Protein Chain Initiation Research Articles

Toxic heavy metal ions activate the heme-regulated eukaryotic initiation factor-2 alpha kinase by inhibiting the capacity of hemin-supplemented reticulocyte lysates to reduce disulfide bonds

Addition of toxic heavy metal ions (Cd2+, Hg2+, and Pb2+) to hemin-supplemented rabbit reticulocyte lysate brings about the activation of the heme-regulated eukaryotic initiation factor 2 alpha kinase (HRI) and the inhibition of protein chain initiation. In this report we examined the effects of monothiol and dithiol compounds, metal ion-chelating agents, and metallothioneins (MT) on metal ion-induced inhibition of protein synthesis. The dithiol compounds dithiothreitol and 2,3-dimercaptopropane sulfonic acid prevented and relieved the inhibition of protein synthesis caused by Cd2+ and Hg2+ in hemin-supplemented lysates, but the monothiol compounds 2-mercaptoethanol, cysteamine, D-(-)penicillamine, and glutathione had no effect. The inhibition of protein synthesis caused by Cd2+ was reversed by the addition of excess EDTA but not by the addition of excess nitrilotriacetic acid. Toxic heavy metal ions inhibited the capacity of hemin-supplemented lysate to reduce disulfide bonds. Addition of excess EDTA to Cd(2+)-inhibited lysates restored the capacity of the lysate to reduce disulfide bonds and inhibited the phosphorylation of eukaryotic initiation factor eIF-2. MTs and their apoproteins (apoMTs) inhibited the activation of HRI and protected protein synthesis from inhibition by Cd2+, Hg2+, and Pb2+. Addition of apoMTs to heavy metal ion-inhibited lysates restored the capacity of lysates to reduce disulfide bonds. The restoration of the lysate's thioredoxin/thioredoxin reductase activity was accompanied by the inactivation of HRI and the resumption of protein synthesis, indicating that apoMTs can "detoxify" metal ions already bound to proteins. Several observations presented in this report suggest that the binding of metal ions to the alpha-domain of MT is responsible for the ability of MT to sequester bound metal in a non-toxic form. Addition of glucose 6-phosphate or NADPH had no effect on protein synthesis in metal ion-inhibited lysates, and NADPH concentrations in Cd(2+)-inhibited and hemin-supplemented control lysates were equivalent. The data suggest that the metal ions cause the inhibition of protein synthesis by binding to vicinal sulfhydryl groups present in some critical protein(s), possibly the dithiols present in the active site of thioredoxin and (or) thioredoxin reductase, which leads to the activation of HRI.

Correlation between the distribution of the reversing factor and eukaryotic initiation factor 2 in heme-deficient or double-stranded RNA-inhibited reticulocyte lysates

The recycling of eukaryotic initiation factor eIF-2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit-bound eIF-2 · GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme-deficient and dsRNA-inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF-2(α- 32P). RF and eIF-2(αP) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K +. The association of eIF-2(α- 32P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F −, indicating the presence of an endogenous ribosome-associated phosphatase activity which is capable of dephosphorylating eIF-2(αP) in the absence of F −. These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S-bound eIF-2 · GDP complex to promote the dissociation of GDP from eIF-2 and the release of eIF-2 from the 60 S subunit as a complex with RF.

Inhibition of rabbit reticulocyte lysate protein synthesis by heavy metal ions involves the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2.

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.

Phosphorothioated binary complex of eukaryotic initiation factor eIF-2 and GDP inhibits protein synthesis in hemin-supplemented reticulocyte lysates

The rabbit reticulocyte heme-regulated eIF-2α kinase (HRI) utilizes adenosine-5′-0-(3-thiotriphosphate) (ATP-γ-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [ eIF-2(α-[ 35S]P)·GDP ], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2·[ 3H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF-2(α-[ 35S]P)·GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2·GDP alone, through the ability of eIF-2(α-P)·GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate.

