Biochemical Analysis of the eIF2beta gamma Complex Reveals a Structural Function for eIF2alpha in Catalyzed Nucleotide Exchange*

Joseph Nika, Scott Rippel, and Ernest M. HannigDagger

From the Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083

Received for publication, August 14, 2000, and in revised form, October 5, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNAiMet to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2·GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The alpha -subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2beta gamma ) completely devoid of the alpha -subunit to further study the role of eIF2alpha in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2alpha structural gene (SUI2). Removal of the alpha -subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNAiMet ligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2beta gamma and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the alpha -subunit increased Km for eIF2beta gamma ·GDP by an order of magnitude, with a smaller increase in Vmax. These data indicate that eIF2alpha is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange. We suggest that this function contributes to the ability of the alpha -subunit to control the rate of nucleotide exchange through reversible phosphorylation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic translation initiation factor eIF21 is a heterotrimeric GTP-binding protein that in yeast is encoded by the essential genes SUI2, SUI3, and GCD11. eIF2 binds charged initiator tRNA (Met-tRNAiMet) in a GTP-dependent manner to form a stable ternary complex that interacts with the small ribosomal subunit and additional initiation factors (including eIF3 and eIF1A) to form a 43 S initiation complex (reviewed in Refs. 1 and 2-5). This complex binds at or near the 5'-end of capped mRNAs and "scans" the mRNA in a 5' to 3' direction until an AUG start codon in proper context is encountered. Genetic analyses in yeast have demonstrated a function for eIF2 in the selection of the translational start site (6, 7). Recognition of the start codon is accompanied by eIF5-mediated hydrolysis of eIF2-bound GTP, which facilitates release of an inactive eIF2·GDP binary complex as well as several other initiation factors. The 40 S/mRNA/Met-tRNAiMet complex interacts with a 60 S ribosomal subunit to form an 80 S initiation complex that can then enter the elongation phase of protein synthesis. Conversion of the inactive eIF2·GDP binary complex to eIF2·GTP, which is capable of rebinding initiator tRNA, is catalyzed by the nucleotide exchange factor eIF2B.

To execute its functions in translation initiation, eIF2 must interact with multiple ligands as well as other components of the translational apparatus. The former include guanine nucleotides and initiator tRNA, whereas the latter may include ribosomes, mRNA, and other initiation factors including eIF2B and eIF5. Several studies have been conducted to characterize the function of individual eIF2 subunits. Both yeast and mammalian eIF2gamma proteins exhibit a high degree of amino acid sequence similarity to bacterial EF1A (formerly EF-Tu) and SELB proteins (8-11), and in vitro cross-linking studies have implicated the gamma -subunit in binding of both guanine nucleotide and Met-tRNAiMet ligands (8, 12, 13). The implied functional similarity with EF1A-like proteins was substantiated by mutational analysis, where point mutations in the structural gene encoding yeast eIF2gamma (GCD11) conferred specific biochemical defects in either nucleotide or tRNA binding in the altered eIF2 proteins (14). These defects were predicted by the EF1A structural model, confirming the notion that eIF2gamma provides EF1A-like function to the eIF2 complex. Other studies demonstrated cross-linking of the gamma -subunit to 18 S rRNA, suggesting an involvement in ribosome interaction and/or 43 S complex formation (15). Although the role of eIF2gamma in GTP binding implies a further role in GTP hydrolysis and nucleotide exchange, there is currently no data to support direct contact between the gamma -subunit of eIF2 and either eIF5 or eIF2B.

Cross-linking studies have also implicated the beta -subunit of eIF2 in nucleotide- and Met-tRNAiMet binding (8, 12, 16, 17), indicating at the very least a proximity of this subunit to binding sites present on eIF2gamma . A role for eIF2beta in tRNA binding was suggested by analysis of partially purified eIF2 preparations from yeast strains harboring mutations in the eIF2beta structural gene (SUI3) (18). The function of eIF2beta has also been examined using preparations of mammalian eIF2alpha gamma obtained as a result of proteolysis of the beta -subunit either during purification or purposefully at the hands of the experimenter (Ref. 19 and references therein). These studies demonstrated little effect on the binding of guanine nucleotides in the absence of intact eIF2beta . However, the eIF2alpha gamma complex was severely defective in the formation of ternary complexes, suggesting the beta -subunit was involved in Met-tRNAiMet binding. The eIF2alpha gamma preparations also showed reduced rates of eIF2B-catalyzed exchange in vitro, suggesting that the beta -subunit plays a role in catalyzed nucleotide exchange. This is consistent with recent reports demonstrating interaction between the C terminus of eIF2beta and the delta - and epsilon -subunits of mammalian eIF2B (20) as well as between the N terminus of eIF2beta and the epsilon -subunit (Gcd6p) of yeast eIF2B (21). The beta -subunit of eIF2 also appears to interact directly with eIF5 (21-24). Curiously, both eIF5 and eIF2Bepsilon contain C-terminal bipartite motifs rich in aromatic and acidic amino acids that are important for interactions with eIF2beta (21).

