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. 1999 Aug;19(8):5557-64.
doi: 10.1128/MCB.19.8.5557.

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

C L Neff  1 , A B Sachs
Affiliations

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

C L Neff et al. Mol Cell Biol. 1999 Aug.

Abstract

The initiation of translation in eukaryotes requires several multisubunit complexes, including eukaryotic translation initiation factor 4F (eIF4F). In higher eukaryotes eIF4F is composed of the cap binding protein eIF4E, the adapter protein eIF4G, and the RNA-stimulated ATPase eIF4A. The association of eIF4A with Saccharomyces cerevisiae eIF4F has not yet been demonstrated, and therefore the degree to which eIF4A's conserved function relies upon this association has remained unclear. Here we report an interaction between yeast eIF4G and eIF4A. Specifically, we found that the growth arrest phenotype associated with three temperature-sensitive alleles of yeast eIF4G2 was suppressed by excess eIF4A and that this suppression was allele specific. In addition, in vitro translation extracts derived from an eIF4G2 mutant strain could be heat inactivated, and this inactivation could be reversed upon the addition of recombinant eIF4A. Finally, in vitro binding between yeast eIF4G and eIF4A was demonstrated, as was diminished binding between mutant eIF4G2 proteins and eIF4A. In total, these data indicate that yeast eIF4G and eIF4A physically associate and that this association performs an essential function. VSports手机版.

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Figures

FIG. 1
FIG. 1
Alleles of TIF4632 used in this study. The position and type of amino acid substitution corresponding to each of the alleles are shown. The numbering of amino acids shown here includes the extra two amino acids introduced at the N terminus of the recombinant protein (26).
FIG. 2
FIG. 2
Overexpression of eIF4A suppresses the temperature sensitivity of the tif4632-1, -6, and -8 mutants. The yeast eIF4A gene TIF1 in a multicopy (2μ) plasmid or the plasmid with no insert (empty 2μ) were transformed into the indicated tif4632 mutants to yield strains YAS2096, YAS2099, YAS2100, YAS2103, YAS2429, and YAS2430. The ability of the transformants to grow on minimal medium plates after 5 days at 37°C is shown.
FIG. 3
FIG. 3
Expression and initiation factor association of mutant eIF4G2 proteins in the presence and absence of excess eIF4A. (A) Excess eIF4A does not lead to overproduction of eIF4G2. Crude lysates prepared from yeast strains YAS2456 to -2463 grown at 37°C were analyzed by Western analysis for their content of the indicated epitope-tagged HA-eIF4G2. Each strain either lacked (−) or contained (+) the multicopy plasmid containing TIF1. All strains contained non-epitope-tagged eIF4G2 as well and therefore were viable at 37°C. Extracts from a strain (YAS2106) lacking an HA-tagged eIF4G (non HA-tagged) were used as a negative control. (B) Association of mutant forms of eIF4G2 with eIF4E and Pab1p. The epitope-tagged HA-eIF4G2 proteins in the crude lysates described for panel A were immunoprecipitated, resolved by SDS-PAGE, and visualized by Western analysis. The degree of coimmunoprecipitation of Pab1p and eIF4E with eIF4G2 was determined by Western analysis of the immunoprecipitate with Pab1p- or eIF4E-specific antibodies.
FIG. 4
FIG. 4
Characterization of eIF4G2 mutant and wild-type translation extracts. (A) Heat inactivation of tif4632-8 mutant extracts. TIF4632- or tif4632-8-derived translation extracts were heated at 30°C for the indicated times, frozen, thawed, and then mixed with LUCpA mRNA and incubated at 26°C for the indicated times under conditions permissive for translation. The amount of LUC protein produced in each of these mixtures was measured by using a luminescence assay. (B) Restoration of translation in heat-inactivated tif4632-8 extracts by the addition of recombinant GST-eIF4G2. The indicated translation extract was heated at 30°C for 12 min in the presence of the indicated amount of GST-eIF4G2 protein. After freezing, thawing, and mixing of the extracts with capLUCpA mRNA, they were incubated at 26°C for 30 min under conditions permissive for translation. The amount of LUC protein produced in each of these mixtures was measured by using a luminescence assay.
FIG. 5
FIG. 5
Stimulation of translation in tif4632-8 extracts by the addition of eIF4A. (A) Stimulation by eIF4A. The indicated extracts were heated at 30°C for 12 min in the presence of the indicated amounts of eIF4A and then assayed for translation of the capLUCpA mRNA as described in the legend to Fig. 4. (B) Rates of LUC production in extracts lacking and containing excess eIF4A. The indicated extracts were heated as described for panel A in the presence (squares) or absence (diamonds) of 2 μg of eIF4A. Aliquots of translation reaction mixtures incubated at 26°C and programmed with capLUCpA mRNA were withdrawn at the indicated times and assayed for LUC protein with a luminescence assay. (C) eIF4E does not stimulate translation in tif4632-8 extracts. Extracts were heated and assayed for LUC protein production from capLUCpA mRNA as described above in the presence of the indicated amounts of eIF4E.
FIG. 6
FIG. 6
In vitro binding of eIF4A to eIF4G. (A) Yeast eIF4A binds to eIF4G1 and eIF4G2 in vitro. Purified GST or GST-eIF4G1 or GST-eIF4G2 fusion protein immobilized on glutathione-Sepharose resin was incubated with recombinant eIF4A and then washed extensively. Bound proteins were eluted with SDS, resolved on an SDS–10% polyacrylamide gel, and detected by either Coomassie brilliant blue staining (GST-eIF4G) or Western analysis (eIF4A). (B) Differential in vitro binding of eIF4A to wild-type and mutant eIF4G2 proteins. The indicated GST-eIF4G2 fusion proteins were immobilized on glutathione-Sepharose resin and analyzed for eIF4A binding as described for panel A. The eIF4G protein was detected by Coomassie brilliant blue staining, and the eIF4A protein was detected by Western analysis. WT, wild type. (C) Association with eIF4E. The procedure was the same as for panel A, except eIF4E was incubated with the indicated GST-eIF4G fusion proteins and was detected by Western analysis with anti-eIF4E antibodies.
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