Identification of Intersubunit Domain Interactions within Eukaryotic Initiation Factor (eIF) 2B, the Nucleotide Exchange Factor for Translation Initiation*

Background: eIF2B is a critical translation factor and regulator of protein synthesis that is implicated in human disease. Results: Protein-protein interactions within the five-subunit eIF2B complex are identified. Conclusion: eIF2B complex formation requires extensive intersubunit domain interactions. Significance: The first experimental model is proposed for eIF2Bγ and -ϵ subunit interactions.

In protein synthesis, the translation initiation factor 2 (eIF2) binds GTP and initiator methionyl tRNA (tRNA i Met ) 2 to deliver the latter to initiating ribosomes for each translation initiation event (1). A G protein, eIF2 cycles between inactive GDP and active GTP-bound states (2). These transitions are governed by eIF5 and eIF2B. eIF5 is a dual function GTPase-accelerating protein and GDP dissociation inhibitor (3). eIF2B is a guanine nucleotide exchange factor (GEF) that promotes GDP release and GTP binding to eIF2, enabling tRNA i Met binding (4). Maintaining this eIF2 cycle is critical for continued active protein synthesis in all cells. Controlling eIF2B confers critically important regulation of the eIF2 cycle and translation initiation. In mammalian cells, four protein kinases (termed EIF2AK1-4, also known as HRI, PKR, PERK, and GCN2) each phosphorylate a serine at position 51 in the eIF2 ␣ subunit in response to different signals. Phosphorylated eIF2 (eIF2(␣P)) binds to eIF2B with higher affinity and acts as a competitive inhibitor of eIF2B, instead of its substrate (5). This reduces the activity of eIF2B and the availability of active eIF2⅐GTP⅐tRNA i Met in cells and has two divergent effects. For a majority of transcripts, eIF2(␣P) causes reduced translation; however, for a subset of translationally controlled RNAs, their expression is enhanced. Many of the regulated mRNAs characterized are transcription factors important for mediating cellular reprogramming of gene expression in response to stress, for example ATF4 in mammalian cells and GCN4 in yeast (6).
The study of eIF2B has gained a new focus in the last decade as mutations within any of its five subunits have been associated with a fatal inherited brain disorder commonly called leukoencephalopathy with vanishing white matter or childhood ataxia with central nervous system hypomyelination. To date, more than 100 separate missense mutations have been described (7). Where analyzed biochemically, in assays using extracts from patient-derived cells, they typically reduce eIF2B activity by between 20 and 80% of control values (8).
eIF2B has five subunits ␣-⑀, in increasing size order, encoded by separate genes (EIF2B1-5 in humans and named GCN3, GCD7, GCD1, GCD2, and GCD6, respectively, in the yeast Saccharomyces cerevisiae). Although they were initially characterized biochemically using proteins purified from mammalian sources, much of our understanding of the roles of individual subunits has come from genetic and biochemical analyses of the yeast factor. eIF2B␣␤␦ all share sequence similarity (see Fig.  1A) and bind together to form a subcomplex that can interact with eIF2␣, with higher affinity for eIF2(␣P). This was termed the regulatory subcomplex (9), and remarkably, even single amino acid substitutions in any of these subunits can disrupt this regulatory mechanism (10). Where tested, the mutants reduced affinity for eIF2(␣P) (11,12). The ⑀ subunit is the catalytic subunit, and the extreme C-terminal region (⑀cat) is sufficient for nucleotide exchange in vitro (13) via interaction with two eIF2 subunits (14). Consistent findings were reported for mammalian and Drosophila eIF2B⑀ (15,16); however, eIF2B⑀ activity is at least ϳ10-fold lower than the full eIF2B complex, indicating that the other subunits act to enhance eIF2B activity as well as regulate its function. How this is achieved is not yet clear, but it is known that eIF2B⑀ binds to the ␥ subunit in the absence of the ␣␤␦ subunits (5) and that this ␥⑀ catalytic subcomplex enhanced GEF activity similarly to that seen with the full five-subunit complex in assays using yeast cell lysates as a source of eIF2B.
