Med9 Is a Novel eIF4E-Interacting Protein in Saccharomyces cerevisiae

The cap-binding protein eukaryotic initiation factor (eIF) 4E is a key protein for mRNA metabolism. The biological role of eIF4E is defined by the specific protein it interacts with. Thus, eIF4E binds to a constellation of partners across eukaryotes. The best-characterized role of eIF4E is to promote mRNA translation through interaction with the translation factor eIF4G. To seek 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. Moreover, the in vivo interaction was also detected. Surprisingly, the eIF4E W75A mutation, which impairs binding to 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 recognition. Mutants F65A/I66A and F65A/I66A/H68N did not interact with eIF4E. Our data suggest that the eIF4E-Med9 complex may be formed in both the cytoplasm and the nucleus.