INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The protein kinases that phosphorylate the  subunit of eukaryotic translation initiation factor 2 (eIF2) on serine-51 down-regulate protein synthesis in response to starvation or stress. In both mammalian and yeast cells, phosphorylation of eukaryotic translation initiation factor 2 (eIF2) converts it from a substrate to an inhibitor of its guanine nucleotide exchange factor, eIF2B, decreasing the recycling of eIF2 from the GDP-bound to the GTP-bound state. Only the GTP-bound form of eIF2 is competent to form a ternary complex with charged initiator tRNA^Met and interact with the 43S preinitiation complex; thus, phosphorylation of eIF2 impairs a key step in translation initiation (14, 22). The mammalian eIF2 kinase HRI is activated in erythroid cells by hemin deficiency as a means of reducing globin synthesis when hemin is limiting. The eIF2 kinase PKR is thought to be activated in virus-infected mammalian cells, where it inhibits total protein synthesis by phosphorylation of eIF2 and thereby limits virus proliferation (21).

The eIF2 kinase of Saccharomyces cerevisiae, known as GCN2, becomes activated in response to starvation for one or more amino acids and specifically induces the translation of GCN4 mRNA, encoding a transcriptional activator of amino acid biosynthetic genes. The newly synthesized GCN4 protein derepresses numerous amino acid biosynthetic enzymes, alleviating the amino acid starvation that triggered its induction. Four short upstream open reading frames (uORFs) in the leader of GCN4 mRNA cause its translation to be inversely coupled to the levels of eIF2-GTP-Met-tRNA_i^Met ternary complexes in the cell. Under nonstarvation conditions, where ternary complexes are plentiful, ribosomes scanning from the 5' end of GCN4 mRNA translate the uORFs and fail to reinitiate at the GCN4 start codon downstream. When ternary complex levels are decreased by phosphorylation of eIF2 in starved cells, ribosomes bypass the inhibitory uORFs and initiate translation at GCN4 instead (15, 16).

Mutations that lead to constitutive activation of kinase function in the absence of amino acid starvation have been isolated in GCN2. Some of these GCN2^c alleles confer such high levels of eIF2 phosphorylation in vivo that general translation initiation is inhibited and cell growth is impaired (7, 25, 32). Because GCN2 is expressed constitutively (32) and its activation has the potential to arrest cell growth, the activity of the protein kinase (PK) domain must be tightly regulated to restrict its function to starvation conditions, where GCN4 is induced. GCN2 is a large protein (190 kDa) containing multiple regulatory domains that are required to couple its eIF2 kinase activity to the levels of amino acids in the cell (32).

Uncharged tRNA appears to be the activating ligand for GCN2 because mutations in aminoacyl-tRNA synthetases lead to increased eIF2 phosphorylation by GCN2 (33), with attendant derepression of GCN4 (18) and genes under its control (23, 30, 33). GCN2 contains a C-terminal (C-term) regulatory domain similar in sequence to histidyl-tRNA synthetase (HisRS) (5, 31), and it is thought that binding of uncharged tRNA to this HisRS domain produces in the adjacent kinase domain a conformational change that increases its ability to phosphorylate eIF2 (31). In support of this model, it was shown that a conserved sequence characteristic of authentic class II aminoacyl-tRNA synthetases (motif 2) found in the HisRS domain of GCN2 is required for kinase function in vivo and in vitro as well as for binding of uncharged yeast tRNA to the HisRS domain of GCN2 in vitro (33, 35). In addition, numerous GCN2^c activating mutations alter residues in the HisRS region, including two conserved positions in motif 2 (7, 25, 32).

There is biochemical evidence that GCN2 can interact with translating ribosomes and free ribosomal subunits and that this association is dependent on the C-term 120 amino acids of the protein (24). The C-term domain is required for GCN2 function in vivo (24), leading to the suggestion that GCN2 is activated by uncharged tRNA bound to ribosomes during translation elongation (25). Interestingly, the GCN2^c alleles which activate GCN2 most effectively alter residues in the C-term domain (7, 25, 32); thus, this segment may function both in anchoring GCN2 to the ribosome and in facilitating activation of the kinase domain by uncharged tRNA. In addition to the HisRS and C-term segments, GCN2 contains a regulatory domain N terminal to the kinase moiety that is required in vivo (32) and in vitro (35) for kinase activity. This region contains sequence similarity to multiple subdomains of eukaryotic protein kinases, although critical residues in putative subdomains I, II, and VI important for ATP binding and catalysis appear to be lacking (12, 31). The function of this degenerate kinase domain, referred to here as the pseudokinase (PK) domain, in regulating the authentic PK domain of GCN2 is unknown.