Since they were first sequenced, it was realized that eIF2B⑀ and -␥ share extensive primary sequence similarity with each other (17) and with a family of enzymes called NTP-hexose sugar pyrophosphorylases (18). ADP-glucose pyrophosphorylase (AGP) binds ATP and glucose to catalyze the formation of ADP-glucose, required for starch (in plants) or glycogen synthesis (in bacteria) (19). Escherichia coli N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) catalyzes a similar reaction with UTP and N-acetylglucosamine 1-phosphate necessary for peptidoglycan synthesis (20). The crystal structures of GlmU and potato tuber AGP have both been solved, and each reveals two domains, an amino-terminal globular pyrophosphorylase domain and a separate carboxyl-terminal domain sometimes referred to as "hexapeptide" repeat or "I-patch" because of a repetitive sequence motif with the loose consensus XXIGXX. This domain adopts a left-handed ␤-helix (L␤H) structure (see Fig. 1, B and C). The structures of eIF2B␥ and the N-terminal 470 residues of eIF2B⑀ have not yet been determined, but due to sequence homology, each subunit is predicted by available modeling software tools to share similar domain architecture with these enzymes (21,22). Both eIF2B subunits share a conserved N-terminal region, here termed pyrophosphorylase-like domain (PLD), and the L␤H region. In this work, we set out to uncover the significance of these conserved domains for eIF2B GEF function and domain organization, with the aim of better understanding the relationship between the structural organization and function of this large protein complex that is critical for protein synthesis and its control. Our findings provide important structural and functional insight into the domain organization of this protein.
Strains were grown at 30°C in Synthetic Complete (SC) medium containing glucose (2% w/v) but lacking nutrients required for plasmid selection or in Synthetic Dextrose minimal medium (SD) with necessary supplements added to complement auxotrophies (23). When inducing P Gal-dependent protein expression, glucose was replaced with galactose and raffinose (2% w/v each) to make SCGal medium. For growth on solid medium, cells were typically grown to A 600 ϭ 0.1 and serially diluted before 3 l of each dilution was spotted on the appropriate medium and grown at 30°C for the indicated time.
Plasmid Constructions-PCR was used to amplify the desired fragments of GCD6 (yeast eIF2B⑀) with added BamHI or stop-SalI tails to enable cloning in-frame with glutathione S-transferase (GST) in pEG(KT) (30), a high copy number yeast P Gal -GST URA3 leu2d expression plasmid, to create GST-fused fragments. Similarly, PCR was used to clone GCD1 (yeast eIF2B␥) fragments with added tandem C-terminal 2ϫFLAG and His 6 tags (␥-FLAG) into pBEVY-GT, a yeast high copy number TRP1 plasmid with both P Gal1 and P Gal10 promoters to drive regulated expression (31). Site-directed mutagenesis of pAV1265 to introduce R39A, R49A, and D138A mutations into GCD6 employed QuikChange (Stratagene). See Table 1 for plasmid details.
GST Purifications and FLAG Immunoprecipitations (IP)-Strains were grown in SCGal medium with 0.2% glucose to an A 600 between 0.2 and 0.6 and an equivalent volume containing 10 A 600 units were harvested by centrifugation at 5000 ϫ g. Cells were washed in sterile ice-cold water with 1 mM PMSF (10 ml) and then in 1 ml of buffer IP (150 mM NaCl, 20 mM TRIS-HCl, pH 7.4, 2 mM MgCl 2 , 0.5% Triton X-100, 5 mM 2-mercaptoethanol) before being resuspended in 250 l of buffer IPϩPI (IP ϩ mini EDTA-free protease inhibitor tablets (Roche Applied Science)). Cell suspensions were added to tubes containing 200 l of 1-mm zirconia/silica beads (Thistle Scientific) and lysed by agitation using a FastPrep (MP Bio) for 3 ϫ 25 s at 6.5 ms Ϫ1 with cooling on iced water for 5 min between cycles. Cell debris was removed from the cell extract by centrifugation at 10,000 ϫ g for 15 min at 4°C. The extracts were precleared with Sepharose 4B beads (GE Healthcare), and the affinity resin was blocked with 1 mg/ml BSA for 1 h in IPϩPI buffer at 4°C. For FLAG purifications, 250 l of 1 mg ml Ϫ1 extract was incubated with 40 l of packed volume of anti-FLAG M2 affinity resin (Sigma-Aldrich). For GST purifications, 50 l of packed volume of anti-GST magnetic resin (Pierce) was used. Extractresin mixes were incubated with rotation for 2 h at 4°C. The supernatant was removed, and the beads were washed 3 ϫ 10 min in IPϩPI. The beads were resuspended in 125 l of 1ϫ Laemmli sample buffer. 2ϫ Laemmli sample buffer was added to each input and supernatant. Samples were heated at 95°C for 5 min before loading onto a 10% SDS-PAGE analysis followed by Western blotting and probing with relevant antibodies. Input and supernatant samples contained 5 g of protein, whereas immunoprecipitated samples contained extract from an equivalent of 20 g of input. Anti-GST, -His, and -FLAG and rabbit polyclonal antibodies to yeast eIF2B subunits were used as described previously (32). Detection used HRP-linked secondary antibodies and chemiluminescence detection (Pierce).