Whereas many functions ascribed to eIF2 can be attributed to the beta - and gamma -subunits, comparatively little is known about the contribution of the alpha -subunit to eIF2 function. Two prior biochemical studies using alpha -deficient forms of eIF2 (i.e. containing low levels of contaminating eIF2alpha protein) yielded different results in comparing specific activities of alpha -deficient and wild-type eIF2 preparations (16, 25). The alpha -subunit has been cross-linked to 18 S rRNA, suggesting an involvement in 43 S complex formation (15), and also appears to contain conserved sequences similar to the S1-RM repeated sequence motif present in bacterial ribosomal protein S1 and thought to be involved in RNA binding (26). However, the best-characterized aspect of eIF2alpha is its role in regulating eIF2 function (reviewed in Refs. 5, 27, 28, and 29). Phosphorylation of the alpha -subunit at a conserved serine residue (Ser-51) converts eIF2 to a competitive inhibitor of eIF2B-catalyzed nucleotide exchange (30, 31). This results in functional sequestration of limiting amounts of eIF2B and leads to decreased levels of active eIF2 and global protein synthesis. Regulation of eIF2 activity in this manner is conserved across species (29, 32) and occurs in response to various physiological stresses including viral infection (33), heat shock (34, 35), heme deprivation (36), amino acid starvation (37-39), and endoplasmic reticulum stress (40).

We recently demonstrated2 that lethality associated with deletion of the yeast SUI2 gene (eIF2alpha ) could be suppressed by cooverexpressing wild-type SUI3, GCD11, and IMT (initiator methionyl-tRNA). Suppression was more efficient when the wild-type GCD11 allele was replaced with gcd11-K250R, which altered the NKXD element conserved in GTP-binding proteins and led to increased dissociation of guanine nucleotides in vitro (14). Because this effect mimics the function of eIF2B in nucleotide exchange, co-overproduction of eIF2gamma K250R and initiator tRNA also bypassed eIF2B function in vivo and, as with bypass of SUI2, was more efficient than overproduction of wild-type eIF2. This suggested that the nucleotide exchange reaction was a rate-limiting step in translation initiation in these strains. Based upon these observations, we proposed that the eIF2beta gamma complex was sufficient for providing eIF2 function, whereas eIF2B and the alpha -subunit of eIF2 served primarily to regulate eIF2 activity.

The construction of strains completely lacking eIF2alpha provides a unique opportunity to address basic aspects of eIF2alpha function. In this study, we purified both eIF2beta gamma and eIF2beta gamma K250R and compared their biochemical activities in vitro with heterotrimeric wild-type eIF2 and eIF2gamma K250R. Removal of the alpha -subunit had little effect on dissociation of guanine nucleotides and GTP-dependent tRNA binding (ternary complex formation). In vitro, ternary complexes formed with eIF2beta gamma interacted with the AUG codon and ribosomes to form 43 S preinitiation complexes, which served as substrates for eIF5-dependent GTP hydrolysis. Kinetic analysis of eIF2B-catalyzed nucleotide exchange using eIF2beta gamma ·GDP as substrate revealed a 16-fold increase in Km and an 8-fold increase in Vmax when compared with wild-type eIF2·GDP. These data suggest that the alpha -subunit of eIF2 plays a crucial role in structural interactions with eIF2B that determine wild-type rates of nucleotide exchange in vivo.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Reagents-- [8,5'-3H]GDP (5-15 Ci/mmol) and L-[methyl-3H]methionine (70-85 Ci/mmol) were from PerkinElmer Life Sciences; [gamma -32P]GTP (800 Ci/mmol) was from ICN Pharmaceuticals. Protease inhibitors, GDP, and Gpp(NH)p were purchased from Sigma. Bulk yeast tRNA was from Roche Molecular Biochemicals. 40 S ribosomal subunits were purified from brine shrimp eggs (Artemia; San Francisco Bay Brands) as described (41) and stored at 0.1-0.4 A260 units/µl. All buffers and reagents were prepared with purified water (MilliQ; Millopore Corp.).

Aminoacylation of Yeast Initiator tRNA-- Yeast methionyl initiator tRNA was aminoacylated with L-[3H]methionine as described previously (14), except that methionine was present at 3 µM, and tRNA was present at 12.5 µg/µl. Typically, 200-400 pmol (specific activity 7.8-9.0 × 104 cpm/pmol) of trichloroacetic acid-precipitable [3H]methionine was obtained from a 1-ml reaction.

Purification of eIF2 and eIF2beta gamma Isoforms-- His8-tagged (C terminus of eIF2gamma ) eIF2 and eIF2gamma K250R were purified from overexpressing yeast strains, as described previously (14). The His8-tagged eIF2beta gamma isoforms were overproduced in a derivative of EY841 (MATalpha leu2-3 leu2-112 trp1-Delta 63 sui2Delta gcd11::hisG GAL2+)2 and purified similarly with the following modifications. After ammonium sulfate precipitation, the protein pellet was suspended 100 ml of nickel column buffer (20 mM Tris-HCl, pH 7.5, 600 mM KCl, 1 µM GDP, 6.25 mM beta -mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 µg/ml aprotinin) and dialyzed overnight against 4 liters of nickel column buffer. Insoluble material and ribosomes were pelleted by centrifugation at 200,000 × g for 2 h at 4 °C. Dialysis after chromatography on nickel nitrilotriacetic acid-agarose was reduced to 2 h against two liters of heparin buffer. These modifications reduced the purification time from 3 days to 2 and also resulted in increased specific activity of the eIF2beta gamma preparations.

Purification of eIF2B and Guanine Nucleotide Exchange Assay-- Purification of yeast eIF2B from an overexpressing yeast strain (H2649) and steady-state kinetic analysis of eIF2B-catalyzed guanine nucleotide exchange were performed as described previously (42). In preliminary experiments, the presence of 5 mM GDP as chase was determined to be saturating for the eIF2beta gamma substrate, since GDP at 10 mM did not lead to further increase in initial reaction velocities.