There is limited genetic and biochemical evidence that the dimerization of GCN2 is important for its activation or catalytic function. The GCN2^c-E1525K allele encodes a hyperactive kinase that inhibits translation initiation so severely that cell growth is impaired. This slow-growth phenotype was suppressed by coexpression of wild-type GCN2 (7), and similar observations have been made for other GCN2^c alleles (8a). To account for this phenomenon, it was proposed that the hyperactive mutant and wild-type GCN2 proteins form oligomers that are less active for eIF2 phosphorylation than oligomers containing only GCN2^c proteins. In addition, much of the GCN2 protein eluted from a sizing column with apparent molecular masses much larger than the mass of monomeric GCN2 (7). It is also noteworthy that the HisRS domain of GCN2 contains a relatively good match to sequence motif I, which contributes to dimer formation in class II aminoacyl-tRNA synthetases (5). It was suggested that a GCN2 homodimer might bind to a single tRNA molecule and that intermolecular autophosphorylation by the two GCN2 protomers would activate the eIF2 kinase function (7).

We present here genetic and biochemical evidence that GCN2 contains at least two domains that mediate dimerization, the PK domain and the C-term ribosome-binding domain, and show that the latter is more critically required for self-interaction by full-length GCN2 molecules in vivo. We also detected independent interactions between the C-term region and the PK and HisRS domains and found that the PK domain interacts strongly with the HisRS and PK domains. Our observations suggest that the regulation of GCN2 kinase activity involves both protein dimerization and physical interactions between the kinase domain and each of its flanking regulatory regions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Yeast strains and plasmids. EGY48 (MAT trp1 ura3 his3 lexAop-LEU2) (11) was used for all two-hybrid interaction assays. HQY132, a gcn2 derivative of EGY48, was used for all coimmunoprecipitation experiments and glutathione S-transferase (GST)-binding assays in which yeast extracts were the source of LexA-GCN2 fusions and was constructed as follows. A 3.8-kb hisG::URA3::hisG fragment was inserted at the BglII site of plasmid p1141 (a gift from T. Dever), from which the GCN2 open reading frame was deleted, to produce plasmid pHQ414, bearing a gcn2 hisG::URA3::hisG allele. pHQ414 was digested with EcoRI and XbaI, and the EcoRI-XbaI fragment containing gcn2 hisG::URA3::hisG was used to transform EGY48 to Ura⁺. The deletion of GCN2 in the resulting transformants was confirmed by PCR with a pair of primers complementary to the sequence of the GCN2 promoter region (that was not deleted) and URA3. Finally, strain HQY132 was obtained by selecting for loss of the URA3 marker via homologous recombination between the hisG repeats on medium containing 5-fluoro-orotic acid (2). Strain H1613 was described previously (25).

gcn2-1498-1517

gcn2-1518-1537

gcn2-1538-1557

gcn2-1558-1577

gcn2-1578-1597

gcn2-1598-1617

gcn2-1618-1637

gcn2-1638-1659

gcn2-324-538

gcn2-324-538

gcn2-572-999

gcn2-572-999

gcn2-1161-1570

gcn2-1161-1246

gcn2-1536-1570

gcn2-1536-1659

Two-hybrid interaction assay. Plasmids encoding the appropriate LexA- and B42-GCN2 fusions were cotransformed into yeast strain EGY48 by use of a modified lithium acetate method (9). The transformants were selected on minimal SD plates (28) supplemented with uracil and leucine (SD+Ura+Leu) and replica printed to SD+Ura and SGal/Raf+Ura media, where the 2% dextrose in SD was replaced with 2% galactose and 1% raffinose in the latter medium. Two-hybrid interactions were indicated by growth on SGal/Raf+Ura but not on SD+Ura plates, indicating a galactose-dependent Leu⁺ phenotype. When needed, the lexAop-lacZ reporter plasmid pSH18-34 was introduced into transformants carrying lexA- and B42-GCN2 fusion plasmids, and three or more independent transformants were analyzed for -galactosidase activities in cell extracts. For these assays, cells were grown for ~38 h to saturation in synthetic complete medium lacking uracil, histidine, and tryptophan (SCUraHisTrp) and were diluted 1:50 into the same medium containing galactose (2%) and raffinose (1%) as carbon sources (SC/Gal/RafUraHisTrp). Cells were harvested in the mid-logarithmic phase after 6 h of growth. -Galactosidase assays were carried out as described previously (19), and -galactosidase activities are expressed as nanomoles of o-nitrophenyl--D-galactopyranoside hydrolyzed per minute per milligram of protein.

