Minimum Requirements for the Function of Eukaryotic Translation Initiation Factor 2

Corresponding author: Ernest M. Hannig, Department of Molecular and Cell Biology, University of Texas at Dallas, Mailstop FO3.1, P.O. Box 830688, Richardson, TX 75083-0688., hannig{at}utdallas.edu (E-mail)

Communicating editor: M. HAMPSEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Eukaryotic translation initiation factor 2 (eIF2) is a G protein heterotrimer required for GTP-dependent delivery of initiator tRNA to the ribosome. eIF2B, the nucleotide exchange factor for eIF2, is a heteropentamer that, in yeast, is encoded by four essential genes and one nonessential gene. We found that increased levels of wild-type eIF2, in the presence of sufficient levels of initiator tRNA, overcome the requirement for eIF2B in vivo. Consistent with bypassing eIF2B, these conditions also suppress the lethal effect of overexpressing the mammalian tumor suppressor PKR, an eIF2 kinase. The effects described are further enhanced in the presence of a mutation in the G protein () subunit of eIF2, gcd11-K250R, which mimics the function of eIF2B in vitro. Interestingly, the same conditions that bypass eIF2B also overcome the requirement for the normally essential eIF2 structural gene (SUI2). Our results suggest that the eIF2ß complex is capable of carrying out the essential function(s) of eIF2 in the absence of eIF2 and eIF2B and are consistent with the idea that the latter function primarily to regulate the level of eIF2·GTP·Met-tRNA_i^Met ternary complexes in vivo.

IN the current model for eukaryotic translation initiation (HINNEBUSCH and LIEBMAN 1991 ; MERRICK 1992 ; PAIN 1996 ), initiator methionyl-tRNA is delivered to the 40S ribosomal subunit in the form of a eukaryotic translation initiation factor 2 (eIF2)·GTP·Met-tRNA_i^Met ternary complex. The resulting 43S complex, which also includes eIF3 and eIF1A, binds at or near the 5' end of capped eukaryotic messenger RNAs in a process that appears to involve interactions between eIF3 and proteins bound at the mRNA 5' cap (SACHS et al. 1997 ). Once bound, the ribosome traverses the mRNA in a 5'-to-3' direction and locates the AUG codon representing the translational start site. Recognition of the start site is accompanied by GTP hydrolysis, which releases Met-tRNA_i^Met to the ribosomal peptidyl site and converts eIF2 to an eIF2·GDP binary complex. The eIF2·GDP complex must be converted to the GTP form to rebind Met-tRNA_i^Met and to participate in another cycle of translation initiation. Mammalian eIF2·GDP is extremely stable in vitro in the presence of physiological concentrations of magnesium and requires the guanine nucleotide exchange factor eIF2B to promote rapid conversion of eIF2·GDP to eIF2·GTP. Although the latter complex is less stable, GTP binding is stabilized upon binding Met-tRNA_i^Met.

In the yeast Saccharomyces cerevisiae, subunits of the eIF2 heterotrimer are encoded by the single-copy essential genes SUI2 (), SUI3 (ß), and GCD11 (). The primary structures of the eIF2 subunits are conserved between yeast and mammals (ERNST et al. 1987 ; DONAHUE et al. 1988 ; PATHAK et al. 1988 ; CIGAN et al. 1989 ; ERICKSON et al. 1997 ). The -subunit of eIF2 is a member of the GTP-binding (G) protein superfamily and is highly similar to eubacterial elongation factor EF1A (formerly EF-Tu; HANNIG et al. 1993 ; GASPAR et al. 1994 ). Functional similarity between EF1A and eIF2 proteins is further suggested by genetic and biochemical data that indicate the -subunit plays a significant role in binding nucleotide and tRNA ligands (ERICKSON and HANNIG 1996 ). The guanine nucleotide exchange factor for eIF2, eIF2B, is a heteropentamer that, in yeast, is encoded by four essential genes (GCD1, GCD2, GCD6, and GCD7) and one nonessential gene (GCN3) that are also conserved in mammals (BUSHMAN et al. 1993A ; CIGAN et al. 1993 ; KOONIN 1995 ; PRICE et al. 1996A , PRICE et al. 1996B ). The exchange reaction is regulated indirectly via phosphorylation of the -subunit of eIF2, which converts eIF2 into a competitive inhibitor of the exchange reaction (ROWLANDS et al. 1988 ; DEVER et al. 1995 ; KIMBALL et al. 1998 ). In vivo, this leads to reduced global rates of translation initiation and, in some cases, gene-specific enhancement of translation (HERSHEY 1991 ; HINNEBUSCH 1993 ; RHOADS 1993 ). A mammalian eIF2 kinase, PKR, has been proposed to function as a tumor suppressor and underlies the importance of regulating the exchange reaction for normal homeostasis in higher eukaryotic organisms (KOROMILAS et al. 1992 ; MEURS et al. 1993 ; BARBER et al. 1995A , BARBER et al. 1995B ; DONZE et al. 1995 ).

We previously described a mutation, gcd11-K250R, that conferred phenotypes consistent with reduced eIF2 function, i.e., reduced growth rates and increased expression of GCN4 (ERICKSON and HANNIG 1996 ). This mutation alters the lysine residue within the NKXD nucleotide-binding motif of eIF2 that is conserved in G proteins (DEVER et al. 1987 ; BOURNE et al. 1991 ). Both phenotypes were suppressed by increased dosage of the yeast initiator tRNA gene (IMT). In vitro, gcd11-K250R led to increased dissociation rates for both eIF2·GDP and eIF2·GTP, while the binding of Met-tRNA_i^Met to eIF2·GTP complexes stabilized GTP binding for both the wild-type and _K250R forms of eIF2 (ERICKSON and HANNIG 1996 ). Together, these data suggested that the _K250R alteration might also promote more rapid formation of ternary complexes and thereby reduce the requirement for eIF2B in vivo. In this article, we confirm these predictions. Furthermore, sufficient levels of wild-type eIF2 also reduce the requirement for eIF2B in vivo, though much less efficiently than in the presence of gcd11-K250R. This suggests that increasing the rate of dissociation of eIF2·GDP complexes is an essential function provided by eIF2B. Interestingly, the -subunit of eIF2, which appears to play an important role in eIF2/eIF2B interactions (VAZQUEZ DE ALDANA and HINNEBUSCH 1994 ; PAVITT et al. 1997 , PAVITT et al. 1998 ; NIKA et al. 2001 ), is no longer required for viability under conditions that lead to bypass of eIF2B. Our combined data suggest that the eIF2ß complex is capable of carrying out all essential eIF2 functions, including essential interactions with additional components of the eukaryotic translational machinery. We propose that eIF2B and the -subunit of eIF2 comprise an elaborate regulatory system for modulating levels of ternary complex that, although not essential per se for growth, plays a critical role in maintaining homeostasis and viability in wild-type cells.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmids:
pSB32 (LEU2), YCp50 (URA3; ROSE and BROACH 1991 ), and pRS316 (URA3; SIKORSKI and HIETER 1989 ) are yeast-Escherichia coli shuttle vectors containing yeast centromere sequences for maintenance in low copy number in yeast. YEp24 (URA3), YEp13 (LEU2; ROSE and BROACH 1991 ), and YEplac112 (TRP1; GIETZ and SUGINO 1988 ) are yeast-E. coli shuttle vectors maintained in high copy in yeast due to the presence of the 2µ origin of replication. Construction of YEp13/SUI2/SUI3/GCD11_His8 (Ep847), YEp13/SUI2/SUI3/gcd11_His8-K50R (Ep922), and YEplac112/IMT (Ep1013) were described previously (ERICKSON and HANNIG 1996 ). Unless otherwise noted, the GCD11 alleles used in this study contain a C-terminal octyl-histidine tag. The tag does not appear to alter the function of wild-type eIF2 in vivo or affect ligand binding in vitro (ERICKSON and HANNIG 1996 ). The galactose-inducible PKR construct (in YCp50) was a generous gift from T. Dever (National Institutes of Health, Bethesda, MD).