Purification of eIF5-- The wild-type TIF5 (yeast eIF5) open reading frame plus 290 base pairs of 3'-flanking sequence was subcloned as a 1.5-kilobase NdeI/BamHI polymerase chain reaction product into a pET-3A expression vector (Novagen) such that the NdeI site included the authentic eIF5 start codon. The entire polymerase chain reaction fragment was sequenced to ensure the absence of polymerase chain reaction-derived mutations. The expression construct or the pET-3A vector alone (for preparation of a control extract) was introduced into Escherichia coli strain BL21(DE3/pLysS) (43). The purification procedure was modified from Chaudhuri et al. (23). Cultures for protein purification (500 ml) were grown at 37 °C, 300 rpm to an A600 of 1.25, induced with 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside, grown an additional 3 h, harvested, and rinsed with 0.9% ice-cold NaCl. Cells (15 g wet weight) were resuspended in 36 ml of buffer (20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 30 mM KCl, 1 mM Na2EDTA, 10 mM beta -mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride) and then lysed by one cycle of freezing at -20 °C followed by thawing at room temperature. All subsequent steps were performed at 4 °C using 18 ml of lysate. Cell debris was removed by centrifugation at 11,000 rpm (SS34 rotor) for 10 min, and 50 µl of a freshly prepared DNaseI (1 mg/ml stock) was added to the supernatant. After a 30-min incubation on wet ice, the sample was centrifuged at 200,000 × g for 150 min, and the pellet was discarded. KCl (2.5 M) was slowly added to supernatant with constant stirring to bring the salt concentration to 250 mM. The sample was applied to a DE-52 column (Whatman, 28-ml bed volume) at a linear flow rate of 56.5 cm/h with buffer A (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM Na2EDTA, 10% glycerol) containing 250 mM KCl. The flow-through (140 ml) was collected and fractionated with ammonium sulfate. The 50-65% ammonium sulfate pellet was resuspended in 7 ml of buffer B (buffer A with 0.5 mM phenylmethylsulfonyl fluoride), 70 mM KCl and dialyzed overnight against 1 liter of the same buffer. The dialysate was applied to a DE-52 column (6.5-ml bed volume) at a linear flow rate of 33 cm/h and washed with buffer B, 90 mM KCl until the absorbance of the eluate was below 0.1 A254. The column was developed with a 50-ml linear KCl gradient (90 to 450 mM) in buffer B. Peak fractions containing eIF5, identified by SDS-polyacrylamide gel electrophoresis, were pooled (10 ml) and dialyzed against 1 liter of buffer B, 100 mM KCl for 3 h. The dialysate was applied to a heparin-Sepharose (Amersham Pharmacia Biotech) column (6.4-ml bed volume) at a linear flow rate of 33 cm/h in buffer B, 100 mM KCl. The column was washed with four column volumes of the same buffer, then developed using a 52-ml linear KCl gradient (100 to 800 mM) in buffer B. eIF5 eluted from the column as a single peak at ~280 mM KCl. Peak fractions containing eIF5 were dialyzed against 1 liter of storage buffer (20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM Na2EDTA, 20% glycerol, 100 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride), spin-concentrated (Centricon 30, Millipore Corp.) to a concentration of 2 mg/ml, then frozen on dry ice and stored at -70 °C. Preparations of eIF5 were 80-85% pure, as judged by SDS-polyacrylamide gel electrophoresis (data not shown).

Nucleotide Binding Assays-- Assays to determine intrinsic dissociation of guanine nucleotides from eIF2 complexes were performed at 0 °C as described previously (14).

43 S Complex Stability Assay-- The 43 S complex stability assay was conducted in a three-stage reaction at 22 °C. The first stage consisted of ternary complex formation by incubation of 4.4 µg of yeast eIF2 with 24 pmol of [3H]Met-tRNAiMet for 15 min in a total volume of 110 µl (20 m Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM Na2EDTA, 1 mg/ml creatine kinase, 1 mM dithiothreitol, 1 mM GTP (or Gpp(NH)p), 5 mM MgCl2, 5% glycerol). Stage two consisted of the addition of 1.0 A260 unit of 40 S Artemia ribosomes and 0.2 A260 units of AUG codon (Oligos, Etc., Wilsonville, OR) followed by a 10-min incubation. For stage 3, the reaction mixture was split into 2 equal aliquots, and 1 µg of eIF5 or an equal volume of a control extract without eIF5 (see above) was added. After a 10-min incubation, the reaction mixture was chilled on wet ice, layered onto a 5-ml 5-25% sucrose gradient (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM Na2EDTA, 1 mM dithiothreitol, 5 mM MgCl2), and centrifuged at 200,000 × g for 2 h 20 min. Fractions (300 µl) were collected from the top of the gradient using an ISCO model 640 gradient fractionator with continuous monitoring at 254 nm. The percentage of each fraction was measured for radioactivity by scintillation counting in 5 ml of UltimaGold (Packard Instrument Co.).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Absence of the eIF2alpha Subunit Does Not Alter Dissociation of Guanine Nucleotides-- We purified eIF2beta gamma and eIF2beta gamma K250R heterodimers from overexpressing yeast strains in which the eIF2alpha structural gene (SUI2) was deleted. Yields were comparable with those of heterotrimeric eIF2 proteins reported previously (14). The purified proteins were 80-85% pure, as judged by densitometric analysis of stained gels (Fig. 1). In addition, the two subunits (beta  and gamma ) were present in an approximate 1:1 stoichiometry (Fig. 1, lanes 2 and 4), suggesting that the absence of the alpha -subunit does not affect association of beta - and gamma -subunits.