Coimmunoprecipitation and immunoblot analysis. For coimmunoprecipitation of wild-type or mutant GCN2 proteins with LexA-GCN2 fusion proteins from yeast cell extracts, fresh transformants of strain HQY132 bearing the appropriate high-copy-number plasmids encoding the GCN2 and LexA-GCN2 fusion proteins under consideration were grown in SCUraHis medium to the exponential phase (optical density at 600 nm, ~1.2 to 1.5). Cells from 20 ml of culture were harvested by centrifugation, washed with ice-cold water, and transferred to a 1.5-ml microcentrifuge tube. Cell pellets were resuspended in 250 µl of breaking buffer (50 mM Tris-HCl [pH 7.5], 50 mM NaCl, 0.1% Triton X-100, 5 µg each of pepstatin A, leupeptin, and aprotinin per ml, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) and broken by vortexing with glass beads, and the extracts were clarified by centrifugation for 15 min in a microcentrifuge. Protein concentrations in the extracts were determined as described previously (3). For immunoprecipitation, 40 µl (10-µl bed volume) of protein A-agarose beads (Santa Cruz) was washed with breaking buffer and resuspended in 200 µl of breaking buffer, after which 10 µg of anti-HA antibody (Boehringer) was added and incubated at room temperature with rocking for 1 h. The beads were collected by brief centrifugation and washed with 500 µl of breaking buffer. Aliquots of cell extracts containing 50 µg of protein were diluted to a final volume of 200 µl with breaking buffer and incubated with 20 µl of protein A-agarose beads suspended in breaking buffer for 1 h at 4°C with rocking. The beads were removed by centrifugation, and the supernatant was added to beads prebound with anti-HA antibody and incubated at 4°C for 2 h with rocking. The beads were collected by centrifugation, washed three times with 500 µl of breaking buffer, and resuspended in 40 µl of Laemmli sample buffer (17). Proteins in the immune complexes were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes (29), and probed with antibodies against GCN2 or LexA. The immune complexes were visualized by enhanced chemiluminescence (ECL; Amersham) according to the vendor's instructions. Anti-LexA antibodies were a gift from Clyde Denis. Preparation of the anti-GCN2 antibodies will be described elsewhere.

Preparation of GST and GST fusion proteins. Transformants of Escherichia coli BL21 carrying plasmid pGEX-5x-1 (containing GST) or derivatives containing different GST-GCN2 fusion proteins were cultured overnight at 37°C in Luria-Bertani medium containing ampicillin. Overnight cultures were diluted (1:100) into fresh Luria-Bertani-ampicillin medium and grown at 30°C. When the optical density at 600 nm reached ~0.8, isopropyl--D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM and incubation was continued for 2.5 h to induce the expression of GST and GST fusion proteins. Cells were harvested, and GST and GST fusion proteins were purified as described in the GST Gene Fusion System Manual from Pharmacia (23a) with the following modification. After the glutathione-Sepharose 4B beads containing bound proteins were washed, the proteins were eluted with elution buffer (100 mM Tris-HCl [pH 8.0], 120 mM NaCl, 0.1% Triton X-100, 20 mM glutathione) and dialyzed against buffer A (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol) containing 12.5% glycerol.

In vitro transcription and translation. In vitro transcription and translation with ³⁵S-labeled methionine were carried out by use of the TNT T7 Coupled Reticulocyte Lysate System (Promega) according to the vendor's instructions. In vitro-translated proteins were partially purified by ammonium sulfate precipitation as described previously (1). The protein precipitates were resuspended in 50 µl of buffer A containing 12.5% glycerol.

GST-binding assays. Immobilization of GST fusion proteins on glutathione-Sepharose 4B beads was carried out by incubating the purified fusion proteins at 1 µg/µl of beads (bed volume) in buffer A containing 0.1% Triton X-100 at room temperature for 30 min with rocking. The beads were washed and resuspended in the same buffer. Five microliters of in vitro-translated ³⁵S-labeled proteins prepared as described above or yeast cell extracts containing 50 µg of proteins prepared as described above for coimmunoprecipitation assays was added to beads (10-µl bed volume) containing 10 µg of bound GST fusion proteins, and the volume was increased to 200 µl with buffer A. The mixtures were incubated at 4°C for 2 h with rocking. The beads were collected by brief centrifugation in a microcentrifuge, washed three times with 500 µl of buffer A, resuspended in 40 µl of Laemmli sample buffer (17), and fractionated by SDS-PAGE. For detecting ³⁵S-labeled proteins, the gels were fixed with destaining buffer (25% methanol, 12% acetic acid), stained with Coomassie blue, treated with Amplify (Amersham), dried, and subjected to fluorography at 70°C. The results of Coomassie blue staining confirmed that similar amounts of each GST protein were bound to the beads in the different reaction mixtures. For detecting unlabeled proteins from cell extracts, fractionated proteins were transferred to nitrocellulose membranes and subjected to immunoblot analysis with anti-LexA antibodies (1:7,000 dilution), and the ECL system was used to visualize immune complexes.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Yeast two-hybrid analysis suggests that GCN2 dimerizes through the PK and C-term domains. GCN2 contains several domains flanking the PK domain that are required for its ability to phosphorylate eIF2 and derepress GCN4 translation in amino acid-starved cells (31, 32) (Fig. 1A). We used the yeast two-hybrid assay to investigate whether the different domains in GCN2 can physically interact with one another. Two sets of plasmids carrying various segments of GCN2 either fused to bacterial LexA and expressed from the yeast constitutive ADH1 promoter or fused to the B42 transcriptional activation domain and expressed from the galactose-inducible GAL1 promoter were constructed. These plasmids were introduced in all pairwise combinations into yeast strain EGY48, containing a LEU2 allele with LexA operators in place of the native transcriptional enhancer. Interaction between a pair of LexA and B42 fusions leads to activation of LEU2 transcription and growth on medium lacking leucine. The results shown in Fig. 1B indicated that about two-thirds of the C-term kinase domain ['PK(720-999)] fused to LexA could interact with itself, with a slightly smaller kinase domain segment ['PK(750-999)], and with the C-term domain [C-term (1498-1659)], all fused to the B42 activation domain (Fig. 1B, row 4). Similar results were obtained with a LexA fusion containing 'PK(750-999), except that this smaller kinase domain segment did not interact with itself (Fig. 1B, row 5). A LexA fusion containing the C-term segment showed a strong interaction with itself and a moderate interaction with the larger of the two B42-PK fusions ['PK(720-999)] (Fig. 1B, row 8). The B42 fusions containing both 'PK segments and the C-term segment also interacted with full-length LexA-GCN2 (Fig. 1B, row 9). These results provided in vivo evidence for both homomeric and heteromeric interactions involving the PK and C-term domains of GCN2.