RESULTS
Mutations Affecting Potential Nucleotide-binding Site in eIF2B⑀ PLD Have Minimal Impact on eIF2B Activity-As outlined in the Introduction, it has been noted previously that the eIF2B␥/⑀ subunits share sequence similarity with pyrophosphorylases such as GlmU and AGP (18). We wished to explore its significance. Multiple sequence alignments of the full-length proteins indicated that the amino-terminal half of the eIF2B␥/⑀ PLD was most conserved (data not shown, see "Experimental Procedures"). In GlmU and AGP and other similar enzymes, this region is responsible for nucleotide binding. Single GlmU missense mutations at Arg-18 and Lys-25 dramatically impaired uridyltransferase activity (20). In addition, the same study identified Asp-105 as a residue important for nucleotide binding, being necessary for coordination of a stabilizing magnesium ion. These residues are conserved in AGP and in human and yeast eIF2B⑀ (residues Arg-39, Arg-49, and Asp-138 in Gcd6p; Figs. 1 and 2A), raising the possibility that this sequence similarity reflected a shared nucleotide-binding region. Although the minimal eIF2B catalytic domain (⑀cat) resides at the C terminus of eIF2B⑀ (Fig. 1A), the full complex has enhanced activity (5). Additionally, one proposed mechanism for eIF2B GEF activity, based upon enzyme kinetic analyses, is a sequential mechanism, whereby GTP binds eIF2B before GDP is released from eIF2 (33,34). This differs from the more widely accepted substituted enzyme mechanism, where GDP is released from the G protein, forming a nucleotide-free state, allowing GTP to bind. Taken together, these observations prompted speculation that binding a nucleotide, or nucleotide derivative, to this region of eIF2B may be important for regulating eIF2B GEF activity. To evaluate this idea experimentally, we used site-directed mutagenesis to introduce single alanine residues at Arg-39, Arg-49, and Asp-138 in a plasmid clone of GCD6.
Transformation of a gcd6⌬ strain and plasmid shuffling generated strains bearing each allele as the sole source of eIF2B⑀. Each strain grew indistinguishably from the wild-type strain on standard growth medium, indicating that no significant impairment to the essential function of eIF2B was conferred by these mutations (Fig. 2A). Western blotting confirmed no change in expression levels (data not shown). Further analyses revealed no defect at 10 or 36°C or growth phenotypes using alternate carbon sources: raffinose, galactose, or maltose (data not shown). The transcription factor GCN4 is translationally controlled by amino acid starvation and highly sensitive to even eIF2B Interdomain Interactions MARCH 9, 2012 • VOLUME 287 • NUMBER 11 minor alterations in eIF2B activity. Growth following amino acid starvation, or in the presence of the histidine inhibitor 3AT or the proline analog azetidine-2-carboxylic acid, requires the eIF2␣ kinase Gcn2p. gcn2⌬ cells cannot normally grow on medium containing 3AT or azetidine-2-carboxylic acid; however, mutations that reduce eIF2B GEF activity facilitate growth (see Ref. 35). We used these assays and found that both drugs could enable growth of the D138A mutant in gcn2⌬ cells, whereas 3AT also allowed weak growth of both R39A and R49A mutants ( Fig. 2B and data not shown). Taken together, these results are consistent with the idea that each mutant confers only a modest reduction in eIF2B activity, not significant enough to affect growth rate in the absence of amino acid starvation. These results are not consistent with the hypothesis that these three residues are critical for eIF2B function and suggest that the sequence conservation does not imply that nucleotide or nucleotide-analog binding to this region is critical for eIF2B essential nucleotide exchange function. The implication of these results for proposed eIF2B catalytic mechanisms is discussed later.
