The phylogenetic dendrogram was constructed using the neighbor-joining method as implemented by PAUP from an alignment of the archaeal sequences listed in Table 1 as MTNA-like (aMTNA), RBPI-like (aRBPI), or aIF2B-like (aIF2B), together with sequences of eukaryotic MTNAs (eMTNA) and eIF2B regulatory subunits (eIF2Bα, eIF2Bβ, or eIF2Bδ) identified in Supplementary Table S3. Significant bootstrap values (>70) are shown above the branches. The abbreviations used to identify archaeal sequence origins are defined in Table 1. The eukaryotic sequences are identified as follows: At: Arabidopsis thaliana; Os: Oryza sativa; Sc: Saccharomyces cerevisiae; Sp: Schizosaccharomyces pombe; Af: Aspergillus fumigatus; Dm: Drosophila melanogaster; Hs: Homo sapiens.

(A) Ribbons depiction of the co-crystal structure of human PKR (orange) and the N-terminal amino acids 3-175 of region of yeast eIF2α (PDB ID 2a19). Strands β1-β5 of the S1domain of eIF2α are colored blue, black, green, red, and yellow, respectively. The locations of Gcn^- mutants R88T and L84F, analyzed in (Fig. 5B) are indicated. (B) Full-length wild-type GST-eIF2α (WT 1-304 aa) and the indicated derivatives truncated at the C terminus (designated by the amino acids [aa] remaining) were immobilized on glutathione-Sepharose beads and assayed for binding T. acidophilum His-aIF2B as described for Fig. 3A, except that 100, 200, or 300 μg of WCE containing the GST fusions were employed. Lanes 1, 5, 9, 13, and 17 contain 5% of total input (for the highest amounts of WCE containing GST fusions) used in the assays (C) GST pull down assays conducted as in (B) except using 200 or 300 μg of WCE containing WT GST-eIF2α or the indicated derivatives truncated at the N terminus (designated by the amino acids remaining). The star indicates GST produced by partial degradation of the GST-eIF2α fusions visible in lanes 1-6. (D) WT GST-eIF2α (lanes 6-9), the indicated truncations described in (B or C) (lanes 10-17), or GST alone (lanes 18 and 19) in two different amounts (200 and 400 μg) of bacterial extract were immobilized on glutathione-Sepharose beads and incubated with (+) or without (-) purified PKR in kinase assay buffer. After washing, immobilized proteins were incubated with 500 μg of WCE from yeast strain H4 over-expressing all three eIF2B regulatory subunits from a high-copy plasmid (HC 2/7/3). After washing, the bound proteins (Pellet, lanes 6-19) were analyzed by Western blotting using antibodies against GCD2, GCD7, GCN3, GST, or Ser51-phosphorylated eIF2α, as indicated. Lane 1 contains 10% of the input yeast WCE, whereas lanes 2-5 contain 5% of the total input (I) amounts of bacterial WCEs containing the GST proteins. Species in lanes 12-14 detected with GCD2 and GCD7 antibodies do not exhibit the same mobilities as GCD2 and GCD7 in lanes 6-7 and, hence, are regarded as non-specific, cross-reacting species.

(A) Transformants of yeast strain H4 harboring plasmids pGAL-Ta0910 or pEGKT encoding, respectively, T. acidophilum GST-aIF2B or GST alone under the GAL promoter, were grown at 30°C to A₆₀₀ of ∼ 0.8 in SC medium containing 2% raffinose as carbon source and shifted to SC medium containing 2% raffinose and 2% galactose and an additional 4 h of incubation. The GST-aIF2B or GST proteins in three different amounts (200, 300 and 400 μg) of yeast WCE were immobilized on glutathione-Sepharose beads. After extensive washing, bound proteins (Pellet, lanes 2-4 and 6-8) were analyzed by Western blotting using antibodies against eIF2B subunits GCD6, GCD1, GCD2, GCD7, and GCN3, eIF2α, eIF2β, eIF2γ, and GST, as indicated. Lane 1 and 5 contains 10% of the input (I) amounts of WCE. (B) Yeast WCE (500 μg) from gcn3Δ strain H1331 grown in SC medium and the WCEs from (A) were immunoprecipitated using polyclonal antibodies against GCD6 and protein A Sepharose beads. After extensive washing, immunocomplexes (P, lanes 2, 4 and 6) were analyzed by Western blotting using antibodies against GCD6, GCD1, GCD2, GCD7, GCN3, and eIF2α. Lanes 1, 3 and 5 represent 10% of the input (I) amounts. (C) Alignment of the NUCT domains in eIF2Bε/GCD6, TK0955, and the consensus NUCT domain sequence defined previously 32 using the following symbols: (.) any residue allowed; U or u, highly or moderately conserved bulky hydrophobic residue, respectively; O or o, highly or moderately conserved small residue; J or j, highly or moderately conserved positively charged residue; $, Ser or Thr. Identities (|) and conservative replacements (*) are indicated.

