Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.     

Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates.

We have obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.     

Phosphorylation and guanine nucleotide exchange on polypeptide chain initiation factor-2 from Artemia embryos

Eukaryotic initiation factor-2 (eIF-2) from Artemia embryos is able to exchange guanine nucleotides at the same rate in the presence or absence of Mg2+ when the reaction is carried out with either purified eIF-2 at 30 degrees C or less purified preparations at any temperature (10-30 degrees C). No exchange factor appears to catalyze this reaction. However, with purified eIF-2 at lower temperatures (10 degrees C) the exchange is clearly impaired by Mg2+ and this impairment is overcome by the guanine nucleotide exchange factor (GEF) of rabbit reticulocytes. Thus, Artemia eIF-2 is able to exchange guanine nucleotides by two alternative mechanisms that may reflect two states of the protein. Phosphorylation of the eIF-2 alpha subunit by the heme-controlled inhibitor (HCI) of rabbit reticulocytes abolishes the GEF-dependent reaction, but has no effect on the factor-independent one. The search for eIF-2 alpha kinases in Artemia embryo led to the detection of only one such enzyme, which was identified as a casein kinase type II. None of the exchange reactions is affected by the phosphorylation of the eIF-2 alpha subunit by this kinase, suggesting that, irrespective of the kind of mechanism for guanine nucleotide exchange that is actually operating in Artemia, it might not be a target for regulation by eIF-2 alpha phosphorylation.     

Evidence for a second phosphorylation site on eIF-2 alpha from rabbit reticulocytes

Ser 51 in the NH2-terminal sequence of the alpha-subunit of eukaryotic peptide initiation factor 2 (eIF-2) has been identified as a second phosphorylation site for the heme-controlled eIF-2 alpha kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the alpha-subunit of spectrin. A synthetic peptide corresponding to eIF-2 alpha (41-54) is phosphorylated only in Ser 51 by the eIF-2 alpha kinase.     

Casein kinase I phosphorylates the 25-kDa mRNA cap-binding protein

The 25-kDa mRNA cap-binding protein (eIF-4E) exists in both phosphorylated and dephosphorylated forms in eukaryotic cells. Phosphorylated eIF-4E appears to be preferentially associated with 48 S initiation complexes and with the 220-kDa subunit of eIF-4F. In addition, dephosphorylation of eIF-4E has been observed during heat shock and mitosis which are accompanied by decreased protein synthesis. However, the control of eIF-4E phosphorylation and its regulatory role remain poorly understood. Using eIF-4E as a substrate we have identified and purified from rabbit reticulocytes a protein kinase that phosphorylates eIF-4E in vitro. This enzyme phosphorylated eIF-4E on both serine and threonine residues with an apparent Km of 3.7 microM. The molecular mass of the enzyme and specificity for substrates other than eIF-4E suggested that this enzyme was a species of casein kinase I. This was confirmed by comparing the phosphopeptide map of the purified reticulocyte enzyme with that of rabbit skeletal muscle casein kinase I and by comparing phosphopeptide maps of eIF-4E phosphorylated in vitro by each enzyme. We conclude that casein kinase I phosphorylates eIF-4E in vitro and suggest that eIF-4E may be phosphorylated by casein kinase I in intact cells under some physiologic conditions.     

Identification of spectrin-related peptides associated with the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 kinase and of Mr 95,000 peptide that appears to be the catalytic subunit

An isolation procedure for the reticulocyte heme-controlled alpha subunit of eukaryotic translational initiation factor 2 (eIF-2 alpha) kinase is described which yields different fractions with kinase activity. Each is associated with a different spectrin-related peptide as identified by anti-spectrin monoclonal antibodies. The most abundant of these peptides is the Mr 90,000 species characterized previously (Kudlicki, W., Fullilove, S., Kramer, G., and Hardesty, B. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5332-5336). Association with the spectrin-related peptides appears to account for the heterogeneity of the enzyme during its isolation and for its highly asymmetric structure. Isolated alpha or beta spectrin subunits as well as the separated homogeneous Mr 90,000 peptide cause an increase in the initial rate of eIF-2 alpha phosphorylation that is related to a decrease in Km with little or no effect on Vmax for the phosphorylation reaction. Fractionation of highly purified eIF-2 alpha kinase preparations using affinity chromatography on monoclonal anti-spectrin antibodies has separated eIF-2 alpha kinase activity from the Mr 100,000 phosphopeptide which copurifies with the kinase during all other purification steps. A Mr 95,000 peptide, detectable only by photoaffinity labeling with 8-azido-[alpha 32P]ATP, is shown to be distinct from the Mr 100,000 phosphopeptide and appears to be the catalytic subunit of the eIF-2 alpha kinase.     

