INTRODUCTION

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In the process of translation initiation as it occurs in eukaryotes, the methionyl-initiator tRNA (Met-tRNA) is transferred to the 40S ribosomal subunit in a ternary complex consisting of Met-tRNA, the heterotrimeric initiation factor 2 (eIF2), and GTP. The resulting 43S preinitiation complex binds to the mRNA, scans for the AUG start codon, and triggers hydrolysis of the GTP bound to eIF2 upon base pairing between Met-tRNA and the AUG. After release of eIF2-GDP, the 60S ribosomal subunit joins to form the 80S initiation complex. The eIF2-GDP is inactive for binding Met-tRNA and must be converted to eIF2-GTP to regenerate the ternary complex. This recycling reaction is stimulated by the guanine nucleotide exchange factor (GEF) eIF2B and is a major target of translational control by a conserved mechanism involving phosphorylation of eIF2. The eIF2 phosphorylated on serine 51 of its  subunit [eIF2(P)] is a competitive inhibitor of eIF2B. As eIF2 generally occurs in excess of eIF2B, and phosphorylation of eIF2-GDP increases its affinity for eIF2B, the recycling of eIF2 can be inhibited by phosphorylation of only a fraction of eIF2 (12).

Four different eIF2 kinases that are activated by different starvation or stress conditions (shown in parentheses) have been identified in mammalian cells: HRI (heme deprivation), PKR (double-stranded RNA produced in virus-infected cells), PERK (unfolded proteins in the endoplasmic reticulum), and GCN2 (amino acid starvation) (3, 12, 14, 25). Activation of the mammalian kinases PKR and HRI leads to a high level of eIF2 phosphorylation sufficient to inhibit general translation initiation as an adaptive response to virus infection or heme starvation, respectively. GCN2 is the sole eIF2 kinase in budding yeast. When activated in amino acid-starved cells, GCN2 induces the translation of GCN4 mRNA, encoding a transcriptional activator of amino acid biosynthetic genes. Translational control of GCN4 involves four short open reading frames (uORFs) in the leader of the mRNA. In nonstarvation conditions, ribosomes translate uORF1, reinitiate at uORF2 to 4, and fail to reach the GCN4 start codon. In starved cells, phosphorylation of eIF2 by GCN2 reduces eIF2B function and lowers the concentration of ternary complexes. Consequently, many ribosomes that resume scanning after translating uORF1 fail to rebind the ternary complex until scanning past uORF4 and thus reinitiate at the GCN4 start site instead. General translation and cell growth are inhibited when eIF2 is phosphorylated at higher levels than occurs in amino acid-starved wild-type yeast, as in GCN2^c mutants bearing constitutively activated forms of the kinase (12).

eIF2B contains five subunits (Table 1) and is found in a 1:1 complex with its substrate, eIF2 (6, 23). The , , , and  subunits of yeast eIF2B (encoded by GCD6, GCD2, GCD1, and GCD7, respectively) are essential, and nonlethal mutations in these genes lead to temperature-sensitive growth (Ts phenotype) and derepression of GCN4 translation (Gcd phenotype), indicative of reduced ternary complex levels. In contrast, deletion of GCN3 (encoding eIF2B) has no effect on cell growth and confers a Gcn phenotype (failure to induce GCN4) (11), suggesting that GCN3 is required primarily for inhibition of eIF2B by eIF2(P). GCD2 and GCD7 have sequence similarity to GCN3, and when all three proteins were overexpressed in yeast, they formed a stable subcomplex that reduced the inhibitory effect of eIF2(P) on translation initiation (29). This subcomplex had no GEF activity in vitro but could bind to purified eIF2 holoprotein in a manner stimulated by phosphorylation of Ser-51 on the  subunit (21). Hence, it was proposed that the overexpressed GCD2-GCD7-GCN3 subcomplex sequestered the inhibitor eIF2(P)-GDP and allowed native eIF2B to recycle the unphosphorylated eIF2-GDP (21).

Additional evidence implicating GCD2 and GCD7 as regulatory subunits in eIF2B was provided by the isolation of point mutations in these proteins that eliminate the effects of eIF2(P) on translation in yeast, conferring a Gcn phenotype and suppressing the growth inhibition of GCN2^c alleles (22, 28). These mutations could decrease the affinity of eIF2B for eIF2(P)-GDP or allow eIF2B to accept eIF2(P)-GDP as a substrate. Evidence for the latter mechanism came from in vitro GDP-GTP exchange assays using purified eIF2(P)-[³H]GDP and cell extracts containing overexpressed eIF2B subunits. Unlike wild-type eIF2B, the mutant complexes containing Gcn substitution GCD7-S119P (22) or GCD7-I118T,D178Y (28) catalyzed nucleotide exchange at nearly identical rates on phosphorylated or unphosphorylated eIF2-[³H]GDP. Thus, these GCD7 mutations allowed eIF2B to utilize the competitive inhibitor eIF2(P)-GDP as a substrate (21).

Remarkably, the two-subunit complex comprised of eIF2B subunits GCD6 and GCD1 has GEF activity greater than that of five-subunit eIF2B and can accept phosphorylated and unphosphorylated eIF2-GDP as equivalent substrates. Thus, GCD6 and GCD1 comprise an unregulated catalytic subcomplex in eIF2B. Accordingly, we proposed that the GCD2-GCD7-GCN3 regulatory subcomplex is required to inhibit the GCD6-GCD1 catalytic subcomplex when the substrate is phosphorylated (21). We envisioned that binding of phosphorylated eIF2-GDP to the eIF2B regulatory subcomplex would preclude its interaction with the active site in the GCD1-GCD6 catalytic subcomplex. The Gcn mutations in GCD7 would overcome this nonproductive interaction and allow binding of eIF2(P)-GDP to eIF2B in the manner required for nucleotide exchange (21).