eIF2B⑀ Catalytic Domain Is Not Required for eIF2B Complex Formation-Next, we decided to examine the role of the PLD and L␤H domains in eIF2B complex formation. Previously, we isolated several mutations in yeast eIF2B⑀ (GCD6) that affected its activity. Three of these truncated the protein (Thr-518*, Gln-500*, and Gln-452*) and eliminated the catalytic ⑀cat domain (26). In addition, we found that an internal deletion ⌬93-358 that interrupted both the PLD and the L␤H domains disrupted eIF2B complex formation between eIF2B␥ and any other subunit. Our published results suggested that the eIF2B⑀ PLD and L␤H regions contributed to holo-complex formation, but that ⑀cat was not required. To demonstrate this directly, we eIF2B Interdomain Interactions co-expressed from high copy plasmids eIF2B␣␤␦, ␥-FLAG, and either wild-type or mutated eIF2B⑀ in yeast cells, precipitated the ␥-FLAG from cell extracts, and assessed which eIF2B subunits were co-precipitated. Overexpression was necessary for our approach as GCD6 is an essential gene and the mutants used do not complement gcd6⌬ (26). As shown in Fig. 3, the wild-type eIF2B complex was efficiently precipitated with ␥-FLAG, but not with an untagged control (lanes 6 and 7). As reported previously, ⌬93-358 disrupted interactions between ␥-FLAG and all eIF2B subunits. In contrast, Gln-452* and Thr-518* efficiently formed five-subunit complexes, showing that the ⑀cat domain is not required for eIF2B complex formation.
eIF2B⑀ PLD and L␤H Domains Independently Contribute to Interactions with eIF2B␥, but Both Are Required for eIF2B Complex Formation-The experiment shown in Fig. 3 suggests that eIF2B⑀ PLD and L␤H domains shared with eIF2B␥, GlmU, and AGP are sufficient for eIF2B complex formation. GlmU forms a homotrimer, with extensive contacts between the L␤H domains and no contact between the pyrophosphorylase domains (Fig. 1C). In contrast, the crystal structure for potato tuber AGP shows that the L␤H domain is rotated around to make contacts with the pyrophosphorylase domain (Fig. 1D) and forms a homotetramer with extensive contacts between both domains and each monomer (Fig. 1E). Thus from homology modeling alone, it is not possible to infer a structure for the eIF2B␥⑀ catalytic subcomplex. To examine the interactions directly, we made a series of constructs fusing the PLD, L␤H, or both domains to GST (Fig. 4A) and expressed from a galactoseinducible promoter in a high copy number yeast vector. We used a combination of multiple sequence alignments and a protein structure models database (ModBase: Database of Comparative Protein Structure Models; Ref. 22) to predict the domain boundaries. In addition, we made three further constructs, two bearing approximately each half of the PLD (termed PLD⌬1 and ⌬2; Fig. 4A) and one with both PLD⌬2 and L␤H combined. We also used a GST-⑀cat construct made previously (13).
We noted that several constructs conferred a galactose-dependent slow growth to the yeast cells. The most severe slow growth phenotype was with the construct containing both the PLD and the L␤H domains (Fig. 4A). Anti-GST Western blotting confirmed that all constructs were expressed; importantly, overexpression levels did not correlate with slow growth (Fig.  4B). One interpretation of the slow growth phenotype is that the excess ⑀PLDϩL␤H protein forms an eIF2B complex lacking GEF activity by replacing endogenous eIF2B⑀ from a proportion of the eIF2B complexes, thereby slowing translation and cell growth.
To assess complex formation between these GST domains and eIF2B␥, cells were co-transformed with a high copy GCD1-FLAG 2 -His 6 plasmid overexpressing tagged eIF2B␥. Reciprocal precipitations were performed to ask which GST-eIF2B⑀ domains would interact with full-length eIF2B␥ using FLAG affinity resin (Fig. 5A) or glutathione resin (Fig. 5B). These experiments showed that three fragments of eIF2B⑀ could interact strongly with eIF2B␥. The PLD and L␤H domains individually or combined together are shown in Fig. 5 (panel A,  lanes 11, 14, and 12, respectively; and panel B, lanes 11, 15, and  12, respectively). These experiments show that both domains of eIF2B⑀ independently contribute to binding to eIF2B␥. This strongly suggests that the eIF2B␥⑀ interdomain interactions do FIGURE 2. Mutations that impair pyrophosphorylase catalytic function have minimal effect on eIF2B⑀ essential function in yeast. A, segments of a sequence alignment between the pyrophosphorylase domains of E. coli GlmU, potato tuber AGP, and yeast eIF2B␥ and -⑀ subunits. * indicates the residues mutated. Identical residues are shaded. These residues are also highlighted in Fig. 1 as green spheres (N-terminal Arg, pale green; middle Lys/Arg, bright green; and conserved Asp, dark green). B, yeast growth assay for indicated single substitution mutants. Serial dilutions of cultures were spotted on minimal medium (SD ϩ U) or amino acid starvation medium (ϩ 3AT). not resemble those in GlmU, but possibly resemble some of those found in the AGP homotetramer (Fig. 1).