Ep1037 is a pSB32/GCD6/GCD7 plasmid. A 3.3-kb SpeI fragment from pJB6 (BUSHMAN et al. 1993A ) containing GCD6 was converted to a HindIII fragment following treatment with Klenow enzyme and ligation with HindIII linkers. This fragment was inserted into HindIII-cleaved pSB32 to create Ep1033. The GCD7 fragment was obtained from pJB100 (BUSHMAN et al. 1993A ) by first converting the EcoRI site to an EagI/NotI site using oligonucleotide linkers as described above. A 2.1-kb NotI fragment containing GCD7 was removed from the modified pJB100, ligated to EagI-cleaved YEp24 to create Ep673, and then subcloned as a 2.1-kb EagI fragment into EagI-cleaved Ep1033. The resulting plasmid, Ep1037, was cleaved with AatII (in vector sequences), flush-ended, and converted to an AscI site using oligonucleotide linkers to create Ep1066.

The pSB32/GCD1/GCD2/GCD6/GCD7 plasmid Ep1127 was constructed by inserting an AscI fragment containing GCD1 and GCD2 into AscI-cleaved Ep1066. The GCD1 fragment was obtained as a 2.4-kbp BamHI fragment from YCp50-Sc4014 (HILL and STRUHL 1988 ) that was initially subcloned into BamHI-cleaved pBluescript (Promega, Madison, WI) to create Ep1082. The GCD2 fragment was obtained from pY26 (DEVER et al. 1995 ) as a 2.6-kbp EagI fragment following conversion of the ClaI site to an EagI site as above. This fragment was subcloned into EagI-cleaved Ep1082. Vector XhoI and SacI sites were sequentially converted to AscI sites by linker tailing to create Ep1120. A 5.0-kb AscI fragment from Ep1120 containing GCD1 and GCD2 was then subcloned into AscI-cleaved Ep1066 to create Ep1127.

Additional plasmids containing the four essential eIF2B subunit genes, with or without GCD11_His8, were constructed as follows. Ep1174 (pBluescript with XhoI and SacI sites altered to AscI) was cleaved with BamHI and HindIII and ligated with a 2.1-kb BamHI/HindIII fragment containing GCD11_His8. The resulting plasmid (Ep1246) was digested with BamHI and ligated with the 2.4-kbp BamHI GCD1 fragment from YCP50-Sc4014. The resulting plasmid was cleaved with EagI and ligated with the 2.6-kbp EagI GCD2 fragment to create Ep1247. A 7.1-kb AscI fragment, containing GCD1, GCD2, and GCD11_His8, was ligated to AscI-cleaved Ep1067 and Ep1066 to create Ep1250 (pRS316/GCD1/GCD2/GCD6/GCD7/GCD11_His8) and Ep1262 (pSB32/GCD1/GCD2/GCD6/GCD7/GCD11_His8), respectively. The pRS316/GCD1/GCD2/GCD6/GCD7 plasmid Ep1125 was constructed by first inserting the 2.1-kb EagI GCD7 fragment from Ep673 into EagI-cleaved pJB5 to create Ep1042. The 5.1-kbp GCD1/GCD2 AscI fragment from Ep1120 was then inserted at the SalI site in Ep1042, which had been modified using AscI oligonucleotide linkers, to create Ep1125.

Plasmids used in the sui2 suppression experiments contained the 2.1-kbp HindIII/SnaBI GCD11 fragment (where the SnaBI site was altered to a BamHI site by linker tailing; HANNIG et al. 1993 ) and/or a 2.5-kbp BamHI fragment from pD14-6 containing SUI2 (a gift from T. Dever, National Institutes of Health). Construction of YEp13/GCD11_His8 (Ep832) and YEp13/gcd11-K250R_His8 (Ep921) were described previously (ERICKSON and HANNIG 1996 ). YEp13/GCD11_His8/SUI3 (Ep1064) and YEp13/gcd11-K250R_His8/SUI3 (Ep1065) were constructed by removing the 2.5-kbp BamHI SUI2 fragment from Ep847 and Ep922, respectively.

