Regulation of protein synthesis in rabbit reticulocyte lysates: Additional initiation factor required for formation of ternary complex (eIF-2·GTP·Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.     

Polypeptide chain initiation in eukaryotes: reversibility of the ternary complex-forming reaction.

In the last step of polypeptide chain initiation in eukaryotes, the interaction of the 40S preinitiation complex eIF-2.GTP.Met-tRNAi.40S [the complex between the 40S ribosomal subunit and the ternary complex containing equimolar amounts of eukaryotic initiation factor 2 (eIF-2), GTP, and eukaryotic initiator methionyl tRNA (Met-tRNAi)] with a 60S ribosomal subunit in the presence of mRNA, cap binding protein (with "capped" messengers), ATP, and the initiation factors eIF-3, eIF-4a, -4b, -4c, and eIF-5, results in the formation of an 80S initiation complex (Met-tRNAi.80S.mRNA) with concomitant hydrolysis of GTP and liberation of eIF-2 for recycling in subsequent initiation events. However, at physiological Mg2+ concentrations, GDP is known to have approximately equal to 100-fold greater affinity than GTP for eIF-2 and eIF-2 is believed to be released in the form of an eIF-2.GDP complex. Previously, we have shown that initiation factor SP (for eIF-2-stimulating protein) promotes the exchange of eIF-2-bound GDP for GTP and catalyzes ternary complex formation in the presence of Met-tRNAi. Binding of GDP by eIF-2 is indeed so tight that, as we now show, homogeneous preparations of eIF-2 contain upward of 0.5 mol of GDP/mol of eIF-2. We further show that, in the presence of Mg2+ and catalytic amounts of SP, ternary complex formation conforms to the overall reversible reaction eIF-2.GDP + GTP + Met-tRNAi in equilibrium eIF-2.GTP.Met-tRNAi + GDP.     

Binding and release of eukaryotic initiation factor eIF-2 and GTP during protein synthesis initiation.

The eukaryotic initiation factor eIF-2 forms a ternary complex with Met-tRNAf and GTP. This complex binds to the 40S ribosomal subunit in the absence of mRNA and mRNA binding factors. Highly purified eIF-2 from rabbit reticulocytes was labeled with 125I by using the Bolton-Hunter reagent or with [gamma-32P]ATP by using the heme-regulated translational inhibitor protein kinase. The labeled eIF-2 was bound, together with equimolar amounts of Met-tRNAf and GTP, to the 40S subunit. In the presence of mRNA, mRNA binding factors, and 60S ribosomal subunits (complete initiation assay), eIF-2 was released from the 40S initiation complex in the subunit joining reaction. GTP also was released in this step and probably was hydrolyzed in the reaction that is dependent upon eIF-5 and the 60S subunit. The function of phosphorylated eIF-2 in initiation of protein synthesis is discussed.     

Removal of beta subunit of the eukaryotic polypeptide chain initiation factor 2 by limited proteolysis.

It is generally considered that the eukaryotic polypeptide chain initiation factor 2 (eIF-2) from rabbit reticulocytes consists of three nonidentical subunits termed alpha, beta, and gamma, in order of increasing molecular weight. However, a recent report [Stringer, E. A., Chaudhuri, A., Valenzuela, D. & Maitra, U. (1980) Proc. Natl. Acad. Sci. USA 77, 3356-3359] suggested that this factor is made up of only two subunits. In this paper we show that limited proteolysis of rabbit reticulocyte eIF-2 leads to loss of the beta subunit. This modified eIF-2 has the same activity as the native factor in promoting ternary (eIF-2.GTP.Met-tRNAi) and 40S (eIF-2.GTP.Met-tRNAi.40S ribosome) initiation complex formation. Like native eIF-2, the protease-treated factor can restore translation in heme-deficient lysates. On the other hand, the treated factor is less stable than the native protein.     

