It is now well established that hydrolysis of GTP during translation initiation occurs only when eIF5 interacts with GTP bound to eIF2 as a Met-tRNAf
–eIF2–GTP ternary complex in the 40S initiation complex (Met-tRNAf
–eIF2–GTP–eIF3–40S–AUG) and that eIF5 by itself does not hydrolyze either free GTP or GTP bound to eIF2 as a Met-tRNAf
–eIF2–GTP ternary complex (4
). These results suggest that eIF5 interacts with one or more components of the 40S initiation complex to effect the hydrolysis of bound GTP. Subsequent studies, showing that eIF5 forms a specific complex with eIF2 (7
) by interacting with the β subunit of eIF2 (1
), led us to hypothesize that protein-protein interaction between eIF5 and the 40S subunit-bound eIF2 may be critical for the hydrolysis of GTP bound to the 40S initiation complex (11
). To demonstrate such a correlation, we carried out mutational analysis of eIF5 to identify the amino acid residues in the protein critical for its interaction with eIF2β. The eIF5 mutants defective in such interactions were then analyzed for the ability to promote the hydrolysis of GTP during translation initiation.
In the work presented in this paper, we first carried out deletion analysis of eIF5 to demonstrate that the C-terminal region of rat eIF5 binds eIF2β (Fig. ). In view of our previous observation (11
) that a 22-amino-acid region at the N-terminal of mammalian eIF2β containing stretches of conserved lysine residues is involved in the binding of eIF2β to eIF5, we reasoned that a stretch of acidic amino acid residues in the C-terminal eIF2β-binding region of eIF5 may be involved in its interaction with mammalian eIF2. Alanine substitution mutagenesis within this region defined several glutamic acid residues, which are highly conserved between species, as important for binding to eIF2β. The E346A,E347A and E384A,E385A double-point mutations each caused a profound decrease in the specific binding of eIF5 to eIF2β, while eIF5 mutant M5, in which all six glutamic acid residues in the two halves of the bipartite motif were mutated to alanine, showed barely detectable binding to eIF2β. It is therefore likely that these conserved glutamic acid residues which constitute a bipartite motif in eIF5 make direct contacts with the conserved lysine residues in the polylysine stretches of eIF2β and is indeed a component of the eIF2β binding site of eIF5. Further characterization of these two eIF5 mutants showed that the purified expressed proteins containing each of the two double-point mutations were severely defective in eIF5-dependent hydrolysis of GTP bound to the 40S initiation complex and consequently defective also in 80S initiation complex formation. These mutants were also defective in stimulating translation of yeast mRNAs in an eIF5-dependent yeast cell-free translation system. The importance of eIF5-eIF2β interaction in eIF5 function was further confirmed by our demonstration that while wild-type rat eIF5 can substitute for yeast eIF5 function in ΔTIF5
haploid yeast cells, the mutant rat eIF5 proteins M1 and M2, when expressed in such ΔTIF5
yeast cells, showed severe growth defects. It appears that the mutant M1 is much more defective in growth (doubling time of 13.4 h) than the mutant M2 (doubling time of 7 h), indicating that the first motif comprising glutamic acid residues 345 to 347 plays a more important role in eIF5-eIF2β interaction and consequently eIF5 function than the second motif comprising the glutamic acid residues 384 to 386. It is interesting to note here that the first motif is more conserved than the second one (Fig. ). Furthermore, mutant eIF5 M5 containing alanine substitution mutations in all six glutamic acid residues in the bipartite motif that showed barely detectable binding to eIF2β was unable to maintain cell growth and viability of such yeast cells. These findings suggest that interaction of eIF5 with eIF2β is required for eIF5 function in vivo and in vitro.
