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Originally published In Press as doi:10.1074/jbc.M003718200 on June 13, 2000

J. Biol. Chem., Vol. 275, Issue 34, 26011-26017, August 25, 2000
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Purification and Kinetic Analysis of eIF2B from Saccharomyces cerevisiae*

Joseph NikaDagger , Weimin Yang§, Graham D. Pavitt§, Alan G. Hinnebusch§, and Ernest M. HannigDagger ||

From the Dagger  Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75080 and the § Laboratory of Eukaryotic Gene Regulation, NICHHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, May 2, 2000, and in revised form, May 31, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine nucleotide exchange factor for translation initiation factor 2 (eIF2). Recent studies in the yeast Saccharomyces cerevisiae have served to characterize genetically the exchange factor. However, enzyme kinetic studies of the yeast enzyme have been hindered by the lack of sufficient quantities of protein suitable for biochemical analysis. We have purified yeast eIF2B and characterized its catalytic properties in vitro. Values for Km and Vmax were determined to be 12.2 nM and 250.7 fmol/min, respectively, at 0 °C. The calculated turnover number (Kcat) of 43.2 pmol of GDP released per min/pmol of eIF2B at 30 °C is approximately 1 order of magnitude lower than values previously reported for the mammalian factor. Reciprocal plots at varying fixed concentrations of the second substrate were linear and intersected to the left of the y axis. This is consistent with a sequential catalytic mechanism and argues against a ping-pong mechanism similar to that proposed for EF-Tu/EF-Ts. In support of this model, our yeast eIF2B preparations bind guanine nucleotides, with an apparent dissociation constant for GTP in the low micromolar range.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic translation initiation factor 2 (eIF2)1 is a heterotrimeric G-protein that binds charged initiator tRNA (Met-tRNAiMet) in a GTP-dependent manner to form a stable ternary complex (1-3). Joining of this complex, along with eIF1A and eIF3, with the 40 S ribosomal subunit generates a 43 S preinitiation complex. In cap-dependent translation, this 43 S complex binds at or near the 5' end of capped mRNAs and "scans" the mRNA in a 5' to 3' direction until an AUG initiation codon in proper context is encountered (4). Initiation codon recognition is accompanied by hydrolysis of eIF2-bound GTP, which releases an eIF2·GDP binary complex and leaves Met-tRNAiMet bound at the ribosomal peptidyl site. The 60 S ribosomal subunit then joins, leading to the elongation phase of protein synthesis.

The eIF2·GDP binary complex is inactive in initiating protein synthesis and must be converted to the GTP-bound form in order to bind charged initiator tRNA and participate in subsequent cycles of initiation. This recycling process is facilitated in both yeast and mammals by the nucleotide exchange factor, eIF2B (5, 6). eIF2B is a 250-300-kDa heteropentamer whose subunits are conserved in yeast and mammals (6-9). In the yeast Saccharomyces cerevisiae, eIF2B subunits are encoded by the essential genes GCD1(gamma ), GCD2(delta ), GCD6(epsilon ), GCD7(beta ), and the non-essential gene GCN3(alpha ) (6, 7, 10).

The exchange reaction is tightly regulated (1-3, 5, 11). The best characterized mechanism is indirect and involves phosphorylation of the eIF2 alpha -subunit at a serine residue (Ser-51). The mammalian heme-regulated protein kinase, pancreatic eIF2alpha kinase/PKR-like endoplasmic reticulum kinase, and PKR kinases (12, 13), as well as mammalian, Drosophila, and yeast homologues of the yeast Gcn2p kinase (14-18) appear to phosphorylate eIF2alpha at Ser-51 in response to various physiological stresses that include heat shock (19, 20), viral infection (21), serum deprivation (20), heme deprivation (22), amino acid starvation (14, 23, 24), and endoplasmic reticulum stress (25). The ability of eIF2B to catalyze the exchange reaction on the phosphorylated eIF2·GDP substrate is severely limited (26-29). Furthermore, phosphorylated eIF2 acts as a potent competitive inhibitor of the exchange reaction due to its estimated 150-fold higher affinity for eIF2B (27, 30). These combined effects lead to an overall reduction of initiation rates in vitro and in vivo. Regulation of the exchange reaction is crucial for maintaining normal homeostasis, as transfection of NIH 3T3 cells with dominant negative PKR isoforms leads to malignant transformation (31, 32). This suggests further that regulation of the exchange reaction is required for maintenance of the cell cycle.