View larger version (44K): [in this window] [in a new window]   FIG. 1.   Yeast two-hybrid analysis of interactions between regulatory domains in GCN2. (A) Location of protein kinase and regulatory domains in GCN2. The rectangular box represents the GCN2 polypeptide chain from the amino (N) to carboxyl (C) terminus with the amino acid residues numbered from 1 to 1659. The N-terminal 69 amino acids (stippled region in the box) were appended to 1,590 residues published previously (31) based on recent findings indicating that the GCN2 open reading frame begins at the 5'-most-in-frame ATG codon at position 1 (unpublished observations). As a result, all GCN2 amino acid positions in this report, including those in GCN2 allele designations, are increased by 69 from the values indicated in previous publications. The different domains, including a highly charged region in the N terminus (/+), a degenerate protein kinase domain (PK), the PK domain, a HisRS-related region, and a C-term region required for ribosome association and dimerization by GCN2 (RB/DD), are delineated in the rectangular box. The filled boxes above the GCN2 schematic indicate the positions of three sequence motifs (M1 to M3) conserved among class II aminoacyl-tRNA synthetases. (B) Yeast two-hybrid analysis of domain interactions in GCN2. Two sets of plasmids carrying the indicated segments of GCN2 fused to either bacterial LexA or the B42 transcriptional activation domain were introduced in all pairwise combinations (indicated in rows 2 to 9 and columns B to J) into yeast strain EGY48. Interaction between a pair of LexA and B42 fusion proteins leads to activation of LEU2 transcription and growth on medium containing galactose as a carbon source and lacking leucine. The + symbol in column A and row 1 indicates that the fusion proteins were expressed, as determined by immunoblot analysis of whole-cell extracts with anti-LexA antibodies for LexA fusion proteins or anti-HA antibodies for B42 fusion proteins. The +, +/, /+, and  symbols in the other columns and rows designate strong, moderate, weak, and no growth, respectively, on medium lacking leucine, indicative of two-hybrid interactions. Boldface type was used to indicate growth responses that were significantly above background levels obtained with the negative controls in row 2 and column C. Segments of GCN2 contained in the LexA and B42 fusion constructs are indicated by amino acid (aa.) positions in full-length GCN2 and the GCN2 domains they encompass. Full-length GCN2 includes amino acids from 27 to 1659. ND, not determined. Additional LexA fusion proteins (not shown) containing GCN2 segments from residues 82 to 551, 574 to 998, and 1150 to 1497 were found to possess intrinsic activation function, conferring a Leu+ phenotype to EGY48 when present in combination with the empty B42 vector, and were not analyzed further.