Strain construction:
The parent strain for EY878 (MAT leu2-3, 112 trp1-63 ura3-52 gcd1::hisG gcd2::hisG gcd6 gcd7::hisG gcn3::hisG <Ep1125>) is EY809 (MAT leu2-3, 112 trp1-63 ura3-52 gcd6 gcd7::hisG <Ep1042 (pRS316[URA3]/GCD6/GCD7>)). The gcd7::hisG allele (from pJB110; BUSHMAN et al. 1993A ) removes 66% (residues 75–325) of the 381-amino-acid GCD7 open reading frame (ORF). The gcd6 allele was from pJB96 (BUSHMAN et al. 1993A ), and removes 87% (residues 93–713) of the 713-amino-acid GCD6 ORF. The remainder of the eIF2B subunit genes were deleted in EY809 after replacing the pRS316-based plasmid with the low-copy LEU2 plasmid Ep1127 that contains GCD1, GCD2, GCD6, and GCD7. The gcd1::hisG-URA3-hisG allele (Ep1191) was derived from Ep175 (a URA3 disruption version of Ep174; HANNIG and HINNEBUSCH 1988 ) by replacing the disrupting URA3 fragment with the hisG-URA3-hisG cassette from pNKY51 (ALANI et al. 1987 ). This removes 52% (residues 1–299) of the 578-amino-acid GCD1 ORF. The gcd2::hisG-URA3-hisG allele (Ep1145) removes 93% (residues 26–632 on a PvuII/EcoRI restriction fragment) of the 651-amino-acid GCD2 ORF (PADDON et al. 1989 ). The gcn3::hisG-URA3-hisG allele was derived from Ep308 (HANNIG et al. 1990 ) by replacing the disrupting LEU2 fragment with hisG-URA3-hisG as above to create Ep545. This construct removes the entire 305-amino-acid GCN3 ORF. DNA fragments used for gene disruptions were obtained following digestion of the corresponding plasmids with BamHI (GCD1), NotI (GCD2), or BglI/PvuII (GCN3) and purification by agarose gel electrophoresis. Gene disruptions were confirmed by Southern blot analysis of Ura⁺ transformants, using appropriate probes to distinguish chromosomal and plasmid-borne (in Ep1127) alleles (data not shown). Chromosomal disruptants were plated on 5-fluoroorotic acid (5-FOA) medium to select for recombination between the direct hisG repeats, an event that evicts the URA3 gene, leaving behind a single copy of the hisG repeat. GCD2, GCD1, and GCN3 were disrupted sequentially in the EY809 background. The URA3 plasmid Ep1125 was then used to replace Ep1127 to create EY878. The absence of each essential eIF2B subunit gene in EY878 was also demonstrated genetically by the inability of plasmids containing only three of the four essential eIF2B subunit genes, in all possible combinations, to complement in the absence of Ep1125.

Disruption of GCD11 in EY878 utilized a derivative of EY878 in which Ep1262(LEU2) replaced Ep1125(URA3). GCD11 was then disrupted using the gcd11::hisG-URA3-hisG allele from Ep523 as described (DORRIS et al. 1995 ), followed by growth on 5-FOA. The URA3 plasmid Ep1250 was used to replace the LEU2 plasmid Ep1262 to create EY923.

EY740 (MATa leu2-3, -112 ura3-52 trp1-63 gcd11::hisG GAL2⁺ <Ep293; YCp50/GCD11>) was obtained as a meiotic segregant from a cross between a GAL2⁺ derivative of H1515 (MARTON et al. 1993 ) and EY585 (MAT leu2-3, -112 ura3-52 gcd11::hisG GAL2⁺ <Ep293>). The gcd11::hisG allele lacks the entire GCD11 open reading frame (HANNIG et al. 1993 ). EY835 (MAT leu2-3, -112 ura3-52 trp1-63 sui2 gcd11::hisG GAL2⁺ <Ep1130; Ycp50/GCD11/SUI2>) was obtained from a cross between EY740 and EY779 (MAT leu2-3, -112 ura3-52 trp1-63 sui2 <pSB32/SUI2>). The sui2 allele removes the N-terminal two-thirds of the SUI2 ORF (DEVER et al. 1992 ). EY779 is a derivative of H1816 (VAZQUEZ DE ALDANA and HINNEBUSCH 1994 ).

Growth rate determination:
Doubling times at 30° were determined for log-phase cultures grown in minimal (SD) media supplemented as necessary (SHERMAN et al. 1986 ).

eIF2B assay:
Reactions (30 µl) contained 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 0.1 mM Na₂EDTA, 1 mM dithiothreitol, 5% glycerol, 1 mg/ml creatine kinase (as carrier), 10 µM GDP, 100 µM GTP, and 300 nM [³H]Met-tRNA_i^Met (65 kcpm/pmol)³. eIF2 preparations (3 µg, >80% pure; ERICKSON and HANNIG 1996 ) were prebound to GDP for 10 min at 23° in the absence or presence of 1 µg yeast eIF2B (NIKA et al. 2001 ) and then transferred to a 10° water bath for an additional 5 min. Under these conditions, both wild-type eIF2 and eIF2_K250R are maximally bound to GDP (ERICKSON and HANNIG 1996 ). GTP and [³H]Met-tRNA_i^Met were then added, and 5-µl aliquots were withdrawn and filtered through nitrocellulose as described previously (ERICKSON and HANNIG 1996 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast eIF2 exhibits an intrinsic nucleotide exchange activity:
We measured nucleotide exchange activity by testing the ability of GDP-bound yeast eIF2 to form eIF2·GTP·Met-tRNA_i^Met ternary complexes in vitro (Fig 1). In mammalian systems, this reaction requires eIF2B to dissociate the eIF2·GDP complex (reviewed in MERRICK 1992 ). In contrast to the mammalian system, wild-type yeast eIF2·GDP readily formed ternary complexes in the absence of eIF2B, with 40% of maximal complex formation seen after 16 min. We attribute this effect to an appreciable intrinsic GDP off-rate for yeast eIF2 that is not seen for the mammalian factor (AHMAD et al. 1985 ; ERICKSON and HANNIG 1996 ). Addition of catalytic amounts of yeast eIF2B further increased ternary complex formation by wild-type eIF2 to 64% of maximum over the same time period. In the absence of eIF2B, eIF2_K250R displayed an apparent rate of ternary complex formation (at early time points) that was greater than that for wild-type eIF2 alone and similar to the eIF2B-promoted reaction. The possibility that the eIF2_K250R preparation was contaminated with eIF2B is unlikely, since the addition of eIF2B to the eIF2_K250R reaction did not further enhance ternary complex formation (i.e., reaction rates at early time points are similar). The ability of the _K250R alteration to mimic eIF2B activity in vitro suggested that gcd11-K250R strains might demonstrate a reduced requirement for eIF2B function in vivo.

View larger version (17K): In this window In a new window Download PPT slide   Figure 1. Effect of the K250R alteration on formation of ternary complexes by eIF2. The amount of [3H]Met-tRNAiMet bound by eIF-2 was determined by nitrocellulose filter binding (ERICKSON and HANNIG 1996 ) and reported as a percentage of maximal Met-tRNAiMet bound. , wild-type eIF2; , eIF2K250R; , wild-type eIF2 and eIF2B; , eIF2K250R and eIF2B. The data shown are the average of two experiments wherein data points differ by <15% at each time point. GTP-independent [3H]Met-tRNAiMet binding was minimal (<0.1 pmol in all cases) and was not subtracted. Reactions were repeated in the absence of added GDP to determine maximal [3H]Met-tRNAiMet binding. The figure 100% corresponds to the following amount of [3H]Met-tRNAiMet bound in each 5-µl aliquot: eIF2, 1.2 pmol; eIF2K250R, 1.0 pmol; eIF2 + eIF2B, 1.2 pmol; eIF2K250R + eIF2B, 1.1 pmol.

