Med9 Interacts with eIF4E Dependent on the Carbon Source in Saccharomyces cerevisiae

The cap-binding protein eIF4E is a key protein for mRNA metabolism. The eIF4E biological role is defined by the specific protein it interacts with. The best characterized role of eIF4E is in promoting mRNA translation through its interaction with eIF4G. To seek for new interactors in the ascomycete Saccharomyces cerevisiae, we performed a genomic yeast two-hybrid screen using eIF4E as bait. In addition to the already reported p20 and Eap1, we identified Med9, a component of the RNA polymerase II Mediator complex. A physical interaction between eIF4E and Med9 was confirmed using recombinant proteins prepared in E. coli and further isolating the eIF4E―Med9 complex both by size-exclusion chromatography and by m7GTP-Sepharose pull-down experiments. Surprisingly, the eIF4E W75A mutation, which impairs the interaction with eIF4G, p20, and Eap1 only slightly affected the interaction with Med9 in the two-hybrid system. We further performed random mutagenesis to identify the Med9 amino acids involved in eIF4E interaction. Mutants F65A/I66A and F65A/I66A/H68N did not interact with eIF4E. We also demonstrated that the interaction eIF4E―Med9 depended on the carbon source for cell growth and that it might happen within the nucleus. Finally, we found that the eIF4E―Med9 interaction is conserved in the yeast Saccharomyces kudriavzevii.