A wheat germ cap-site factor functional in protein chain initiation

Component C1 from wheat germ, a factor that functions in attaching ribosomes to mRNA, has been resolved into a fraction that does not bind to m 7 GDP-agarose (referred to as eIF4B) and one that binds and is eluted specifically by m 7GDP. Both components are required for the attachment of ribosomes to [ 3H]methyl-labeled reovirus RNA and for the translation of a number of mRNAs, including the noncapped RNA of satellite tobacco necrosis virus. The component that binds to m 7GDP-agarose, referred to as CSF (cap-site factor), contains primarily proteins of M r 24,000,26,000, and 75,000. Crosslinking studies with oxidized [ 3H]methyl-labeled reovirus RNA show that one of the lower molecular weight polypeptides of CSF interacts specifically with the 5′-cap of the mRNA in the absence of any other components. Incubation of component C1 and eIF4A in the presence of ATP results in the additional crosslinking of a 51- and a 65-kDa protein. In the absence of eIF4A, there is only the crosslinking of the lower molecular mass polypeptide (24 or 26 kDa). Attempts to reconstitute the C1 reaction with CSF and eIF4B result in a considerably diminished reaction. Crosslinking of eIF4A, however, is obtained in an incubation containing only CSF and eIF4A, suggesting that CSF may bring about an initial interaction of eIF4A with the 5′ end of the mRNA.

Initiation factors elF4A and C1 from wheat germ and the formation of mRNA · ribosome complexes

The binding of ribosomes to mRNA is analyzed in a fractionated system from wheat germ with [ 3H]uridine-labeled poly(A)+ RNA prepared from germinating wheat embryos. The reaction requires factors eIF3, eIF4C, and eIF5; Met-tRNA and the Met-tRNA binding system; either GTP or GMP-PNP; ATP; and factors C1 and eIF4A. These requirements are identical to those previously found to be necessary for formation of ribosome · Met-tRNA i Met complexes, with the exception of ATP, and factors C1 and eIF4A. The function of factors C1 and eIF4A is therefore specifically related to the mRNA attachment reaction. The presence of GTP in the mRNA binding reaction results in the formation of 80 S ribosome complexes, while with GMP-PNP only 40 S ribosome complexes are formed. Ribosome binding to native reovirus RNA in the fractionated wheat germ system is similar to the reaction with poly(A)+ RNA, strongly requiring ATP and factors C1 and eIF4A. Binding to inosine-substituted reovirus RNA, however, is only partially dependent upon ATP, and both the ATP-dependent and the ATP-independent binding reactions strongly require factor C1 and are substantially stimulated by factor eIF4A. The ATP-independent reaction is inhibited by pm 7GDP, has a strong requirement for Met-tRNA i Met, and the 40 S ribosome complex is stable to RNase. These results indicate that the ATP-independent binding of ribosomes to inosine-substituted reovirus RNA proceeds through the normal initiation process. They further suggest that neither factor C1 nor eIF4A function exclusively to unwind mRNA secondary structure. Since eIF4A is required for the ATP-independent binding to inosine mRNA, and at the same time interacts with ATP in the reaction with ATP-requiring mRNAs, this factor may have two roles in protein chain initiation, one related to the mRNA · ribosome interaction, and one related to the function of ATP.

Eukaryotic initiation factor 4A is the component that interacts with ATP in protein chain initiation.

Protein synthesis in a resolved homogenate of wheat germ requires ATP and eight factors functioning at the level of protein chain initiation. To identify the component(s) interacting with ATP, the different factors were treated with the ATP affinity analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and tested for their function in protein synthesis. The activity of eukaryotic initiation factor 4A (eIF4A) was strongly curtailed, whereas all other factors were unaffected. At a concentration of 250 microM, AMP, ADP, and ATP protected eIF4A against FSBA inactivation, whereas at a concentration 50 microM, protection was afforded only by ATP. GTP did not protect at a concentration of 250 microM. In another approach, the substrate analogue 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) was found to inhibit protein synthesis in a manner, at least in part, competitive with ATP. Supplementing a TNP-ATP inhibited reaction with eIF4A substantially reversed the inhibition. Except for a small effect by factor C1, no reversal was obtained with any other component. Finally, a preincubation of ribosomes with ATP, mRNA, and eIF4A resulted in the formation of a complex capable of TNP-ATP-resistant amino acid incorporation. These data are interpreted to indicate that the primary interaction of ATP is with eIF4A. A model is proposed reconciling this conclusion with other observations relevant to the mRNA . ribosome attachment reaction.