Previously, we suggested that the homologous regulatory segments in GCN3, GCD2, and GCD7 are juxtaposed to form a binding site for the phosphorylated N-terminal portion of eIF2 (22). Consistent with this idea, Gcn mutations were obtained in yeast eIF2 (encoded by SUI2) in residues surrounding Ser-51 that reduce the inhibitory effect of eIF2(P)-GDP on eIF2B activity in vivo. These mutations alter residues Ile-58, Leu-84, Arg-88, and Val-89 (27). Alanine substitution of Ser-48 has a similar effect in mammalian cells (4, 7, 15, 18). Moreover, addition of recombinant human eIF2-S48A to rabbit reticulocyte lysates reduced the abundance of 15S complexes containing eIF2, thought to represent inactive eIF2B-eIF2(P)-GDP complexes stabilized by Ser-51 phosphorylation (26). This last finding suggests that mutation of Ser-48 to Ala reduces the affinity of eIF2(P)-GDP for eIF2B as a means of overcoming the inhibition by Ser-51 phosphorylation.

At odds with our proposal that eIF2B regulatory subunits interact directly with eIF2 (22, 27), no binding was detected between eIF2B holoprotein and phosphorylated recombinant rat eIF2. By contrast, recombinant eIF2 showed significant binding to eIF2B holoprotein, and it also bound to the isolated  and  subunits of eIF2B (GCD2 and GCD6, respectively, in yeast) (17). Based on these findings, it was suggested that eIF2 does not contact eIF2B and that Ser-51 phosphorylation elicits a conformational change in eIF2 that enhances interaction between eIF2 and the  and  subunits of eIF2B (17).

In this study, we show for the first time that the GCD2-GCD7-GCN3 regulatory subcomplex of yeast eIF2B can form a stable complex in vitro with a recombinant form of the  subunit of eIF2. This glutathione S-transferase (GST)-SUI2 fusion protein formed a stable complex with the regulatory subcomplex, but not with the catalytic subcomplex of eIF2B, in a manner stimulated by phosphorylation of Ser-51 in the recombinant protein. None of the individual eIF2B subunits was capable of this stable interaction, consistent with the idea that the binding domain for phosphorylated SUI2 [SUI2(P)] in eIF2B requires contributions from all three regulatory subunits. We present genetic data that SUI2(P) competes effectively with eIF2(P) for association with the GCD2-GCD7-GCN3 subcomplex in vivo, providing evidence that binding of SUI2(P) to the eIF2B regulatory subunits is a physiological interaction. Furthermore, in vitro binding of GST-SUI2(P) to the eIF2B regulatory subunits was impaired by Gcn mutations in SUI2 and also by those in GCD7 shown previously to permit eIF2(P)-GDP to be accepted as a substrate by eIF2B in vitro. These last findings provide compelling evidence that tight binding of SUI2(P) to the eIF2B regulatory subunits is crucial for the negative regulation of eIF2B function when the substrate eIF2-GDP is phosphorylated.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Yeast strains and plasmids. Yeast strains and plasmids used in this study are shown in Tables 2 and 3, respectively; details of their construction will be made available on request.

Purification. His₆-eIF2B was overexpressed from plasmids pTK1.11 and p1871 in yeast strain H2767. Cells were grown at 30°C in YPD to an optical density at 600 nm (OD₆₀₀) of 8 to 10, harvested, washed with ice-cold distilled water, resuspended in breaking buffer (75 mM Tris-HCl [pH 7.5], 100 mM KCl, 1 mM Na₂EDTA, 14 mM 2-mercaptoethanol, 10 µM GDP, 50 mM NaF) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 1 µg each of pepstatin A, leupeptin, and aprotinin per ml), and broken using the French press or a bead beater. For the French press, 1 ml of lysis buffer was used per g cells, whereas twice the volume was used for the bead beater. The whole-cell extract (WCE) was centrifuged at 4°C for 15 min at 8,000 × g to remove cellular debris, and the supernatant was subjected to ultracentrifugation at 200,000 × g for 2 h to pellet the ribosomes. Ribosomes were resuspended gently in buffer A (20 mM Tris-HCl [pH 7.5], 0.1 mM MgCl₂, 10% glycerol, 10 µM GDP, 2 mM 2-mercaptoethanol, 50 mM NaF, protease inhibitors) containing 500 mM KCl (buffer A-500) and incubated for 1 h at 4°C with slow stirring. The ribosomal salt wash (RSW) was obtained by ultracentrifugation of the ribosomal suspension at 200,000 × g for 2 h. Ni-silica resin (Qiagen) was washed twice in buffer A-500 containing 5 mM imidazole, and 1.2 ml of a 50% slurry in the same buffer was added to the RSW and incubated overnight at 4°C. The mixture was centrifuged at 1,000 rpm for 2 min, the supernatant was discarded, and the resin was washed five times, each with 10 volumes of buffer A-500 containing 5 mM imidazole. The resin was washed three more times with 10 volumes of buffer A containing 1 M KCl and 5 mM imidazole and twice with buffer B (Tris-buffered saline, 10% glycerol, 10 mM imidazole, 0.5 mM PMSF, complete protease inhibitor tablets without EDTA [Boehringer Mannheim]). eIF2B was eluted three times, each with 1 ml of buffer B containing 250 mM imidazole. Protein concentrations of the eluates were determined using the Bradford assay (Bio-rad), and the peak fractions were pooled.