Activation of hemin-regulated initiation factor-2 kinase in heat-shocked HeLa cells

Protein synthesis was drastically inhibited in HeLa cells incubated for 5 min at 42.5 degrees C, but it resumed after 20 min at a rate about 50% that of control cells. After 10 min of heat shock, the binding of Met-tRNAf to 40 S ribosomal subunits was greatly reduced and a polypeptide identified by immunoprecipitation with the alpha subunit of eukaryotic initiation factor-2 (eIF-2) was phosphorylated. Extracts prepared from control and heat-shocked cells were assayed for in vitro protein synthesis. Both extracts were active when supplemented with hemin, but the extract from heat-shocked cells had little initiation activity without this addition. A Mr 90,000 polypeptide and eIF-2 alpha were phosphorylated in this extract, but hemin or an antibody which inhibits the protein kinase designated heme-controlled repressor reduced this phosphorylation. These findings implicated heme-controlled repressor as the kinase at least in part responsible for eIF-2 alpha phosphorylation. Furthermore, the initial inhibition of protein synthesis and eIF-2 alpha phosphorylation after heat shock were reduced by adding hemin to intact HeLa cells. These cells synthesized heat-shock proteins with some delay relative to cells without added hemin. The binding of Met-tRNAf to 40 S ribosomal subunits was inhibited by about 50% in extracts prepared from cells heat-shocked for 40 min, and eIF-2 alpha phosphorylation was increased in these cells. These results suggest that heme-controlled repressor is activated in heat-shocked cells and that eIF-2 alpha phosphorylation limits mRNA translation even after partial recovery of protein synthesis.     

Phorbol esters stimulate phosphorylation of eukaryotic initiation factors 3, 4B, and 4F

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, was isolated by m7 GTP-Sepharose affinity chromatography from rabbit reticulocytes incubated with [32P]orthophosphate. Following treatment of reticulocytes with phorbol 12-myristate 13-acetate (PMA) for 30 min, stimulation of phosphorylation of both the p25 and p220 subunits was observed (2.5-5-fold). Two variants were observed for p25 in the absence and presence of PMA when analyzed by two-dimensional gel electrophoresis. Only the more acidic of these was phosphorylated, with the level of phosphorylation increased upon PMA treatment. One main variant was observed for p220; following PMA stimulation, in addition to increased labeling of this variant, two more acidic phosphorylated variants were observed. Low levels of eIF-3 and -4B were associated with purified eIF-4F, and PMA treatment stimulated phosphorylation of eIF-3 (p170) by 2-4-fold and eIF-4B by 1.5-2.5 fold. Two-dimensional phosphopeptide mapping of p25 phosphorylated in the absence or presence of PMA generated a single tryptic phosphopeptide, suggesting a single phosphorylation site. A more complex phosphopeptide map was observed with p220 subunit. The maps for both subunits contained the same phosphopeptides as those obtained when eIF-4F was phosphorylated in vitro by the Ca2+/phospholipid-dependent protein kinase, indicating this protein kinase directly modulated eIF-4F in response to PMA.     

The 90-kilodalton peptide of the heme-regulated eIF-2 alpha kinase has sequence similarity with the 90-kilodalton heat shock protein