PKR-mediated growth inhibition is suppressed by increased eIF2 gene dosage:
We reasoned that yeast cells less dependent upon eIF2B might show decreased sensitivity to overexpression of the mammalian eIF2 kinase PKR, which confers a severe slow-growth phenotype in wild-type yeast (DEVER et al. 1993 ). This phenotype appears to result from an increase in phosphorylated eIF2 (at residue serine 51), which converts eIF2 to a competitive inhibitor of the exchange reaction (ROWLANDS et al. 1988 ; CHONG et al. 1992 ; DEVER et al. 1995 ). Because eIF2B is typically present at reduced levels relative to eIF2 (PRICE and PROUD 1994 ), the resulting functional sequestration of eIF2B leads to decreased levels of ternary complex and thus reduced growth rates. We constructed yeast strains harboring a high-copy plasmid containing GCD11 or gcd11-K250R in combination with SUI2 and SUI3, or a low-copy plasmid containing the GCD11 allele alone. Co-overexpression of the three eIF2 subunit genes has been shown to increase the level of this initiation factor complex at least fivefold in vivo (HANNIG et al. 1993 ; DEVER et al. 1995 ; ERICKSON and HANNIG 1996 ). The chromosomal GCD11 allele was deleted in these strains, which, in addition, harbored a plasmid containing a galactose-inducible PKR construct, as well as a high-copy IMT plasmid (or the empty vector). All strains grew well on dextrose media (Fig 2). In the absence of the high-copy IMT plasmid, all strains failed to grow upon induction of PKR expression on galactose media (data not shown). However, in the presence of increased IMT gene dosage, gcd11-K250R suppressed the PKR-mediated growth defect in the presence of elevated levels of eIF2 and eIF2ß (Fig 2), suggesting that suppression is mediated by the eIF2 complex. Under these same conditions wild-type eIF2 was a much weaker suppressor, as would be predicted on the basis of its slower intrinsic off-rate for GDP. eIF2_Y142H, which demonstrates a wild-type GDP off-rate in vitro (ERICKSON and HANNIG 1996 ), suppresses at a level similar to wild-type eIF2. The effects of overexpression of eIF2 are likely not due to inhibition of phosphorylation of the -subunit by PKR, as these conditions have been shown to increase the level of phosphorylated eIF2 in the cell (DEVER et al. 1995 ). Our results suggest that elevated levels of eIF2 and initiator tRNA reduce the requirement for eIF2B at a level proportional to the intrinsic nucleotide off-rate for yeast eIF2.

View larger version (34K): In this window In a new window Download PPT slide   Figure 2. Suppression of lethality associated with PKR expression in yeast. Three plasmids were introduced into yeast strain EY740 (MATa ura3-52 trp1-63 leu2-3 leu2-112 gcd11::hisG GAL2+): (i) a high-copy LEU2 plasmid (YEp13) containing genes encoding the (SUI2), ß(SUI3), and (GCD11) subunits of eIF-2, or a low-copy LEU2 plasmid (pSB32) encoding the -subunit only; (ii) a high-copy TRP1 plasmid (YEplac112; GIETZ and SUGINO 1988 ) containing the yeast IMT gene; and (iii) a low-copy URA3 plasmid (YCp50; ROSE and BROACH 1991 ) with (PKR) or without (vector) the mammalian PKR gene expressed from the inducible yeast GAL1 promoter. Strains representing independent isolates were grown to saturation in liquid minimal media containing 2% dextrose (SHERMAN et al. 1986 ), diluted, and 104 cells were spotted onto minimal media plates containing either 2% dextrose or 2% galactose and were then incubated for 2 days at 30°.

Bypass of the essential function of eIF2B:
If the only essential function provided by eIF2B is to promote the rapid dissociation of eIF2·GDP complexes, we reasoned that gcd11-K250R might suppress a deletion of some or all of the four essential eIF2B subunit genes. To test this hypothesis, we constructed a yeast strain lacking the chromosomal GCD1, -2, -6, and -7 genes that encode the essential eIF2B subunits (CIGAN et al. 1991 , CIGAN et al. 1993 ), as well as the nonessential eIF2B subunit gene GCN3 (HANNIG and HINNEBUSCH 1988 ). The resulting strain (EY878) is viable because it contains a low-copy URA3 plasmid harboring the four essential eIF2B subunit genes. To test the requirement for eIF2B in gcd11-K250R strains, we constructed LEU2 plasmids containing either gcd11-K250R or GCD11 in combination with SUI2 and SUI3. These plasmids were introduced into EY878 that also harbored either a high-copy IMT plasmid or the empty vector. Transformants were then plated on 5-FOA medium to examine the ability of the eIF2 plasmids to suppress the complete loss of essential eIF2B genes. 5-FOA selects for Ura^- cells that have lost the URA3 plasmid (BOEKE et al. 1987 ), which, in the case of EY878, contains the only copies of the essential eIF2B subunit genes. As shown in Fig 3, the gcd11-K250R/SUI2/SUI3 combination was an effective suppressor in the eIF2B quintuple-deletion strain. Suppression was dependent upon increased IMT gene dosage and, in addition, required high-copy expression of all three eIF2 subunit genes (Fig 3; data not shown). Western blot analysis confirmed the absence of detectable eIF2B subunits in the suppressed strain (data not shown). Overexpression of wild-type eIF2 also suppressed the complete absence of eIF2B in an IMT-dependent manner, albeit at a reduced level, consistent with the intrinsic nucleotide off-rate seen for wild-type eIF2·GDP complexes. Enhanced dissociation of eIF2·GDP resulting from the gcd11-K250R mutation (Fig 1) is consistent with more efficient bypass of the nucleotide exchange function of eIF2B. Our results lend strong support to the notion that the rapid dissociation of eIF2·GDP complexes is an essential eIF2B function.

View larger version (28K): In this window In a new window Download PPT slide   Figure 3. Suppression of the lethal effect associated with deletion of the five eIF2B subunit genes. Low-copy (L.C.; pSB32) or high-copy (H.C.; YEp13) LEU2 plasmids indicated to the left of the figure were introduced into the eIF2B quintuple deletion strain EY878, which also harbored either YEplac112 [TRP1] (Vector) or YEplac112/IMT (Vector + IMT). Plasmid transformants were selected, grown in liquid media containing uracil, and 5 µl of a 1:10 dilution was spotted onto minimal media containing uracil + 5-FOA (5-FOA) or uracil alone (SD + URA). Each spot corresponds to an independent isolate. This photograph was taken following incubation at 30° for 5 days. Low-copy LEU2 plasmids harboring any combination of three of the four essential eIF2B subunit genes failed to complement in EY878 (data not shown).