In situ phosphorylation of the α subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase

Protein synthesis initiation in reticulocyte lysates is inhibited by heme deficiency, low levels of double-stranded RNA (dsRNA), oxidized glutathione (GSSG), or the purified kinase (HRI) that acts on the alpha polypeptide of eukaryotic initiation factor 2 (eIF-2alpha). The phosphoprotein profiles produced in lysates in response to these various conditions have been monitored directly in lysates after labeling for brief periods with pulses of [gamma-(32)P]ATP. The [(32)P]phosphoprotein profiles were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide slab gels under conditions in which the HRI and eIF-2alpha polypeptides were clearly distinguished. All four modes of inhibition produced a rapid phosphorylation of eIF-2alpha compared to control lysates, which displayed little or no phosphorylation of eIF-2alpha. In heme-deficient lysates, phosphorylation of eIF-2alpha occurred rapidly both before and after the shut-off of protein synthesis; the delayed addition of hemin to these lysates resulted in a decrease in the phosphorylation of eIF-2alpha and the subsequent restoration of protein synthesis. These data suggest that rapid turnover of phosphate occurs at the site(s) of eIF-2alpha phosphorylation. In lysates inhibited by heme deficiency, GSSG, or added HRI, the phosphorylation of eIF-2alpha was accompanied by the rapid in situ phosphorylation of HRI. The inhibition of initiation induced by dsRNA was accompanied by the phosphorylation of eIF-2alpha and a 67,000-dalton polypeptide but not HRI. These observations in situ indicate that (i) the phosphorylation of eIF-2alpha is the critical event in these inhibitions of protein chain initiation, and (ii) the phosphorylation of HRI is associated with its activation in heme deficiency.     

Protein synthesis in rabbit reticulocytes: characteristics of a postribosomal supernatant factor that reverses inhibition of protein synthesis in heme-deficient lysates and inhibition of ternary complex (Met-tRNAfMet.eIF-2.GTP) formation by heme-regulated inhibitor.

During heme deficiency in reticulocyte lysates, a translational inhibitor (heme-regulated inhibitor, HRI) that blocks polypeptide chain initiation is activated. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAfMet binding factor, eIF-2. Phosphorylation of eIF-2 by HRI prevents its interaction with at least two additional factors, resulting in a net reduction in formation of ternary complex (Met-tRNAfMet.eIF-2.GTP) and AUG-dependent transfer of Met-tRNAfMet to 40S ribosomal subunits. A factor (sRF) that reverses protein synthesis inhibition in heme-deficient lysates has been purified from reticulocyte postribosomal supernatant. sRF also reverses the inhibition of ternary complex formation by HRI in a fractionated system. The ternary complex inhibition reversal activity and the protein synthesis inhibition reversal activity cosediment at 12.5 S upon glycerol density gradient centrifugation, and both activities are sensitive to heat or N-ethylmaleimide. Purified sRF does not dephosphorylate eIF-2 whose phosphorylation has been catalyzed by HRI, nor does the sRF prevent the phosphorylation of eIF-2 by HRI in a fractionated system. sRF stimulates ternary complex formation by both phosphorylated and nonphosphorylated eIF-2. These observations suggest that the sensitivity of protein synthesis to phosphorylation of eIF-2 by HRI may be modulated by the concentration and activity of sRF.     

Relationship between phosphorylation and activity of heme-regulated eukaryotic initiation factor 2 alpha kinase.

In heme-deficient reticulocytes and their lysates, a heme-regulated inhibitor of protein synthesis is activated; this inhibitor is a cyclic AMP-independent protein kinase that specifically phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF-2 alpha). Heme regulates this kinase by inhibiting its activation and activity. The purified heme-regulated kinase (HRI) undergoes autophosphorylation; at least 3 mol of phosphate can be incorporated per HRI subunit (Mr 80,000). The phosphorylation of HRI, its eIF-2 alpha kinase activity, and its ability to inhibit protein synthesis are diminished by hemin (5 microM) and increased by N-ethylmaleimide (MalNEt). Treatment of MalNEt-activated HRI with hemin reduces its autophosphorylation and its ability to inhibit protein synthesis . These findings demonstrate a correlation of the phosphorylation of HRI, its eIF-2 alpha kinase activity, and its inhibition of protein synthesis. The mechanism of hemin regulation of HRI activity was studied by examining the binding of hemin to purified HRI. Significant binding was demonstrable by difference spectroscopy which revealed a pronounced shift in the absorption spectrum of hemin with the appearance of a peak at 418 nm, a shift similar to that observed with proteins known to bind hemin. These findings are consistent with a direct effect of hemin on HRI.     

Protein synthesis in rabbit reticulocytes: characteristics of the protein factor RF that reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates.