Next, we extended our analysis to examine whether these individual eIF2B⑀ domains are sufficient for eIF2B complex formation. We transformed our cells with a plasmid co-expressing eIF2B␣␤␦ and used FLAG immune precipitation to assess this. In contrast to results presented in Fig. 5, but in agreement with findings shown in Fig. 3, we find that both PLD and L␤H domains of eIF2B⑀ are required to stably bind eIF2B␣␤␦ to eIF2B␥ (Fig. 6).

eIF2B␥ PLD and L␤H Domains Independently Contribute to Interactions with eIF2B⑀, but Both Are Required for Full eIF2B
Complex Formation-We then assessed domain interaction requirements for eIF2B␥. Using a similar strategy to that described above, constructs expressing ␥-FLAG domains from galactose-inducible promoters were made and introduced into yeast, either singly or as a pair (Fig. 7). Only the construct expressing both domains independently conferred a modest slow growth phenotype, although all fragments were expressed (Fig. 7, A and B). These cells were co-transformed with eIF2B␣␤␦⑀ genes, and anti-FLAG immune precipitations were performed. The results were similar to those obtained with the equivalent eIF2B⑀ domains (Fig. 7C) because each ␥ domain could independently form a complex with the ⑀ subunit, albeit at reduced levels when compared with full-length ␥. When both domains were present, the ␣␤␦ subunits could also join, forming a five-subunit complex. This was true whether full-length ␥ was used, or remarkably, even when both separate domains were co-expressed (compare lanes 7 and 10). These results are consistent with the idea that each domain independently contributes to ␥⑀ complex formation, but that both ␥ domains together are required to recruit ⑀ and the ␣␤␦ subcomplex.
Extensive Interactions between ␥ and ⑀ PLD and L␤H Domains-To determine which individual ⑀ domains the eIF2B␥ PLD and L␤H domains interact with, we co-expressed pairs of GST-⑀ and ␥-FLAG domains in yeast and performed anti-FLAG IPs. As controls, we showed that GST alone would not interact with either ␥ domain or the FLAG resin alone (Fig.  8A, right panel, lanes 1, 3, and 7) and that both ␥ domains could interact with the GST-⑀PLDϩL␤H construct (lanes 6 and 10). The experiments showed that either ␥ domain could interact with both GST-⑀PLD and GST-⑀ L␤H separately (lanes 4, 5, 8,  and 9). Quantification of the relative interaction efficiency of GST domains with ␥-FLAG was consistent with the idea that the ␥PLD domain interacts strongly with ⑀PLD but very weakly with ⑀L␤H. In contrast, the ␥L␤H construct interacted equally    4, 5, and  6). Taken together, these data suggest that there are extensive interactions between the ␥ and ⑀ structural domains that are necessary for eIF2B complex formation. These observations allow us to propose a model for domain interactions within this protein complex (Fig. 8B; see "Discussion").