Bypass of the essential function of SUI2 (eIF2):
In cells that no longer require eIF2B, it is possible that certain eIF2 subunit(s) with which eIF2B interacts are not required. We chose the -subunit of eIF2 to test this idea, on the basis of previous genetic evidence that suggested a direct interaction between the -subunit of eIF2 and eIF2B (VAZQUEZ DE ALDANA et al. 1993 ; PAVITT et al. 1997 , PAVITT et al. 1998 ). We constructed a gcd11 sui2 strain (EY835) that harbored a URA3/GCD11/SUI2 plasmid and used the plasmid shuffle technique described above to examine the ability of GCD11 and gcd11-K250R constructs (LEU2) to suppress the sui2 mutation by conferring viability in the absence of the resident URA3 plasmid. To demonstrate the absence of chromosomal SUI2 and GCD11 in this strain, a low-copy LEU2 plasmid containing both SUI2 and GCD11, but not plasmids harboring either gene alone, supported the viability of EY835 in the absence of the URA3/GCD11/SUI2 plasmid (Fig 4). High-copy plasmids containing gcd11-K250R, either alone or in combination with SUI3 (eIF2ß), suppressed the sui2 mutation, increasing doubling times 1.5- to 2.2-fold compared with controls (Fig 5, bottom panel). Suppression by gcd11-K250R was independent of the presence of a multi-copy IMT plasmid, although suppression was more efficient with the IMT plasmid (20–40% decrease in doubling times). This result suggests that the two-subunit form (ß) of eIF2 is functional in this strain, but does not rule out the additional possibility that the -subunit alone is functional. A low-copy plasmid containing gcd11-K250R also suppressed sui2, albeit less efficiently (data not shown). Overexpression of wild-type GCD11 weakly suppressed sui2 (5-fold increase in doubling time) and suppression required co-overexpression of SUI3 and IMT. Our results suggest that the contribution of the -subunit to eIF2 function is not essential for ligand binding or the interaction of eIF2 with additional components of the translational apparatus.

View larger version (74K): In this window In a new window Download PPT slide   Figure 4. The -subunit of eIF2 is dispensable in gcd11-K250R strains. LEU2 plasmids harboring alleles listed to the left were introduced into yeast strain EY835 (MAT leu2-3, -112 trp1-63 ura3-52 sui2 gcd11::hisG <YCp50/SUI2/GCD11>) containing either YEplac112 (Vector) or YEplac112/IMT (Vector + IMT). Transformants were grown in minimal media containing uracil for 2 days at 30°, plated as described in Fig 2 (either undiluted or at a 1:10 dilution) on minimal media (SD) containing uracil and 5-FOA (5-FOA) or uracil alone (SD + URA), and grown for 4 days at 30°. Results are representative of analysis of a larger number of independent transformants (data not shown). L.C., plasmids based on the low-copy vector pSB32; the remainder of the plasmids listed to the left are based upon the high-copy vector YEp13.

View larger version (32K): In this window In a new window Download PPT slide   Figure 5. Growth rates of suppressed strains lacking eIF2B or eIF2. Growth rates were determined in EY923 (eIF2B bypass; see Fig 6) or EY835 (eIF2 bypass; see Fig 4) following growth on 5-FOA as described in RESULTS. Low-copy (L.C.) or high-copy (H.C.) LEU2 plasmids present in each strain containing eIF2 or eIF2B subunit genes are indicated to the left. All strains contained a high-copy TRP1 vector or the same vector containing IMT as indicated. Doubling times were determined in triplicate as described in MATERIALS AND METHODS.

Comparative requirements for bypass of essential eIF2B and eIF2 functions:
Examination of results presented in Fig 3 and Fig 4 reveals a difference in the requirements for suppression in the eIF2B deletion strain compared with the sui2 strain. In the former instance, suppression in all cases required increased IMT gene dosage, whereas suppression of sui2 by gcd11-K250R is independent of, though enhanced by, the presence of additional copies of IMT. A trivial explanation for this difference may be related to the presence of the chromosomal GCD11 allele in EY878 used in the eIF2B bypass experiments (Fig 3). In this case, the presence of wild-type eIF2 complexes may compete with eIF2_K250R and thereby reduce the efficiency of suppression in these strains. To test this idea, we created a gcd11 strain (EY923) isogenic with EY878 and repeated the eIF2B bypass experiments. The results, shown in Fig 6, are essentially identical to those shown in Fig 3; i.e., bypass of the essential function of eIF2B requires overexpression of both eIF2 and initiator tRNA and is independent of the presence of a chromosomal GCD11 allele in the host strain. Again, eIF2_K250R is a more efficient suppressor, resulting in a 1.4-fold increase in doubling time (vs. the control) compared with a 3-fold increase for wild-type eIF2 (Fig 5, top).

View larger version (45K): In this window In a new window Download PPT slide   Figure 6. Increased IMT gene dosage is required for bypass of eIF2B. Low-copy (L.C.; pSB32) or high-copy (H.C.; YEp13) LEU2 plasmids indicated to the left of the figure were introduced into EY923, which also harbored either YEplac112 [TRP1] (Vector) or Ep1013 (Vector + IMT). EY923 lacks all five chromosomal eIF2B subunit genes as well as GCD11, but harbors a URA3 plasmid (Ep1250) containing GCD11 and the essential eIF2B subunit genes. Independent transformants were grown, diluted, and plated on media as described in the legend to Fig 3. This photograph was taken following growth at 30° for 5 days.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Previous biochemical studies using mammalian factors indicated that eIF2 and eIF2B play critical roles in the initiation of eukaryotic protein synthesis (reviewed in MERRICK 1992 ; PRICE and PROUD 1994 ; PAIN 1996 ). Genetic analyses in yeast provided evidence that both factors are required for growth and viability (HINNEBUSCH 1997 ). In yeast, each of the three conserved eIF2 subunits and four of the five conserved eIF2B subunits are encoded by essential genes. We previously described a mutation in the gene encoding the -subunit of eIF2, gcd11-K250R, that increased the intrinsic rate of dissociation of guanine nucleotides from binary complexes in vitro (ERICKSON and HANNIG 1996 ). Although eIF2_K250R showed increased dissociation for both GDP and GTP in vitro, GTP binding by both eIF2_K250R and wild-type eIF2 could be stabilized by forming ternary complexes with charged initiator tRNA. These results were consistent with in vivo experiments that demonstrated increased IMT gene dosage suppressed both the slow growth and increased expression of GCN4 (i.e., the Gcd^- phenotype) in gcd11-K250R strains. The latter are indicative of at least partial restoration of eIF2 function (HINNEBUSCH 1994 ). These combined results suggested to us that gcd11-K250R strains might exhibit a reduced dependence upon eIF2B, resulting in partial or complete bypass of the requirement for eIF2B dependent upon (or enhanced by) increased IMT gene dosage. The data presented here demonstrate that overexpression of either the wild-type or _K250R form of eIF2 suppresses deletion of the four essential eIF2B subunit genes and that bypass of essential eIF2B function(s) requires co-overexpression of initiator tRNA. Consistent with a reduced requirement for eIF2B function, strains grown under bypass conditions but containing all eIF2B subunit genes show a reduced sensitivity to the eIF2 kinases PKR (Fig 2) and Gcn2p (DEVER et al. 1995 ). The efficiency of suppression of both the PKR-induced growth phenotype and the absence of eIF2B correlated directly with the rate of dissociation of guanine nucleotides determined previously with purified eIF2 preparations; i.e., gcd11-K250R strains were more efficient than strains harboring the wild-type GCD11 allele. Our data imply that enhancing the rate of nucleotide dissociation from eIF2 is an essential function of eIF2B.