Other conditions that affect initiation rates appear to be independent of changes in the state of eIF2 phosphorylation and have been suggested to involve a more direct regulation of eIF2B function in vivo. These conditions include treatment of cells in tissue culture with growth factors (33-35), fertilization of oocytes (36), and exposure to ultraviolet radiation (37). In vitro, phosphorylation of eIF2B by casein kinase II leads to increased activity (38, 39), whereas phosphorylation by glycogen synthase kinase 3 antagonizes exchange factor activity, apparently by inhibiting phosphorylation by casein kinase II (40, 41).

The kinetics of the exchange reaction has been studied in mammalian systems (5, 27, 40, 42), but detailed information concerning the functions of individual eIF2B subunits is not available due to the lack of a suitable genetic system. Mutations in eIF2B subunit genes have been isolated and characterized in the yeast S. cerevisiae (7, 10, 43-45). The yeast enzyme has been purified and shown to catalyze nucleotide exchange in vitro (6). However, insufficient amounts of purified protein were obtained for detailed kinetic analysis. In this report, we describe a procedure for purification of yeast eIF2B from an overexpressing yeast strain. We define basic kinetic parameters for the yeast enzyme using standard Michaelis-Menton analysis, and we have applied these analyses to address the mechanism of the exchange reaction in vitro. The latter is a subject of controversy as a result of two recent studies of mammalian eIF2B (27, 42). Conclusions based upon these studies have been called into question by Manchester (46) in a recent theoretical discussion, thus inviting a renewed study of the kinetic mechanism for the eIF2B-catalyzed reaction. In agreement with Dholakia and Wahba (42), our kinetic analysis of the yeast enzyme supports a sequential (ternary complex) mechanism and appears to exclude a substituted enzyme ("ping pong") mechanism. Biochemical characterization of yeast eIF2B provides a framework for studies that, combined with genetic analysis, will provide for a more complete understanding of the function of eIF2B proteins in eukaryotic organisms.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Reagents-- [3H]GDP (10-15 Ci/mmol) and L-[methyl-3H]methionine (70-85 Ci/mmol) were from NEN Life Science Products; [gamma -32P]GTP (800 Ci/mmol) was from ICN Pharmaceuticals. Protease inhibitors and GDP were purchased from Sigma. Bulk yeast tRNA (Roche Molecular Biochemicals) was aminoacylated using radiolabeled methionine under conditions that preferentially charge the initiator methionyl-tRNA as described previously (47). All reagents were prepared with purified (MilliQ; Millipore Corp.) water.

Strains and Plasmids-- Yeast strain H2649 overexpressing wild-type eIF2B subunits Gcd6p, Gcd2p, Gcd7p, and Gcn3p and a 6-histidine-tagged form of Gcd1p was constructed as follows. Strain BJ1995 (MATalpha ura3-52 leu2 trp1 pep4-3 prb1-1122 gal2) (48) was transformed to Ura+ with MluI-digested pJB96 (7), a URA3-integrating plasmid bearing an unmarked gcd6Delta allele, and subsequently transformed with p1998, a low copy plasmid bearing GCD6 and TRP1, and p1305, a low copy plasmid bearing GCD7 and LEU2. (Plasmid p1998 contains the 2.6-kilobase pair XhoI-BamHI fragment bearing GCD6 from pJB85 (7) inserted between the corresponding sites in the polylinker of pRS314 (49); p1305 contains the 2.1-kilobase pair XhoI-NotI fragment from pJB99 (7) inserted between the corresponding sites in pRS315 (49).) A Ura- derivative, containing gcd6Delta in place of GCD6, was isolated by growth on medium containing 5-fluoroorotic acid (50) and transformed to Ura+ with the EcoRI-SphI fragment of pJB110 (7) bearing a gcd7Delta ::hisG::URA3::hisG allele. A Ura- derivative of this strain containing the unmarked gcd7Delta ::hisG allele was selected on 5-fluoroorotic acid medium and transformed with the high copy URA3 plasmid p1871 (30) bearing GCD2, GCD7, and GCN3. A Leu- segregant lacking p1305 was obtained after growth on minimal medium supplemented with leucine and then transformed with p2337, a high copy LEU2 plasmid bearing GCD6 and GCD1-6xHis (29), yielding H2649.

Purification of eIF2-- Yeast eIF2 was purified from an overproducing yeast strain as described previously (47).