Highly purified preparations of the heme-controlled eIF-2 alpha (eukaryotic peptide initiation factor 2 alpha subunit) kinase of rabbit reticulocytes contain an abundant 90-kilodalton (kDa) peptide that is immunologically cross-reactive with spectrin and that modulates the activity of the enzyme [Kudlicki, W., Fullilove, S., Read, R., Kramer, G., & Hardesty, B. (1987) J. Biol. Chem. 262, 9695-9701]. The amino-terminal sequence of the 90-kDa protein has a high degree of similarity with the known amino-terminal sequences of the Drosophila 83-kDa heat shock protein (20 out of 22 residues) and with other related heat shock proteins. The amino acid sequence of a tryptic phosphopeptide isolated by high-performance liquid chromatography from the eIF-2 alpha kinase associated 90-kDa protein after phosphorylation by casein kinase II is shown to be identical with a 14 amino acid segment of the known sequence of the Drosophila 83-kDa heat shock protein. Results of hydrodynamic studies indicate a highly elongated structure for the reticulocyte protein, characteristic of a structural protein. Additional structural similarities between the eukaryotic heat shock proteins, the reticulocyte eIF-2 alpha kinase associated 90-kDa peptide, and spectrin are discussed.     

Phosphorylation of protein synthesis initiation factor-2. Identification of the site in the alpha-subunit phosphorylated in reticulocyte lysates.

The data presented here show that serine-51 of the alpha-subunit of eukaryotic initiation factor eIF-2 is the only residue phosphorylated by the eIF-2 alpha-specific kinases HCR (haem-controlled repressor) and dsI (double-stranded RNA-activated inhibitor) in vitro. This confirms our earlier finding that serine-48 is not labelled by either kinase. Methodology appropriate for the examination of phosphorylation sites in eIF-2 alpha in whole cells and their extracts has been developed, and used to study the site(s) in eIF-2 alpha labelled in reticulocyte lysates. Only serine-51 became phosphorylated under conditions of haem-deficiency or in the presence of double-stranded RNA. No evidence for a second phosphorylation site on the alpha-subunit was obtained with the lysates and conditions used here.     

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. $\text{48}$, 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.     

Regulation of protein synthesis in rabbit reticulocyte lysates: the hemeregulated 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.     

Purification of a novel heat-stable translational inhibitor from rabbit reticulocyte lysates

We have purified to apparent homogeneity a novel heat-stable (HS) factor from postribosomal supernatants of rabbit reticulocyte lysates by heating for 10 min at 80 degrees C, fractionation on Sephadex, anion-exchange chromatography on QMA Accell, and gel filtration HPLC. The apparent molecular mass of HS is 500-1000 Da on the basis of its behaviour on gel filtration. Like a factor from bovine heart [(1982) Proc. Natl. Acad. Sci. USA 79, 3134-3137], the reticulocyte HS inhibits translation in hemin-supplemented lysates with biphasic kinetics similar to hemin deficiency and promotes phosphorylation of the alpha-subunit of the eukaryotic initiation factor eIF-2. It is active at nanomolar concentrations. Reticulocyte HS appears to be neither a peptide nor an oligonucleotide since HS activity was insensitive to proteolytic or nucleolytic digestion.     

Interaction of wheat germ translation initiation factor 2 with GDP and GTP.

The wheat germ translation initiation factor 2 (WGeIF-2) was isolated in a homogeneous state by an efficient procedure and characterized. Its molecular mass, as determined by a gel-filtration method is approximately 150,000 Da. According to SDS-PAGE WGeIF-2 consists of four subunits with M(r) 37,000 (alpha), 40,000 (beta), 42,000 (gamma) and 52,000 (delta). The beta- and gamma-subunits (but not the alpha-subunit) of WGeIF-2 can be readily phosphorylated by the double-stranded RNA activated kinase isolated from rabbit reticulocytes. Dissociation constants for WGeIF-2 complexes with GDP and GTP were measured. In our evaluation the WGeIF-2 affinity for GDP (KdGDP = 1.5 x 10(-7) M) was only 10 times higher than for GTP (KdGTP = 1.5 x 10(-6) M), while for rabbit reticulocyte eIF-2 (RReIF-2) the difference has been estimated as as much as two orders of magnitude in accordance with the literature. Close values of dissociation constants for WGeIF-2 complexes with guanine nucleotides suggest that at a sufficiently high [GTP]/[GDP] ratio the nucleotide exchange in wheat cells may take place without the participation of specific factor (eIF-2B) which catalyzes the nucleotide exchange on eIF-2 from mammalian cells.     

Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, gamma-hordothionin, from barley endosperm

A new sulfur-rich and basic polypeptide, designated as gamma-hordothionin, has been isolated from barley endosperm by a semi-preparative purification consisting of extraction with a volatile salt solution followed by high-performance liquid chromatography using a reversed-phase C4 column. The isolated polypeptide was found to be homogeneous by micro-two-dimensional gel electrophoresis in the presence of sodium dodecyl sulfate. The complete primary structure of gamma-hordothionin was determined by automatic degradation of the intact, S-carboxymethylated and S-pyridylethylated gamma-hordothionin and fragments obtained by proteolytic cleavage. gamma-Hordothionin consists of a single polypeptide chain of 47 amino acids with a calculated molecular mass of 5250 Da and contains four disulfide bridges. gamma-Hordothionin inhibits translation in cell-free systems derived from mammalian (rabbit reticulocyte, mouse liver) as well as non-mammalian (Artemia embryo) cells, at several levels. At low concentrations (1-10 microM) the protein seems to affect mainly the polypeptide-chain-initiation process, although it might also act at the elongation level. At higher concentrations (20-80 microM) this inhibitor induces activation of an eukaryotic polypeptide-chain initiation factor 2 alpha-subunit (eIF-2 alpha) kinase in hemin-supplemented reticulocyte lysates, as does hemin deficiency. The presence of the disulfide bridges in gamma-hordothionin appears to be essential for the eIF-2 alpha kinase activation. Based on its similarity at both the structural and functional level with the different genetic variants of thionins (alpha and beta-thionins, from wheat and barley), gamma-hordothionin is a putative member of the thionin family.     

Protein synthesis in Drosophila melanogaster embryos. Two mechanisms for guanine nucleotide exchange on eukaryotic initiation factor 2

The mechanism for guanine nucleotide exchange with eukaryotic initiation factor-2 (eIF-2) from Drosophila melanogaster embryos was studied using the reaction eIF-2 X [3H]GDP + GDP (GTP) in equilibrium eIF-2 X GDP (GTP) + [3H]GDP. When highly purified eIF-2 is used the rate of nucleotide exchange is greatly reduced by Mg2+ and this reduction is overcome by the guanine-nucleotide-exchange factor (GEF) of rabbit reticulocytes. This GEF-dependent exchange is inhibited when Drosophila eIF-2 is either phosphorylated by the hemin-controlled inhibitor (HCI) of rabbit reticulocytes or treated with phosphatidylserine or a rabbit eIF-2 X phosphatidylserine complex. The Mg2+ impairment of guanine nucleotide exchange is less severe when highly purified eIF-2 is incubated at a higher temperature (37 degrees C) and is not observed at any temperature if partially purified eIF-2 is used instead of the highly purified factor. In the latter two cases the exchange is not inhibited by either phosphorylation with HCI or phospholipid treatment of Drosophila eIF-2, possibly suggesting that the observed exchange is not mediated by a GEF-like factor. Our data support two possible mechanisms for GDP/GTP exchange with Drosophila embryos eIF-2: a GEF-dependent exchange, similar to that described in rabbit reticulocytes, which may be regulated by phosphorylation of eIF-2, and a factor-independent exchange which appears to be insensitive to this type of control.     

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.     

Regulation of protein synthesis in eukaryotes. Eukaryotic initiation factor eIF-2 and eukaryotic recycling factor eRF from neuroblastoma cells

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.     

Specific phosphorylation of pig liver initiation factor eIF-2 by the N-ethylmaleimide-treated hemin-controlled translational inhibitor

The specific phosphorylation of pig liver initiation factor 2(eIF-2) by the N-ethylmaleimide (NEM)-treated hemin-controlled translational inhibitor (HCI) from rabbit reticulocytes was investigated. The inhibitor phosphorylated the serine residue of the alpha subunit of eIF-2 (eIF-2 alpha) and 1 mol of phosphate was incorporated into 1 mol of eIF-2 alpha by the inhibitor on maximal phosphorylation, even when eIF-2 was pretreated with alkaline phosphatase prior to phosphorylation. The 32P-labeled eIF-2 alpha was subjected to tryptic digestion and the tryptic digest was analyzed by two-dimensional peptide mapping on a cellulose thin-layer sheet. After 94 h digestion, the autoradiograph of the peptide map showed a single 32P-labeled band with a molecular weight of approximately 1,200. These findings suggest that one specific serine residue of pig liver eIF-2 alpha was phosphorylated by the NEM-treated HCI.