KINZY and WOOLFORD 1995 demonstrated previously that additional copies of the TEF2 gene, encoding elongation factor eEF1A (previously eEF1), were sufficient to bypass the requirement for its exchange factor, eEF1B, when provided on a low-copy-number plasmid. The requirement for elevated levels of both wild-type eIF2 and initiator tRNA in bypassing eIF2B function suggests that eIF2 and/or initiator tRNA are normally maintained at limiting levels such that eIF2B is essential for promoting levels of ternary complex required in rapidly growing cells. Such a mechanism would also allow for rapid and effective changes in the level of ternary complexes by modulating eIF2B activity in response to various stimuli and, as such, may play an important role in regulating cell growth. Our results make the prediction that cells harboring mutations analogous to gcd11-K250R may be less sensitive to growth regulation mediated through protein kinases that phosphorylate the -subunit of eIF2 (SAMUEL 1993 ; WEK 1994 ; SHI et al. 1998 ; SOOD et al. 2000A , SOOD et al. 2000B ). Additional strategies developed to circumvent this regulatory mechanism, such as specific alterations in eIF2 that render it refractory to phosphorylation (DONZE et al. 1995 ) or dominant negative mutations in PKR (KOROMILAS et al. 1992 ; MEURS et al. 1993 ; BARBER et al. 1995A ), have been shown to promote tumor formation in mammals, implying that PKR may function as a tumor suppressor (LENGYEL 1993 ).

Conditions required to suppress a sui2 mutation differed somewhat from those required to suppress the deletion of essential eIF2B subunit genes. In the latter case, increased IMT gene dosage was absolutely required, whereas suppression of sui2 in gcd11-K250R strains was more efficient in the presence of, but did not require, additional copies of IMT. The difference in gene dosage requirements for IMT raises the possibility that eIF2B provides a function, in addition to nucleotide exchange, that is substituted (in the eIF2B bypass experiments) by elevated levels of initiator tRNA. It is possible that catalyzed nucleotide exchange proceeds through an eIF2·GTP·eIF2B intermediate that facilitates the interaction of eIF2 with initiator tRNA, perhaps by increasing the on-rate for tRNA relative to eIF2·GTP binary complexes. MANCHESTER and STASIKOWSKI 1990 proposed a similar model based upon theoretical considerations of association and dissociation rate constants under physiological conditions and the reaction rates for protein synthesis initiation. If this is indeed the preferred pathway for ternary complex formation in vivo, increasing the level of initiator tRNA may overcome the requirement for this eIF2B function via mass action. However, this model does not fully explain the requirement for increased IMT gene dosage in gcd11-K250R strains in the absence of eIF2B. In the presence of eIF2B, the viability of gcd11-K250R sui2 strains does not require additional copies of IMT, despite the fact that these conditions would be expected to bypass the eIF2B nucleotide exchange function. In fact, co-overexpression of gcd11-K250R, SUI3, and IMT is sufficient to bypass eIF2B in vivo (J. NIKA and E. M. HANNIG, unpublished observations). Furthermore, we demonstrated recently that eIF2 is required to promote efficient interaction between eIF2 and eIF2B in vitro (NIKA et al. 2001 ). These combined observations suggest that eIF2B may contribute to the formation of eIF2_K250R ternary complexes in a manner that does not appear to require catalyzed nucleotide exchange and that is independent of (or less dependent upon) direct eIF2/eIF2B interaction. An alternative means through which eIF2B may facilitate ternary complex formation is by increasing local concentrations of tRNA, perhaps through a channeling type of mechanism. Such a mechanism may be direct or indirect, would not require direct interaction between eIF2 and eIF2B, and may be facilitated by a ribosomal localization of at least a portion of the eIF2B pool (MATTS et al. 1988 ; CIGAN et al. 1991 ; CHAKRABARTI and MAITRA 1992 ; RAMAIAH et al. 1992 ; BUSHMAN et al. 1993B ; MUELLER et al. 1998 ). However, we cannot rule out completely the involvement of at least some form of an eIF2·eIF2B intermediate. In this respect, it is interesting to note recent data demonstrating genetic as well as physical interaction between GCD11 and LOS1 (HELLMUTH et al. 1998 ; GROSSHANS et al. 2000 ). LOS1 encodes a member of the ß-importin family that plays a nonessential role in transport of tRNA across the yeast nuclear membrane. However, it is unclear whether this interaction is important in the specific transport and/or localization of initiator tRNA. On the other hand, eEF1A (the functional homolog of prokaryotic EF1A) does appear to be required for efficient nuclear export of certain noninitiator tRNAs and has been suggested to function in coordinating translation and tRNA export in yeast (GROSSHANS et al. 2000 ).

Our combined data predict that eIF2ß carries out all eIF2 functions required for translation initiation, including interactions with ribosomes and other translational factors, start site recognition, nucleotide exchange (in the presence or absence of eIF2B), and formation of ternary complexes. This suggests a model in which eIF2ß comprises the eIF2 functional core, whereas the -subunit of eIF2 and the eIF2B heteropentamer form a regulatory core that modulates the level of eIF2 function by regulating nucleotide exchange and formation of ternary complexes in vivo. The availability of yeast strains lacking normally essential subunits of eIF2 and eIF2B should provide valuable tools for dissecting the functions of individual polypeptides in these multisubunit complexes. Such functions could include roles in catalysis, as well as regulatory functions involved in cellular responses to stress or other environmental stimuli (WELSH and PROUD 1992 ; ENGELBERG et al. 1994 ; KIMBALL and JEFFERSON 1994 ; BROSTROM et al. 1996 ; GALLIE et al. 1997 ; QU et al. 1997 ; SCHEPER et al. 1997 ). In this regard, results of previous studies have indicated a role for Gcd6p in catalysis, whereas Gcd2p, Gcd7p, and Gcn3p appear to form a regulatory subcomplex (YANG and HINNEBUSCH 1996 ; FABIAN et al. 1997 ; PAVITT et al. 1997 , PAVITT et al. 1998 ; GOMEZ and PAVITT 2000 ). Biochemical analysis of individual eIF2B polypeptides and subcomplexes that are devoid of contaminating subunits, purified from strains using the genetic backgrounds developed in this article, in addition to further genetic analyses of these strains, will allow these questions to be addressed more directly.