(A). The image on the left shows the predicted sequence conservation on the conserved face of the hypothetical β/GCD7 homodimer, exactly as depicted in Fig. 7B, with the addition of stars indicating the positions of single amino acid substitutions conferring a Gcn^- phenotype in yeast 7. The locations of these Gcn^- mutations are shown in shades of blue on the model on the right, which depicts the same view of the homodimer but without sequence conservation coloration. The amino acid substitutions in β/GCD7 are given in blue type in parentheses under the corresponding PH0440 residues in black type. Pairs of residues colored black on the model are at the borders of inserts found in eIF2Bβ/GCD7 (i-1 to i-4) with no corresponding sequences in PH0440 (see alignments in Fig. S1). These inserts contain the Gcn^- mutations listed in parentheses in blue type. Note that amino acid residues have been colored blue or black in both protomers, but labeled in only one of the two protomers. (B-C). The β/GCD7 homodimer model in (A) has been rotated successively 90° about the vertical axis to show the locations of the two Gcn^- mutations that do not map to the highly conserved face visible in (A). The view in (C), designated II, is identical to view II in the right-hand panels of Fig. 7.

(A) Homohexameric structure of PH0440 viewed from below along the crystallographic 3-fold axis (reproduced from 25). The three homodimer interfaces labeled “1” are identical to those indicated in Fig. 7; those labeled “2” represent interactions among the three homodimers, involving the long helix α5 in each protomer, that would stabilize the proposed homohexamer. (B) Hypothetical model for eIF2B. The aIF2B-related domains of δ/GCD2, β/GCD7, and α/GCN3 are depicted in surface representations with sites of Gcn^- mutations colored blue and Gcd^- mutations colored orange, as in Figs. 8A, 9A and 9C, and arranged in the manner of the three aIF2B protomers delineated in panel A by dotted outlining, to produce a hypothetical regulatory subcomplex. The subunits γ/GCD1 and ε/GCD6 of the catalytic subcomplex interact with the δ/GCD2 and β/GCD7 subunits of the regulatory subcomplex. (C) The same model in panel B but showing the predicted binding surface for eIF2α on the conserved face of the eIF2B regulatory subcomplex and the proposed heterodimer formation between aIF2B and α/GCN3 using the dimer interface “1” defined in (A). (see text for further details.)

A multiple sequence alignment was constructed using ClustalW for 24 archaeal sequences retrieved in a BLAST search using PH0440 as the query sequence. The sequences are identified on the far left by the abbreviations of their species of origin (listed below) followed by the UniProtKB/Swiss-Prot accession numbers. As explained in the text, the sequences have been grouped according to their classification as RBPI-like (top 8), MTNA-like (middle 9), or aIF2B-like (bottom 7) sequences, based on the presence (or absence) of the following sequence motifs (mI to mVIII) which are highly conserved in eukaryotic and bacterial MTNAs: [QxxLP]_I; [VRGAPxI]_II; [LxxRPTA]_III; [TxCNxGxxATxxxGTA]_IV; [ETRPxxQGxxLTxxE(x)₁₂D]_V; [GAD(x)₅GDxANKxGTxxLA(x)₉F]_VI; [GxxIxxExRxxxE(x)₅G]_VII; and [FDxTPxxLI(x)₇G]_VIII23; 24 The locations of mI to mVIII are indicated on the line immediately below the MTNA-like sequences. The underlined residues in the motifs are proposed to make contacts with the bound phosphate in yeast MTNA 23. Residues in the archaeal sequences matching the motifs are shown in red. Positions within these motifs that are identical, highly conserved or moderately conserved in all 24 archaeal sequences are indicated below the aIF2B-like sequences with asterisks, colons or periods, respectively. The species abbreviations are: ARCFU, Archaeoglobus fulgidus; HALMA, Haloarcula marismortui; METJA, Methanococcus jannaschii; META3, Methanococcus aeolicus; METHJ, Methanospirillum hungatei; PYRFU, Pyrococcus furiosus; PYRHO, Pyrococcus horikoshii; TKO, Thermococcus kodakaraensis; PYRAB, Pyrococcus abyssi; THEAC, Thermoplasma acidophilum.

