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. 2000 Jun;20(11):3965-76.
doi: 10.1128/MCB.20.11.3965-3976.2000.

Identification of domains and residues within the epsilon subunit of eukaryotic translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation

E Gomez  1 , G D Pavitt
Affiliations

Identification of domains and residues within the epsilon subunit of eukaryotic translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation

E Gomez et al. Mol Cell Biol. 2000 Jun.

Abstract

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2Bepsilon). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2Bepsilon in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiae gene encoding eIF2Bepsilon (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2Bepsilon (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2Bepsilon without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2Bepsilon disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2Bepsilon alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2Bepsilon alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2Bepsilon and the other eIF2B subunits. This suggests that the N-terminal region of eIF2Bepsilon is an activation domain that responds to eIF2B complex formation.

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Figures

FIG. 1
FIG. 1
Genetic characterization of novel mutations in yeast eIF2Bɛ. (A) eIF2Bγ and eIF2Bɛ subunits encoded by yeast genes GCD1 and GCD6 are shown schematically from N to C termini. The patterns indicate regions of significant sequence similarity both between these proteins and with other protein families as shown in the key. The amino acids at the boundaries of these regions are indicated by numbers. The relative positions and nature of missense and nonsense mutations identified in GCD6 are indicated below the eIF2Bɛ schematic. (B) A segment of a multiple-sequence alignment of eIF2Bγ and eIF2Bɛ proteins from diverse organisms. The region around the mutations at N249 and F250 is shown. These mutant changes are indicated with arrows pointing down. Residues identical in all eIF2Bɛ sequences are shown in reverse type. Residues identical in at least three eIF2Bɛ sequences are shaded, as are residues in eIF2Bγ sequences that are identical to those of any eIF2Bɛ sequence. Other residues shared by three or more eIF2Bγ sequences are boxed. The sequences used are as follows (GenBank accession numbers given in brackets): S. cerevisiae (S. cere) GCD6 [Z68195] and GCD1 [Z75168], Schizosaccharomyces pombe (S. pomb) eIF2Bɛ (ɛ) [P56287] and eIF2Bγ (γ) [P56288], Arabidopsis thaliana (A. thal) ɛ [AAC12836], Rattus norvegicus (R. rat) ɛ [Q64350] and γ [P70541], Caenorhabditis elegans (C. eleg) ɛ [CAA91063.1] and γ [P80361], and D. melanogaster (D. melan) ɛ [AL021086]. The number after each sequence abbreviation indicates the position in the protein of the first residue of each sequence shown in the alignment. (C) The segment of the multiple-sequence alignment of eIF2Bɛ proteins from the region around the mutations at T552 and S576. All shading and other information are as described above for panel B. (D) Rates of growth of cells transformed with mutant alleles of GCD6. Rates of growth are scored on a linear scale from 6+ (wild type, maximal growth rate) to − (no visible growth) for growth on SD or SGal. Medium supplemented with 25 mM 3-aminotriazole (3AT) was used to assess response to amino acid starvation. Growth tests were performed in three genetic backgrounds. Columns 2 to 4 show results following transformation of strain GP3667 (gcn2Δ) with galactose-inducible GCD6-only plasmids carrying the indicated allele. Column 5 shows results of cooverexpressing mutant alleles of GCD6 with all other eIF2B subunits from high-copy-number (h.c.) plasmids transformed into strain GP3667. Column 6 shows the ability of low-copy-number (l.c.) GCD6-only plasmid-borne alleles to complement a deletion of GCD6 in strain GP3751 (gcd6Δ gcn2Δ).
FIG. 2
FIG. 2
Purification and GEF activity of eIF2Bɛ mutants. (A) SDS–12.5% polyacrylamide gel of the indicated nickel affinity gel-purified eIF2Bɛ polypeptides (lanes 1 to 9) stained with Coomassie brilliant blue. eIF2Bɛ polypeptides were added to the lanes of the gel as follows; 2.5 μg was loaded in lanes 2, 5, and 6, while 1.25 μg was loaded in lanes 1, 3, 4, 7, 8, and 9. In lane 9, the polypeptide corresponding to eIF2BɛΔ93-358 is indicated with an arrow. All proteins were purified with Triton X-100 (0.1%) added to the buffer, except for proteins shown in lanes 2 and 7. Lane 10 contains prestained molecular mass markers (M) (New England BioLabs) with the approximate masses (in kilodaltons) indicated to the right. (B) The initial rates of nucleotide exchange for the mutant polypeptides are shown as a percentage of the wild-type protein activity. Initial rates of [3H]GDP release were determined from exponential curves fitted to the data using a computer program (Cricketgraph 3.0) of time course nucleotide exchange assays performed using a standard filter binding assay. The eIF2BɛΔ144-230 mutant was expressed very poorly, resulting in high copurification of contaminating proteins. Partially purified cell extract (15 μg) was used for its assay. It is likely that this mutant retains full activity. (C) Nucleotide exchange assay results for selected purified proteins. Some of the primary data used in panel B is shown. In these experiments, 2.5-μg samples of nickel-purified extract were