Inhibition of eukaryotic protein chain initiation by vanadate.

Vanadate inhibits protein chain initiation in rabbit reticulocyte lysates. The evidence that supports this conclusion is as follows: (i) the biphasic kinetics of inhibition in which protein synthesis is maintained at the control rate for 1-2 min is followed by an abrupt decline in the rate of synthesis; (ii) inhibition is associated with a marked disaggregation of polyribosomes and a concomitant increase in 80S ribosomes; and (iii) vanadate concentrations that inhibit protein chain initiation do not inhibit polypeptide chain elongation or the aminoacylation of tRNA. In partial reactions of protein chain initiation, vanadate concentrations that inhibit protein synthesis have no detectable effect on the formation of eukaryotic initiation factor eIF-2-promoted ternary complex with Met-tRNAf and GTP and on the assembly of 40S ribosomal subunit-Met-tRNAf complexes. On the addition of mRNA, the 40S ribosomal subunit-Met-tRNAf complexes also are transformed into 80S ribosome-mRNA-Met-tRNAf complexes, termed 80S initiation complexes. In vanadate-treated samples, however, these 80S initiation complexes are defective and unable to proceed beyond this step.

Regulation of protein synthesis in rabbit reticulocyte lysates by guanosine triphosphate

GTP (2 mM) promotes protein synthesis in rabbit reticulocyte lysates in which protein chain initiation is inhibited by the activation of specific adenosine 3′:5′ cyclic monophosphate independent protein kinases in: 1) heme deficiency; or 2) in hemin-supplemented lysates by the addition of the purified heme-regulated protein kinase (HRI); or 3) oxidized glutathione; or 4) by low levels of double stranded RNA. The molecular basis for the promotion of protein synthesis by GTP under these various conditions was investigated by examining the in, situ state of eIF-2 phosphorylation. The results show that GTP (2 mM) blocks eIF-2 phosphorylation and also promotes the dephosphorylation of phosphorylated eIF-2. These findings suggest that GTP restores protein synthesis by a common mechanism that involves the relief of eIF-2 from phosphorylation. The nonphosphorylated eIF-2 is, therefore, available for the maintenance and the restoration of protin chain initiation cycle.

Effects of skeletal muscle protein phosphatase inhibitor-2 on protein synthesis and protein phosphorylation in rabbit reticulocyte lysates.

Reticulocyte lysates contain two major classes of protein phosphatase activities, designated type 1 and type 2. These designations are based on criteria derived from the analyses of protein phosphatase species in other tissues. The criteria include (i) chromatographic elution profiles on DEAE-cellulose; (ii) specificity of lysate phosphatases toward [(32)P]phosphorylase a and [(32)P]phosphorylase kinase; (iii) sensitivity of lysate phosphatases to Mg(2+) ATP; and (iv) sensitivity to the heat-stable protein phosphatase inhibitor-2. The lysate phosphatase species are similar to those described in rabbit skeletal muscle and rabbit liver. Reticulocyte protein phosphatase type 1, but not type 2, is inhibited by heat-stable protein phosphatase inhibitor-1 and -2 which have been characterized from rabbit skeletal muscle. We have initiated a study on the function and specificity of lysate protein phosphatase activities involved in the regulation of protein synthesis by examining the effects of protein phosphatase inhibitor-2 on reticulocyte protein synthesis and protein phosphorylation. Our findings are as follows. (a) Protein phosphatase inhibitor-2 inhibits protein chain initiation in hemin-supplemented lysates. (b) Inhibition is characterized by biphasic kinetics and is reversed by the delayed addition of purified reticulocyte eukaryotic initiation factor 2 (eIF-2). (c) Inhibition of protein synthesis by inhibitor-2 is accompanied by the phosphorylation of the alpha-subunit (38,000 daltons) of eIF-2 (eIF-2alpha) and of two heat-stable polypeptides of 29,000 and 44,000 daltons. (d) The 29,000-dalton component is phosphorylated in lysates under conditions of protein synthesis and appears to be inhibitor-2, but the physiological significance of this modification of inhibitor-2 is not clear. (e) Inhibitor-2 has no effect on the activation in vitro of isolated heme-regulated or double-stranded RNA-dependent eIF-2alpha kinases. We propose that the inhibition of protein synthesis in hemin-supplemented lysates by added inhibitor-2 is due at least in part to the inhibition of a type 1 eIF-2alpha phosphatase activity, which permits a basal eIF-2alpha kinase activity to be expressed leading to the accumulation of phosphorylated eIF-2alpha and an inhibition of protein synthesis.