Yeast WCE preparation. WCEs used for GST pull-down assays were prepared from the transformants of yeast strain BJ1995 overexpressing the appropriate eIF2B subunits as described previously (21), except that the cells were broken by vortexing with acid-washed glass beads five times for 1 min each, with 1-min intervals on ice, and including 75 mM Tris-HCl (pH 7.5), 1 mM EDTA, and complete protease inhibitor tablets in the breaking buffer. To prepare WCEs for Ni⁺²-silica pull-down assays, 0.1 mM EDTA and 5 mM 2-mercaptoethanol were used instead of 1 mM EDTA and 1 mM DTT in the breaking buffer.

GST pull-down assays. GST-SUI2 fusions were expressed in Escherichia coli BL21(DE3) (Novagen), using 0.4 mM isopropyl-B-D-thiogalactopyranoside to induce the fusion proteins. WCEs were prepared in lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM KCl, 2.5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, protease inhibitors, complete protease inhibitor tablets without EDTA) by sonication six times for 2 min each with 30-s intervals on ice. Glutathione-Sepharose beads (Amersham) were prewashed in binding buffer (BB; same as lysis buffer but including 0.1% Triton X-100, 5 mM NaF, and 0.1 mM ATP) and 30 µl of a 50% slurry was mixed with WCE in BB for 40 min at 4°C on a nutator. The beads were washed thrice with 500 µl of ice-cold BB and resuspended in 50 µl of BB. The immobilized fusion proteins were incubated with or without PKR for 10 min at room temperature. In control assays, 5 to 10 µCi of [³²P]ATP was added prior to addition of PKR. The kinase reaction was stopped by addition of 150 µl of BB on ice. Radioactive beads were washed with BB, mixed with 12 µl of 2× Laemmli's sample buffer (NOVEX), boiled, resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), stained with Coomassie blue, dried, and exposed to X-ray film, and the film was developed.

Nickel pull-down assays with His-tagged eIF2. His₆-tagged eIF2 (2.5 µg) was purified as described above and incubated in 50 µl of modified binding buffer BB-1 (in which 5 mM 2-mercaptoethanol replaced 1 mM DTT) with or without PKR (1 µg) for 10 min at RT. The reaction was stopped by addition of 50 µl of BB-1 on ice and added to 100 µg of WCEs prepared from yeast transformants overexpressing the appropriate eIF2B subunits and incubated for 10 min at 10°C. The His₆-tagged eIF2 and bound proteins were recovered using 10 µl of Ni-silica beads (prewashed in BB-1) as described previously (21) except that BB-1 was used instead of PD (21). The samples were resolved by SDS-PAGE on a 10 to 20% gradient gel and subjected to immunoblot analysis as described above.

	RESULTS

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GST-SUI2 binds purified eIF2B in vitro dependent on Ser-51 phosphorylation To determine whether the  subunit of yeast eIF2 interacts directly with eIF2B, we purified a full-length GST-SUI2 fusion protein from E. coli by using glutathione-Sepharose beads and tested the immobilized protein for interaction with partially purified yeast eIF2B. In parallel, we examined a mutant fusion protein containing Ala in place of Ser at position 51 (GST-SUI2-S51A). Prior to the binding reactions, both immobilized fusion proteins were treated with purified human PKR and ATP to phosphorylate Ser-51 in the wild-type protein. In control experiments where [-³²P]ATP was included in the kinase reactions, we confirmed that GST-SUI2, but not GST-SUI2-S51A, was phosphorylated by PKR in vitro, confirming PKR's specificity for Ser-51 (Fig. 1A). The eIF2B used in the binding assays was purified by nickel chelation chromatography from a yeast strain expressing a polyhistidine-tagged form of eIF2B (GCD1). The purification was carried out at a high salt concentration to dissociate eIF2 from eIF2B, as the presence of eIF2 would complicate the interpretation of binding data. Western analysis confirmed the absence of eIF2 subunits in the His₆-eIF2B preparation (Fig. 1B, lane I; Fig. 1C). After incubation of purified His₆-eIF2B with the immobilized fusion proteins, with or without pretreatment with PKR, the beads were washed extensively and the bound proteins were analyzed by Western blotting using antibodies against eIF2B subunits. As shown in Fig. 1B, His₆-eIF2B bound to wild-type GST-SUI2 in a manner stimulated by incubation with PKR. In contrast, little or no His₆-eIF2B bound to GST-SUI2-S51A regardless of PKR treatment. These results provide strong evidence that eIF2B interacts directly with the  subunit of eIF2 and that this interaction is greatly enhanced by phosphorylation of Ser-51.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   GST-SUI2 binds to purified eIF2B in a manner stimulated by phosphorylation of Ser-51. GST-SUI2, GST-SUI2 S51A, or GST alone was expressed in E. coli, immobilized on glutathione-Sepharose beads, and incubated with (+) or without () 1 µg of purified PKR in buffer BB. In panel A, 5µCi of [-32P]ATP was added with the PKR, and the reactions were resolved by SDS-PAGE, stained with Coomassie blue (lower panel), and subjected to autoradiography (upper panel, 32P). In panel B, 2.0 µg of partially purified His6-eIF2B was incubated with the immobilized GST-SUI2, GST-SUI2-S51A, or GST proteins treated with or without PKR. After extensive washing, the bound proteins were resolved by SDS-PAGE and analyzed by Western blotting using antibodies against GCD6, GCD7, and GCD11 (GST pull-down assay). Two different amounts of bound proteins differing by a factor of 3 were loaded in successive lanes for each fusion protein. The input (I) lane contains 25% of the input amount of purified eIF2B used in the pull-down assays shown in lanes 1 to 10. (C) Western blot comparing the levels of SUI2 and GCD6 in 1 µg of yeast WCE (lane 1) and 3 µg of the purified His6-eIF2B (lane 2) used in panel B. wt, wild type.