used, except for the Q452* mutant where 5 μg was used. The wild-type and buffer-only control curves are shown as broken lines, and the mutant curves are shown as solid lines. Experiments were done in duplicate and replicated two to eight times. Typical data are shown with error bars indicating the standard deviation (ς3) where ς3 ≤ 5.65 for each time point. (D) Western blot of eIF2B subunits in purified fractions from the wild type (lanes 1 to 3) and gcd6Δ93-358 mutant (lanes 4 to 6). Blots were probed with the antisera indicated to the right of each panel to detect the eIF2B subunits indicated to the left. For eIF2Bɛ, 62.5 ng (lanes 2 and 4) and 125 ng (lanes 3 and 5) were loaded. For detection of eIF2Bδ and -γ, 2.5 μg (lanes 1 and 4), 5 μg (lanes 2 and 5), and 7.5 μg (lanes 3 and 6) were loaded. For detection of eIF2Bβ and -α, 1 μg (lanes 1 and 4), 1.5 μg (lanes 2 and 5), and 2 μg (lanes 3 and 6) were loaded.
FIG. 3
FIG. 3
In vitro binding between eIF2 and eIF2Bɛ proteins. (A) Titration of interaction between a fixed concentration (200 nM) of FLAG-tagged eIF2Bɛ (even-numbered lanes) or 200 nM FLAG peptide as a control (odd-numbered lanes) and the indicated concentration of eIF2. Proteins remaining bound after washing were identified by SDS-PAGE and Western blotting. Subunits indicated to the left of each panel were identified with the antisera shown to the right. Pellet fractions (33%) were loaded for probing with eIF2 antibodies, and 10% was used for eIF2Bɛ. (B) Binding of mutants at saturating concentrations of eIF2. Binding reactions were performed with a 200 nM concentration of the indicated FLAG-tagged eIF2Bɛ protein (lanes 6 to 14) or 200 nM control FLAG peptide (lane 5) and 20 nM eIF2. Proteins were visualized as described above for panel A. Lanes 1 to 4 show decreasing concentrations of input eIF2 (equivalent 20, 10, 5, and 2.5% of the 20 nM used in the reaction mixtures) and a single concentration input eIF2Bɛ (5%) (lane 4). (C) Binding of mutants at limiting concentrations of eIF2. Binding reactions were performed with a 200 nM concentration of the indicated FLAG-tagged eIF2Bɛ protein (lanes 2 to 7) or 200 nM control FLAG peptide (lane 1) and 5 nM eIF2. Proteins were visualized as described above for panel A.
FIG. 4
FIG. 4
In vivo analysis of eIF2Bɛ mutants. (A) Immunoprecipitation of FLAG-tagged eIF2Bγ and associated eIF2B subunits from extracts of yeast strain GP3667 overexpressing all five subunits of wild-type or mutant eIF2B as indicated. Cell extract (10 μg) was loaded in the input lanes (lanes 1 to 4), and the equivalent of 20 μg was loaded in the immune precipitated (IP) lanes (lanes 5 to 8) and unbound supernatant (SUP) lanes (lanes 9 to 12). Proteins were visualized as described in the legend to Fig. 3. (B) Three missense mutations complement a deletion of GCD6. Low-copy-number plasmids bearing the indicated alleles of GCD6 were introduced into strain GP3751 (gcd6Δ), and plasmid shuffling was used to make the indicated alleles the only source of GCD6. Growth on rich medium YPD is shown. (C) Immunoprecipitation of eIF2Bɛ and associated eIF2B and eIF2 polypeptides from extracts of cells shown in panel B using anti-GCD6 (αGCD6) antiserum. Pellets from 300 μg of cell extract were loaded in each lane.
FIG. 5
FIG. 5
Analysis of polysome profiles from gcd6 mutant yeast strains using low-salt sucrose density gradient centrifugation. (A) Extracts prepared from cells grown in YPD medium at 30°C were centrifuged on low-salt 7 to 47% sucrose gradients. Gradients were fractionated while scanning at 254 nm, and the resulting profiles are shown. The positions of ribosomal subunits, 80S monosomes, and polysomes are indicated. The ratio of polysomes to 80S monosomes was determined by measuring the area under the peaks using NIH Image software. (B) Extracts from the same yeast strains were centrifuged on low-salt 15 to 35% sucrose gradients. This provides greater separation of the top portion of the gradient. Proteins collected in fractions from the gradients were analyzed by SDS-PAGE and Western blotting using the antisera indicated to the right.
FIG. 6
FIG. 6
In vitro analysis of purified mutant eIF2B five-subunit complexes. (A) Nucleotide exchange assays comparing activities for mutant eIF2B complexes containing N249K (filled triangle) and F250L (filled circle) alleles of eIF2Bɛ with wild-type eIF2B (eIF2Bwt) (filled square) (1 μg each) and eIF2Bɛ alone (2.5 μg, open diamond). Assays were done in triplicate, with a standard deviation of less than 2.5 for each time point. (B) Analysis of rates of nucleotide exchange activity for mutant eIF2B complexes and isolated eIF2Bɛ subunits relative to wild-type eIF2B activity (percent initial activity). Analysis was performed as described in the legend to Fig. 2. (C) Analysis of binding between the indicated concentration of purified eIF2 and FLAG-tagged wild-type eIF2B complex (lanes 7 to 10) or eIF2BɛF250L (lanes 11 to 14). Also shown are control lanes using FLAG peptide (lane 4) or wild-type eIF2Bɛ alone (lanes 5 and 6) in place of eIF2B. Lanes 1 to 3 contain inputs; 5% of each eIF2B preparation used in the reaction mixtures was loaded and 6.25 ng of eIF2 was also loaded in lane 3 (representing 10% of 5 nM used in the reaction mixtures). Detection as described in the legend to Fig. 3, with 33% of each reaction pellet loaded to probe for eIF2 and 10% loaded to probe for each eIF2B subunit.
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