Biochemical evidence for homomeric and heteromeric interactions involving the PK and C-term domains of GCN2. To obtain biochemical evidence for interactions involving the PK and C-term segments, we carried out in vitro protein-binding assays with purified GST fusion proteins expressed in E. coli and some of the LexA fusion proteins used in two-hybrid assays described above. Equivalent amounts of GST fusion proteins containing the C-term segment (1498 to 1659) or the full-length PK domain (568 to 998) immobilized on glutathione-Sepharose beads were incubated with yeast extracts containing the LexA fusion proteins listed across the top of Fig. 2. The LexA fusion proteins that remained bound to the GST proteins after extensive washing of the beads were visualized by immunoblot analysis with antibodies against LexA. As expected, LexA alone did not interact detectably with any of the GST fusion proteins (Fig. 2, lanes 2 to 4), nor did any of the LexA-GCN2 segments interact with GST alone (Fig. 2, lanes 6, 10, 14, and 18). In contrast, significant fractions of LexA-'PK(720-999) bound to the GST-PK(568-998) and GST-C-term(1498-1659) fusion proteins (Fig. 2, lanes 11 and 12). A very small fraction of input LexA-C-term(1498-1659) was recovered with GST-PK(568-998), but a much larger proportion of this LexA fusion protein bound to GST-C-term(1498-1659) (Fig. 2, lanes 19 and 20). These findings are in accordance with the combined results of the two-hybrid analyses shown in Fig. 1B and Table 2 in demonstrating a strong self-interaction of the C-term segment and relatively weaker PK self-interaction and PK-C-term heteromeric interaction.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   In vitro binding of LexA fusion proteins containing different GCN2 segments, expressed in yeast, to GST fusion proteins containing the GCN2 PK and C-term domains. Aliquots of whole-cell extracts (50 µg of total protein) from transformants of yeast gcn2 strain HQY132 carrying plasmids containing the LexA fusion proteins indicated across the top were incubated with approximately equivalent amounts of GST, GST-C-term(1498-1659), or GST-PK(568-998) fusion proteins immobilized on glutathione-Sepharose beads. After extensive washing, LexA fusion proteins bound to the beads were resolved by SDS-PAGE and detected by immunoblot analysis with anti-LexA antibodies. Transformants of strain HQY132 contained the following plasmids: pEG202 (LexA), pHQ385 [LexA-PK(230-604)], pHQ433 [LexA-'PK(720-999)], pHQ426 [LexA-HisRS(970-1497)], and pHQ311 [LexA-C-term(1498-1659)]. Whole-cell extract (12.5 µg) (input, lanes I) or the bound fraction recovered from 25 µg of extract was loaded in each lane. aa, amino acids. Numbers at the left of gel are kilodaltons.

Evidence that interactions between the PK and C-term segments of GCN2 occur in the absence of other yeast proteins. It was conceivable that the interactions that we detected for the C-term and PK domains of GCN2 with themselves and with one another were indirect and were mediated by some other yeast protein(s), such as GCN1 or GCN20 (20). To establish that these segments can interact with one another in the absence of all other yeast proteins, we carried out in vitro protein-binding assays with the GST-PK(568-998) and GST-C-term(1498-1659) fusion proteins and different GCN2 segments translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine. As expected, none of the labeled GCN2 segments interacted with GST alone (Fig. 3A to D), and in vitro-translated luciferase showed no detectable interaction with the GST-PK and GST-C-term fusion proteins (Fig. 3E). Quantitation of the binding data showed that small fractions of the input labeled PK(568-998) protein were precipitated with the GST-C-term (0.5%) and GST-PK (0.6%) fusion proteins; similarly, a small fraction of the labeled C-term(1498-1659) segment was recovered with the GST-PK fusion protein (1.9%) (Fig. 3F). Although the amounts of the bound fractions were small, they exceeded the background levels of binding to GST alone by more than 1 order of magnitude (Fig. 3F). A much larger fraction of the labeled C-term(1498-1659) segment was recovered with the GST-C-term fusion protein (20%) (Fig. 3D and G). The labeled PK segment interacted strongly with the GST-PK fusion protein (8% recovery; Fig. 3A and H), and the labeled HisRS(970-1497) fragment showed strong interactions with the GST-PK and GST-C-term fusion proteins (23 and 39% recoveries, respectively; Fig. 3C and I). The interaction of the labeled PK segment with the GST-C-term fusion protein was the weakest of all the interactions shown in Fig. 3, with the amount of bound fraction being only 2.5-fold larger than the background binding to GST alone (Fig. 3H). Together, these results are in agreement with the results of the GST pull-down experiments in Fig. 2 in showing relatively strong C-term self-interaction, strong PK-PK, PK-HisRS, and HisRS-C-term heteromeric interactions, and weaker but significant PK-PK and PK-C-term interactions.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   In vitro binding of GCN2 segments translated in vitro to GST fusion proteins containing the GCN2 PK and C-term domains. (A to E) The GCN2 segments indicated to the left of panels A to D or the control protein luciferase (panel E) was translated in vitro with [35S]methionine and incubated with GST, GST-C-term(1498-1659), or GST-PK(568-998) immobilized on glutathione-Sepharose beads. After a wash, the precipitated proteins were resolved by SDS-PAGE and visualized by fluorography. The leftmost lane in each panel contains the indicated fraction of the input radiolabeled protein used in the binding experiments. (F to I) Densities of bands in the autoradiograms in panels A to D were quantitated with a scanner (Silverscanner III) and NIH Image software (version 1.61). The data were plotted as the percentage of input radioactivity recovered in the fraction bound to the GST fusion protein.