	FOOTNOTES

¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

We thank Juan Gonzalez and Larry Reitzer for thoughtful comments on the manuscript, Alan Hinnebusch for GCD6/GCD7 plasmids and Gcd6p/Gcd7p antibodies, Tom Dever for PKR plasmid constructs, and Weimin Yang for construction of the yeast strain used for purification of eIF2B. This work was supported by a grant from the American Cancer Society (RPG-97-061-01-NP).

Manuscript received May 24, 2000; Accepted for publication February 9, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AHMAD, M. F., N. NASRIN, M. K. BAGCHI, I. CHAKRAVARTY, and N. K. GUPTA, 1985  A comparative study of the characteristics of eIF-2 and eIF-2-ancillary factor activities from yeast Saccharomyces cerevisiae and rabbit reticulocytes. J. Biol. Chem. 260:6960-6965[Abstract/Free Full Text].

ALANI, E., L. CAO, and N. KLECKNER, 1987  A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116:541-545[Abstract/Free Full Text].

BARBER, G. N., R. JAGUS, E. F. MEURS, A. G. HOVANESSIAN, and M. G. KATZE, 1995a  Molecular mechanisms responsible for malignant transformation by regulatory and catalytic domain variants of the interferon-induced enzyme RNA-dependent protein kinase. J. Biol. Chem. 270:17423-17428[Abstract/Free Full Text].

BARBER, G. N., M. WAMBACH, S. THOMPSON, R. JAGUS, and M. G. KATZE, 1995b  Mutants of the RNA-dependent protein kinase (PKR) lacking double-stranded RNA binding domain I can act as transdominant inhibitors and induce malignant transformation. Mol. Cell. Biol. 15:3138-3146[Abstract].

BOEKE, J. D., J. TRUEHEART, G. NATSOULIS, and G. R. FINK, 1987  5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154:164-175[Medline].

BOURNE, H. R., D. A. SANDERS, and F. MCCORMICK, 1991  The GTPase superfamily: conserved structure and molecular mechanism. Nature 349:117-127[Medline].

BROSTROM, C. O., C. R. PROSTKO, R. J. KAUFMAN, and M. A. BROSTROM, 1996  Inhibition of translational initiation by activators of the glucose-regulated stress protein and heat shock protein stress response systems. Role of the interferon-inducible double-stranded RNA-activated eukaryotic initiation factor 2 kinase. J. Biol. Chem. 271:24995-25002[Abstract/Free Full Text].

BUSHMAN, J. L., A. I. ASURU, R. L. MATTS, and A. G. HINNEBUSCH, 1993a  Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.. Mol. Cell. Biol. 13:1920-1932[Abstract/Free Full Text].

BUSHMAN, J. L., M. FOIANI, A. M. CIGAN, C. J. PADDON, and A. G. HINNEBUSCH, 1993b  Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3. Mol. Cell. Biol. 13:4618-4631[Abstract/Free Full Text].

CHAKRABARTI, A. and U. MAITRA, 1992  Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex. J. Biol. Chem. 267:12964-12972[Abstract/Free Full Text].

CHONG, K. L., L. FENG, K. SCHAPPERT, E. MEURS, and T. F. DONAHUE et al., 1992  Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2. EMBO J. 11:1553-1562[Medline].

CIGAN, A. M., E. K. PABICH, L. FENG, and T. F. DONAHUE, 1989  Yeast translation initiation suppressor sui2 encodes the subunit of eukaryotic initiation factor 2 and shares sequence identity with the human subunit. Proc. Natl. Acad. Sci. USA 86:2784-2788[Abstract/Free Full Text].

CIGAN, A. M., M. FOIANI, E. M. HANNIG, and A. G. HINNEBUSCH, 1991  Complex formation by positive and negative translational regulators of GCN4.. Mol. Cell. Biol. 11:3217-3228[Abstract/Free Full Text].

CIGAN, A. M., J. L. BUSHMAN, T. R. BOAL, and A. G. HINNEBUSCH, 1993  A protein complex of translational regulators of GCN4 mRNA is the guanine nucleotide-exchange factor for translation initiation factor 2 in yeast. Proc. Natl. Acad. Sci. USA 90:5350-5354[Abstract/Free Full Text].

DEVER, T. E., M. J. GLYNIAS, and W. C. MERRICK, 1987  GTP-binding domain: three consensus sequence elements with distinct spacing. Proc. Natl. Acad. Sci. USA 84:1814-1818[Abstract/Free Full Text].

DEVER, T. E., L. FENG, R. C. WEK, A. M. CIGAN, and T. F. DONAHUE et al., 1992  Phosphorylation of initiation factor 2 by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell 68:585-596[Medline].

DEVER, T. E., J. J. CHEN, G. N. BARBER, A. M. CIGAN, and L. FENG et al., 1993  Mammalian eukaryotic initiation factor 2 kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast. Proc. Natl. Acad. Sci. USA 90:4616-4620[Abstract/Free Full Text].

DEVER, T. E., W. YANG, S. ASTROM, A. S. BYSTROM, and A. G. HINNEBUSCH, 1995  Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met- tRNA(iMet) ternary complexes. Mol. Cell. Biol. 15:6351-6363[Abstract].

DONAHUE, T. F., A. M. CIGAN, E. K. PABICH, and B. C. VALAVICIUS, 1988  Mutations at a Zn(II) finger motif in the yeast eIF-2ß gene alter ribosomal start-site selection during the scanning process. Cell 54:621-632[Medline].

DONZE, O., R. JAGUS, A. E. KOROMILAS, J. W. HERSHEY, and N. SONENBERG, 1995  Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J. 14:3828-3834[Medline].

DORRIS, D. R., F. L. ERICKSON, and E. M. HANNIG, 1995  Mutations in GCD11, the structural gene for eIF-2 in yeast, alter translational regulation of GCN4 and the selection of the start site for protein synthesis. EMBO J. 14:2239-2249[Medline].

ENGELBERG, D., C. KLEIN, H. MARTINETTO, K. STRUHL, and M. KARIN, 1994  The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals. Cell 77:381-390[Medline].