Purification of Yeast eIF2B-- Strain H2649 was grown in 12 liters of rich medium (YEPD) (51) at 30 °C to an A600 of 4-8. (It is not necessary to maintain selection based on the nutritional markers present on each plasmid, since each plasmid contains one essential gene that is deleted from the chromosome of the host strain.) All subsequent manipulations were carried out at 4 °C. Cells were harvested by centrifugation, washed with 1 liter of ice-cold distilled de-ionized water (Millipore Corp.), and resuspended in 100 ml of lysis buffer (75 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM Na2EDTA, 12.5 mM 2-ME, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride). The suspension was combined with 175 ml of 0.5-mm acid-washed glass beads in a 350-ml stainless steel chamber (Biospec Products), and cells were lysed in a Bead-Beater (Biospec Products) for 4 cycles of 1 min breaking/1 min rest (chamber continuously cooled in ice/water bath). The lysate was removed, and beads were washed twice with 50 ml of lysis buffer. The pooled lysate was clarified via centrifugation for 20 min at 13,000 × g at 4 °C. The supernatant was centrifuged again at 200,000 × g for 2 h at 4 °C to pellet ribosomes. The supernatant was discarded, and ribosomes were resuspended in 100 ml of high salt buffer (20 mM Tris-HCl, pH 7.5, 600 mM KCl, 0.1 mM MgCl2, 25 µM GDP, 10% v/v glycerol, 6.25 mM 2-ME, and protease inhibitors as described above) and stirred for 30 min on ice. High salt-washed ribosomes were pelleted as above, and the supernatant was dialyzed overnight against 4 liters of P-11/150 Buffer (20 mM Tris-HCl, pH 7.5, 150 mM KCl, 0.1 mM MgCl2, 25 µM GDP, 6.25 mM 2-ME 10% glycerol, and protease inhibitors as described above). Insoluble material was cleared from the dialysate by centrifugation at 100,000 × g at 4 °C for 15 min. The supernatant was applied to a 1.5 × 23-cm phosphocellulose column (40-ml bed volume; P-11, Whatman) that had been pre-cycled as per the manufacturer's specifications. The column was washed with 10 volumes of P-11/150 buffer and eluted using a 500-ml 150 mM to 1 M linear KCl gradient in P-11 buffer. eIF2B eluted between 300 and 720 mM KCl. Fractions containing eIF2B activity were pooled, and imidazole concentration was adjusted to 10 mM. A 15-ml bed volume of pre-washed nickel NTA-agarose beads (Qiagen) was added to the pooled fractions, mixed for 1 h on ice, and transferred to a 1.5-cm ID column. Beads were packed by gravity flow (open column) and washed with 10 column volumes of wash buffer (20 mM Tris-HCl, pH 7.5, 600 mM KCl, 0.1 mM MgCl2, 100 µM GDP, 10 mM imidazole, 6.25 mM 2-ME, 10% glycerol, and protease inhibitors as above). Bound protein was eluted with wash buffer containing 200 mM imidazole, and protein-containing fractions were pooled (33 ml) and dialyzed against 2 liters of heparin/150 buffer (20 mM Tris-HCl, pH 7.5, 150 mM KCl, 0.1 mM MgCl2, 25 µM GDP, 1 mM DTT, 10% glycerol, 0.5 µg/ml pepstatin A, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride). Insoluble material was cleared by centrifugation, and the supernatant was applied to a heparin-Sepharose CL-6B column (1 × 12 cm; 10-ml bed volume; Amersham Pharmacia Biotech) that had been pre-cycled as per the manufacturer's instructions. The column was washed with 10 column volumes of heparin/300 buffer (heparin buffer with 300 mM KCl), and protein was eluted with a 50-ml linear 300-600 mM KCl gradient in heparin buffer. eIF2B eluted between approximately 460 and 530 mM KCl. Fractions containing eIF2B were identified by SDS-PAGE and Coomassie staining, pooled, and concentrated to a volume of 0.5 ml using a spin concentrator (Centri-Plus 100; Amicon). The protein was applied to a Superdex 200 HR10/30 FPLC gel filtration column (Amersham Pharmacia Biotech) that was pre-cycled as per the manufacturer's instructions. The column was developed with 2 column volumes of sizing buffer (20 mM Tris, pH 7.5, 600 mM KCl, 0.1 mM MgCl2, 25 µM GDP, 1 mM DTT). Fractions containing eIF2B, identified by SDS-PAGE, were pooled and dialyzed against storage buffer (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM MgCl2, 1 mM DTT, 50% glycerol). Protein was stored in small aliquots at -70 °C.

