The Yeast Eukaryotic Initiation Factor 4G (eIF4G) HEAT Domain Interacts with eIF1 and eIF5 and Is Involved in Stringent AUG Selection

Abstract

Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap- and poly(A) tail-binding proteins eIF4E and Pap1p. eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel α-helices. The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined. Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1. eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5. In vivo, cooverexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts⁻ mutation by eIF4A overexpression. In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by cooverexpression of eIF4A. Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro. Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon. These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex.

ACKNOWLEDGMENTS

We are greatly indebted to Alan Sachs for generous gifts of materials and permission to use YAS1998, YAS1999, and YAS2000 prior to publication and to Ernie Hannig and Cynthia Curtis for a SUI5 LEU2 plasmid prior to publication and technical advice on β-galactosidase assays. We also thank Tom Donahue and Stu Peltz for timely gifts of plasmids, Ashik Srinivasan for technical help, and Beth Montelone and other members of the KSU MCDB Program for advice and discussion.

This work was supported by NIH COBRE award 1 P20 RR15563, matching support from the State of Kansas and KSU, NIH grant GM64781 to K.A., and Wellcome Trust funding to J.E.G.M.

Hui He, Tobias von der Haar, C. Ranjit Singh, and Miki Ii contributed equally to this work.