Biochemical evidence that GCN2 dimerizes in vivo through self-interactions in the PK and C-term domains. To provide more direct evidence that GCN2 can dimerize in vivo, we examined whether native GCN2 could be immunoprecipitated from yeast whole-cell extracts with LexA fusion proteins containing different segments of GCN2. To this end, we fused the coding sequences for two tandem copies of the HA epitope to the genes encoding LexA and the LexA fusion proteins bearing GCN2 segments PK(230-604), 'PK(720-999), HisRS(970-1497), and C-term(1498-1659), used above for the two-hybrid analysis. A high-copy-number plasmid encoding wild-type GCN2 was introduced into strains expressing LexA-HA alone or LexA-HA fused to the different GCN2 segments. Extracts prepared from the resulting strains were immunoprecipitated with anti-HA antibodies, and the immune complexes were probed by immunoblot analysis with antibodies against LexA or GCN2. The results shown in Fig. 4 indicate that a substantial fraction of native GCN2 was coimmunoprecipitated with LexA-HA-'PK(720-999), whereas little or no GCN2 was recovered with LexA-HA-PK(230-604), LexA-HA-HisRS(970-1497), or LexA-HA alone. A small fraction of GCN2 was coimmunoprecipitated with LexA-HA-C-term(1498-1659), and this amount was reproducibly larger than the background amount obtained with LexA-HA alone. These findings are consistent with the idea that GCN2 can dimerize through homomeric interactions involving the PK and C-term domains. The much greater recovery of GCN2 with 'PK(720-999) than with the C-term domain in these experiments is ostensibly at odds with the previous experiments indicating a stronger homomeric interaction of isolated C-term than of PK segments. Perhaps a weaker self-interaction of the LexA-HA-'PK protein than of the LexA-HA-C-term protein results in relatively lower amounts of free LexA-HA-C-term being available for dimerization with GCN2. Interdomain interactions in full-length GCN2 may increase the ability of the PK domain to dimerize with Lex-HA-'PK or decrease the ability of the C-term domain to interact with LexA-HA-C-term, relative to the self-interactions of the isolated segments in LexA fusions.

View larger version (42K): [in this window] [in a new window]   FIG. 4.   Coimmunoprecipitation of native GCN2 with LexA-HA-GCN2 segments from yeast cell extracts. Transformants of gcn2 strain HQY132 bearing high-copy-number plasmid p630 encoding wild-type GCN2 and plasmids encoding the LexA fusion proteins indicated to the right of each panel were grown in liquid SCUraHis medium, and whole-cell extracts were prepared. Aliquots of extracts containing 50 µg of protein were incubated with protein A-agarose beads prebound with anti-HA antibody. Whole-cell extract (5 µg) (Input), immune complexes precipitated from 25 µg of extract (Pellet), and supernatant fractions corresponding to 5 µg of starting extract (Supernatant) were resolved by SDS-PAGE (8 to 16%) and transferred to nitrocellulose membranes. One portion of the blots was probed with anti-GCN2 antibodies (left panels), and the other portion was probed with anti-LexA antibodies (right panels). The following plasmids encoding LexA fusion proteins were used: p2247 (LexA-HA), pHQ385 [LexA-HA-PK(230-604)], pHQ587 [LexA-HA-'PK(720-999)], pHQ588 [LexA-HA-HisRS(970-1497)], and pHQ589 [LexA-HA-C-term(1498-1659)].

View larger version (54K): [in this window] [in a new window]   FIG. 5.   Coimmunoprecipitation of GCN2 with LexA-HA-PK requires the PK domain in GCN2. (A) Whole-cell extracts were prepared from transformants of gcn2 strain HQY132 bearing high-copy-number plasmid pHQ587 containing LexA-HA-'PK(720-999) and plasmids containing wild-type GCN2 (p630) or one of the following deletion mutants: gcn2-324-538(PK) (p2327), gcn2-572-999(PK) (pB82), gcn2-1161-1570(HisRS+1/2C-term) (p2463), or gcn2-1536-1659(C-term) (p2461). Aliquots of extracts containing 50 µg of protein were immunoprecipitated with anti-HA antibodies, and the immune complexes were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-GCN2 antibodies (upper panel) or anti-LexA antibodies (lower panel), all as described in the legend to Fig. 4. Lanes 1 to 5 contain 5 µg of extract used for the immunoprecipitations, lanes 6 to 10 contain amounts of supernatants corresponding to 5 µg of starting extract from the immunoprecipitations, and lanes 11 to 15 contain amounts of pellets corresponding to 25 µg of starting extract from the immunoprecipitations. (B) Whole-cell extracts prepared from transformants of HQY132 harboring plasmid p2247 containing LexA-HA and either p630, p2327, pB82, p2463, or p2461 were immunoprecipitated with anti-HA antibodies, and the immune complexes were analyzed by immunoblot analysis with anti-GCN2 antibodies (upper panel) or anti-LexA antibodies (lower panel), with the same proportions of samples loaded as in panel A. (C) Densities of the bands in panel A for the input and pellet fractions were calculated with a scanner (Silverscanner III) and NIH Image software (version 1.61). The percentage of the input amount that was coimmunoprecipitated with LexA-HA-PK was calculated from two independent experiments, and the average was plotted for each GCN2 protein (scale on the left; averages shown above the bars). The percentage of the mutant protein that was coimmunoprecipitated with LexA-HA-GCN2 relative to wild-type GCN2 was also plotted (scale on the right; values shown inside the bars). wt, wild type.