Antibodies-- All antibodies were polyclonal and were raised in female New Zealand White rabbits. Gcn3p (10), Gcd6p (45), Gcd7p (45), and Gcd1p (52) antibodies have been described previously. Purified GST-Gcd2p fusion protein expressed in Escherichia coli was used as antigen to raise Gcd2p antibodies. The GST-Gcd2p expression plasmid Ep1059 contained a 1.3-kilobase pair HindIII/EcoRV restriction fragment encoding residues 254-651 at the carboxyl terminus of Gcd2p. The HindIII site was flush-ended with Klenow enzyme, and the resulting fragment was ligated to SmaI-cleaved pGEX-2TK (Amersham Pharmacia Biotech). The insoluble fusion protein was harvested as described previously (53) and purified by preparative SDS-PAGE (54). Rabbits were immunized using 0.5-mg aliquots of protein as described previously (43).

SDS-PAGE and Western Blotting-- SDS-PAGE analysis (29.8:0.2 acrylamide/methylene-bisacrylamide) was as described (43). Western blots were performed as described previously (55) and developed using alkaline phosphatase-conjugated secondary antibody (Bio-Rad) and a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate system (Kirkegaard & Perry Laboratories).

Nucleotide Exchange Reactions-- Exchange reactions contained 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM Na2EDTA, 1 mg/ml creatine kinase (as carrier), 1 mM DTT, 5% glycerol (exchange buffer), indicated concentrations of eIF2B, eIF2, [3H]GDP, and GDP in a total volume of 120 µl. eIF2 was preincubated with [3H]GDP in a total volume of 96 µl of exchange buffer for 5 min at room temperature, transferred to a 0 °C water bath, and incubated for 15 min. In a second mixture, eIF2B was preincubated (in exchange buffer) with unlabeled GDP as above in a total volume of 20 µl. After the 15-min incubation in ice water, 16 µl of the eIF2 mixture was removed, transferred to 1 ml of ice-cold wash buffer (exchange buffer with no GDP), and vacuum-filtered through a pre-wetted nitrocellulose filter (0.45 micron; MSI, Westboro, MA; catalogue number E04WP02500). The filter was washed with 5 ml of ice-cold wash buffer, dried, and counted in 5-ml Econofluor2 (Packard Instruments). This was used as the zero time point. Twenty microliters of the eIF2B mixture was added to the remaining eIF2 mixture, and 20-µl aliquots (to correct for the dilution due to addition of eIF2B and GDP) were withdrawn and filtered at various time points and counted as above. All measurements were performed in triplicate; [3H]GDP retained (picomoles) on the filters at each time point varied by less than 5%. Velocities are reported femtomoles of [3H]GDP released per min. All velocities were determined at time points at which no more than 10% of the eIF2·[3H]GDP substrate had been consumed.

eIF2B Nucleotide Binding Assay-- Nitrocellulose filter-binding assays to determine nucleotide binding by eIF2B were performed as described previously (42).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of Yeast eIF2B-- We purified yeast eIF2B from a protease-deficient yeast strain that contained the five eIF2B subunit genes on two high copy plasmids. Typical yields from 12 liters of late log phase culture were 500-800 µg of eIF2B protein of approximately 85% purity, as judged by densitometric analysis of stained gels (Fig. 1). A supernatant from high salt-washed ribosomes was considered as starting material in the purification table (Table I), since crude extract induced an off-rate from eIF2·[3H]GDP at 0 °C independent of the addition of unlabeled GDP (data not shown). The supernatant was fractionated successively on phosphocellulose, nickel-NTA-agarose, heparin-Sepharose, and Superdex 200, resulting in a 1600-fold enrichment in eIF2B activity. The entire fractionation procedure was completed in 3 days. The purification shown in Table I is typical for three independent purifications. The presence of each of the eIF2B subunits in the final preparation was confirmed by Western blot analysis using subunit-specific antisera (Fig. 1A, lanes 2-6). The final material was assayed for exchange factor activity using eIF2·[3H]GDP as substrate. Fig. 1B demonstrates that exchange activity required the presence of both eIF2B and unlabeled GDP; no activity was detected in the absence of added unlabeled nucleotide.


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  		Fig. 1.   Purity and activity of yeast eIF2B. A, lane 1, 5 µg of purified yeast eIF2B was fractionated by SDS-PAGE (10%), followed by staining with Coomassie Brilliant Blue R250; lanes 2-6, 100 ng of purified yeast eIF2B was fractionated as above and analyzed by Western blot analysis using antibodies specific for Gcd6p (lane 2), Gcd2p (lane 3), Gcd1p (lane 4), Gcd7p (lane 5), or Gcn3p (lane 6). Molecular mass markers (Mr) include soybean trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), and phosphorylase B (97.4 kDa) (Bio-Rad catalogue number 161-0304). Mobilities of eIF2B subunits are shown to the right of the figure. B, release of [3H]GDP from eIF2·[3H]GDP was measured at 0 °C in the presence of 2.5 mM unlabeled GDP (open circle ), 3.5 pmol eIF2B (), or 2.5 mM unlabeled GDP and eIF2B (Delta ) as described under "Experimental Procedures." For all reactions, eIF2·[3H]GDP was pre-formed by incubation of 35 pmol of eIF2 with 200 pmol of [3H]GDP.