(A) Aliquots of E. coli WCE containing GST-aIF2α from T. acidophilum (300 and 400 μg, lanes 2 and 3), P. horikoshii (200, 300 or 400 μg, lanes 8-10), or P. furiosus (200, 300, or 400 μg, lanes 16-18) or equal amounts of GST alone (lanes 5 and 6, 12-14, 20-22) were incubated with glutathione-Sepharose beads to immobilize the GST proteins. After washing, the beads were incubated with 400 μg bacterial WCE containing the His-aIF2B of the corresponding species. After extensive washing, the bound polypeptides (P, pellet) were separated by SDS-PAGE and analyzed by Western blotting using antibodies against GST or the hexa-histidine tag. Lanes 1, 4, 7, 11, 15, and 19 contain 5% of the total input (I) amounts of WCEs used for the reactions. (B) T. acidophilum GST-aIF2α or GST alone from 400 μg of bacterial WCE were immobilized on glutathione-Sepharose beads and incubated with 400 μg WCE from cells containing the vector plasmid (P, lane 4) or a plasmid expressing His₆-aIF2B (P, lane 5), as in (A). Bound proteins were resolved by SDS-PAGE and visualized by Coomassie Blue (CBB-R250) staining. Lanes 1-3 contain 10% of the input (I) fractions for GST alone, GST-aIF2α, and His-aIF2B respectively. Stars indicate the respective full-length proteins, as confirmed by Western blotting. Molecular size markers (kDa) are indicated on the right. (C) GST-aIF2α from P. horikoshii (lanes 4-11) or GST alone (lanes 12-17) from 300 or 400 μg of E. coli WCEs were immobilized on glutathione-Sepharose beads and incubated with 400 μg bacterial WCE containing P. horikoshii His-aIF2B in the presence of 100, 200 or 300 mM NaCl, as indicated. Bound proteins (Pellet) were analyzed as in (A). Lanes 1-3 contain 5% of the input (I) amounts of WCEs used for the reactions.

(A) Wild-type GST-eIF2α in three different amounts (200, 300 and 400 μg) of bacterial WCE were immobilized on glutathione-Sepharose beads and phosphorylated by PKR (+), or left unphosphorylated (-), before incubating with either 500 μg of yeast WCE containing the over-expressed eIF2B regulatory subunits or 400 μg bacterial WCE containing T. acidophilum His-aIF2B, and the bound proteins were detected by Western blot analysis, all as described in Figs. 4B and D. Lanes headed by P contain the bound fractions. Lanes 1 and 5 contain 10%, whereas lanes 9 and 13 contain 5%, of the input (I) WCEs. (B) Pull-down assays were done as described in Fig. 4B, except that 200 or 300 μg of WCEs containing either WT GST-eIF2α, GST-eIF2α X-R88T, GST-eIF2α-L84F, or GST alone and 400 μg of WCE containing T. acidophilum His-aIF2B were used. Lanes 1-5 contain 5% of the input (I) amounts of WCE.

(A) Ribbons depiction of the crystal structure of the anti-parallel homodimer of P. horikoshii aIF2B, PH0440 (PDB ID 1vb5). The black line indicates the dimer interface between the two protomers. The N-terminal domain (NTD, residues 1-95) is connected to C-terminal domain (CTD, residues 96-276) by a long α5-helix (residues 78-106). In (II), on the right-hand side, the dimer was rotated 180° about the vertical axis from the view in (I). (B). Model predicting the sequence conservation of surface-exposed residues among eIF2Bβ/GCD7 homologs. Highly conserved residues are colored in red shades, whereas highly variable residues are colored in green shades as indicated in the conservation scale at the very bottom. Multiple sequence alignment of eIF2Bβ/GCD7 homologs and PH0440 was performed (supplementary Fig. S1) and the degree of sequence conservation at amino acid positions that can be aligned with the PH0440 sequence is projected on the surface of the PH0440 homodimer. Highly conserved residues (red) are clustered on one surface (I), whereas most of the variable residues (blue) are on the opposite face (II). (C) and (D). Models predicting the sequence conservation of surface-exposed residues among eIF2Bδ/GCD2 (C) and eIF2Bα/GCN3 homologs, constructed from the alignments shown in Figs. S2 and S3, respectively, just as in (B).

(A-B) The locations of spontaneous Gcn^- mutations in yeast δ/GCD2 7 are depicted in views I and II, identical to those shown in Fig. 7C, following the same conventions described in Fig. 8 for β/GCD7 mutations. (C-D) The locations of Gcn^- (blue) and Gcd^- (orange) mutations in yeast α/GCN3 7; 34 are depicted in views I and II, identical to those shown in Fig. 7D, following the same conventions described in Fig. 8 for β/GCD7 mutations. Surface conservation on the left panel is as shown in Fig. 7D. The amino acid residues of GCN3 which produce Gcd^- phenotype are colored in orange shades, whereas the one that correspond to Gcn^- phenotype are colored in blue (right panel). The aIF2B amino acid residues are numbered in black, followed by yeast mutations in parenthesis. (D) The dimer has been rotated 180° about the vertical axis in opposite direction showing the least conserved face.