View larger version (71K): [in this window] [in a new window]   FIG. 2.   (A) SUI2 residues 1 to 245 are sufficient for binding of GST-SUI2(P) to eIF2B stimulated by phosphoserine 51. Full-length GST-SUI2 (wild type [wt]) and the indicated derivatives truncated at the C terminus (designated by the amino acids [aa] remaining) were immobilized on glutathione-Sepharose beads, treated with (+) or without () 3 µg of PKR in buffer BB, and incubated with 4 µg of purified eIF2B. Binding of eIF2B to the GST-SUI2 fusions in pull-down assays was analyzed by SDS-PAGE and Western blotting (upper panel) as described in Fig. 1B. The lower panel shows Ponceau S staining of the bound proteins, with asterisks indicating the full length GST-SUI2 fusions. The input (I) lane contained 50% of the eIF2B used in each reaction. (B) Deletion of the C terminus (amino acids 140 to 304) of SUI2 abolished phosphorylation of GST-SUI2 by PKR. The experiment was carried out exactly as described for the upper panel of Fig. 1A for the indicated GST-SUI2 proteins, using the same amounts designated 3X in panel A.

GST-SUI2(P) binds to the regulatory subcomplex of eIF2B. The eIF2B regulatory subcomplex can be overexpressed in yeast from a high-copy-number plasmid bearing GCD2, GCD7, and GCN3 (under the control of their native promoters) and coimmunoprecipitated with eIF2 from WCEs (29). Previously, we showed that the overexpressed regulatory subcomplex in WCEs could bind to exogenously added eIF2 holoprotein in a manner stimulated ~3-fold by prior phosphorylation of the eIF2 on Ser-51 (21). The catalytic subcomplex, comprised of GCD1 and GCD6, also bound to eIF2 but did not discriminate between phosphorylated and unphosphorylated eIF2 (21). To determine whether SUI2 alone can interact with these eIF2B subcomplexes, and in a manner stimulated by phosphorylation on Ser-51, we incubated the immobilized GST-SUI2 and GST-SUI2-S51A fusions (pretreated with PKR) with WCEs containing the appropriate overexpressed eIF2B subunits and analyzed the bound proteins by Western blotting.

View larger version (49K): [in this window] [in a new window]   FIG. 3.   The eIF2B regulatory subcomplex in cell extracts binds to GST-SUI2(P). Wild-type (wt) GST-SUI2 and GST-SUI2-S51A fusions were immobilized on glutathione-Sepharose beads and treated with 1 µg of PKR in buffer BB, followed by incubation with the appropriate yeast WCE and 800 µg of bovine serum albumin in buffer BB. (A) The pull-down assays contained 100 or 200 µg of GST-SUI2 (lanes 1 to 14), or 150 or 300 µg of GST-SUI2-S51A (lanes 15 to 20), and 600 µg of WCE from transformants of yeast strain BJ1995 overexpressing the regulatory subcomplex GCD2-GCD7-GCN3 (from plasmid p1871; lanes 5, 6, 19, and 20), GCD2 (from plasmid p2297; lanes 8 and 22), GCD7 (from plasmid p2305; lanes 10, 11, 24, and 25), or GCN3 (from plasmid p2304; lanes 13, 14, 27, and 28) or carrying the empty vector (from plasmid pRS426; lanes 2, 3, 16, and 17). The bound proteins were analyzed by SDS-PAGE (on an 8 to 16% gradient gel) and Western blotting as described for Fig. 1B. Input (I) lanes contained 10% of the WCE used in each reaction. For the binding reactions in lanes 8 and 22, only the larger amounts of the GST-SUI2 fusion proteins described above were used. In panel B, the pull-down assays contained 25 or 100 µg of wild-type GST-SUI2 or of GST-SUI2-S51A and 200 µg of WCE from transformants of yeast strain BJ1995 overexpressing GCD1 and GCD6 (from plasmid p2302; lanes 7 to 10) or carrying the empty vector (from plasmid pRS426; lanes 2 to 5). The bound proteins were analyzed as described for Fig. 1B. Input (I) lanes contained 5% of the WCE used in each reaction.

Gcn regulatory mutations near Ser-51 eliminate binding of GST-SUI2(P) to both eIF2B and the GCD2-GCD7-GCN3 regulatory subcomplex. Previously, we isolated point mutations in the amino terminus of SUI2 that suppressed the growth-inhibitory effects of eIF2 phosphorylation and prevented derepression of GCN4 translation (Gcn phenotype) (27). Recently, additional Gcn mutations were isolated in SUI2, and it was shown that they did not diminish Ser-51 phosphorylation by GCN2 in vivo or in vitro (T. E. Dever, unpublished results). Thus, the latter mutations most likely abrogate the inhibitory effect of eIF2(P) on eIF2B activity in vivo. One possibility is that these mutations weaken the direct interaction between phosphorylated SUI2 and the eIF2B regulatory subcomplex. To test this idea, we introduced the newly identified Gcn mutations into the GST-SUI2 fusion and investigated whether they reduce binding of GST-SUI2(P) to eIF2B. The GST-SUI2 fusion proteins containing the mutations indicated in Fig. 4A were purified, phosphorylated with PKR, and incubated with purified His₆-eIF2B. Except for GST-SUI2-D83A, the mutant proteins were phosphorylated efficiently by PKR (Fig. 4A, ³²P); however, none bound to eIF2B at the high levels observed for wild-type GST-SUI2(P). In fact, all of them displayed the low-level nonspecific binding characteristic of GST alone (Fig. 4A, Western).