DISCUSSION
eIF2B is the GEF for eIF2 during eukaryotic protein synthesis. With five different subunits, it has a significantly greater structural complexity than GEFs for other G proteins, many of which are single subunit factors (29,36,37). In this study, we have focused our attention on the roles of conserved domains within the ⑀ and ␥ subunits. Both subunits share homology with sugar nucleotide pyrophosphorylase enzymes found in all organisms. These domains are the PLD and L␤H regions (Fig.  1). We first examined whether residues important for nucleotide binding in this enzyme family and conserved in eIF2B⑀ PLD were critical for eIF2B essential function. Site-directed mutagenesis and genetic assays revealed only modest defects (Fig. 2). These findings are not consistent with the idea that binding a nucleotide or a nucleotide derivative to this motif significantly influences an essential eIF2B function. These results are in contrast to mutations characterized previously within conserved residues of the PLD at ⑀Asn-249 and ⑀Phe-250. Both N249K and F250L severely impaired growth and eIF2B activity, with N249K identified as a lethal allele (26). These new results are of interest because previous enzyme kinetic analyses of eIF2B have proposed two competing mechanisms to account for eIF2B GEF activity: i) a standard substi-tuted enzyme mechanism where GDP is released, allowing GTP to bind a nucleotide-free eIF2; or ii) a sequential mechanism where GTP binds before GDP is released (33,34). The latter, more unusual mechanism would require a second nucleotidebinding site. The best candidate for this site is the PLD of eIF2B⑀. However, the mutations to this motif that we have analyzed here, known to significantly impair nucleotide binding function in other proteins, have only minimal impact on yeast eIF2B function. Therefore a model where GTP binds directly to this motif before being transferred to eIF2 could probably be discounted (38). Similarly, ideas that nucleotide binding here may have an allosteric activation effect on eIF2B GEF function, as suggested for mammalian eIF2B, seem unlikely for the yeast protein, based on our findings (39).
Experiments examining the role of eIF2B⑀ domains for complex formation revealed that ⑀cat was not required (Fig. 3). Instead the PLD and L␤H regions were implicated, so we examined the roles of these domains in mediating protein-protein interactions between eIF2B⑀ and -␥ in the catalytic subcomplex. We overexpressed GST-fused eIF2B⑀ domains or FLAG-His-tagged eIF2B␥ domains in yeast cells along with the fulllength partner subunit. Results were similar for both sets of experiments as interactions between eIF2B␥ and -⑀ were mediated by either PLD alone or L␤H alone as well as PLDϩL␤H domains combined (Figs. 5 and 7). We further showed that there are interactions possible between all four domains (Fig.  8A). The domains appear to form homologous interacting pairs as the PLDs appear to interact more strongly with each other than they do with the L␤H domains, and similarly, the L␤H domains bind more strongly to each other than to the PLDs. This domain interaction arrangement differs from that found in GlmU, where only L␤H domains interact together (Fig. 1C) (20). However, these interactions are possibly broadly compatible with one of the two possible dimer pair combinations that comprise the potato tuber AGP homotetramer structure (Fig.  1E) (19). One dimer pair interacts exclusively through pyrophosphorylase interactions (e.g. Fig. 1E, the left upper and lower monomers). Our data do not favor this arrangement. A second monomer pair (as exemplified in Fig. 1E by the lower left and right pair) combine through interactions between all domains. Our data for eIF2B␥/⑀ are broadly compatible with these domain interactions. These ideas are shown in the model in Fig.  8B, but we do acknowledge that other domain arrangements or orientations are possible.
In contrast to the requirements for ␥⑀ complex formation, we found that only constructs comprising both pairs of PLD and L␤H domains together were able to bind stably to the ␣, ␤, and ␦ subunits to form a five-subunit eIF2B complex (Figs. 6 and 7C). Interestingly, when we co-expressed both ␥PLD and ␥L␤H as separate polypeptides together in the same strain, this was also capable of forming a five-subunit complex similar to full-length eIF2B␥ (Fig. 7C, lanes 7 and 10). Previous work has shown that the ␣␤␦ subunits can bind together to form a three-subunit subcomplex that can bind eIF2␣ with high affinity when it is phosphorylated (9,11). However, how these subunits interact with each other and with the ␥⑀ subcomplex is less clear. The eIF2B␣ crystal structure formed dimers (Fig. 1A) and higher order octamer arrangements (40) and is highly similar to archaeal proteins, some of which form hexamers rather than octamers within their crystals. This hexameric arrangement has prompted the suggestion that the ␣␤␦ trimer may resemble one-half of the hexamer (41). Other experiments indicate that the ␣ and ␦ subunits may be more peripheral in the complex than in the ␤ subunit. Firstly, the ␣ subunit is nonessential, and a ␤-⑀ complex forms that retains GEF function (5,12,42). Next, recent experiments depleting the yeast ␦ subunit genetically, using a "degron" strain, could also form an ␣␤␥⑀ complex (43). In contrast, similar degron depletion of eIF2B␤ caused co-depletion of eIF2B␦. A similar result was obtained previously with a missense mutation (eIF2B␤-V341D). The mutation destabilized both eIF2B␤ and eIF2B␦ binding to the eIF2B complex (35). These results are consistent with eIF2B␤ being more central in the complex, or at least stabilizing eIF2B␦ binding. Yeast genetic evidence also suggested that eIF2B␣␤␥ can form an independent subcomplex (43), although this complex was not directly shown, and we have not been able to demonstrate significant levels of interaction between eIF2B␣␤␥ in our experiments (data not shown). Taken together, these data suggest that the ␣ and ␦ subunits can be more readily excluded from the eIF2B complex and therefore likely occupy more peripheral positions in it. Experiments with mammalian eIF2B subunits also provide some support to these ideas. Purification of human subunits from 293 cells identified stable ␥⑀ and ␤␥␦⑀ complexes (44). Experiments purifying rat eIF2B from an insect cell co-expression system suggested that the N-terminal half of the ⑀PLD (1-164) could bind to eIF2B␤ and that the remaining part (⑀159 -716) could bind ␤␥␦, but not ␣ (45). As studies have used a variety of expression and purification systems, sources of eIF2B subunits, and differing washing stringencies in their experimental systems, these elements may FIGURE 7. Both eIF2B␥ PLD and L␤H regions are necessary for holo-complex formation with eIF2B␣␤␦⑀. A, top, graphic of eIF2B␥ fragments fused to FLAG and His 6 (FH 6 ) cloned into pBEVY-GT (31) and transformed into yeast strain GP4597. Bottom, growth assay of 5-fold serially diluted cultures on noninducing (SC-TRP) and inducing (GAL RAF-TRP) media. B, Western blotting of the strains in panel A grown in medium containing galactose and raffinose carbon sources. C, Western blotting of co-immune precipitation experiments using total cell extracts from strain GP4597 co-expressing full-length eIF2B⑀ (Gcd6p) and regions of eIF2B␥-FLAG 2 His 6 (Gcd1p) and eIF2B␣␤␦ (Gcn3p, Gcd7p, and Gcd2p). Co-immune precipitation used anti-FLAG M2 affinity resin. eIF2B Interdomain Interactions account for any apparently different findings. When taken together, the experiments suggest that eIF2B␤ is central to the ␣␤␦ subcomplex. We suggest that our data (Figs. 6 and 7C) are compatible with two possible modes for ␣␤␦ subcomplex binding to the ␥⑀ subcomplex. One option is that ␣␤␦ directly bind to all four interacting domains within the ␥⑀ subcomplex. A second possibility is that all four domains are necessary to adopt or constrain a specific ␥⑀ conformation and that this facilitates stable binding of ␣␤␦ to only one of the other subunits, possibly eIF2B␥.
An independent study conducted in parallel and examining domain interactions within human eIF2B has led to similar conclusions to our work (48). Using a transient transfection approach, cDNAs were co-expressed in HEK293 cells, and complexes were purified from these cells. By examining the effects of missense mutations in ⑀ or ␥ and by co-expression of domain deletion mutants, the contribution of each domain to protein-protein interactions is evaluated. In general, the experiments nicely complement the findings reported here. They confirm our results that mutations within human eIF2B⑀ equivalent to our R39A and D138A have no significant negative impact on eIF2B activity as measured in vitro. In addition, they also find that the ␥⑀ PLDs are important for domain interac-tions with each other and that both ⑀PLDϩL␤H domains are necessary for complex formation with the ␣␤␥␦ subunits. The only apparent difference is that human ␥-L␤H was not necessary for human ␣␤␥␦⑀ complex formation but was required for the yeast intersubunit interactions. This most likely reflects experimental differences in the extent of the ␥-L␤H domain. Yeast ␥-L␤H is defined here as 220 amino acids, whereas human ␥-L␤H domain deleted in the companion study is only 82 residues. A simple explanation for the apparent difference between experiments is that our yeast ␥-L␤H deletion removed an element critical for complex formation, which is retained in the human ␥PLD construct used.
In summary, our experiments suggest that potential nucleotide-binding elements within eIF2B⑀ have minimal impact on the rate of eIF2B nucleotide exchange. This finding is at odds with one proposed mechanism for nucleotide exchange (the sequential mechanism) but is consistent with the substituted enzyme mechanism: an issue of long controversy (33). In addition, by protein-protein interaction studies, we provide insight into the domain interactions between eIF2B⑀ and -␥ necessary for eIF2B complex formation. Although further studies will be necessary to resolve precisely how the catalytic and regulatory subcomplexes bind each other, our work provides important insight toward resolving this conundrum.