View larger version (51K): [in this window] [in a new window]   FIG. 6.   Coimmunoprecipitation of mutant and wild-type GCN2 proteins with full-length LexA-HA-GCN2. (A) Whole-cell extracts were prepared from transformants of gcn2 strain HQY132 bearing high-copy-number plasmid pHQ400 containing LexA-HA-GCN2 and plasmids containing wild-type GCN2 (p630) or one of the following deletion mutants: gcn2-324-538(PK) (p2327), gcn2-572-999(PK) (pB82), gcn2-1161-1570(HisRS+1/2C-term) (p2463), or gcn2-1536-1659(C-term) (p2461). Aliquots of extracts containing 50 µg of protein were immunoprecipitated with anti-HA antibodies, and the immune complexes were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-GCN2 antibodies, all as described in the legend to Fig. 4. Lanes 1 to 5 contain 5 µg of extract used for the immunoprecipitations, lanes 6 to 10 contain amounts of supernatants corresponding to 5 µg of starting extract from the immunoprecipitations, and lanes 11 to 15 contain amounts of pellets corresponding to 25 µg of starting extract from the immunoprecipitations. (B) Whole-cell extracts prepared from transformants of HQY132 harboring plasmid p2247 containing LexA-HA and either p630, p2327, pB82, p2463, or p2461 were immunoprecipitated with anti-HA antibodies, and the immune complexes were analyzed by immunoblot analysis with anti-GCN2 antibodies (upper panel) or anti-LexA antibodies (lower panel), with the same proportions of samples loaded as in panel A. (C) Densities of the bands in panel A for the input and pellet fractions were calculated with a scanner (Silverscanner III) and NIH Image software (version 1.61). The percentage of the input amount that was coimmunoprecipitated with LexA-HA-GCN2 was calculated from three or more independent experiments, and the average was plotted for each GCN2 protein (scale on the left; averages ± standard deviations shown above the bars). The percentage of the mutant protein that was coimmunoprecipitated with LexA-HA-GCN2 relative to wild-type GCN2 was also plotted (scale on the right; values shown inside the bars). wt, wild type.

Mapping the residues in the C-term domain required for self-interaction and interaction with the PK domain. We used the two-hybrid assay in an effort to identify specific segments of the C-term region of GCN2 that mediate its strong self-interaction. We began by introducing into both the LexA-C-term and the B42-C-term fusion proteins point mutations shown previously to alter the regulatory function of native GCN2. These included two different two-codon insertions at positions 1571 and 1656 that inactivate GCN2 function (gcn2-1571SS and gcn2-1656EL) (31) and a substitution at Glu-1591 that leads to constitutive activation of GCN2 function in vivo (25). Neither these point mutations nor a two-codon insertion at position 1536 that does not affect GCN2 function (31) detectably altered the C-term self-interaction, as judged by the Leu phenotype of the yeast transformants (Fig. 7, no. 2 to 5, "Self"-LexA fusion). We next analyzed the effects of nested N-terminal and C-term deletions in the LexA- and B42-C-term constructs on self-interaction in the two-hybrid assay. The results obtained with the N-terminal deletions indicated that self-association of the C-term segment required residues between positions 1498 and 1535 (Fig. 7, no. 6 to 9). Truncation from the C terminus to position 1635 had no effect on the interaction (Fig. 7, no. 10), whereas the protein truncated at position 1610 had intrinsic activation function and could not be analyzed (no. 11). The protein with the 1585-1659 truncation failed to interact (Fig. 7, no. 12); however, this protein was expressed at relatively low levels (Fig. 8, lane 6), making it unclear whether an important interaction domain is located C terminal to position 1585. 