                              
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Table I
Purification of eIF2B
Exchange assays were carried out at 0 °C, as described under "Experimental Procedures," in the presence of 400 µM unlabeled GDP. 1 µg of total protein was assayed at each step except for the heparin and Superdex eluates, where 0.5 µg of total protein was assayed. Protein concentrations were determined by the method of Bradford (67), using bovine serum albumin (Sigma) as a standard. Velocities were determined as described under "Experimental Procedures." Specific activity is reported as picomoles of [3H]GDP released per min/total protein (mg).

Kinetic Parameters of the Exchange Reaction-- Initial studies to characterize kinetic parameters for the yeast exchange reaction were performed at 0 °C, as eIF2·[3H]GDP is stable under these conditions over the time course of our assay in the absence of eIF2B (Fig. 1B). These studies were performed as described above using a filter-binding assay to measure the release of [3H]GDP from pre-formed eIF2·[3H]GDP complexes. Double-reciprocal plots of velocity versus substrate concentration were linear over a range of eIF2·[3H]GDP concentrations between 2.4 and 117.6 nM (Fig. 2, A-C) at saturating concentrations of unlabeled GDP, indicating that under these conditions the exchange reaction followed typical Michaelis-Menten kinetics. Velocities measured at 0 °C at eIF2·[3H]GDP concentrations ranging from 2.4 to 28.6 nM were plotted in double-reciprocal format (Fig. 2A). Apparent Km and Vmax values were extrapolated from the x and y intercepts and determined to be 12.2 nM and 250.7 fmol of [3H]GDP released per min, respectively (Fig. 2A). Similar values have been reported for mammalian eIF2B at 0 °C (6.7 nM and 420 fmol/min, respectively) (27).


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  		Fig. 2.   Determination of kinetic parameters of exchange reaction. For all reactions, eIF2·[3H]GDP binary complexes were pre-formed by incubation with 800 nM [3H]GDP. Unlabeled GDP (minimum 50-fold molar excess) was used as the chase. Exchange reactions were performed using 0.70 pmol of eIF2B (5.8 nM) at 0 °C (A), 0.14 pmol of eIF2B (1.16 nM) at 10 °C (B), or 0.07 pmol of eIF2B (0.58 nM) at 20 °C (C). Data were corrected for intrinsic loss of bound radioactivity at 10 (B) and 20 °C (C).

Because yeast eIF2·GDP preparations exhibit a significant off-rate at temperatures as low as 10 °C (47), it was possible that nucleotide exchange involving yeast eIF2 would be less dependent upon eIF2B at physiological temperatures when compared with mammalian factors, where eIF2·GDP complexes are relatively stable at 37 °C (27). This effect might be seen as a reduced turnover number (Kcat) for yeast eIF2B relative to the mammalian exchange factor, which was previously estimated as Vmax divided by moles of eIF2B required to attain that Vmax (27). We determined Kcat values for the yeast exchange reaction at 0, 10, and 20 °C after correcting for GDP dissociation in the absence of eIF2B. Velocities were determined at 10 and 20 °C at substrate concentrations ranging from 5.2 to 117.6 nM (Fig. 2, B and C). Double-reciprocal plots yielded Vmax values of 495.5 and 1091.7 fmol of GDP released per min at 10 and 20 °C, respectively (Fig. 2, B and C). Kcat values were calculated for all three temperatures (Table II) and plotted as described by the Arrhenius equation (Fig. 3). Extrapolation of this plot gave estimated Kcat values for yeast eIF2B of 43.2 and 110.2 pmol of GDP released per min·pmol eIF2B at 30 and 37 °C, respectively. This compares with 495 and 2300 pmol of GDP released per min·pmol eIF2B for mammalian factors at 30 and 37 °C, respectively (27). At either temperature, the turnover number for the yeast enzyme is reduced by an order of magnitude and is consistent with the notion that, relative to the mammalian factor, yeast eIF2 has a decreased requirement for eIF2B.

                              
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Table II
Kinetic parameters of exchange reactions at various temperatures
Kcat = Vmax/mol eIF2B and is similar to the turnover number for enzymatic reactions (27). Kcat is expressed as pmol of [3H]GDP released per min/pmol of eIF2B. NA, not applicable.


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  		Fig. 3.   Estimation of Kcat at physiological temperatures. Kcat values from Table II were plotted as described by the Arrhenius equation.