During heme deficiency in reticulocyte lysates, the heme-regulated translational inhibitor of protein synthesis (HRI) is activated and shuts off protein synthesis. In partial reactions, HRI phosphorylates the Mr 38,000 subunit (alpha subunit) of eukaryotic initiation factor 2 (eIF-2), which forms a ternary complex, Met-tRNAf X eIF-2 X GTP. The eIF-2 alpha (P) thus formed is not recognized by two eIF-2 ancillary factors, Co-eIF-2B (which promotes the dissociation of the ternary complex at high Mg2+) and Co-eIF-2C (which reverses the inhibition of ternary complex formation), and thus, is presumably inactive in peptide chain initiation. A protein factor, designated RF, which reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates, has been purified from reticulocyte cell supernatant. RF is a high molecular weight (Mr approximately equal to 450,000) protein complex composed of multiple polypeptides. An active RF preparation contains Co-eIF-2B and Co-eIF-2C activities, and these two activities in RF preparation are not inhibited by HRI and ATP--i.e., eIF-2 alpha (P) is recognized. During purification, RF remains associated with eIF-2 activity (eIF-2 X RF) and can be freed of this eIF-2 activity by CM-Sephadex chromatography. Both eIF-2 X RF and RF contain a Mr 38,000 polypeptide component that is indistinguishable from the Mr 38,000 subunit of eIF-2 by two-dimensional gel electrophoresis. It has been observed that a significant part of this Mr 38,000 polypeptide component in eIF-2 X RF and almost the entire Mr 38,000 polypeptide component in RF remain unphosphorylated after prolonged incubation with HRI and ATP. A possible role of this free Mr 38,000 polypeptide in RF action is discussed.     

Roles of a 67-kDa polypeptide in reversal of protein synthesis inhibition in heme-deficient reticulocyte lysate.

During heme deficiency in reticulocyte lysates, the heme-regulated protein synthesis inhibitor, HRI, phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF-2) and thus inhibits protein synthesis. Two factors, eIF-2 and a reticulocyte-lysate supernatant factor that we term RF, reverse this inhibition. We now report the following. (i) An active eIF-2 preparation contained, in addition to the three subunits (alpha, beta, and gamma), a 67-kDa polypeptide. Pretreatment of eIF-2 with polyclonal antibodies against either isolated alpha subunit or 67-kDa polypeptide almost completely inhibited the reversal activity. Upon further fractionation, three-subunit eIF-2 and the 67-kDa polypeptide were resolved. Neither the three-subunit eIF-2 nor the 67-kDa polypeptide alone was active in protein synthesis inhibition reversal. The activity was, however, restored by combining both the three-subunit eIF-2 and the 67-kDa polypeptide. (ii) Active RF preparations contained eIF-2 alpha (unphosphorylated) and beta subunits and the 67-kDa polypeptide. As with eIF-2, prior treatment of the RF preparation with antibodies to either the alpha subunit or the 67-kDa polypeptide almost completely inhibited the reversal activity. The RF preparation devoid of eIF-2 gamma subunit did not form ternary complex (Met-tRNA(fMet).eIF-2.GTP). The eIF-2 gamma subunit in the free form was isolated, and addition of this isolated gamma subunit to RF promoted significant ternary-complex formation. (iii) Purified HRI efficiently phosphorylated the alpha subunit in the three subunit eIF-2. However, the extent of such phosphorylation was significantly reduced when eIF-2 containing the 67-kDa polypeptide was used. The 67-kDa polypeptide apparently protected eIF-2 alpha subunit from HRI-catalyzed phosphorylation but did not inhibit HRI activity. Based on these results, we suggest that the protein synthesis inhibition reversal activity in both eIF-2 and RF is due to the same components--namely, eIF-2 alpha subunit and the 67-kDa polypeptide. The 67-kDa polypeptide protects eIF-2 alpha subunit from HRI-catalyzed phosphorylation and may also be a necessary component of the functioning eIF-2 molecule.     

Molecular cloning and expression of cDNA for mammalian translation initiation factor 5.

Eukaryotic translation initiation factor 5 (eIF-5) catalyzes the hydrolysis of GTP bound to the 40S ribosomal initiation complex (40S.AUG.Met-tRNAf-eIF-2.GTP) with the subsequent joining of a 60S ribosomal subunit resulting in the formation of a functional 80S initiation complex. A rat cDNA that encodes eIF-5 has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. The 3.55-kb cDNA encodes a protein of 429 amino acids (calculated M(r) 48,926) with properties that are similar to eIF-5 isolated from rabbit reticulocyte lysates. The deduced amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.     

Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis.

