Fig. 1. GCN2 can be co-immunoprecipitated in a complex with GCN1 and GCN20 from cell extracts. (A) GCN2 was immunoprecipitated with antibodies against GCN2 from 500 µg of whole-cell extracts prepared from gcn2Δ strain H1894 transformed with either the vector YEp24 (gcn2Δ) or high-copy plasmids containing wild-type GCN2 (WT) or a catalytically inactive mutant (K628R). Following SDS–PAGE separation of the proteins and transfer to nitrocellulose, the membranes were probed for the different proteins indicated on the left. I and S indicate one-tenth of the input extract and supernatant, respectively, and P represents the total pellet. (B) GCN1 antibodies were used to co-immunoprecipitate GCN2 and GCN20 with GCN1 from 500 µg of whole-cell extracts prepared from strain H1402 (wild-type) and the isogenic strains H2081 (gcn1Δ) and H2563 (gcn20Δ). Western blotting was performed with antibodies against the proteins indicated on the left.

Fig. 3. Precipitation of GCN1 and GCN20 in cell extracts with bacterially expressed GST–GCN2 fusions. (A) The segments of GCN2 contained in different GST fusions are shown as numbered black bars below the schematic depiction of GCN2, along with a summary of their GCN1–GCN20 binding abilities based on the data shown in (B) and (C). (B and C) Similar amounts of the indicated GST–GCN2 fusion proteins, or GST alone, expressed in E.coli were used to precipitate native GCN1 and GCN20 from yeast cell extracts prepared from gcn2Δ strain H2557. Selected fusions were examined at two different relative concentrations (1× or 4×) to verify that binding of GCN1–GCN20 was dose dependent at the selected concentrations of GST fusions. Lanes labeled Input contain one-tenth or one-twentieth of the amount of cell extract used in each binding assay, as indicated. The precipitated proteins were subjected to Western analysis using antibodies against GCN1 (top panel), GCN20 (middle panel) or GST (bottom panel). White dots in the bottom panel mark the predicted sizes of the full-length GST–GCN2 fusions. (D) Binding experiments were performed using bacterially expressed GST–GCN2(1–598) (lanes 2 and 3, and 5 and 6) or GST alone (lanes 1 and 4) and 500 µg of yeast cell extracts prepared from wild-type strain H1511 (GCN20, lanes 1–3) or isogenic gcn20Δ strain H2563 (gcn20Δ, lanes 4–6).

Fig. 5. Dominant-negative effect of overexpressing the N-terminal 572 amino acids of GCN2. (A) Schematic representation of GST–GCN2 fusions containing the indicated GCN2 residues that were expressed in a GCN2 yeast strain under the control of a galactose-inducible promoter. The second construct contains residues 1–504 of D.melanogaster GCN2, which are similar in sequence to yeast GCN2 residues 1–572. The second and third columns contain a summary of the Gcn phenotypes of the fusion constructs measured as described in (B), and the protein levels measured by Western analysis (data not shown), respectively, under galactose-inducing growth conditions. The fourth column contains a qualitative summary of the binding data shown in Figure 6. ND, not determined due to the failure of the fusion to bind the glutathione–Sepharose beads efficiently [GST–GCN2(230–572)] or its low level of expression [GST–GCN2(70–312)]. (B) A transformant of gcn2Δ strain H2557 bearing vector pEGKT (Mitchell et al., 1993) encoding GST alone (top row) and transformants of GCN2 strain H1511 bearing pEGKT or plasmids encoding the indicated GST–GCN2 fusions described in (A) were grown to late logarithmic phase, diluted to an OD₆₀₀ of 0.15 (first column) and 0.015 (second column) and spotted onto minimal medium containin 2% glucose as carbon source (SD) or minimal medium containing 2% galactose (to induce the GST–GCN2 fusions) and 30 mM 3AT to impose histidine starvation (Sgal + 3AT). The SD and Sgal + 3AT plates were incubated at 37°C for 1 and 3 days, respectively.

Fig. 7. Suppression of the dominant-negative phenotype of GST–GCN2(1–572) by overexpression of GCN2, GCN1 or GCN20. (A) Transformants of GCN2 strain H1511 bearing the TRP1 plasmid pB167 encoding the galactose-inducible GST–GCN2(1–572) fusion and high copy number (HC) plasmids encoding GCN2 (URA3 plasmid p630), GCN1 (URA3 plasmid p1834), GCN20 (URA3 plasmid p1748 or LEU2 plasmid p1747) or empty vectors (URA3 vector pRS426 or LEU2 vector pRS425), as indicated, were streaked on Sgal medium or Sgal containing 50 mM 3AT. The two control strains streaked in the upper left-hand sectors were transformants of H1511 (GCN2 + vectors) or gcn2Δ strain H2557 (gcn2Δ + vectors), both bearing empty vectors. The Sgal and Sgal + 3AT plates were incubated at 30°C for 36 h or 4 days, respectively. (B) The GCN2 strain H1511 was transformed with URA3 plasmid pB165 encoding the galactose-inducible GST–GCN2(1–572) fusion and the high copy number LEU2 plasmid p897 bearing the tRNA^His gene, or the corresponding empty vectors (pEGKT and Yep13, respectively). The transformants were cultured and spotted onto Sgal or Sgal containing 50 mM 3AT, as described in Figure 5B, and incubated at 30°C for 2 and 3.5 days, respectively. The first and third columns contain one-tenth the number of cells spotted in the second and fourth columns. (C) Transformants of the isogenic strains H1511 (GCN2, upper left sector), H2557 (gcn2Δ, lower left sector), H2556 (gcn1Δ, upper right sector) and H2558 (gcn20Δ, lower right sector) harboring YEp13 (vector) or p897 (HC tRNA^His) were cultured and spotted onto Sgal containing 25 mM 3AT, as described in Figure 5B, and incubated for 3.5 days at 30°C.