The Catalytic Mechanism of the Yeast eIF2B Exchange Reaction-- Previous studies to elucidate the catalytic mechanism of the mammalian eIF2B exchange reaction yielded conflicting results. Data from one study (27) supported a ping-pong mechanism (Fig. 4A) and appeared to exclude a sequential mechanism (Fig. 4B), whereas a second study (42) supported a sequential mechanism and excluded a ping-pong mechanism. To gain insight into the mechanism of the reaction catalyzed by the yeast enzyme, we determined reaction velocities at 0 °C using eIF2·[3H]GDP concentrations ranging from 2.4 to 28.6 nM (KeIF2·[3H]GDP ~6 nM; see below). Assays were performed at three fixed concentrations of unlabeled GDP (Fig. 5A), with the unlabeled nucleotide present in at least 50-fold molar excess relative to labeled GDP. In a reciprocal experiment (Fig. 5B), reaction velocities were determined using GDP concentrations ranging from 25 to 500 µM (KGDP ~100 µM; see below) at three fixed concentrations of eIF2·[3H]GDP (range 5.5-66 nM). Double-reciprocal plots in each case yielded a set of lines that intersected to the left of the y axis, consistent with a sequential mechanism, and appear to exclude a ping-pong mechanism, which would predict a series of lines with identical slopes. Replotting the data in Fig. 5 as the reciprocal of the fixed substrate versus the y intercept (1/Vmax(app)) gave estimates for KeIF2·[3H]GDP and KGDP of 6 nM and 100 µM, respectively (data not shown). TheKeIF2·[3H]GDP determined here is similar to the Km value of 12 nM determined in Fig. 2.


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  		Fig. 4.   Alternative proposed catalytic mechanisms for eIF2B. A, ping-pong (enzyme-substituted) mechanism for guanine nucleotide exchange. GDP bound by eIF2 is displaced by eIF2B creating a nucleotide-free eIF2·eIF2B complex. GTP converts eIF2 to its active form, displacing eIF2B in the process. B, sequential (ternary complex) mechanism for guanine nucleotide exchange. eIF2B binds GTP and eIF2·GDP to form a ternary complex (GTP·eIF2B·eIF2·GDP). eIF2B then catalyzes the exchange reaction to release GDP and eIF2·GTP.


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  		Fig. 5.   Double-reciprocal plots for yeast eIF2B activity. Release of [3H]GDP from eIF2·[3H]GDP was measured at 0 °C as described under "Experimental Procedures." For all assays, eIF2 was preincubated with 800 nM [3H]GDP prior to the addition of 0.7 pmol (5.8 nM) of eIF2B. A, eIF2·[3H]GDP concentrations were varied from 2.4 to 28.6 nM; unlabeled GDP was present at 50 µM (), 100 µM (open circle ), or 5 mM (black-down-triangle ). B, unlabeled GDP concentrations were varied from 25 to 500 µM; eIF2·[3H]GDP was present at 5.5 nM (black-down-triangle ), 17.6 nM (open circle ), or 66 nM().

Yeast eIF2B Binds Guanine Nucleotides-- The proposed sequential mechanism predicts a central complex of the form eIF2·GDP/eIF2B/GDP (or GTP; Fig. 4B), that is, the presence of a second nucleotide-binding site on the eIF2·eIF2B complex. We examined this hypothesis by testing the ability of eIF2B to bind either [3H]GDP or [gamma -32P]GTP using standard nitrocellulose filter-binding assays. Results in Fig. 6 demonstrate that purified yeast eIF2B binds guanine nucleotides in this assay, with GTP apparently bound more efficiently than GDP. Equilibrium binding using varying GTP concentrations indicated an apparent Kd of 1 µM (Fig. 7). It is unlikely that this activity represents a contaminant, since similar activities were present both before and after chromatography on Superdex 200. 


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  		Fig. 6.   Nucleotide binding by eIF2B. 4 µg of eIF2B was added in a mixture (120 µl total volume) containing either 800 µM [3H]GDP or 800 µM [gamma -32P]GTP. Samples were incubated 5 min at room temperature and then 15 min at 0 °C. 20-µl aliquots were vacuum-filtered through nitrocellulose disks, and bound radioactivity was measured. Data represent the average value for experiments performed in triplicate; small vertical bars represent standard error.