Regulation of eukaryotic protein synthesis by protein kinases that phosphorylate initiation factor eIF-2: Further evidence for a common mechanism of inhibition of protein synthesis

The role of reversing factor (RF) in the regulation of protein synthesis by inhibitory protein kinases that phosphorylate the 38,000-dalton subunit of initiation factor eIF-2 has been examined. Results show that as with the heme-regulated protein kinase (HRI), RF restores protein synthesis in reticulocyte lysates inhibited by translational inhibitors from rat liver, wheat germ, Krebs ascites cell, by oxidized glutathione, the protein kinase activated by double stranded RNA (dRI), and the interferon-induced double stranded RNA activated protein kinase from Ehrlich ascites and Hela cells. These findings suggest that RF plays an important role in eukaryotic protein chain initiation cycle.

The role of translational inhibitor in ethionine-induced inhibition of protein synthesis

The soluble fraction from rat liver contains an inhibitor of protein chain initiation when tested in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The administration of ethionine to rats increased the inhibitory activity in the liver. This liver inhibitor displayed properties similar to those of hemin-controlled inhibitor found in rabbit reticulocytes: (i) the liver inhibitor inhibited protein chain initiation in rabbit reticulocyte lysate with characteristic biphasic kinetics; (ii) the liver inhibitor disaggregated the reticulocyte polysomes with a concomitant increase in 80 S ribosomes; (iii) the inhibition was prevented or reversed by eIF-2. The activation of the liver inhibitor by ethionine was rapidly and completely counteracted by the subsequent administration of methionine and adenine to rats. The mechanism of inhibition of protein synthesis by ethionine was discussed in the light of these findings.

Isolation of a translational inhibitor from wheat germ with protein kinase activity that phosphorylates initiation factor eIF-2

A translational inhibitor (WGI) has been partially purified from wheat germ extracts. WGI inhibits protein synthesis in rabbit reticulocyte lysates with inhibition kinetics that are similar to those observed in heme-deficiency or by the addition of purified heme-regulated translational inhibitor (HRI). Initiation factor eIF-2 from rabbit reticulocytes overcomes this inhibition. This finding suggests that WGI inhibits protein chain initiation. WGI induced inhibition is enhanced by ATP (2 mM), and overcome by GTP (2 mM) and cyclic-AMP (10 mM). WGI preparations contain a cyclic-AMP independent protein kinase activity that phosphorylates the 38,000-dalton subunit of rabbit reticulocyte eIF-2. The phosphopeptide analyses of eIF-2 phosphorylated by WGI or HRI show that they phosphorylate the same site(s) of eIF-2. HRI phosphorylates the corresponding 38,000-dalton subunit of wheat germ eIF-2. These results obtained with WGI are similar to that of HRI. HRI has been identified as a cyclic-AMP independent protein kinase that phosphorylates the 38,000-dalton subunit of eIF-2 [for review see Ochoa, S. and de Haro, C. (1979) Ann. Rev. Biochem. 48 , 549]. Hence, these findings with wheat germ-a phylogenetically distant eukaryote, raise further the possibility that phosphorylation-dephosphorylation of eIF-2 may be an important general mechanism in the regulation of eukaryotic protein biosynthesis.