Asano et al. (1
) have previously demonstrated that the yeast eIF2β-binding region of yeast eIF5 contains a bipartite motif at the C terminus of eIF5. Our observation that eIF2β-binding region of rat eIF5 also contains a bipartite motif is in agreement with their work. We show that this bipartite motif consists of two regions, one surrounding glutamic acid residue 345 to 347 and the other surrounding glutamic acid residues 384 to 386. While mutagenesis of the glutamic acid residues in any one region caused profound decrease both in the binding of eIF5 to eIF2β as well as in eIF5-mediated GTP hydrolysis from the 40S initiation complex, neither of these two reactions was completely abolished under these conditions. However, when the glutamic acid residues in both regions of the bipartite motif were mutated, the resulting mutant eIF5 was virtually inactive in eIF5-dependent GTP hydrolysis and in 80S initiation complex formation. These observations suggest that in the native eIF5 molecule, these two regions of the bipartite motif must come together in interacting with the polylysine-rich region of the eIF5-binding site of eIF2β. Presumably, mutagenesis of any one region of the bipartite motif still allows eIF5 to bind weakly to the polylysine-rich eIF5-binding region of eIF2β via the glutamic acid residues of the other region and allow slow GTP hydrolysis. Additionally, it should be noted that the assays used to study binding of eIF5 to eIF2β measure stoichiometric interaction between the two initiation factors and are at best semiquantitative. In contrast, eIF5-dependent hydrolysis of GTP bound to the 40S initiation complex measures the rate of GTP hydrolysis in which eIF5 is known to act catalytically (12
). This may explain our observation that eIF5 mutants M1 and M2, which bind very weakly to eIF2β, still exhibit slow GTP hydrolysis activity that is about 20% of the activity of wild-type eIF5.
An important property of eIF5-dependent GTP hydrolysis reaction is that in addition to eIF2 and eIF5, 40S ribosomal subunits also play a key role in GTP hydrolysis during translation initiation. It is likely that when the ternary complex is transferred to the 40S ribosomal subunits, eIF2 acquires a conformation such that its interaction with eIF5 via the β subunit of eIF2 activates the latent GTPase activity of eIF2. Alternatively, 40S ribosomes may play a more direct role in GTP hydrolysis and could be a coeffector. Kozak has postulated (16
) that the 40S ribosomal subunit may have a “GTPase-activating center,” analogous to the presence of a similar domain in the 50S ribosomal subunit of prokaryotes that mediates GTP hydrolysis by the prokaryotic initiation factor IF2 and elongation factors EFTu and EFG. In prokaryotes, both IF2 and EFTu have been shown to have a weak GTPase activity (16
) that is markedly stimulated by 50S ribosomal subunits. It remains unknown whether eIF2 possesses an intrinsic GTPase activity. In analogy with proteins of the GTPase superfamily, the γ subunit of eIF2, which contains the consensus GTP-binding domains (14
) and is presumably involved in binding of GTP by eIF2, may also possess latent GTPase activity, although this has not been demonstrated experimentally. We postulate that the interaction of eIF5 with the β subunit of eIF2 bound as the Met-tRNAf
–eIF2–GTP ternary complex on the 40S ribosomal subunit induces a conformational change in eIF2 resulting in the activation of the latent GTPase activity of the γ subunit of eIF2. In this respect, eIF5 acts as a GTPase-activating protein (GAP). It should, however, be noted that typical GAPs e.g., Rho GAPs and Ras GAPs, that have been characterized extensively contain sequence motifs that are necessary for their GTPase-stimulating activity, in addition to motifs that are necessary for interacting with their G proteins (25
). However, eIF5 has no apparent homology with any member of the GAP family. It remains to be determined whether eIF5 possesses any additional sequence motifs that are required for its GTPase-activating function.
Finally, Asano et al. (1
) have shown that the C-terminal one-third of yeast eIF5 contains an acidic and aromatic amino acid-rich bipartite motif that is necessary for the binding of both eIF2β and the Nip1p subunit of yeast eIF3. Mutations (one mutant containing 7 mutations and the other containing 12 mutations of conserved amino acid residues) in this motif which disrupt eIF5-eIF2β interaction also disrupt eIF5-Nip1p interaction (1
). Data presented in this paper suggest, however, that the amino acid residues critical for eIF5-eIF2β interaction may be distinct from those that are critical for eIF5-Nip1p interaction although the binding domains of eIF2β and eIF3-Nip1p may be in the same region of eIF5 and may in fact overlap. It is quite likely that the interactions between eIF5-eIF2β and eIF5-Nip1p are not mutually exclusive and each interaction may play a distinct role in eIF5 function. Clearly additional structure-function studies will be necessary to establish the nature of the eIF5-Nip1p interaction and its role in eIF5 function.