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. 2010 Nov;30(21):5218-33.
doi: 10.1128/MCB.00265-10. Epub 2010 Aug 30.

The beta/Gcd7 subunit of eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor, is crucial for binding eIF2 in vivo

Kamal Dev  1 , Hongfang Qiu, Jinsheng Dong, Fan Zhang, Dominik Barthlme, Alan G Hinnebusch
Affiliations

The beta/Gcd7 subunit of eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor, is crucial for binding eIF2 in vivo

Kamal Dev et al. Mol Cell Biol. 2010 Nov.

Abstract

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor (GEF) for eukaryotic translation initiation factor 2, which stimulates formation of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) in a manner inhibited by phosphorylated eIF2 [eIF2(αP)]. While eIF2B contains five subunits, the ε/Gcd6 subunit is sufficient for GEF activity in vitro. The δ/Gcd2 and β/Gcd7 subunits function with α/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(αP)-GDP, but the essential functions of δ/Gcd2 and β/Gcd7 are not well understood. We show that the depletion of wild-type β/Gcd7, three lethal β/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in β/Gcd7 and eIF2α all impair eIF2 binding to eIF2B without reducing ε/Gcd6 abundance in the native eIF2B-eIF2 holocomplex. Additionally, β/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2α (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, β/Gcd7 can overcome the toxicity of eIF2(αP) and rescue native eIF2B function when overexpressed with δ/Gcd2 or γ/Gcd1. In aggregate, these findings provide compelling evidence that β/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (αP).