View larger version (57K): [in this window] [in a new window]   FIG. 4.   Gcn mutations proximal and distal to phosphoserine 51 in SUI2 disrupt binding of eIF2B holoprotein and the eIF2B regulatory subcomplex to GST-SUI2(P). (A) Wild-type GST-SUI2 and the indicated mutant derivatives were immobilized on glutathione-Sepharose beads and treated with 1 or 2 µg of PKR in buffer BB. The immobilized proteins were incubated with His6-eIF2B (4 µg) and bovine serum albumin (1 mg) in buffer BB, and the pull-down assays were analyzed as described for Fig. 1B, with the results shown in the upper two panels (Western). The input (I) lane contained 25% of the His6-eIF2B used in each reaction. The results in the lower two panels were obtained exactly as described for the panels labeled 32P and Coomassie in Fig. 1A, respectively. Amounts of the fusion proteins used for the pull-down assays were the same as shown in the bottom panel (Coomassie) used for the kinase assays. (B) Pull-down assays were carried out using 84 or 168 µg of GST, 100 or 200 µg of GST-SUI2, 150 or 300 µg of either GST-SUI2-E49N or GST-SUI2-R88T fusion protein, and 800 µg of WCE from transformants of strain BJ1995 overexpressing the regulatory subcomplex GCD2-GCD7-GCN3 (from plasmid p1871; lanes 11 to 17) or carrying the empty vector (from plasmid pRS426; lanes 2 to 9). Bound proteins were analyzed as described for Fig. 1B. Input (I) lanes contained 10% of the WCE used in each reaction. wt, wild type.

Gcn mutations in GCD7 reduce binding of the eIF2B regulatory subcomplex to GST-SUI2(P) Previously, we described mutant alleles of GCD7 with a Gcn phenotype (22, 28) and showed that the substitutions in two such alleles (GCD7-S119P and GCD7-I118T, D178Y [Fig. 5A]) allowed eIF2B to accept eIF2(P)-GDP as a substrate using in vitro assays for guanine nucleotide exchange (21). Accordingly, we investigated here whether these GCD7 mutations would weaken association between GST-SUI2(P) and the eIF2B holoprotein. We prepared three different WCEs containing overexpressed amounts of all five eIF2B subunits, containing either wild-type GCD7, GCD7-S119P (mutant *M1), or GCD7-I118T,D178Y (*M2), and incubated them with GST-SUI2(P) and GST-SUI2-S51A. As shown in Fig. 5B, the eIF2B*M1 and eIF2B*M2 complexes showed moderate (*M1) or severe (*M2) reductions in binding to GST-SUI2(P) compared to the wild-type eIF2B complex (lanes 4 to 6 versus 7 to 12). As expected, the two mutant eIF2B complexes did not bind to GST-SUI2-S51A (data not shown).

View larger version (39K): [in this window] [in a new window]   FIG. 5.   Gcn mutations in GCD7 decrease binding of the eIF2B holoprotein and the eIF2B regulatory subcomplex to GST-SUI2(P). (A) Schematic showing the sequence similarities among the eIF2B regulatory subunits and the point mutations in GCD7 that were analyzed in this study. (B) Wild-type (wt) GST-SUI2 fusion was immobilized on glutathione-Sepharose beads and treated with 1 µg of PKR in buffer BB, followed by incubation with the appropriate yeast WCE and 800 µg of bovine serum albumin in buffer BB. The pull-down assays were carried out with 100 or 200 µg of GST-SUI2 and 600 µg of yeast WCE from transformants of strain BJ1995 overexpressing all five wild-type eIF2B subunits (from plasmids p1873 and p1871; lanes 5 and 6), wild-type GCD1-GCD6-GCD2-GCN3 and GCD7-S119P (from plasmids p1873 and pAV1139, designated eIF2B*M1; lanes 8 and 9), or wild-type GCD1-GCD6-GCD2-GCN3 and GCD7-I118T, D178Y (from plasmids p1873 and pAV1140; designated eIF2B*M2; lanes 11 and 12) or carrying the empty vectors (from plasmids pRS425 and pRS426; lanes 2 and 3). Bound proteins were analyzed as described for Fig. 1B. Input (I) lanes contained 10% of the WCE used in each reaction. (C) Pull-down assays were done exactly as described for panel B except that the WCEs were from transformants of strain BJ1995 overexpressing wild-type GCD2-GCD7-GCN3 (from plasmid p1871; lanes 1 to 3), wild-type GCD2-GCN3 and GCD7-S119P (from plasmid pAV1139; lanes 4 to 6), or wild-type GCD2-GCN3 and GCD7-I118T, D178Y (from plasmid pAV1140; lanes 7 to 9). Input (I) lanes contained 10% of the WCE used in each reaction. (D) Histograms showing the results of densitometric quantification of the binding data in panel C relative to the input signal in percentage.

Gcn mutations in GCD7 decrease interaction between the eIF2B and eIF2 holoproteins. GCD6, the principal catalytic subunit of yeast eIF2B, has a strong binding domain for eIF2 (1); hence, it was unclear how much the enhanced interaction between SUI2(P) and the eIF2B regulatory subunits would contribute to the stability of the complex formed between the eIF2 and eIF2B holoproteins. To address this issue, we purified eIF2 holoprotein containing a polyhistidine-tagged form of the  subunit (His₆-eIF2), phosphorylated the eIF2 in vitro with PKR, and incubated it with WCEs containing overexpressed amounts of all five eIF2B subunits, containing either wild-type GCD7, GCD7-*M1, or GCD7-*M2. Following incubation, the His₆-eIF2 was recovered on nickel-silica resin and probed for bound eIF2B subunits by Western blotting. The overexpressed wild-type eIF2B holoprotein bound to His₆-eIF2 in a manner enhanced by phosphorylation of Ser-51 (Fig. 6A, lanes 7 and 8), to a degree similar to that observed previously (21). Interestingly, binding of eIF2B to both phosphorylated and unphosphorylated His₆-eIF2 was reduced by the *M1 and *M2 mutations (Fig. 6A, lanes 11, 12, 15, and 16 versus 7 and 8; Fig. 6B). These data imply that the GCD7 Gcn mutations impair the interaction between SUI2 and the eIF2B regulatory subunits in the context of the eIF2-eIF2B holocomplex. Apparently, loss of these contacts destabilizes the eIF2-eIF2B holocomplex even when SUI2 is unphosphorylated.