ERICKSON, F. L. and E. M. HANNIG, 1996  Ligand interactions with eukaryotic translation initiation factor 2: role of the -subunit. EMBO J. 15:6311-6320[Medline].

ERICKSON, F. L., L. D. HARDING, D. R. DORRIS, and E. M. HANNIG, 1997  Functional analysis of homologs of translation initiation factor 2 in yeast. Mol. Gen. Genet. 253:711-719[Medline].

ERNST, H., R. F. DUNCAN, and J. W. HERSHEY, 1987  Cloning and sequencing of complementary DNAs encoding the -subunit of translational initiation factor eIF-2. Characterization of the protein and its messenger RNA. J. Biol. Chem. 262:1206-1212[Abstract/Free Full Text].

FABIAN, J. R., S. R. KIMBALL, N. K. HEINZINGER, and L. S. JEFFERSON, 1997  Subunit assembly and guanine nucleotide exchange activity of eukaryotic initiation factor-2B expressed in Sf9 cells. J. Biol. Chem. 272:12359-12365[Abstract/Free Full Text].

GALLIE, D. R., H. LE, C. CALDWELL, R. L. TANGUAY, and N. X. HOANG et al., 1997  The phosphorylation state of translation initiation factors is regulated developmentally and following heat shock in wheat. J. Biol. Chem. 272:1046-1053[Abstract/Free Full Text].

GASPAR, N. J., T. G. KINZY, B. J. SCHERER, M. HUMBELIN, and J. W. HERSHEY et al., 1994  Translation initiation factor eIF-2. Cloning and expression of the human cDNA encoding the -subunit. J. Biol. Chem. 269:3415-3422[Abstract/Free Full Text].

GIETZ, R. D. and A. SUGINO, 1988  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527-534[Medline].

GOMEZ, E. and G. D. PAVITT, 2000  Identification of domains and residues within the -subunit of eukaryotic translation initiation factor 2B (eIF2Bepsilon) required for guanine nucleotide exchange reveals a novel activation function promoted by eIF2B complex formation. Mol. Cell. Biol. 20:3965-3976[Abstract/Free Full Text].

GROSSHANS, H., E. HURT, and G. SIMOS, 2000  An aminoacylation-dependent nuclear tRNA export pathway in yeast. Genes Dev. 14:830-840[Abstract/Free Full Text].

HANNIG, E. M. and A. G. HINNEBUSCH, 1988  Molecular analysis of GCN3, a translational activator of GCN4: evidence for posttranslational control of GCN3 regulatory function. Mol. Cell. Biol. 8:4808-4820[Abstract/Free Full Text].

HANNIG, E. M., N. P. WILLIAMS, R. C. WEK, and A. G. HINNEBUSCH, 1990  The translational activator GCN3 functions downstream from GCN1 and GCN2 in the regulatory pathway that couples GCN4 expression to amino acid availability in Saccharomyces cerevisiae.. Genetics 126:549-562[Abstract].

HANNIG, E. M., A. M. CIGAN, B. A. FREEMAN, and T. G. KINZY, 1993  GCD11, a negative regulator of GCN4 expression, encodes the subunit of eIF-2 in Saccharomyces cerevisiae.. Mol. Cell. Biol. 13:506-520[Abstract/Free Full Text].

HELLMUTH, K., D. M. LAU, F. R. BISCHOFF, M. KUNZLER, and E. C. HURT et al., 1998  Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for tRNA. Mol. Cell. Biol. 18:6374-6386[Abstract/Free Full Text].

HERSHEY, J. W., 1991  Translational control in mammalian cells. Annu. Rev. Biochem. 60:717-755[Medline].

HILL, D. E. and K. STRUHL, 1988  Molecular characterization of GCD1, a yeast gene required for general control of amino acid biosynthesis and cell-cycle initiation. Nucleic Acids Res. 16:9253-9265[Abstract/Free Full Text].

HINNEBUSCH, A. G., 1993  Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2. Mol. Microbiol. 10:215-223[Medline].

HINNEBUSCH, A. G., 1994  Translational control of GCN4: an in vivo barometer of initiation-factor activity. Trends Biochem. Sci. 19:409-414[Medline].

HINNEBUSCH, A. G., 1997  Translational regulation of yeast GCN4. A window on factors that control initiator-TRNA binding to the ribosome. J. Biol. Chem. 272:21661-21664[Free Full Text].

HINNEBUSCH, A. G., and S. W. LIEBMAN, 1991 Protein synthesis and translational control in Saccharomyces cerevisiae, pp. 627–735 in The Molecular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, edited by J. R. BROACH, J. R. PRINGLE and E. W. JONES. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

KIMBALL, S. R. and L. S. JEFFERSON, 1994  Mechanisms of translational control in liver and skeletal muscle. Biochimie 76:729-736[Medline].

KIMBALL, S. R., J. R. FABIAN, G. D. PAVITT, A. G. HINNEBUSCH, and L. S. JEFFERSON, 1998  Regulation of guanine nucleotide exchange through phosphorylation of eukaryotic initiation factor eIF2. Role of the - and - subunits of eIF2B. J. Biol. Chem. 273:12841-12845[Abstract/Free Full Text].

KINZY, T. G. and J. L. WOOLFORD, JR., 1995  Increased expression of Saccharomyces cerevisiae translation elongation factor 1 bypasses the lethality of a TEF5 null allele encoding elongation factor 1ß. Genetics 141:481-489[Abstract].

KOONIN, E. V., 1995  Multidomain organization of eukaryotic guanine nucleotide exchange translation initiation factor eIF-2B subunits revealed by analysis of conserved sequence motifs. Protein Sci. 4:1608-1617[Abstract].

KOROMILAS, A. E., S. ROY, G. N. BARBER, M. G. KATZE, and N. SONENBERG, 1992  Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science 257:1685-1689[Abstract/Free Full Text].

LENGYEL, P., 1993  Tumor-suppressor genes: news about the interferon connection. Proc. Natl. Acad. Sci. USA 90:5893-5895[Abstract/Free Full Text].

MANCHESTER, K. L. and P. STASIKOWSKI, 1990  Kinetic parameters governing the formation of eIF-2.methionyl-tRNAi complexes in protein synthesis. Biochem. Int. 20:257-265[Medline].

MARTON, M. J., D. CROUCH, and A. G. HINNEBUSCH, 1993  GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2. Mol. Cell. Biol. 13:3541-3556[Abstract/Free Full Text].

MATTS, R. L., N. S. THOMAS, R. HURST, and I. M. LONDON, 1988  Correlation between the distribution of the reversing factor and eukaryotic initiation factor 2 in heme-deficient or double-stranded RNA-inhibited reticulocyte lysates. FEBS Lett.