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Figures

FIG. 1.
FIG. 1.
Degron alleles of GCD2, GCD7, and GCD6 confer inviability and reduced translation initiation under nonpermissive conditions. The WT (yKD1) and gcd2-td (yKD2), gcd6-td (yKD6), and gcd7-td (yKD7) degron mutants were streaked in parallel on the following media and grown for 5 days: (A) SC containing 2% raffinose and 100 μM CuSO4 at 25°C (permissive conditions) and (B) SC containing 2% galactose, 2% raffinose, and the copper-chelating agent BCS and lacking CuSO4 at 36°C (nonpermissive conditions). (C to F) Polysome profiles of the WT and the indicated degron mutants grown for 16 h under nonpermissive conditions.
FIG. 2.
FIG. 2.
Depletion of Gcd7 in gcd7-td mutant cells codepletes Gcd2. (A to C) Strains of the indicated genotypes described in Fig. 1 were cultured under permissive conditions to an A600 of 1.5 and then shifted to nonpermissive conditions for the indicated times. WCEs were subjected to Western analysis using antibodies against the indicated proteins, including eIF3g (Tif35), which was examined as a loading control.
FIG. 3.
FIG. 3.
Codepleting Gcd2 and Gcd7 or depleting only Gcd6 weakens eIF2-eIF2B interactions and reduces TC levels in vivo. (A) The WT and degron strains described in Fig. 1 were cultured under nonpermissive conditions for 16 h, and WCEs were immunoprecipitated with antibodies against Gcd6 (lanes 6 to 8) or Gcd2 (lanes 5 and 9). Twenty percent of the input WCE (lanes 1 to 4) and the entire pellet fractions (lanes 5 to 9) were subjected to Western analysis for the indicated proteins. (B) Transformants of the WT and degron mutant strains described above harboring SUI3-FLAG on plasmid p3153 (lanes 1 to 4 and 6 to 9) and WT SUI3 strain yKD1-5 containing an empty vector (lanes 5 and 10) were cultured as described for panel A, and WCEs were immunoprecipitated with anti-FLAG antibodies and analyzed as described above, except that 10% of the input WCE was analyzed. Note that two exposures are shown for ɛ/Gcd6 in the pellet lanes, a short exposure (Sh. exp.) and a longer exposure; however, only the short exposure is shown for the input lanes. (C) Total RNA was extracted from input and pellet samples prepared as for panel B from the same four (WT or degron mutant) strains harboring SUI3-FLAG described in panel B (lanes 2 to 5 and 9 to 12). Comparable samples derived from transformants of strains J294 (GCD11) and J295 (GCD11-N135D) containing SUI3-FLAG on pKD3153 (lanes 6 and 7 and lanes 13 and 14, respectively) and a transformant of J294 (GCD11) with the empty vector (lanes 1 and 8) were analyzed in parallel as controls. RNA was subjected to Northern analysis using a 32P-labeled probe for tRNA iMet. (D) Northern signals from the pellet fractions in panel C were normalized for the input signals, and the resulting mean ratios and standard errors of the means calculated from four independent experiments were expressed as a percentage of the WT value derived from lane 12 of panel C for the degron mutants and lane 13 of panel C for the gcd11-N135D mutant. **, P ≤ 0.01 (Student's t test).
FIG. 4.
FIG. 4.
Overexpressing tRNA iMet, but not eIF2, specifically suppresses the lethality of Gcd2 depletion. Serial dilutions of transformants of the indicated WT and degron mutant strains harboring high-copy-number plasmids for overexpressing TC (A), eIF2 (B), or tRNA iMet (C) or the empty vector (VEC) were spotted in parallel to permissive (I) or nonpermissive (II) medium and incubated at 25°C for 4 days (I) or 36°C for 6 days (II).
FIG. 5.
FIG. 5.
Co-overexpression of β/Gcd7, γ/Gcd1, and α/Gcn3 suppresses the toxicity of constitutively activated Gcn2 in vivo. (A) Transformants of GCN2c mutant strain H1608 or GCN2 mutant strain H1402 harboring high-copy-number plasmids with the indicated genes or empty vectors (V) were streaked in parallel on SD with minimal supplements and incubated at 30°C for 5 days. (B) Western analysis of WCEs of strains from panel A with two different loadings (1× and 2×) for each strain.
FIG. 6.
FIG. 6.
Lethal substitutions in β/Gcd7 impair eIF2 interaction with intact eIF2B. (A, left panels) Model predicting the sequence conservation of surface-exposed residues among eIF2Bβ homologs, projected on one protomer of the P. horikoshii aIF2B homodimer (PH0440) as described previously (11, 37). (Right panels) Predicted locations of β/Gcd7 residues substituted with alanines (red, in parentheses) and cognate aIF2B residues (black) for gcd7-151, gcd7-262-263, and gcd7-358-360. (B) Western analysis of transformants of gcn2Δ gcd7Δ mutant strain H2218 harboring a GCD7 URA3 plasmid and LEU2 plasmids containing the indicated mutant or WT GCD7-HA alleles, cultured in SC lacking Leu and Ura at 30°C. Increasing amounts of WCE (1×, 2×, and 3×) were loaded in successive lanes, and blots were probed with anti-HA or anti-Gcd6 antibodies. (C) Strains from panel B containing GCD7-HA alleles were cultured as described for panel B, and coimmunoprecipitation analysis was conducted as described for Fig. 3A, except using anti-HA antibodies.
FIG. 7.
FIG. 7.
gcd7-164-165 confers Slg− and Gcd− phenotypes and impairs translation initiation without reducing eIF2-eIF2B association. (A) Surface projection of sequence conservation and predicted locations of β/Gcd7 residues substituted with alanines (orange) and the cognate aIF2B residues (black) for gcd7-164-165, generated as described for Fig. 6A. (B) Serial dilutions of derivatives of gcn2Δ gcd7Δ mutant strain H2218 containing GCD7-HA or gcd7-HA-164-165 as the only GCD7 allele and a transformant of the isogenic GCN2 strain (H2217) containing WT GCD7-HA were spotted in parallel on SC lacking Leu or SC containing 30 mM 3-AT and incubated for 4 days at 30°C. (C) Polysome profiles of the gcn2Δ mutant strains from panel B grown in SC lacking Leu at 30°C and analyzed as in Fig. 1C. (D) GCD7-HA and gcd7-HA-164-165 mutant strains from panel B and GCD7 strain H2218 were subjected to coimmunoprecipitation analysis as described for Fig. 3A, except using anti-HA antibodies.
FIG. 8.
FIG. 8.
Allele-specific synthetic phenotypes of mutants combining Gcd− gcd7 alleles and Gcn− sui2 alleles. (A) gcn2Δ gcd7Δ sui2Δ mutant strains containing a URA3 GCD7 plasmid and a TRP1 plasmid with SUI2 (WT), sui2-Y81S, sui2-L84F, or sui2-R88T were transformed with a LEU2 gcd7-164-165A plasmid and replica plated in parallel to SC lacking Ura and Leu (SC) and SC containing 5-FOA (to evict the URA3 GCD7 plasmid). The 5-FOA-resistant segregants were subsequently replica plated to SC lacking His and containing 30 mM 3-AT. (B) The Ura− 5-FOAR segregants from panel A were streaked in parallel on SC lacking Leu and incubated for 5 days at 30°C. (C) Coimmunoprecipitation analysis of strains described in panel A prior to plasmid shuffling on 5-FOA-containing medium, harboring untagged GCD7, the WT or 164-165 allele of GCD7-HA, and the indicated SUI2 allele (lanes 1 to 3 and 5 to 7). Strain H2507 containing only untagged GCD7 and SUI2 was analyzed as a control (lanes 4 and 8). WCEs were immunoprecipitated with anti-HA antibodies as described for Fig. 6C.
FIG. 9.
FIG. 9.
Hypothetical model explaining the effects of gcd7 and sui2 mutations on productive and nonproductive modes of eIF2 binding to eIF2B. (A) GDP-GTP exchange on unphosphorylated eIF2 by WT eIF2B. eIF2 (three subunits in red, orange, and yellow) has two binding surfaces in eIF2B that promote the reaction. The regulatory subcomplex (three green subunits) binds eIF2α in a productive manner that allows the catalytic subcomplex (two blue subunits) to interact properly with the G domain of eIF2γ to catalyze efficient exchange of GDP (ball labeled “D”) for GTP (ball labeled “T”) (thick solid black arrows). (B) Phosphorylation of eIF2α (ball labeled “P”) inhibits GDP-GTP exchange by strengthening a nonproductive mode of eIF2α binding to the eIF2B regulatory subcomplex, which prevents proper interaction between ɛ and Gcd6 in the catalytic subcomplex and eIF2γ-GDP. (C) The lethal substitution in eIF2Bβ of gcd7-358-360 disrupts productive binding of eIF2α to the regulatory subcomplex as the means of impairing nucleotide exchange. (D and E) The R88T Gcn− substitution in eIF2α modestly impairs productive binding by unphosphorylated eIF2α, having little effect on the exchange reaction on its own (D); R88T strongly reduces nonproductive binding by phosphorylated eIF2α to restore nucleotide exchange on eIF2(αP)-GDP (E). (F and G) The L84F Gcn− substitution in eIF2α does not affect productive binding of eIF2α (F) and only impairs nonproductive binding to restore GDP-GTP exchange on eIF2(αP)-GDP (G). (H, I, and J) The gcd7-164-165 substitution mimics eIF2α phosphorylation, as in panel B, to stabilize nonproductive eIF2α binding (H). This nonproductive interaction is exacerbated by destabilization of productive binding by eIF2α-R88T, as in panel D, to confer a lethal reduction in exchange (I) but is eliminated by eIF2α-L84F to restore exchange to WT levels (J). Thin and gray dotted arrows indicate moderate and strong reductions in exchange, respectively; substitution mutations are indicated by asterisks.
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