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Fig. 1.   Purification of eIF2 isoforms. 1.5 µg of eIF2 (lane 1) or eIF2gamma K250R (lane 3) and 1.0 µg of eIF2beta gamma (lane 2) or eIF2beta gamma K250R (lane 4) were subjected to SDS-polyacrylamide gel electrophoresis on a 12.5% acrylamide gel (29.8:0.2 acrylamide to bisacrylamide ratio) followed by staining with Coomassie Brilliant Blue R-250. The approximate masses (kDa) of pre-stained molecular weight markers are indicated to the left (Mr, Bio-Rad pre-stained calibrated SDS-polyacrylamide gel electrophoresis standards, catalog number 161-0318; soybean trypsin inhibitor, 28.9 kDa; carbonic anhydrase, 34.8 kDa; ovalbumin, 49.1 kDa; bovine serum albumin, 80 kDa; beta -galactosidase, 124 kDa; myosin, 209 kDa); eIF2 subunits are indicated on the right.

To determine the requirement for the alpha -subunit in guanine nucleotide binding, we measured nucleotide off-rates for eIF2, eIF2beta gamma , eIF2gamma K250R, and eIF2beta gamma K250R using a nitrocellulose filter binding assay. Reactions were performed at 0 °C to retard the rapid dissociation of nucleotide from gamma K250R-containing complexes. At 0 °C, wild-type eIF2 complexes showed little or no dissociation of eIF2·[3H]GDP (Fig. 2A), consistent with previously reported results (42). The absence of the alpha -subunit did not further enhance dissociation of the GDP-containing eIF2 binary complexes. Dissociation of GTP (Fig. 2B) from wild-type eIF2 complexes, which is more rapid than GDP, was unaffected by the absence of the alpha -subunit. As expected, the presence of the K250R form of the gamma -subunit increased the intrinsic dissociation rate for both GDP and GTP, and the rate of GDP dissociation was not significantly altered in the absence of eIF2alpha (the GTP reaction is essentially complete at the first time point in both cases). These data suggest that the alpha -subunit of eIF2 does not play a role in guanine nucleotide binding.



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Fig. 2.   Intrinsic dissociation of guanine nucleotides from eIF2 isoforms. 35 pmol of each eIF2 isoform was pre-incubated with 500 pmol (5 µM) of [3H]GDP (A) or [gamma -32P]GTP (B). Bound radioactivity was chased at 0 °C with either water (dotted lines, open symbols) or 5 mM unlabeled GDP (solid lines, closed symbols), wild-type eIF2 (black-diamond , diamond ), eIF2beta gamma (black-triangle, triangle ), eIF2gamma K250R (black-square, ), eIF2beta gamma K250R (, open circle ). Assays were performed in triplicate; vertical bars represent standard error

Removal of the alpha -Subunit Has a Mild Effect on Met-tRNAiMet Binding by eIF2 in Vitro-- We examined the affinity of the different eIF2 isoforms for Met-tRNAiMet by measuring equilibrium binding at various concentrations of [3H]Met-tRNAiMet in the presence of saturating levels of GTP. Both eIF2beta gamma and eIF2beta gamma K250R bound Met-tRNAiMet in a GTP-dependent manner (Fig. 3A). Scatchard analysis was used to calculate apparent dissociation constants of 19, 62, and 107 nM for eIF2, eIF2beta gamma K250R, and eIF2beta gamma , respectively (Fig. 3B). This compares with previously reported apparent Kd values of 15 and 25 nM for eIF2 and eIF2gamma K250R, respectively (14). The largest difference, approximately 5-fold, was observed between eIF2 and eIF2beta gamma , although this difference in our filter binding assay is of questionable significance and is discussed further below. We suggest that the absence of the alpha -subunit has, at best, a very mild effect on tRNA binding in vitro.



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Fig. 3.   Met-tRNAiMet binding by eIF2 isoforms. A, assays were performed as described previously (14) using the indicated concentrations of [3H]Met-tRNAiMet and wild-type eIF2 (black-diamond ), eIF2beta gamma K250R (), and eIF2beta gamma (black-triangle). B, Scatchard analysis of data from A. Apparent dissociation constants (Kd) were estimated to be 19, 62, and 107 nM for wild type eIF2, eIF2beta gamma K250R, and eIF2beta gamma , respectively.

eIF2beta gamma Forms 43 S Complexes That Are Effective Substrates for eIF5-dependent GTP Hydrolysis-- In vitro eIF2·GTP·[3H]Met-tRNAiMet (ternary complex) interacts with the 40 S ribosomal subunit in an AUG codon-dependent fashion to form a stable 43 S preinitiation complex. This complex serves as a substrate for eIF5, which is required for hydrolysis of eIF2-bound GTP. Under these conditions, [3H]Met-tRNAiMet is released from the resulting eIF2·GDP binary complex. We developed an assay to measure this activity based upon similar assays reported by Chakrabarti and Maitra (41) and Chakravarti and Maitra (44) using yeast eIF2, recombinant yeast eIF5, and 40 S ribosomal subunits purified from brine shrimp eggs. Formation of 43 S complexes in this assay was dependent upon the presence of ternary complex, 40 S ribosomes, and AUG codon triplet (data not shown). Complexes were treated using either purified recombinant yeast eIF5 or extract prepared from recombinant bacteria harboring the expression vector alone (control extract), then subjected to centrifugation through sucrose gradients. The addition of control extract (Fig. 4A) or no treatment (data not shown) resulted in cosedimentation of 40 S ribosomes and [3H]Met-tRNAiMet using wild-type ternary complexes. The addition of yeast eIF5 led to the disappearance of radioactivity from the 40 S peak, presumably as a result of GTP hydrolysis. Because eIF2·GDP no longer binds the initiator tRNA, the weak association between the tRNA, AUG triplet, and the ribosome forms an unstable complex that is revealed by centrifugation through the sucrose gradient. That the disappearance of radioactivity from the 40 S complex is due to eIF5-mediated GTP hydrolysis is demonstrated by the persistence of radioactivity in the 40 S peak in eIF5-treated 43 S complexes assembled using ternary complexes containing the nonhydrolyzable GTP analog Gpp(NH)p (Fig. 4C). In the absence of the alpha -subunit, eIF2beta gamma readily formed 43 S complexes that appeared qualitatively identical to those formed using the wild-type eIF2 heterotrimer and were also effective substrates for eIF5-mediated GTP hydrolysis (Fig. 4B). This suggests that the alpha -subunit of eIF2 is not required in vitro for formation of 43 S initiation complexes or for eIF5-mediated GTP hydrolysis.