The reticulocyte reversing factor (RF) isolated as a complex with eukaryotic initiation factor 2 (eIF-2) acts catalytically in restoring protein synthesis in reticulocyte lysates inhibited by heme deficiency. In reconstituted in vitro assay mixtures containing Mg2+ (0.25-0.5 mM), RF catalyzes the formation of the binary complex (eIF-2-GDP) but this effect is inhibited when eIF-2 is phosphorylated by the heme-regulated kinase for the alpha-subunit of eIF-2 (HRI). More significantly, RF catalyzes the rapid dissociation of (eIF-2-GDP), which permits the exchange of GTP for GDP and, in the presence of Met-tRNAf, promotes the formation of the ternary complex (eIF-2-Met-tRNAf X GTP). However, phosphorylation of the binary complex by HRI prevents its dissociation by RF and, as a consequence, ternary complex formation is inhibited. Our results indicate that phosphorylated binary complex [eIF-2(alpha P).GDP] interacts with RF to form a [RF . eIF-2(alpha P)] that is not readily dissociable. This binding of RF renders it unavailable to catalyze the dissociation of unphosphorylated binary complex, thereby blocking the recycling of eIF-2. Since RF is present in lysates at a limited concentration relative to that of eIF-2, the sequestering of RF in this manner could account for the observation that the phosphorylation of a small proportion of eIF-2 in heme-deficient lysates is sufficient to inhibit protein synthesis.     

Protein synthesis in rabbit reticulocytes: a study of the mechanism of action of the protein factor RF that reverses protein synthesis inhibition in heme-deficient reticulocyte lysates.

A eukaryotic initiation factor 2 (eIF-2)-ancillary protein factor Co-eIF-2 promotes displacement of GDP from eIF-2 X GDP and facilitates ternary complex (Met-tRNAf X eIF-2 X GTP) formation in the presence of Mg2+. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the alpha-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF-2 does not displace GDP from eIF-2 alpha (P) X GDP. RF, a high molecular weight cell supernatant factor, reverses protein synthesis inhibition in heme-deficient reticulocyte lysates and also reverses HRI inhibition of ternary complex formation. RF contains Co-eIF-2 activity. In addition, an active RF preparation contains excess alpha-subunit of eIF-2 in the free and unphosphorylated form and this alpha-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess alpha-subunit of eIF-2 and this alpha-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by eIF-2 X [3H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2 X [3H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNAf X 40S initiation complex formation; (v) both the ternary complex and the Met-tRNAf X 40S complex formation in the presence of HRI and ATP are completely inhibited by prior incubation of RF with GDP; (vi) upon further fractionation of an active RF fraction, a preparation can be obtained that contains HRI-sensitive Co-eIF-2 activity. However, this preparation does not efficiently reverse protein synthesis inhibition in heme-deficient reticulocyte lysates and does not contain excess alpha-subunit of eIF-2. Based on these observations, we have suggested (a) RF provides the unphosphorylated alpha-subunit to eIF-2 alpha (P) X GDP and restores eIF-2 activity. This RF activity is inhibited as the alpha-subunit in the RF preparation becomes phosphorylated by HRI and ATP in the presence of GDP; (b) RF contains Co-eIF-2 activity, which has dual functions: (i) stimulation of ternary complex formation by eIF-2 and (ii) GDP displacement from eIF-2 X GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 but does not displace GDP from eIF-2 alpha(P) X GDP.     

Protein synthesis in rabbit reticulocytes: mechanism of protein synthesis inhibition by heme-regulated inhibitor.

Partially purified Met-tRNAf binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography. Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNAf and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg2+ and dissociated by Co-eIF-2B at 5 mM Mg2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-e-IF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNAf binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNAf . 40S . AUG complex to the 60S ribosomal subunit to form Met-tRNAf-80S . AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.     

Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha subunit of initiation factor 2.

Earlier, we isolated eukaryotic initiation factor 2 (eIF-2)-stimulating protein (SP) as a homogeneous complex with eIF-2 (eIF-2-SP) and showed that, in the presence of Mg2+, eIF-2-SP promotes formation of a ternary complex with GTP and eukaryotic initiator methionyl tRNA (Met-tRNAi) (eIF-2-GTP-Met-tRNAi) catalytically. We now show that SP-bound eIF-2 exchanges with eIF-2 (eIF-2 exchange). Furthermore, in the presence of Mg2+, eIF-2-SP catalyzes the exchange of eIF-2-bound [3H]GDP with unlabeled GDP or GTP (GDP exchange) and the release of [3H]GDP when the ternary complex is formed from eIF-2-[3H]GDP, GTP, and [35S]Met-tRNAi. All these reactions are blocked by alpha-subunit, but not by beta-subunit, phosphorylation of eIF-2. The eIF-2 and GDP exchanges are compatible with the reaction eIF-2-GDP + SP in equilibrium EIF-2-SP + GDP reminiscent of the exchange between the Tu and Ts components of prokaryotic elongation factor 1 (EF-Tu and EF-Ts, respectively) EF-Tu-GDP + EF-Ts in equilibrium EF-Tu-EF-Ts + GDP. Due to the high affinity of GDP (approximately 100 times greater than that of GDP) for eIF-2, 40S (eIF-2-GTP-Met-tRNAi-40S) to 80S (Met-tRNAi-mRNA-80S) initiation complex conversion, which is accompanied by GTP hydrolysis, probably releases eIF-2 as eIF-2-GDP. Our results suggest that, in the presence of Mg2+, GDP binding restricts the availability of eIF-2 for chain initiation and that SP relieves this restriction in a catalytic fashion, provided that the alpha subunit of eIF-2 is not phosphorylated.     

Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) in Saccharomyces cerevisiae by the GCN2 protein kinase stimulates the translation of GCN4 mRNA. The protein kinases heme-regulated inhibitor of translation (HRI) and double-stranded RNA-dependent eIF-2 alpha protein kinase (dsRNA-PK) inhibit initiation of translation in mammalian cells by phosphorylating Ser-51 of eIF-2 alpha. We show that HRI and dsRNA-PK phosphorylate yeast eIF-2 alpha in vitro and in vivo and functionally substitute for GCN2 protein to stimulate GCN4 translation in yeast. In addition, high-level expression of either mammalian kinase in yeast decreases the growth rate, a finding analogous to the inhibition of total protein synthesis by these kinases in mammalian cells. Phosphorylation of eIF-2 alpha inhibits initiation in mammalian cells by sequestering eIF-2B, the factor required for exchange of GTP for GDP on eIF-2. Mutations in the GCN3 gene, encoding a subunit of the yeast eIF-2B complex, eliminate the effects of HRI and dsRNA-PK on global and GCN4-specific translation in yeast. These results provide further in vivo evidence that phosphorylation of eIF-2 alpha inhibits translation by impairing eIF-2B function and identify GCN3 as a regulatory subunit of eIF-2B. These results also suggest that GCN4 translational control will be a good model system to study how mammalian eIF-2 alpha kinases are modulated by environmental signals and viral regulatory factors.     

Structure and function of peptide initiation factor 2: differential loss of activities during proteolysis and generation of a terminal fragment containing the phosphorylation sites of the alpha subunit.

A procedure is described by which the 38,000-dalton alpha subunit of native eukaryotic peptide initiation factor 2 (eIF-2) can be cleaved by trypsin to yield a 34,000-dalton fragment and a peptide of about 4,000 daltons after elimination of the beta subunit. Under nondenaturing conditions the 4,000-dalton peptide remains bound to the modified eIF-2 and still can be phosphorylated by the heme-controlled eIF-2 alpha kinase from reticulocytes. All of the phosphorylation sites for this protein kinase are located on the 4,000-dalton peptide. The ability of eIF-2 to form a ternary complex with GTP and Met-tRNAf and the ability to promote binding of Met-tRNAf to 40S ribosomal subunits are lost differentially during the proteolysis. Loss of te latter activity occurs rapidly and appears to be correlated with loss of the beta subunit. Loss of activity for ternary complex formation is correlated with the appearance of the 4,000-dalton peptide.     

Partial reaction of peptide initiation inhibited by phosphorylation of either initiation factor eIF-2 or 40S ribosomal proteins.

Preparations of the hemin-controlled repressor (HCR) from rabbit reticulocytes contain 3':5'-cyclic-AMP-independent protein kinase activity for the smallest subunit of the peptide initiation factor eIF-2 and for proteins of reticulocyte 40S ribosomal subunits. Binding of the ternary complex formed between Met-tRNAf, GTP, and eIF-2 to 40S ribosomal subunits is shown to be inhibited by phosphorylation of either the ribosomal subunits or eIF-2. The protein kinase activity responsible for phosphorylation of eIF-2 has been separated from the activity for phosphorylation of 40S ribosomal subunits and shown to independently block the same partial reaction of peptide initiation. It appears that different enzymes are involved, each capable of regulating peptide initiation at the same step but by a different mechanism.     

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: Inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet·40S ribosomal subunit complexes

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF.We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNA(f) (Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNA(f) (Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF.The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5 mM Mg(2+) at 0 degrees . The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5'-[beta,gamma-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.     

Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.

We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases.     

Molecular mechanisms of translation initiation in eukaryotes

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.