View larger version (64K): [in this window] [in a new window]   FIG. 7.   Deletion mapping of interaction determinants in the C-term region that mediate self-interaction and interaction with the PK domain in the two-hybrid assay. The GCN2 C-term region (residues 1498 to 1659) and deletion derivatives (depicted by gray rectangular boxes) were fused to the B42 activation domain (open box) in plasmid pJG4-5, producing the constructs listed on the left, and to LexA in plasmid pEG202 (not shown). Broken lines represent amino acids (aa) deleted from the C-term segments, as indicated by the numbers above the lines indicating the first and last amino acids deleted in each construct. For determination of two-hybrid interactions between the LexA and B42 fusion proteins containing identical C-term segments (interactions with "Self"-LexA fusion), strain EGY48 was cotransformed with the appropriate LexA and B42 fusion plasmids. For determination of two-hybrid interactions between the LexA-'PK(720-999) and B42-C-term fusion proteins, EGY48 was cotransformed with plasmid pHQ433 containing LexA-'PK(720-999) and the constructs listed on the left containing B42-C-term fusion proteins. The resulting transformants were tested for growth on SGal/Raf+Ura medium, indicative of a two-hybrid interaction that activates the expression of the lexAop-LEU2 reporter (Leu phenotype). +, +/, /+, and , strong, moderate, weak, and no growth, respectively; NA, not applicable, because the LexA fusion proteins conferred a Leu+ phenotype in the absence of a B42 fusion protein. Selected transformants containing LexA and B42 fusion proteins bearing the same C-term segments (self-interactions) were also analyzed for -galactosidase activities in the cell extracts, indicating a two-hybrid interaction that activates the expression of the lexAop-lacZ reporter. The activities shown are averages calculated from results obtained with three or more independent transformants and expressed as nanomoles of o-nitrophenyl--D-galactopyranoside hydrolyzed per minute per milligram of protein. The standard deviations were less than 30% of the averages.

View larger version (65K): [in this window] [in a new window]   FIG. 8.   Expression of selected LexA and B42 fusion proteins bearing different portions of the GCN2 C-term domain used for two-hybrid analysis. Transformants bearing plasmids containing LexA or B42 fused to C-term(1498-1659) or their derivatives with the deletions indicated above the panels were grown to the log phase in liquid synthetic complete medium containing galactose and raffinose as carbon sources and lacking histidine and tryptophan to select for the plasmids. Whole-cell extracts were prepared, and aliquots containing 25 µg of protein were resolved by SDS-PAGE and subjected to immunoblot analysis with anti-LexA antibodies to detect LexA fusion proteins (A) and anti-HA antibodies to detect B42 fusion proteins (B). Immune complexes were visualized by ECL. Numbers at left of panels are kilodaltons.

The entire C-term segment of GCN2 is required for its regulatory function in vivo. If the two regions in the C-term domain required for self-interaction of this domain in the two-hybrid assay are also important for the dimerization of native GCN2 and if dimerization is required for GCN2 activation or catalytic function, then deletions of these regions in full-length GCN2 should reduce its function in vivo. To test this prediction, we introduced into an otherwise wild-type GCN2 allele on a low-copy-number plasmid the eight consecutive 20-codon deletions spanning residues 1498 to 1659 that were analyzed in Fig. 7 (constructs 6, 10, and 13 to 18). The resulting gcn2 alleles were tested for complementation of the 3-aminotriazole (3-AT)-sensitive phenotype of the gcn2 strain H1472. 3-AT inhibits histidine biosynthesis, and gcn2 mutants are 3-AT sensitive because they fail to induce the expression of GCN4 and the histidine biosynthetic genes under GCN4 control. With the exception of gcn2-1498-1517, which was indistinguishable from wild-type GCN2, all of the remaining 20-codon deletion alleles appeared to be completely nonfunctional. Of these, only the deletions from residues 1518 to 1537 and 1578 to 1597 led to significant reductions in protein expression which might have accounted for their 3-AT-sensitive phenotypes (Table 3). To eliminate this possibility, we inserted these alleles into a high-copy-number plasmid from which wild-type GCN2 is overexpressed 20- to 50-fold (32) and retested their phenotypes in the gcn2 strain. Both the gcn2-1518-1537 and gcn2-1578-1597 alleles retained their 3-AT-sensitive phenotypes when present on multicopy plasmids, confirming that the encoded proteins are nonfunctional.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Evidence for dimerization of GCN2 mediated by self-interactions in the C-term and PK domains. Using the yeast two-hybrid assay and in vitro binding studies with recombinant proteins, we found that the isolated PK and C-term domains of GCN2 can interact with themselves and that the self-interaction of the isolated C-term domain appears to be stronger than the PK-PK interaction. Using the two-hybrid assay and coimmunoprecipitation analysis, we showed that LexA fusion proteins containing the isolated PK and C-term domains can also form relatively stable complexes with full-length GCN2 in vivo. Deletion of the PK domain in GCN2 abolished its coimmunoprecipitation with LexA-'PK(720-999), confirming that complex formation between the isolated PK domain and full-length GCN2 was dependent on the PK self-interaction. We also showed by coimmunoprecipitation experiments that a substantial fraction of wild-type GCN2 could form a stable complex in vivo with a full-length LexA-HA-GCN2 fusion protein, providing direct evidence for dimerization by full-length GCN2 molecules. This interaction was abolished by deletion of the C-term domain, indicating that the C-term segment is critical for dimerization by full-length GCN2, whereas deletion of the PK domain led to only a modest reduction in complex formation. This last finding is in accordance with the fact that the self-interaction of isolated PK segments was weaker than that of isolated C-term segments.