Characterization of double-stranded-RNA-activated kinase that phosphorylates alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) in reticulocyte lysates.

Incubation of reticulocyte lysates with low levels of double-stranded (ds) RNA (1-20 ng/ml) activates a cAMP-independent protein kinase (dsI) that phosphorylates the alpha-subunit (M(r) 38,000) of initiation factor 2 (eIF-2) and produces an inhibition of protein chain initiation similar to that caused by heme deficiency. Activation of dsI from its latent precursor takes place on the ribosomes and requires ATP. dsI can also be activated in ribosomal salt washes and in partially purified preparations of the latent precursor of dsI. In all preparations, activation is accompanied by the ds RNA-dependent phosphorylation of a polypeptide doublet that migrates as bands of 67 and 68.5 kilodaltons (67/68.5) in NaDodSO(4)/acrylamide gels. The rate of phosphorylation of these components in a ribosome salt wash is more rapid than the ds RNA-dependent phosphorylation of eIF-2alpha. Other polypeptides in the salt wash also undergo ds RNA-dependent phosphorylation, but their significance is not clear. All of these phosphorylations are prevented by high concentrations of poly(I).poly(C)(20 mug/ml), but not by an antiserum specific for the heme-regulated eIF-2alpha kinase. Both the latent and activated forms of dsI have been partially purified from a 0.5 M KCl wash of reticulocyte ribosomes. The two species have similar M(r)s ( approximately 120,000) and sedimentation coefficients ( approximately 3.75 S), which suggests that activation of dsI probably does not involve extensive changes. By comparison, the heme-regulated eIF-2alpha kinase has an M(r) of approximately 160,000 and sediments at approximately 6.6 S. However, in vitro, dsI and HRI both phosphorylate the same site(s) of eIF-2alpha. Purified dsI inhibits protein synthesis in hemin-supplemented lysates with the same kinetics induced by the addition of ds RNA; both inhibitions are reversed by eIF-2. dsI that has been activated in the salt wash and then purified does not require ds RNA for expression and no longer displays phosphorylation of the 68.5/67 doublet, which appears to occur only during activation. The data support the view that this component(s) may be the eIF-2alpha kinase activated by ds RNA.

Regulation of protein synthesis in rabbit reticulocyte lysates: The heme-regulated protein kinase (HRI) and double stranded RNA induced protein kinase (dRI) phosphorylate the same site(s) on initiation factor eIF-2

Double stranded RNA (dsRNA) induced inhibitor (dRI) has been partially purified (80–100 fold). The dRI inhibits protein synthesis in rabbit reticulocyte lysates; the inhibition is overcome by the initiation factor eIF-2. The dRI preparations phosphorylate the 38,000-dalton subunit of eIF-2. Heme-deficiency in rabbit reticulocyte lysates also induces a translational inhibitor (HRI) which inhibits protein chain initiation by specifically phosphorylating the 38,000-dalton subunit of eIF-2. To establish correlation of the mechanism of inhibition of protein synthesis by dRI and HRI, the phosphopeptide patterns of eIF-2 phosphorylated by using HRI or dRI are compared. Treatment with various proteases of eIF-2 phosphorylated by HRI or dRI yield identical phosphopeptide patterns. This finding suggests that HRI and dRI phosphorylate the same site(s) of the 38,000-dalton subunit of eIF-2 and raises the possibility that dRI may also inhibit protein chain initiation by the mechanism similar to that of HRI.

Regulation of translational initiation ina temperature-sensitive mutant of BHK cells.