View larger version (40K): [in this window] [in a new window]   FIG. 6.   Binding of wild-type and mutant eIF2B holoproteins to His-tagged eIF2 holoprotein. (A) WCEs from transformants of strain BJ1995 overexpressing all five wild-type eIF2B subunits (from plasmids p1873 and p1871, designated h.c.eIF2B wt; lanes 5 to 8), wild-type subunits GCD1-GCD6-GCD2-GCN3 and mutant subunit GCD7-S119P (from plasmids p1873 and pAV1139. designated h.c. eIF2*M1; lanes 9 to 12), or wild-type subunits GCD1-GCD6-GCD2-GCN3 and mutant subunit GCD7-I118T,D178Y (from plasmids p1873 and pAV1140, designated h.c. eIF2*M2; lanes 13 to 16) or carrying the empty vectors (from plasmids pRS425 and pRS426; lanes 1 to 4) were incubated with purified eIF2 phosphorylated in vitro with PKR [eIF2(P)] (lanes 4, 8, 12, and 16), unphosphorylated eIF2 (lanes 3, 7, 11, and 15), or no eIF2 (lanes 2, 6, 10, and 14). The proteins that bound to eIF2 were purified by Ni-silica affinity chromatography and analyzed by SDS-PAGE and Western blotting using antibodies against the proteins listed to the right of each panel. For SUI2(P), antibodies specific for eIF2 phosphorylated on Ser-51 were employed. Input lanes contained 20% of the WCE used in each reaction. (B) Histograms showing densitometry of signals for each eIF2B antibody shown in panel A as a percentage of the input.

Genetic evidence that SUI2 binds to the eIF2B regulatory subcomplex in vivo. To obtain evidence that SUI2 on its own can interact with the eIF2B regulatory subcomplex in vivo, we exploited our previous finding (29) that cooverexpression of GCD2, GCD7, and GCN3 alleviates the slow-growth phenotype associated with the constitutively activated kinase encoded by GCN2^c-M719V-E1537G (Fig. 7A, compare strains 1 and 2). We previously provided evidence that the GCD2-GCD7-GCN3 subcomplex sequestered eIF2(P) and prevented it from inhibiting native eIF2B holoprotein, freeing the latter to catalyze GDP-GTP exchange on the unphosphorylated pool of eIF2-GDP (21, 29) (Fig. 7C, strains 1 and 2). It is known that excess SUI2 in cells overexpressing only this eIF2 subunit is phosphorylated to high levels by GCN2 in vivo (8, 9). Hence, we predicted that if SUI2 and the eIF2B regulatory subcomplex were cooverexpressed in cells containing GCN2^c-M719V-E1537G, the SUI2(P) would compete with eIF2(P) for binding to the regulatory subcomplex. In this event, the eIF2(P) would be released from the overexpressed eIF2B regulatory subcomplex and become available to inhibit the GEF activity of native eIF2B holoprotein (Fig. 7C, strain 3). In accordance with this prediction, overexpressing SUI2 from a high-copy-number plasmid restored the slow-growth phenotype conferred by GCN2^c-M719V-E1537G in the strain cooverexpressing the eIF2B regulatory subunits (Fig. 7A, compare strains 2 and 3). Importantly, overexpressing SUI2 alone did not exacerbate the slow-growth phenotype of the GCN2^c-M719V-E1537G mutant (Fig. 7A). We also verified that overexpressing SUI2 did not reduce the degree of GCD2, GCD7, and GCN3 overexpression (Fig. 7B). These findings provide strong evidence that SUI2(P) can compete effectively with eIF2(P) holoprotein for interaction with the eIF2B regulatory subcomplex in vivo, implying that SUI2(P) on its own makes strong contacts with the regulatory subunits of eIF2B.

View larger version (30K): [in this window] [in a new window]   FIG. 7.   Genetic evidence that SUI2 binds individually to the regulatory subcomplex of eIF2B in vivo. (A) Strain H1608 bearing the chromosomal GCN2c-M719V,E1537G allele was transformed with high-copy-number (H.C.) plasmids encoding GCD2, GCD7, and GCN3 (p1871) or SUI2 (pTK29) or with empty vectors (V, pRS425 and pRS426). Isogenic GCN2 strain H1402 was transformed with the empty vectors to provide a wild-type control (WT). The transformants were streaked on SD medium supplemented with inositol and incubated at 30°C for 4 days. (B) WCEs were prepared from the transformants of strain H1608 overexpressing GCD2-GCD7-GCN3 or GCD2-GCD7-GCN3-SUI2 as described for panel A. Forty micrograms of each WCE was resolved by SDS-PAGE and subjected to immunoblot analysis using antibodies against the indicated proteins. (C) A model explaining the possible protein-protein interactions occurring in the transformants described in panel A. (Strain 1) eIF2B holoprotein (labeled 2, 7, 3, 6, 1) interacts with unphosphorylated eIF2 holoprotein (, , ) to exchange the GDP () present on eIF2 for GTP. As these cells contain an activated GCN2c kinase, much of the eIF2 is phosphorylated (, labeled ~P) and forms inactive complexes with eIF2B, impeding GDP-GTP exchange on the unphosphorylated eIF2-GDP. This leads to a slow-growth phenotype. (Strain 2) In GCN2c cells overexpressing the GCD2-GCD7-GCN3 regulatory subcomplex of eIF2B (labeled 2, 3, 7), the latter competes with eIF2B holoprotein for the inhibitor, eIF2(P)-GDP, allowing the eIF2B to exchange GDP for GTP on unphosphorylated eIF2. This suppresses the slow-growth phenotype associated with the GCN2c allele. (Strain 3) Overexpressed SUI2 is phosphorylated in GCN2c cells and competes with eIF2(P) holoprotein for binding to the eIF2B regulatory subcomplex. This releases eIF2(P) and reinstates inhibition of eIF2B and the attendant slow-growth phenotype of GCN2c cells. (See Fig. 9 for additional details on the relative orientations of eIF2 and eIF2B subunits in the different complexes.)