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  		Fig. 7.   Estimation of Kd for GTP by equilibrium binding. A, 1 µg of eIF2B (3.5 pmol) was incubated with the indicated concentration of [gamma -32P]GTP in a total volume of 50 µl for 5 min at room temperature. Reactions were transferred to a 0 °C ice water bath and incubated for 15 min. The reaction was then vacuum-filtered through nitrocellulose, as described under "Experimental Procedures," and bound radioactivity was determined by scintillation counting. Data represent the average value for experiments performed in triplicate; vertical bars represent standard error. B, Scatchard analysis of data in A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study represents the first kinetic analysis of yeast eIF2B. Our studies were facilitated by the development of a yeast strain containing multiple copies of each of the structural genes encoding the five eIF2B subunits and a purification scheme that resulted in greater than 1600-fold purification of the enzyme. The scheme utilized an initial enrichment to select proteins peripherally associated with ribosomes, followed successively by chromatography on phosphocellulose, nickel-NTA agarose, heparin, and Superdex 200. Phosphocellulose significantly enriched for eIF2B activity and also increased the total activity by greater than 9-fold (Table I), suggesting that this step removed an antagonist of eIF2B activity. Since eIF2 (free and complexed with eIF2B) remained present after this step (data not shown), it is unlikely that the increase in total activity was due to removal of phosphorylated eIF2. The physiological relevance of this potentially novel antagonist is presently unclear. The nickel-agarose column, used to take advantage of an oligohistidine tag engineered on the Gcd1p subunit, was the least effective of the four columns used, yielding 3.6-fold enrichment in activity (Table I). Although a significant amount of eIF2B is lost in this step in the flow-through and wash fractions, eIF2 is depleted beyond detectable levels as a result of the high salt conditions (0.6 M KCl) present in the nickel column buffer (Fig. 1A and data not shown). Heparin and gel filtration chromatography appear to remove contaminating proteins without reducing total activity.

Measurement of kinetic parameters for yeast eIF2B-catalyzed nucleotide exchange required that the assays be carried out 0 °C due to intrinsic dissociation of the eIF2·GDP substrate at higher temperatures. This intrinsic dissociation is apparent both in the presence and absence of magnesium (47, 56), which appears to stabilize mammalian eIF2·GDP complexes (57). Estimated Kcat values for nucleotide exchange catalyzed by yeast eIF2B at 30 and 37 °C were reduced by approximately 1 order of magnitude when compared with published values for mammalian eIF2B. It is possible that the overall rate of nucleotide exchange for yeast eIF2 in vivo is a function of both intrinsic and catalyzed components. The catalyzed component is required for vegetative growth in yeast, since the intrinsic rate alone is apparently insufficient to maintain viability under normal growth conditions.2 Additional factors may modulate the rate of nucleotide exchange in vivo by altering the activity of eIF2B or its substrate. A well known example of this occurs through phosphorylation at the conserved serine 51 on eIF2alpha (1-3, 58). On the other hand, studies of mammalian eIF2B indicate the potential for regulation via both covalent modification (e.g. phosphorylation) or binding of certain co-factors including NAD+ and related molecules, ATP, and GTP (5).

Our data suggest that the exchange reaction catalyzed by the yeast eIF2B enzyme proceeds via a sequential rather than a ping-pong mechanism. Recent attempts to characterize the catalytic mechanism for the mammalian reaction have yielded conflicting results. Studies similar to those shown in Fig. 5 reported by the Henshaw group (27) yielded a series of parallel lines, which appeared to exclude a sequential mechanism in favor of a ping-pong mechanism. The latter suggests that a product (GDP) is released prior to binding of the second substrate (GDP or GTP) and is similar to a mechanism proposed for EF-Ts-catalyzed nucleotide exchange (59). Studies from the Wahba group (42) yielded data similar to those reported here, that is, consistent with the sequential mechanism. Although the two groups studying the mammalian enzyme used different unlabeled nucleotides as the "chase," this presumably should not affect the overall pattern seen in Fig. 5. In fact, we observe a similar pattern using GTP as the chase (data not shown). Manchester (46) noted a potential source of discrepancy in the report of Rowlands et al. (27) as a result of omitting GDP concentrations below KGDP, an issue we have addressed in this report. An additional difference between the earlier reports is the use of eIF2B, free from detectable levels of eIF2, by the Wahba group (42) versus an eIF2B/eIF2 (1:1) complex by the Henshaw group (27). Our eIF2B preparations were free from detectable levels of eIF2. Although our results are consistent with the notion that the mechanism for nucleotide dissociation catalyzed by eIF2B is conserved via a sequential mechanism, Price and Proud (5) have pointed out that these data may also be consistent with a ping-pong mechanism in which GTP binding to eIF2B acts allosterically to release GDP from eIF2.