Fig. 2. N-terminal deletions in GCN2 impair its interaction with the GCN1/20 complex. (A) GCN1 antibodies were used to co-immunoprecipitate GCN2 from 500 µg of whole-cell extracts prepared from gcn2Δ strain H1149 transformed with high-copy plasmids bearing wild-type GCN2 (WT) or the indicated gcn2 deletion alleles. The amino acids removed by each deletion are shown by the numbered boxes beneath the schematic depiction of the GCN2 sequence on the top. The latter shows the amino acid positions of GCN2 functional domains designated as in the text, except for RB/DD which signifies the ribosome-binding/dimerization domain at the C-terminus. For each construct, the results of Western analysis of GCN2 for duplicate immunoprecipitations are shown on the right, with input (I), pellet (P) and supernatant (S) fractions as described in Figure 1. The amount of GCN1 detected in the pellet fractions did not vary significantly from one mutant extract to the next (data not shown). For each gcn2 mutant, a control co-immunoprecipitation was performed in parallel using an extract from a transformant expressing wild-type GCN2 from the high copy number plasmid p630, allowing quantitative comparisons of the recovery of mutant and wild-type GCN2 proteins in immune complexes with GCN1. The amount of each mutant GCN2 protein co-immunoprecipitating with GCN1 is expressed in the graph on the right as a fraction of the amount of wild-type GCN2 that co-immunoprecipitated with GCN1. To the right of the GCN2 schematic at the top is a typical result showing co-immunoprecipitation of wild-type GCN2 with GCN1 from a GCN1 but not a gcn1Δ extract. (B) Lanes 1–6: exponentially growing cultures of transformants of gcn2Δ strain H1149 bearing vector alone (lane 1) or high-copy plasmids containing either wild-type GCN2 (lane 2) or the indicated gcn2 deletion alleles (lanes 4–6), or a transformant of gcn1Δ strain H2081 bearing the high-copy GCN2 plasmid (lane 3), were grown for 1 h in minimal (SD) medium containing 40 mM 3AT. Whole-cell extracts were prepared and subjected to SDS–PAGE and immunoblot analysis using phosphospecific antibodies raised against an eIF2α peptide bearing phosphoserine 51 (top panel) or polyclonal antibodies against eIF2α (bottom panel). Lanes 7–11: extracts were prepared from transformants of GCN2 strain H1511 expressing GST alone or GST–GCN2(1–572), described below in Figure 5B, grown in Sgal medium (lanes 9 and 11) to induce (I) expression of the GST fusions or in uninducing conditions (SD medium; UI; lane 10). Lanes 7 and 8 contain the same extracts as in lanes 2 and 3, re-examined as controls.

Fig. 4. Purified GCN1–GCN20 complex binds specifically to bacterially expressed GST–GCN2 fusions bearing the CNT domain of GCN2. (A) Coomassie staining of 100 ng of affinity-purified GCN1–GCN20 complex. The protein concentration was estimated by comparison with a known amount of purified BSA (fraction V, Boehringer Mannheim) (data not shown). (B) GST pull-down assays were performed using 100 ng of purified GCN1–GCN20 complex shown in (A) and GST–GCN2 fusions bearing the GCN2 residues indicated above each lane. The assays were conducted as described in Figure 3 except that 1 mg of E.coli extract was added to reduce the non-specific binding of GCN1–GCN20 to the GST fusions. The amounts of GCN1 (top panel), GCN20^Flag (middle panel) and the GST fusions (bottom panel) recovered in the precipitates were determined by Western analysis. Asterisks in the bottom panel mark the full-length GST fusion proteins. The lane headed ‘In 1/10’ contains one-tenth of the amount of purified GCN1–GCN20 complex used as input in the binding reactions.

Fig. 6. Dominant-negative GST–GCN2 fusions bind to the GCN1–GCN20 complex in vivo. (A) Extracts were prepared from transformants of the wild-type yeast strain H1511 containing plasmids encoding the GST–GCN2 fusions bearing the GCN2 residues indicated in the column headings, or GST alone, after growth in Sgal medium containing 2% galactose to induce the fusion proteins. Aliquots of extracts containing 50, 200 or 400 µg of protein (1×, 4× or 8×, respectively) were incubated with gluthatione–Sepharose beads to precipitate the GST fusions and the precipitates were probed for GCN1, GCN2 and the GST fusion proteins by Western analysis. Lanes headed ‘1/10’ contain one-tenth of the amount of extract used in the 4× reactions (20 µg). (B) A subset of the extracts employed in (A) were used in co-immunoprecipitation experiments with anti-GCN1 antiserum, exactly as described in Figure 1B.