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In previous experiments, we provided genetic and biochemical evidence that eIF2B contains two independent binding sites for eIF2. The GCD1-GCD6 subcomplex was found to be sufficient to catalyze nucleotide exchange on eIF2-GDP. Unlike eIF2B holoprotein, however, its affinity for eIF2 was not increased, and its GEF activity was not inhibited, by phosphorylation of eIF2 on Ser-51. The GCD2-GCD7-GCN3 regulatory subcomplex had no GEF activity, but it bound to eIF2 holoprotein in a manner stimulated by phosphorylation of Ser-51 (21). Hence, we proposed that interaction of eIF2(P)-GDP with its binding site in the eIF2B regulatory subcomplex would interfere with the ability of the GCD1-GCD6 subcomplex to catalyze nucleotide exchange (22). Consistent with this model, Gcn mutations were obtained in all three regulatory subunits of eIF2B that abolished the inhibitory effect of eIF2 phosphorylation on eIF2B function in vivo (22, 28). Moreover, several such mutations allowed eIF2B to catalyze nucleotide exchange on eIF2(P)-GDP in vitro (22). These findings, combined with the identification of additional Gcn mutations in the N terminus of SUI2 (27), led us to propose that SUI2 interacts directly with the eIF2B regulatory subcomplex dependent on Ser-51 phosphorylation, and that this interaction impedes GDP-GTP exchange by the eIF2B catalytic subcomplex (21). In the context of this model, the Gcn mutations in SUI2 or the eIF2B regulatory subunits might weaken interaction between SUI2(P) and the regulatory subcomplex, neutralizing the effect of Ser-51 phosphorylation on the GEF activity of eIF2B.

A different view of the effect of eIF2 phosphorylation on interaction between eIF2 and eIF2B was proposed by Kimball and colleagues (17). These workers found that the  subunit of rat eIF2 did not interact with any individual subunits of rat eIF2B in vitro, even when the eIF2 was phosphorylated on Ser-51. In contrast, the C-terminal portion of eIF2 bound to the  and  subunits of rat eIF2B. Accordingly, they proposed that eIF2 does not directly interact with eIF2B subunits and that its phosphorylation leads to a conformational change in the eIF2 holoprotein that enhances interactions of eIF2 with eIF2B and eIF2 (17). Presumably, this enhanced interaction would be responsible for inhibiting the GEF activity of eIF2B.

In accordance with our model, we found that the  subunit of eIF2 can interact directly with the regulatory subcomplex of eIF2B. As reported by Kimball et al. (17), we observed no stable association between a recombinant form of eIF2, GST-SUI2, and any individual subunits of eIF2B, with the possible exception of GCD2 (eIF2B). On the other hand, we found that GST-SUI2 formed a tight complex with the eIF2B holoprotein, and also with the GCD2-GCD7-GCN3 regulatory subcomplex, and that both interactions were strongly stimulated by phosphorylation of Ser-51. Hence, we propose that the binding domain for SUI2 in eIF2B requires contributions from all three regulatory subunits. These eIF2B subunits are related in sequence, and most of the Gcn mutations in these proteins are clustered in two regions of strong similarity (22). Single amino acid substitutions in any one of these proteins is sufficient to overcome the effects of Ser-51 phosphorylation on eIF2B function in vivo. Thus, the homologous segments in GCN3, GCD2, and GCD7 that are altered by these Gcn mutations may form a multivalent binding surface for SUI2 rather than providing alternative, redundant binding sites. Presumably, SUI2(P) makes more extensive molecular contacts with this binding surface than does unphosphorylated SUI2.

In contrast to its stable interaction with the regulatory subcomplex, we detected no interaction between GST-SUI2 and the catalytic subunits of eIF2B, irrespective of Ser-51 phosphorylation. Although these are negative results, they are in keeping with our proposal that SUI2 interacts primarily with the regulatory subunits of eIF2B. Previously, we detected stable interaction between the N-terminal half of eIF2 and the C-terminal domain of eIF2B (GCD6) that was important for association between the eIF2 and eIF2B holoproteins in vivo and also for eIF2B function (1). Thus, it appears that eIF2 interacts directly with the catalytic subcomplex in eIF2B. It remains to be seen whether eIF2, which contains conserved motifs for guanine nucleotide binding, also interacts directly with the eIF2B catalytic subunits, or whether interaction of the latter with eIF2 leads to a conformational change in eIF2 that stimulates dissociation of GDP.

