Stepwise assembly of the eukaryotic translation initiation factor 2 complex

Figure 1. Role of N-terminal sequences of yeast eIF2γ in eIF2 assembly.A, schematic domain structure of yeast eIF2γ (Gcd11). Domain I (amino acids 92–310) is shown in light gray including the GTP-binding motifs G–G5 highlighted in red, domain II (amino acids 311–421) in gray, and domain III (amino acids 422–519) in dark gray (11, 12). Arrows indicate the eIF2γ N termini in Homo sapiens (H.s.), Schizosaccharomyces pombe (S.p.), and Sulfolobus solfataricus (S.s.). Numbers indicate amino acid positions of N-terminally truncated versions of Gcd11 used for immunoprecipitation shown in (B). B, WB analysis of interactions between N-terminally truncated and FLAG-tagged variants of Gcd11 and eIF2α (Sui2), eIF2β (Sui3), and Cdc123. Protein levels were analyzed in whole cell extract (WCE, left panel) and after anti-FLAG immunoprecipitation (α-FLAG IP, right panel). A strain lacking a FLAG epitope (N) was included as specificity control. eIF2, eukaryotic translation initiation factor 2; WB, Western blot.

Figure 2. Independent binding of eIF2α and eIF2β to eIF2γ.A, schematic domain structure of yeast eIF2γ (Gcd11) with its binding sites for eIF2α (Sui2), eIF2β (Sui3), and Cdc123. Amino acid positions of Gcd11 involved in binding are highlighted in blue for Sui2, green for Sui3, and yellow for Cdc123 (11, 19, 25). Numbers indicate amino acid positions of mutated and C-terminally truncated versions of Gcd11 used for IP (B–D) and tetrad dissection (E). B–D, WB analysis of interactions between FLAG-tagged derivatives of Gcd11 with Sui2, Sui3, Cdc123, eIF2Bε (Gcd6), and eIF2Bβ (Gcd7). See Fig. S1 for quantification of coprecipitated Cdc123 by eIF2γ variants. Protein levels were analyzed in WCE (left panel) and after α-FLAG IP (right panel); the analysis included a no-tag control (N). E, tetrad dissection of gcd11Δ/GCD11 heterozygous diploid strains expressing the indicated FLAGGcd11 constructs from a gene copy integrated at the HIS3 locus. For each case, the meiotic progeny of two tetrads is shown. G = endogenous GCD11; Δ = GCD11 deletion allele; F = FLAG-tagged GCD11 construct. Complementation is indicated by the viability of haploid gcd11Δ cells carrying a FLAG-tagged GCD11 construct, that is,…

Figure 3. Quantitative analysis of eIF2 assembly intermediates.A and B, Sui2, Sui3, or Gcd11 was endogenously FLAG tagged and analyzed for expression levels (A) and integration into eIF2 complexes (B). C and D, WB quantification of FLAGeIF2 subunits and coprecipitated Cdc123. # indicates immunoglobulin G (IgG) heavy chain. Signals of immunoprecipitated eIF2 subunits and coprecipitated Cdc123 were quantified by use of an infrared imaging system. Cdc123 signals were normalized to the amount of the corresponding FLAGeIF2 subunit in IP samples. No-tag control (N) served as blank value. The coprecipitation of Cdc123 is shown as mean and SD, and coprecipitation of Cdc123 by Gcd11 was set to 100% (n = 3). E and F, WB analysis of interaction between Cdc123FLAG and eIF2γ (Gcd11), eIF2β (Sui3), and eIF2α (Sui2MYC). Protein levels were analyzed in WCE (left panel) and after α-FLAG IP (right panel); the analysis included a no-tag control (N). Signals of immunoprecipitated Cdc123 and coprecipitated eIF2 subunits were quantified by use of an infrared imaging system. eIF2 subunit signals in the IP sample were normalized to the signal in WCE samples. The coprecipitation of eIF2 subunits is shown as mean…

Figure 4. Characterization of Cdc123–eIF2α and Cdc123–eIF2αβ interactions.A, schematic domain structure of eIF2β (Sui3). N-terminal lysine (K) boxes are shown in gray, domain I (amino acids 125–141) in light green, domain II (amino acids 142–233) in green, and domain III (amino acids 234–272) in dark green (33, 43). Numbers indicate amino acid positions of mutated versions of Sui3 used for IP (B and C). B and C, WB analysis of interactions between FLAG-tagged versions of Sui3 with eIF2γ (Gcd11), eIF2α (Sui2), Cdc123, and eIF5 (Tif5) in co-IPs. D, schematic domain structure of eIF2α (Sui2). Domain I (amino acids 1–93) is shown in light blue, domain II (amino acids 94–177) in blue, and domain III (amino acids 178–275) in dark blue (33, 43). Numbers indicate amino acid positions of mutated versions of Sui2 used for IP (E). E, interaction of FLAG-tagged versions of Sui2 with myc-tagged Cdc123, eIF2γ (Gcd11), and eIF2β (Sui3) was analyzed using IP. WCE and α-FLAG-immunoprecipitates were analyzed by WB. F, interaction of Cdc123 with eIF2α (Sui2) and eIF2β (Sui3). GST-Cdc123 and His-tagged eIF2α (HisSui2) or eIF2β (HisSui3) were coexpressed in Escherichia coli. E. coli cells expressing GST…