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Fig. 4.   43S complex formation and eIF5-dependent GTP hydroylsis using eIF2 and eIF2beta gamma ternary complexes. Preinitiation complexes were formed using 40 S ribosomal subunits, AUG codon triplet, and ternary complexes containing [3H]Met-tRNAiMet and eIF2·GTP (A), eIF2beta gamma ·GTP (B), or eIF2·Gpp(NH)p (C) and treated with recombinant yeast eIF5 (closed circles, solid lines) or control extract without eIF5 (open circles, dashed lines). Complexes were subjected to centrifugation through 5-25% sucrose gradients; fractions were collected, and radioactivity was measured by liquid scintillation counting. Curves shown were reproducible in experiments performed in duplicate or triplicate.

eIF2alpha Provides a Structural Function in Catalyzed Nucleotide Exchange-- Progress curves comparing the eIF2B-catalyzed release of [3H]GDP from binary complexes indicated that the absence of the alpha -subunit in eIF2beta gamma appeared to have an effect on the reaction compared with wild-type eIF2. However, neither the addition of eIF2B nor the absence of eIF2alpha had a significant effect on the intrinsic dissociation of [3H]GDP from binary complexes formed using eIF2gamma K250R or eIF2beta gamma K250R (these latter curves were essentially identical to Fig. 2A) (data not shown).

We used steady-state kinetic analysis to better characterize eIF2beta gamma ·[3H]GDP as a substrate in catalyzed nucleotide exchange. Reaction velocities were measured at early time points using substrate concentrations between 18.9 and 250 nM. Extrapolation of x and y intercepts from reciprocal plots of velocity versus substrate concentration (Fig. 5) yielded values for Km and Vmax of 193 nM and 1980 fmol/min, respectively. These data, when compared with our previously reported values of 12 nM and 251 fmol/min using wild-type heterotrimeric eIF2 and the same preparation and concentration of eIF2B (42) indicated a 16-fold increase in Km and a smaller 8-fold increase in Vmax using the eIF2beta gamma ·[3H]GDP substrate. We interpret these data as suggesting that the presence of eIF2alpha is required for structural interactions with the eIF2B enzyme that contribute to catalyzed nucleotide exchange. These interactions may be direct or indirect and are discussed further below. The 8-fold increase in Vmax in the absence of eIF2alpha results in a similar 8-fold increase in kcat and suggests that the presence of the alpha -subunit acts to retard catalyzed nucleotide exchange (i.e. turnover is less efficient) in a manner that is not dependent upon phosphorylation. The combined increase in both Km and Vmax reduces the specificity constant (kcat/Km) for eIF2beta gamma ·[3H]GDP by 50% compared with the wild-type heterotrimer, and thus, it remains a relatively good substrate for catalyzed exchange.



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Fig. 5.   Kinetic analysis of eIF2B-catalyzed exchange using eIF2beta gamma ·[GDP] as substrate. Substrate concentrations (eIF2beta gamma ·[3H]GDP) used were 18.9, 22.2, 27.8, 33.3, 125, 142.9, and 250 nM. Exchange reactions were initiated by the addition of 5.8 nM eIF2B and 5 mM unlabeled GDP. Reaction velocities were determined at time points at which no more than 10% of the substrate had been consumed. All velocities were determined in triplicate and varied by less than 3%.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Considerable progress has been made toward understanding the function of individual eIF2 subunits as relates to the overall activity of the eIF2 heterotrimer. In the case of the alpha -subunit, much effort has been directed toward understanding its role in regulating nucleotide exchange via reversible phosphorylation at the conserved serine 51 (reviewed in Refs. 5, 27, 28, and 29). Genetic analysis in yeast has suggested an additional function for eIF2alpha in selecting the translational start site (45). In this report, we used a biochemical approach to analyze the function of eIF2alpha by examining the activity of purified eIF2beta gamma heterodimers in vitro. Our eIF2beta gamma proteins were purified from yeast strains in which the normally essential SUI2 (eIF2alpha ) gene was deleted but in which lethality was suppressed by increased gene dosage of SUI3 (eIF2beta ), GCD11 (eIF2gamma ), and IMT (initiator tRNA). These preparations were completely devoid of the alpha -subunit and differ from previously reported alpha -subunit-deficient forms of purified eIF2 that contained reduced, but measurable, amounts of the alpha -subunit (16, 25).