We showed previously that overexpression of the GCD2-GCD7-GCN3 regulatory subcomplex overcomes the growth-inhibitory effects of high-level eIF2 phosphorylation in GCN2^c cells (29). Based on the tighter binding of eIF2(P) versus unphosphorylated eIF2 to the regulatory subcomplex (21), we proposed that GCD2-GCD7-GCN3 sequestered eIF2(P)-GDP and prevented it from competing with unphosphorylated eIF2-GDP for binding to native eIF2B. Here we showed that cooverexpressing SUI2 with GCD2, GCD7, and GCN3 neutralized the ability of the eIF2B subcomplex to rescue growth in cells containing high-level eIF2(P). These data are consistent with our in vitro binding data showing that SUI2(P) interacted strongly with GCD2-GCD7-GCN3 independently of the  and  subunits of eIF2. Hence, we propose that the overexpressed SUI2(P) sequestered GCD2-GCD7-GCN3 and reduced its association with eIF2(P) holoprotein, reinstating the inhibition of native eIF2B by eIF2(P)-GDP. These data provide in vivo evidence that the interaction between GST-SUI2(P) and the eIF2B regulatory subcomplex is an important aspect of the regulatory mechanism. Additional support for this conclusion came from the fact that binding of GST-SUI2(P) to eIF2B in vitro was impaired by all of the Gcn mutations that we tested. These included mutations mapping in the N-terminal third of SUI2 and GCD7 mutations shown previously to permit eIF2B to catalyze nucleotide exchange on phosphoryated eIF2(P)-GDP (21, 28). These last results provide strong evidence that tight binding of SUI2(P) to the eIF2B regulatory subunits (in the context of the two haloproteins) is required for inhibition of eIF2B activity by phosphorylated eIF2.

Evidence for multiple contacts between SUI2(P) and the eIF2B regulatory subcomplex. The Gcn mutations in SUI2 that weakened binding of GST-SUI2(P) to eIF2B mapped in Glu-49, two residues away from the phosphorylation site, and in Lys-79, Gly-80, and Arg-88, located 30 or more residues away from Ser-51. These findings suggest that two noncontiguous segments in the N terminus of SUI2 are involved in binding to the regulatory subunits of eIF2B. Interestingly, recent findings indicate that eIF2 kinases also have a bipartite binding domain in the N terminus of SUI2. The K3L protein is a pseudosubstrate inhibitor of PKR encoded by vaccinia virus that is 28% identical to the N-terminal one-third of SUI2 and contains a perfect match to residues ⁷⁹KGYID⁸³ in eIF2. Truncations or mutations of the ⁷⁹KGYID⁸³ sequence in K3L abolished its PKR inhibitory activity (16), suggesting that ⁷⁹KGYID⁸³ is an important binding determinant in SUI2 for its interaction with PKR. In accordance with this hypothesis, mutations that block phosphorylation by GCN2 both in vivo and in vitro have been identified at Glu-49 and ⁷⁹KGYID⁸³ of SUI2 (Dever, unpublished results). Our finding that SUI2 mutations in residues 49, 80, 83, and 88 impaired interaction between GST-SUI2(P) and eIF2B suggests that there is considerable overlap between the binding domains for eIF2B and eIF2 kinases in SUI2. At the same time, the requirements for binding to eIF2B and eIF2 kinases cannot be identical because most of the SUI2 mutations analyzed here impaired its interaction with eIF2B but did not reduce phosphorylation by eIF2 kinases.

View larger version (35K): [in this window] [in a new window]   FIG. 8.   Locations of regulatory mutations in a hypothetical structure of the N-terminal region of SUI2 predicted from the structure of ribosomal protein S1 domain of E. coli PNPase. The three-dimensional structure of the S1 domain of E. coli PNPase (2) is depicted in grey, using the accession code 1SR0 and the program WebLab ViewerLite from Molecular Simulation Inc. Based on a sequence alignment of eIF2 residues 2 to 87 and the S1 domain of PNPase, the Ser-51 phosphorylation site (, labeled with a circled P) falls in the loop connecting  strands 3 and 4, while the eIF2 kinase recognition motif 79KGYID83 (shown in black) resides in the loop connecting  strands 4 and 5 and extending into strand 5. Indicated in the structure are the predicted locations of Gcn mutations in SUI2 (O, labeled with amino acid substitutions) that reduce the inhibition of eIF2B by eIF2(P) in vivo and decrease binding of GST-SUI2(P) to the eIF2B regulatory subcomplex in vitro.

View larger version (50K): [in this window] [in a new window]   FIG. 9.   A mechanistic model for negative regulation of the guanine nucleotide exchange activity of eIF2B by eIF2(P). (A) Unphosphorylated SUI2 promotes the GDP-GTP exchange activity of eIF2B. The heterotrimeric eIF2 (shown as , , ) complexed with GDP () has two binding sites in eIF2B. The GCD2-GCD7-GCN3 regulatory subcomplex in wild-type (WT) eIF2B (labeled 2, 3, 7) binds to the  subunit of eIF2 (SUI2), while the GCD1-GCD6 catalytic subcomplex in eIF2B (labeled 1, 6) interacts with the  and  subunits of eIF2. Based on results with rat proteins, the GCD2 () subunit of eIF2B may also interact with eIF2. The binding interactions shown here position the catalytic subunit of eIF2B (GCD6) in proximity to the bound GDP in the manner required to catalyze exchange of GDP for GTP () on eIF2. (B) SUI2(P) inhibits the GDP-GTP exchange activity of eIF2B. Phosphorylation of SUI2 [, labeled ~P in eIF2(P)-GDP] leads to more extensive interactions between SUI2 and the eIF2B regulatory subcomplex, preventing productive interactions between GCD6 and the  and  subunits of eIF2, inhibiting nucleotide exchange. The arrow depicts the proposed shift in eIF2-eIF2B interactions elicited by phosphorylation. (C) A Gcn mutation in the GCD7 regulatory subunit of eIF2B weakens interaction between SUI2(P) and the regulatory subcomplex of the mutant eIF2B complex (eIF2B*), permitting the interaction between GCD6 and eIF2(P)-GDP necessary for GDP-GTP exchange.