Figure 5. Importance of eIF2α for eIF2 assembly and cell growth.A, WB analysis of interactions between Cdc123FLAG with eIF2γ (Gcd11) in strains overexpressing either eIF2α (Sui2) or eIF2β (Sui3). The analysis included strains with endogenous levels of both proteins (end) and a no-tag control lacking Cdc123FLAG. SUI2 and SUI3 were expressed under control of the GAL1 promoter. Protein levels were analyzed in WCE (left panel) and after α-FLAG IP (right panel). B, WB analysis of interactions between Cdc123FLAG and Sui2, Sui3, and Gcd11 in a SUI2/SUI2 homozygous and a sui2Δ/SUI2 heterozygous diploid strain. C, quantification of coprecipitated Gcd11 and Sui3 in (B). Signals of immunoprecipitated Cdc123 and coprecipitated Gcd11 and Sui3 were quantified by use of an infrared imaging system. Signals of Gcd11 and Sui3 were normalized to the amount of the corresponding Cdc123FLAG signal. The increase of coprecipitated eIF2 subunits in the sui2Δ/SUI2 heterozygous diploid strain is shown as mean and SD (n = 4). D, GCN4-lacZ reporter levels were measured in a WT control and diploid strains heterozygous for the indicated eIF2 subunit gene deletion. β-Galactosidase activity in Miller units is shown as…

Figure 6. eIF2α-mediated Cdc123 release requires binding of eIF2α to eIF2γ.A, schematic domain structure of eIF2α (Sui2). B and C, WB analysis of interactions between Cdc123FLAG with eIF2β (Sui3) and Gcd11 (eIF2γ) in strains overexpressing variants of eIF2α (HASui2). The analyses included strains with endogenous Sui2 levels (end). Strains lacking Cdc123FLAG served as no-tag controls (left lane in all panels). HA-tagged SUI2 variants were expressed under control of the GAL1 promotor. eIF2, eukaryotic translation initiation factor 2; HA, hemagglutinin; WB, Western blot.

Figure 7. Intramolecular interaction between Gcd11 domains dissolved by Cdc123.A, interaction between Gcd11 domain I and domain II + III fragments was analyzed in a Y2H assay using fragment DI fused to the activator domain (AD) and fragment DII + III fused to the LexA DNA-binding domain (DBD). The experiment was performed in a regular reporter strain with endogenous Cdc123 levels (left panel) and a modified reporter strain overexpressing Cdc123 (right panel). For each combination, six independent transformants are shown. Blue color indicates activation of the lacZ reporter gene. B, WB analysis of interaction between DI and DII + III fragments of Gcd11. Protein levels were analyzed in WCE (left panel) and after α-FLAG IP (right panel). The analysis included no-tag controls lacking FLAGGcd11(DI). The interaction was analyzed in yeast strains with natural and increased levels of Cdc123 expression. IP, immunoprecipitation; WB, Western blot; WCE, whole cell extract; Y2H, yeast two-hybrid assay.

Figure 8. Interactions between human eIF2 subunits and human Cdc123.A, schematic domain structure of human eIF2γ. Domain I (G-domain) is shown in light gray, domain II in gray, and domain III in dark gray. B, WB analysis of interactions between human FLAGeIF2γ with human MYCeIF2α, HAeIF2β, and MYCCdc123 proteins in yeast cells. Protein levels were analyzed in WCE and after α-FLAG IP. No tag controls (N) were included. C, WB analysis of interaction between FLAGheIF2γ with heIF2α, heIF2β, and hCdc123 in Flp-In T-REx-293 cells. D, WB analysis of interactions between FLAGhCdc123 with heIF2α, heIF2β, and heIF2γ in Flp-In T-REx-293 cells. C and D, protein levels were analyzed in WCE and after α-FLAG IP, and no-tag controls were included. eIF2, eukaryotic translation initiation factor 2; IP, immunoprecipitation; WB, Western blot; WCE, whole cell extract.

Figure 9. Model of eIF2 assembly. Numbers indicate the distinct steps of the assembly pathway. (1) Cdc123 associates with the C-terminal part of eIF2γ and activates the eIF2α and eIF2β binding sites, possibly via structural change of eIF2γ. This enables eIF2α and eIF2β to bind. (2) eIF2β binds and a detectable eIF2γβ–Cdc123 trimer is formed. (3) eIF2α binds the assembly intermediate and destabilizes the Cdc123–eIF2γ interaction. Therefore, the tetrameric eIF2αγβ–Cdc123 intermediate is a transient structure, and its abundance in vivo is very low. (4) Cdc123 quickly dissociates (as indicated by the double arrow) and leaves behind the trimeric eIF2 complex. Upper part, in a putative secondary reaction pathway, eIF2α, rather than eIF2β, binds the eIF2γ–Cdc123 dimer. Cdc123 dissociates and leaves behind an eIF2γα dimer, to which eIF2β binds. The final product of both reaction pathways is trimeric eIF2. eIF2, eukaryotic translation initiation factor 2.