Removal of the alpha -subunit did not alter the rate of guanine nucleotide dissociation in the presence of either the wild-type gamma -subunit or gamma K250R relative to the corresponding heterotrimeric proteins. This suggests that the alpha -subunit does not participate in guanine nucleotide binding and contradicts results from earlier studies that suggested a direct role for eIF2alpha in binding guanine nucleotide (46). Indeed, nucleotide binding by eIF2 appears to be a direct function of the gamma -subunit, although participation by eIF2beta cannot be excluded (12, 14, 16, 17, 47). Binding of initiator tRNA also appears to be a direct function of eIF2gamma , with additional data suggesting participation by the beta -subunit (8, 14). In the absence of the alpha -subunit, the apparent dissociation constant for Met-tRNAiMet increased 5-fold for eIF2beta gamma (107 nM) compared with the wild-type eIF2 complex (Fig. 3B), with a smaller 2-fold increase for eIF2beta gamma K250R compared with eIF2gamma K250R. The apparent Kd value obtained using eIF2beta gamma falls near the upper limits of values that we have obtained using different wild-type eIF2 preparations (up to 70 nM).3 Thus, the differences observed may represent normal variation or, at best, a mild defect in tRNA binding. In either case, the significance of even a 5-fold difference may be minimal in vivo, as the normal range of concentrations for initiator tRNA in mammalian cells has been estimated at 85-310 nM (48), 40-160 nM (49), and 100-200 nM (50). No such estimates are available for yeast as far as we are aware. Therefore, we cannot exclude the possibility of a mild tRNA binding defect, which may in part explain the requirement for increased IMT gene dosage in some suppressed Delta sui2 strains.2 A mild increase in Kd for tRNA binding would also be consistent with the reduced specific activity of alpha -deficient eIF2 reported by Anthony et al. (16).

Ternary complexes containing eIF2beta gamma , GTP, and Met-tRNAi Met formed stable 43 S preinitiation complexes in the presence of the AUG codon in vitro, indicating the absence of an absolute requirement for the alpha -subunit for interaction between eIF2 and the ribosome. Although genetic analysis in yeast indicates participation of eIF2alpha in start site recognition (45), the viability of our Delta sui2 strains and the formation of AUG-dependent preinitiation complexes in vitro using eIF2beta gamma indicates that the contribution of the alpha -subunit to this function is dispensable. Preinitiation complexes containing eIF2beta gamma ternary complexes were also substrates for eIF5-mediated GTP hydrolysis. Interaction of eIF5 with eIF2 has been shown to require the N-terminal lysine-rich region of eIF2beta (21, 24), and our data suggest that this interaction is not abolished in the absence of eIF2alpha . Because eIF2 GTPase activity is also ribosome-dependent, it appears that interactions between eIF2beta gamma and the ribosome are sufficient to maintain eIF2 in a conformation that is recognized by eIF5.

Although the absence of the alpha -subunit did not appear to alter intrinsic dissociation of guanine nucleotides, the Km for eIF2beta gamma ·GDP in eIF2B-catalyzed nucleotide exchange increased by 18-fold compared with eIF2·GDP. This is the first biochemical demonstration of a contribution of the alpha -subunit to eIF2B-mediated nucleotide exchange that is independent of phosphorylation. Hinnebusch and co-workers (51, 52) proposed a general mechanism for eIF2B-mediated nucleotide exchange based upon analysis of yeast strains either harboring mutations in eIF2B subunits or overexpressing various combinations of eIF2 or eIF2B subunit genes. In their model, an initial interaction between eIF2 and eIF2B is proposed involving eIF2alpha and the alpha (Gcn3p), beta (Gcd7p), and delta (Gcd2p) eIF2B subunits. This is supported by studies of overexpressing strains, in which Gcn3p, Gcd2p, and Gcd7p appear to form an independent subcomplex that is capable of recognizing the phosphorylation status of eIF2alpha (52, 53). In addition, specific mutations in the structural genes encoding these subunits bypass the toxic effects of hyperphosphorylation at serine 51 (51, 54). The proposed initial interaction would be nonproductive but, in the absence of phosphorylation at serine 51, rapidly undergoes isomerization to a productive (catalytic) mode dominated by interactions involving eIF2Bgamma (Gcd1p) and eIF2Bepsilon (Gcd6p). These subunits comprise the eIF2B catalytic core, and evidence exists in both yeast and mammalian systems for the primary role of eIF2Bepsilon in catalysis (52, 55, 56). Although the gamma -subunit of eIF2 is directly involved in guanine nucleotide binding, no evidence exists for a direct interaction between this eIF2 subunit and the eIF2B catalytic core. However, there is evidence from two studies for direct interaction between eIF2beta and the delta - and epsilon -subunits of mammalian eIF2B (20) or the yeast eIF2Bepsilon subunit (21), but the region of eIF2beta required for the interaction remains controversial. Given the effect on Km for the exchange reaction upon removal of eIF2alpha , our results indicate that structural interactions between eIF2B and eIF2 that require the presence of eIF2alpha contribute to wild-type rates of catalysis and are consistent with the ability of eIF2B to recognize the presence of the eIF2alpha subunit. In view of the current model, it is possible that removal of the alpha -subunit bypasses the proposed initial nonproductive interaction, involving eIF2alpha and the eIF2Balpha beta delta regulatory core, and proceeds more or less directly to the productive (catalytic) interaction. This interaction, involving primarily eIF2beta (gamma ?) and eIF2Bgamma epsilon may in fact be weaker than nonproductive or productive interactions involving eIF2alpha . These additional contacts mediated by the alpha -subunit may also be inhibitory to nucleotide exchange, as suggested by the increase in Vmax observed upon their removal as in the eIF2beta gamma ·GDP substrate. The increased Vmax may therefore reflect the absence of a slow step (e.g. involving the nonproductive interaction) in the exchange reaction. Taken together, our data are consistent with the idea that the eIF2 alpha -subunit occupies a key position within the eIF2 complex through which it can mediate eIF2B-catalyzed nucleotide exchange.

