Tight binding of the phosphorylated alpha subunit of initiation factor 2 (eIF2alpha) to the regulatory subunits of guanine nucleotide exchange factor eIF2B is required for inhibition of translation initiation

Abstract

Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNA(Met) to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its alpha subunit [eIF2(alphaP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the alpha subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2alpha (glutathione S-transferase [GST]-SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(alphaP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(alphaP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(alphaP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the beta and gamma subunits of eIF2 in the manner required for GDP-GTP exchange.

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 [γ-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.

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.

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.

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.

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.

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(αP)] (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 eIF2α 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.

FIG. 7

Genetic evidence that SUI2 binds individually to the regulatory subcomplex of eIF2B in vivo. (A) Strain H1608 bearing the chromosomal GCN2^c-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 (α, β, γ) to exchange the GDP (▴) present on eIF2 for GTP. As these cells contain an activated GCN2^c 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 GCN2^c cells overexpressing the GCD2-GCD7-GCN3 regulatory subcomplex of eIF2B (labeled 2, 3, 7), the latter competes with eIF2B holoprotein for the inhibitor, eIF2(αP)-GDP, allowing the eIF2B to exchange GDP for GTP on unphosphorylated eIF2. This suppresses the slow-growth phenotype associated with the GCN2^c allele. (Strain 3) Overexpressed SUI2 is phosphorylated in GCN2^c cells and competes with eIF2(αP) holoprotein for binding to the eIF2B regulatory subcomplex. This releases eIF2(αP) and reinstates inhibition of eIF2B and the attendant slow-growth phenotype of GCN2^c cells. (See Fig. 9 for additional details on the relative orientations of eIF2 and eIF2B subunits in the different complexes.)

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 eIF2α 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 β strands 3 and 4, while the eIF2α kinase recognition motif ⁷⁹KGYID⁸³ (shown in black) resides in the loop connecting β 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(αP) in vivo and decrease binding of GST-SUI2(αP) to the eIF2B regulatory subcomplex in vitro.

FIG. 9

A mechanistic model for negative regulation of the guanine nucleotide exchange activity of eIF2B by eIF2(αP). (A) Unphosphorylated SUI2 promotes the GDP-GTP exchange activity of eIF2B. The heterotrimeric eIF2 (shown as α, β, γ) 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 α subunit of eIF2 (SUI2), while the GCD1-GCD6 catalytic subcomplex in eIF2B (labeled 1, 6) interacts with the β and γ subunits of eIF2. Based on results with rat proteins, the GCD2 (δ) subunit of eIF2B may also interact with eIF2β. 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(αP)-GDP] leads to more extensive interactions between SUI2 and the eIF2B regulatory subcomplex, preventing productive interactions between GCD6 and the β and γ 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(αP)-GDP necessary for GDP-GTP exchange.