-2chl-dependent reaction 3-fold. The binding reaction shows broad monovalent and divalent cation optima. The activity of IF-2chl is stimulated 2-fold by the addition of either Escherichia coli IF-1 or IF-3, and 4-fold by the inclusion of both factors. Chloroplast IF-2 is quite active on the homologous 30 S ribosomal subunits but shows little activity on E. coli 30 S or wheat germ 40 S subunits.     

Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

A protein with specific affinity for the mRNA cap structure was purified both from the postribosomal supernatant and from the ribosomal high-salt wash of Drosophila melanogaster embryos by m7GTP-Sepharose chromatography. This protein had an apparent molecular mass of 35 kilodaltons (kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size very different from those of the cap-binding proteins that have been characterized thus far. Drosophila 35-kDa cap-binding protein (CBP) could also be isolated from the ribosomal high-salt wash as part of a salt-stable protein complex consisting of polypeptides of 35, 72, and 140 to 180 kDa. Polyclonal antibodies against Drosophila 35-kDa CBP neither reacted with eucaryotic initiation factor 4E from rabbit reticulocytes nor affected mRNA translation in a rabbit reticulocyte cell-free system. However, in a cell-free system from Drosophila embryos, mRNA translation was specifically inhibited by these antibodies. The requirement of 35-kDa CBP for mRNA translation in Drosophila was diminished under ionic conditions in which the importance of mRNA cap structure recognition was reduced. Despite the structural differences between Drosophila 35-kDa CBP and mammalian initiation factor 4E, both proteins were functionally interchangeable in the in vitro translation system from Drosophila embryos.     

Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Purification and characterization of a protein factor that reverses the inhibition of protein synthesis by the heme-regulated translational inhibitor in rabbit reticulocyte lysates.

We have purified and partially characterized a supernatant factor which reverses the effect of the heme-regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti-inhibitor restores protein synthesis activity in heme deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF-2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti-inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF-2. A possible mechanism of action is discussed.     

Overview: phosphorylation and translation control

Protein synthesis is controlled by the phosphorylation of proteins comprising the translational apparatus. At least 12 initiation factor polypeptides, 3 elongation factors and a ribosomal protein are implicated. Stimulation of translation correlates with enhanced phosphorylation of eIF-4F, eIF-4B, eIF-2B, eIF-3 and ribosomal protein S6, whereas inhibition correlates with phosphorylation of eEF-2 and the alpha-subunit of eIF-2. Strong evidence for regulatory roles exists for eIF-2, eIF-4F and eEF-2, whereas changes in other factor activities due to phosphorylation remain to be demonstrated. Regulation of the specific activity of the translational apparatus by phosphorylation appears to be a general mechanism for the control of rates of global protein synthesis, and may also play a role in modulating the translation of specific mRNAs.     

Evidence that the requirements for ATP and wheat germ initiation factors 4A and 4F are affected by a region of satellite tobacco necrosis virus RNA that is 3' to the ribosomal binding site

A cDNA containing the complete genome of satellite tobacco necrosis virus (STNV) RNA was constructed and cloned into a plasmid vector containing the T7 polymerase promotor. A second clone containing the first 54 nucleotides from the 5' end, which includes the ribosome binding site, was also constructed. RNAs were transcribed from these plasmids (pSTNV1239 and pSTNV54) and tested for their ability to bind to wheat germ 40 S ribosomal subunits in the presence of wheat germ initiation factors eIF-4A, eIF-4F, eIF-4G, eIF-3, eIF-2, Met-tRNA, ATP, and guanosine 5'-(beta, gamma-imino)triphosphate (GMP-PNP). Maximal binding of the STNV RNA transcribed from pSTNV1239 is obtained only in the presence of all the initiation factors and ATP. In contrast, close to maximal binding of STNV RNA transcribed from pSTNV54 is obtained in the absence of eIF-4A, eIF-4F, eIF-4G, and ATP. A series of deletion clones from the 3' end of the STNV cDNA was prepared, and the requirements for binding to 40 S ribosomal subunits were determined. STNV RNAs containing more than 134 nucleotides from the 5' end require eIF-4A, eIF-4F, eIF-4G, and ATP for maximal binding to 40 S ribosomal subunits, whereas STNV RNAs containing 86 nucleotides or less no longer require ATP and these factors. These findings indicate that a region 3' to the initiation codon affects the requirements for eIF-4A, eIF-4F, eIF-4G, and ATP.     

Activation of double-stranded RNA-activated protein kinase in HeLa cells after poliovirus infection does not result in increased phosphorylation of eucaryotic initiation factor-2.

Protein kinase activity in general is stimulated at least 5- to 10-fold in ribosomal salt wash preparations from poliovirus-infected HeLa cells compared with those from mock-infected cells. The stimulation of kinase activity is manifested by increased phosphorylation of ribosome-associated polypeptides having approximate molecular weights of 135,000, 120,000, 85,000, 68,000, 65,000, 40,000, 28,000, 25,000, and 21,000. The Mr 68,000 phosphoprotein is structurally identical to the interferon-induced, double-stranded RNA-activated protein kinase (P1) which phosphorylates the alpha subunit of eucaryotic initiation factor-2 (eIF-2). A similar protein of Mr 68,000 is more phosphorylated in poliovirus-infected cells than in mock-infected cells. Increased phosphorylation of P1 protein in poliovirus-infected cells, however, does not result in an increased phosphorylation of the alpha subunit of endogenous or exogenously added eIF-2, both in vitro and in vivo. These results suggest that a mechanism must exist in poliovirus-infected HeLa cells which prevents further phosphorylation of eIF-2 by the activated kinase.     

Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.