Regardless of the precise mechanism, our results indicate that the alpha -subunit of eIF2 plays an important role in catalyzed nucleotide exchange independent of its role in regulation in response to phosphorylation. Despite the effect on nucleotide exchange, the overall similarity between the activities of eIF2beta gamma and wild-type heterotrimeric eIF2 complexes in vitro is consistent with the viability of the suppressed Delta sui2 strains and suggests that in vivo eIF2beta gamma is able to form ternary complexes, interact with ribosomes, messenger RNA, and (presumably) other initiation factors, and also serve as a substrate for eIF5-mediated GTP hydrolysis. The effect on catalyzed nucleotide exchange is apparently compensated in Delta sui2 strains by cooverexpression of initiator tRNA and the beta - and gamma -subunits of eIF2 in addition to the intrinsic rate of nucleotide dissociation seen for yeast eIF2 (14, 57). The latter suggestion is supported by the observation of more efficient suppression in the presence of eIF2beta gamma K250R.2 However, the presence of the alpha -subunit results in a more effective interaction with eIF2B in vitro. We suggest that this interaction is critical if eIF2B is to efficiently monitor and respond to the phosphorylation status of the alpha -subunit in vivo. In this respect, it is likely that a subset of the GCD2 and GCD7 mutations that render eIF2B less sensitive to the inhibitory effects of eIF2alpha phosphorylation might likewise increase Km for the eIF2 substrate. These studies provide a framework for further analysis of interactions between eIF2 and eIF2B and, in particular, those interactions specifically involving the catalytic or regulatory eIF2B subcomplexes.


    ACKNOWLEDGEMENTS

We thank Alan Hinnebusch and William Merrick for helpful discussions and valuable comments and suggestions on the manuscript.


    FOOTNOTES

* This work was supported by the American Cancer Society Grant RPG-97-061-01-NP.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, The University of Texas at Dallas, Mail Station FO3.1, P. O. Box 830688, Richardson, TX 75083-0688. Tel.: 972-883-2505; Fax: 972-883-2409; E-mail: hannig@utdallas.edu.

Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M007398200

2 F. L. Erickson, J. Nika, and E. M. Hannig, submitted for publication.

3 S. Rippel and E. M. Hannig, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: eIF2, wild-type heterotrimeric eukaryotic initiation factor 2; eIF2gamma K250R, eIF2 heterotrimer with altered gamma  subunit (K250R); eIF2beta gamma or eIF2beta gamma K250R, heterodimeric eIF2 containing beta - and gamma -(or gamma K250R) subunits; eIF2alpha gamma , heterodimeric eIF2 containing the alpha - and gamma -subunits; Gpp(NH)p, guanosine 5'-(beta ,gamma -imido)triphosphate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Hinnebusch, A. G., and Liebman, S. W. (1991) in The Molecular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics (Broach, J. R. , Pringle, J. R. , and Jones, E. W., eds) , pp. 627-735, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
2. Kozak, M. (1999) Gene 234, 187-208[CrossRef][Medline] [Order article via Infotrieve]
3. Merrick, W. C. (1992) Microbiol. Rev. 56, 291-315[Abstract]
4. Pain, V. M. (1996) Eur. J. Biochem. 236, 747-771[Abstract]
5. Rhoads, R. E. (1993) J. Biol. Chem. 268, 3017-3020[Free Full Text]
6. Castilho-Valavicius, B., Yoon, H., and Donahue, T. F. (1990) Genetics 124, 483-495[Abstract/Free Full Text]
7. Dorris, D. R., Erickson, F. L., and Hannig, E. M. (1995) EMBO J. 14, 2239-2249[Abstract]
8. Gaspar, N. J., Kinzy, T. G., Scherer, B. J., Humbelin, M., Hershey, J. W., and Merrick, W. C. (1994) J. Biol. Chem. 269, 3415-3422[Abstract/Free Full Text]
9. Hannig, E. M., Cigan, A. M., Freeman, B. A., and Kinzy, T. G. (1993) Mol. Cell. Biol. 13, 506-520[Abstract]
10. Erickson, F. L., Harding, L. D., Dorris, D. R., and Hannig, E. M. (1997) Mol. Gen. Genet. 253, 711-719[CrossRef][Medline] [Order article via Infotrieve]
11. Keeling, P. J., Fast, N. M., and McFadden, G. I. (1998) J. Mol. Evol. 47, 649-655[Medline] [Order article via Infotrieve]
12. Bommer, U. A., Kraft, R., Kurzchalia, T. V., Price, N. T., and Proud, C. G. (1991) Biochim. Biophys. Acta 1079, 308-315[Medline] [Order article via Infotrieve]
13. Bommer, U. A., and Kurzchalia, T. V. (1989) FEBS Lett. 244, 323-327[CrossRef][Medline] [Order article via Infotrieve]
14. Erickson, F. L., and Hannig, E. M. (1996) EMBO J. 15, 6311-6320[Abstract]
15. Westermann, P., Nygard, O., and Bielka, H. (1980) Nucleic Acids Res. 8, 3065-3071[Abstract]
16. Anthony, D. D., Jr., Kinzy, T. G., and Merrick, W. C. (1990) Arch. Biochem. Biophys. 281, 157-162[Medline] [Order article via Infotrieve]
17. Dholakia, J. N., Francis, B. R., Haley, B. E., and Wahba, A. J. (1989) J. Biol. Chem. 264, 20638-20642[Abstract/Free Full Text]
18. Donahue, T. F., Cigan, A. M., Pabich, E. K., and Valavicius, B. C. (1988) Cell 54, 621-632[Medline] [Order article via Infotrieve]
19. Flynn, A., Oldfield, S., and Proud, C. G. (1993) Biochim. Biophys. Acta 1174, 117-121[Medline] [Order article via Infotrieve]
20. Kimball, S. R., Heinzinger, N. K., Horetsky, R. L., and Jefferson, L. S. (1998) J. Biol. Chem. 273, 3039-3044[Abstract/Free Full Text]
21. Asano, K., Krishnamoorthy, T., Phan, L., Pavitt, G. D., and Hinnebusch, A. G. (1999) EMBO J. 18, 1673-1688[Abstract/Free Full Text]
22. Das, S., and Maitra, U. (2000) Mol. Cell. Biol. 20, 3942-3950[Abstract/Free Full Text]
23. Chaudhuri, J., Das, K., and Maitra, U. (1994) Biochemistry 33, 4794-4799[Medline] [Order article via Infotrieve]
24. Das, S., Maiti, T., Das, K., and Maitra, U. (1997) J. Biol. Chem. 272, 31712-31718[Abstract/Free Full Text]
25. Mouat, M. F., and Manchester, K. (1998) Mol. Cell. Biochem. 183, 69-78[CrossRef][Medline] [Order article via Infotrieve]
26. Gribskov, M. (1992) Gene 119, 107-111[CrossRef][Medline] [Order article via Infotrieve]
27. Hershey, J. W. (1989) J. Biol. Chem. 264, 20823-20826[Free Full Text]
28. Proud, C. G. (1992) Curr. Top. Cell Regul. 32, 243-369[Medline] [Order article via Infotrieve]
29. Samuel, C. E. (1993) J. Biol. Chem. 268, 7603-7606[Free Full Text]
30. Rowlands, A. G., Panniers, R., and Henshaw, E. C. (1988) J. Biol. Chem. 263, 5526-5533[Abstract/Free Full Text]
31. Dever, T. E., Yang, W., Astrom, S., Bystrom, A. S., and Hinnebusch, A. G. (1995) Mol. Cell. Biol. 15, 6351-6363[Abstract]
32. Wek, R. C. (1994) Trends Biochem. Sci. 19, 491-496[CrossRef][Medline] [Order article via Infotrieve]
33. Petryshyn, R., Levin, D. H., and London, I. M. (1983) Methods Enzymol. 99, 346-362[Medline] [Order article via Infotrieve]
34. Panniers, R. (1994) Biochimie (Paris) 76, 737-747[CrossRef][Medline] [Order article via Infotrieve]
35. Rowlands, A. G., Montine, K. S., Henshaw, E. C., and Panniers, R. (1988) Eur. J. Biochem. 175, 93-99[Abstract]
36. Trachsel, H., Ranu, R. S., and London, I. M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3654-3658[Abstract]
37. Dever, T. E., Feng, L., Wek, R. C., Cigan, A. M., Donahue, T. F., and Hinnebusch, A. G. (1992) Cell 68, 585-596[Medline] [Order article via Infotrieve]
38. Kimball, S. R., Antonetti, D. A., Brawley, R. M., and Jefferson, L. S. (1991) J. Biol. Chem. 266, 1969-1976[Abstract/Free Full Text]
39. Pain, V. M. (1994) Biochimie (Paris) 76, 718-728[CrossRef][Medline] [Order article via Infotrieve]
40. Sood, R., Porter, A. C., Ma, K., Quilliam, L. A., and Wek, R. C. (2000) Biochem. J. 346, 281-293[CrossRef][Medline] [Order article via Infotrieve]
41. Chakrabarti, A., and Maitra, U. (1991) J. Biol. Chem. 266, 14039-14045[Abstract/Free Full Text]
42. Nika, J., Yang, W., Pavitt, G. D., Hinnebusch, A. G., and Hannig, E. M. (2000) J. Biol. Chem. 275, 26011-26017[Abstract/Free Full Text]
43. Studier, F. W. (1991) J. Mol. Biol. 219, 37-44[Medline] [Order article via Infotrieve]
44. Chakravarti, D., and Maitra, U. (1993) J. Biol. Chem. 268, 10524-10533[Abstract/Free Full Text]
45. Cigan, A. M., Pabich, E. K., Feng, L., and Donahue, T. F. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2784-2788[Abstract]
46. Barrieux, A., and Rosenfeld, M. G. (1977) J. Biol. Chem. 252, 3843-3847[Abstract]
47. Naranda, T., Sirangelo, I., Fabbri, B. J., and Hershey, J. W. (1995) FEBS Lett. 372, 249-252[CrossRef][Medline] [Order article via Infotrieve]
48. Cooper, H. L., and Braverman, R. (1981) J. Biol. Chem. 256, 7461-7467[Free Full Text]
49. Safer, B., and Jagus, R. (1981) Biochimie (Paris) 63, 709-717[Medline] [Order article via Infotrieve]
50. Manchester, K. L. (1989) Biochem. Int. 18, 1279-1285[Medline] [Order article via Infotrieve]
51. Pavitt, G. D., Yang, W., and Hinnebusch, A. G. (1997) Mol. Cell. Biol. 17, 1298-1313[Abstract]
52. Pavitt, G. D., Ramaiah, K. V., Kimball, S. R., and Hinnebusch, A. G. (1998) Genes Dev. 12, 514-526[Abstract/Free Full Text]
53. Yang, W., and Hinnebusch, A. G. (1996) Mol. Cell. Biol. 16, 6603-6616[Abstract]
54. Vazquez de Aldana, C. R., and Hinnebusch, A. G. (1994) Mol. Cell. Biol. 14, 3208-3222[Abstract]
55. Gomez, E., and Pavitt, G. D. (2000) Mol. Cell. Biol. 20, 3965-3976[Abstract/Free Full Text]
56. Fabian, J. R., Kimball, S. R., Heinzinger, N. K., and Jefferson, L. S. (1997) J. Biol. Chem. 272, 12359-12365[Abstract/Free Full Text]
57. Ahmad, M. F., Nasrin, N., Bagchi, M. K., Chakravarty, I., and Gupta, N. K. (1985) J. Biol. Chem. 260, 6960-6965[Abstract/Free Full Text]


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