The role of various essential components in mammalian cell protein chain initiation, such as the 40S ribosomal subunit and the initiation factors, have been studied largely in fractionated systems and little is known about the mechanisms of their regulation. The process was studied in an unfractionated system derived from temperature-sensitive (ts) 422E cells. The mutant cells fail to assemble 60S ribosomal subunits at the nonpermissive temperature with little effect on the production of 40S ribosomal subunits. Although the resulting imbalance in ribosomal subunits has little immediate effect on the overall rate of protein synthesis, the nascent 40S subunits in mutant cells appear to be markedly unstable at the nonpermissive temperature. The initial step in protein synthesis, initiation factor elF-2-promoted binding of [35S]Met-tRNAf Met to 40S ribosomal subunits, remains unimparied in ts 422E cells at the nonpermissive temperature. However, most of the nascent 40S ribosomal subunits in ts 422E cells band at a higher density, suggesting their failure to bind initiation factor elF-3. These newly synthesized 40S subunits in the mutant cells are unstable, and may be degraded before their appearance in the polysomes.

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.

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: Inhibition of interaction of Met-tRNA f Met binding factor with another initiation factor in formation of Met-tRNA f Met ·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.

Activity of partially purified protein chain initiation factors from the livers of dimethylnitrosamine-treated rats

Partially purified polypeptide chain initiation factors were prepared from the 0.5 M KCl wash of rat liver microsomes. Their activities in connection with dimethylnitrosamine (DMNA)-induced inhibiton of protein synthesis were studied by use of the following reactions: (1) poly(U)-directed binding of Phe-tRNA to ribosomes, (2) formation of a GTP-dependent ternary initiation complex with Met-tRNA f, (3) binding of Met-tRNA f to 40-S ribosomal subunits, (4) assembly of a Met-tRNA f containing 80-S ribosomal initiation complex and (5) ribosome-dependent GTPase activity. The inhibition of protein synthesis with DMNA was not associated with a loss of factor activity in any of these reactions. In the binding of Met-tRNA f to 40-S subunits there was a noticeable increase, probably related to the stability of the resulting complex. The Met-tRNA deacylase activity was also increased.

The Role of Complexes between the 40-S Ribosomal Subunit and Met-tRNAMetf in the Initiation of Protein Synthesis in the Wheat-Germ System

We have demonstrated the formation of a complex between the 40-S ribosomal subunit and Met-tRNAMetf in the absence of mRNA in an unfractionated wheat-germ system. Two types of experiment indicate that these complexes (40S · Met-tRNAMetf) are true intermediates in the formation of 80-S initiation complexes containing mRNA, Met-tRNAMetf and both ribosomal subunits. Firstly, isolated 40S · Met-tRNAMetf complexes were used preferentially over free Met-tRNAMetf in the formation of 80-S initiation complexes on addition of mRNA. Secondly by the use of methioninol-AMP, an inhibitor of methionyl-tRNA synthetase, to deplete endogenous Met-tRNAMetf pools, we showed that the binding of mRNA to ribosomes was strictly dependent on the presence of Met-tRNAMetf. The 40S · Met-tRNAMetf complexes had other properties consistent with their role as intermediates in initiation. The radioactivity in 40S · Met-tRNAMetf complexes labelled with [35S]Met-tRNAMetf was shifted into 80-S initiation complexes on addition of mRNA. This shift was prevented by aurintricarboxylic acid, which inhibits the binding of mRNA to ribosomes. In the presence of edeine, which blocks the binding of the 60-S subunit, the radioactivity was found in complexes containing mRNA, Met-tRNAMetf and 40-S subunits. These results were confirmed by the use of labelled mRNA. We propose that the mechanism of protein chain initiation is universal to all cells, the first step being the formation of a complex between the small ribosomal subunit and (f)Met-tRNAMetf. This complex can then bind mRNA in a process which must to some extent involve selection of the initiator AUG codon through interactions with the anticodon of the (f)Met-tRNAMetf. The binding of mRNA is followed by the addition of the large ribosomal subunit and the formation of an initiation complex capable of forming the first peptide bond.