Eukaryotic translation initiation factors 4G and 4A from Saccharomyces cerevisiae interact physically and functionally.

The recruitment of the 40S ribosomal subunit to the initiator codon of a eukaryotic mRNA is mediated by the interplay of several different translation initiation complexes (reviewed in reference 18). A key complex in this process is eukaryotic translation initiation factor 4F (eIF4F). The eIF4F complex in higher eukaryotes is composed of three proteins, eIF4G, eIF4A, and eIF4E (7). The eIF4E protein binds to the m7G cap structure at the 5′ end of the mRNA, as well as to eIF4G. The eIF4G protein acts as a bridging factor in that it binds to eIF4E, to the poly(A) tail binding protein Pab1p, and to eIF3, a complex of proteins which is tightly associated with the 40S ribosomal subunit (11, 13, 19, 20, 23). The RNA-stimulated ATPase eIF4A is also associated with eIF4G in higher eukaryotes and is thought to use its ATPase activity to unwind RNA secondary structure in the 5′ leader of each message (21). This presumably allows for the unimpeded scanning of the 40S ribosomal subunit along the 5′ leader as it searches for the initiator codon. The mechanism by which the eIF4F complex mediates translation initiation in the yeast Saccharomyces cerevisiae is slightly more obscure. This organism encodes the eIF4E protein with the CDC33 gene (3), the eIF4A protein with two different genes (TIF1 and TIF2) (17), and the two eIF4G proteins (eIF4G1 and eIF4G2) with the TIF4631 and TIF4632 genes, respectively (8). Although S. cerevisiae expresses all of these putative components of eIF4F, the yeast eIF4A protein has not previously been shown to bind to eIF4G. For example, the purification of eIF4E from higher eukaryotes by cap analog chromatography results in the copurification of eIF4A and eIF4G (reviewed in reference 7), whereas purification of yeast eIF4E through a similar procedure results in the copurification of eIF4G but not eIF4A (9, 26). Furthermore, the initial mapping of the eIF4A binding domain in rabbit eIF4G placed it within the C-terminal third of the protein (16). The yeast eIF4G protein, although homologous to much of mammalian eIF4G, lacks homology to this entire C-terminal region (8). Each of these observations has brought into question whether eIF4A and eIF4G associate in yeast and, more generally, whether the mechanism by which translation initiation occurs in yeast involves the early recruitment of eIF4A to the 5′ end of the mRNA. During the course of our study it was reported that mammalian eIF4G contains a second eIF4A binding site that is distinct from its previously identified C-terminal binding site (14). This site lies in the middle of the protein, which is conserved in all eukaryotic eIF4G proteins. It was shown in that study that the presence of both eIF4A binding sites on mammalian eIF4G was required for eIF4G to stimulate translation in vitro. Based on extensive sequence homology between yeast and mammalian eIF4G proteins, it was also proposed that yeast eIF4A binds to eIF4G within this region. Here we present evidence that yeast eIF4A and eIF4G functionally and physically interact. Overexpression of eIF4A in several different tif4632 mutants led to suppression of their temperature-sensitive phenotype. This suppression was shown to be allele specific. A heat-inactivated translation extract from a tif4632 mutant could have its translational activity significantly stimulated by the addition of excess eIF4A, and this stimulation was also shown to be tif4632 allele specific. Finally, the binding of recombinant yeast eIF4A to eIF4G1 and eIF4G2 in vitro was demonstrated for the first time, as was a decreased ability of the several mutant eIF4G2 proteins to bind to eIF4A. These data show that yeast eIF4G contains an eIF4A binding site, and they indicate that an interaction between yeast eIF4A and eIF4G is required for translation and is essential for yeast cell viability.