A prediction of the sequential model that is not predicted by the ping-pong mechanism is the requirement of the second substrate (GDP or GTP) for dissociation of eIF2·[3H]GDP. As shown in Fig. 1B, both eIF2·[3H]GDP and GDP (or GTP, data not shown) are required to observe nucleotide exchange using the yeast enzyme. However, under our experimental conditions, the inability to observe exchange in the absence of added GDP may be a result of the presence of free labeled nucleotide in our binary complex preparations and the relatively high affinity of eIF2 for this ligand. On the other hand Cigan et al. (6), using "desalted" binary complexes, observed exchange in the presence of cold guanine nucleotide but not adenine nucleotide, implying a requirement for a second substrate (i.e. GDP or GTP). A further prediction of the sequential reaction mechanism is the presence of an additional guanine nucleotide-binding site on the eIF2·GDP·eIF2B complex. By using an equilibrium filter-binding assay, we detected binding of purified eIF2B to both GDP and GTP, with a preference for binding GTP. Assuming a single binding site, approximately 15% of the eIF2B preparation was active in binding GTP under our experimental conditions. Further analysis of GTP binding indicated an apparent dissociation constant of approximately 1 µM, similar to a value of 4 µM determined by the Wahba group (42) for the mammalian enzyme. These results are somewhat unusual for most G proteins (including eIF2), which generally exhibit a preference for GDP (57, 60, 61). In this respect, eIF2B subunits appear to lack the canonical set of guanine nucleotide-binding sequence elements, although the 8-azido analogue of GTP has been cross-linked to the beta (Gcd7p) subunit (62). The gamma (Gcd1p) and epsilon (Gcd6p) subunits of both mammalian and yeast eIF2B contain a putative nucleotide-binding domain related to that found in nucleotidyltransferases (9, 63). In the case of the gamma -subunit, cross-linking studies suggest these residues may be involved in binding pyridine dinucleotides (9, 62).

The ability to purify sufficient amounts of yeast eIF2B for detailed kinetic analysis makes possible more in-depth studies to define the roles of individual subunits in eIF2B function. Genetic analysis in yeast suggests a strictly regulatory function for the non-essential Gcn3p subunit (10, 43, 45). Results from studies of overexpressing yeast strains suggest that Gcn3p can form a complex with the related proteins Gcd2p and Gcd7p and that this subcomplex binds tightly to phosphorylated eIF2 (29, 64, 65). Gcd6p and Gcd1p also appear able to form a subcomplex distinct from the Gcn3p·Gcd2p·Gcd7p complex. By using total yeast cell protein extracts from overexpressing strains as a source of eIF2B, Pavitt et al. (29) provided evidence that Gcd6p is responsible for catalysis and that this function is enhanced in the presence of Gcd1p. Furthermore, this activity is not inhibited by phosphorylated eIF2. The proposed catalytic role for Gcd6p is consistent with results of overexpression studies of mammalian subunit genes in insect cells (66). We have recently constructed yeast strains that bypass the requirement for eIF2B.2 These strains express an altered version of eIF2 that enhances its intrinsic rate of nucleotide exchange and suppresses the otherwise lethal deletion of the four essential eIF2B subunit genes. The use of these strains to express specific subsets of eIF2B subunits will allow us to purify subcomplexes devoid of even low levels of contaminating subunits, thereby avoiding potential problems associated with overexpressing strains. Comparison of biochemical and kinetic properties of these subcomplexes, as well as heteropentameric complexes containing subunit-specific alterations, with those of wild-type complexes determined in this report, will allow for a more detailed analysis of subunit-specific roles in both catalysis and regulation.

    ACKNOWLEDGEMENTS

We thank Les Erickson and Steve Levene for valuable discussions and suggestions during the course of this work.

    FOOTNOTES

* This work was supported by American Cancer Society Grant RPG-97-061-01-NP (to E. M. H.).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.

Present address: Dept. of Anatomy and Physiology, University of Dundee, MSI/WTB Complex, Dow Street, Dundee, DD1 5EH, UK.

|| To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, The University of Texas at Dallas, Mail Station FO 3.1, P. O. Box 8306888, Richardson, TX 75083-0688. Fax: 972-883-2409; E-mail: hannig@utdallas.edu.

Published, JBC Papers in Press, June 13, 2000, DOI 10.1074/jbc.M003718200

2 F. L. Erickson, J. Nika, and E. M. Hannig, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: eIF2, eukaryotic translation initiation factor 2; eIF2B, eukaryotic initiation factor 2B (guanine nucleotide exchange factor); PKR, double-stranded RNA-regulated protein kinase; 2-ME, beta -mercaptoethanol; PAGE, polyacrylamide gel electrophoresis; NTA, nitrilotriacetic acid; DTT, dithiothreitol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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