Redox control of the plant specific cap-binding protein eIFiso4E in Arabidopsis

Eukaryotic initiation factor (eIF) 4E and the plant-specific homolog, eIFiso4E, participate in translation initiation by recognizing the 7‐methylguanosine (m⁷G) cap of mRNAs. All plant cap-binding proteins (eIF4E and eIFiso4E) have two conserved cysteine residues and wheat (Triticum aestivum) eIF4E was observed in crystals to form a disulfide bond between these two residues under oxidizing conditions. To study the effect of these conserved cysteines on Arabidopsis thaliana eIFiso4E activity in vivo or in vitro, one of the conserved residues (Cys-97) was mutated to serine (AteIFiso4E C97S), which abolishes the ability to form a disulfide bond or undergo modification. Under abiotic stress, AteIFiso4E C97S plants showed stress hypersensitivity and slowed ROS (reactive oxygen species) propagation, which suggests an in vivo role of Cys-97 in response to stress induced redox. The in vitro cap binding activity of AteIFiso4E C97S and in vitro translation activity of AteIFiso4F C97S (the complex of eIFiso4E C97S and eIFiso4G) was measured. AteIFiso4E C97S is not able to bind m⁷GTP-Sepharose affinity resin, but its binding can be restored by interaction with its binding partner, AteIFiso4G. However, a capped mRNA (barley α-amylase) was translated in vitro at a 3-fold lower rate by AteIFiso4F C97S compared to AteIFiso4F. These findings suggest that cysteine modification of AteIFiso4E and similarly conserved cysteine residues of other plant cap binding proteins (eIF4E) may have a role in regulation of translation during responses to the environment or other conditions that may alter the cellular redox status. To create a simple in vivo model for studying the differential activities of AteIF4E and AteIFiso4E or their complexes (AteIF4F and AteIFiso4F) towards messenger RNA (mRNA), yeast strains were created to attempt to substitute yeast eIF4E or eIF4G or both for Arabidopsis subunits. Lastly, an Arabidopsis mutant for ascorbic acid peroxidase (apx1) accumulates a high amount of cytosolic H₂O₂ which generates a more oxidizing environment. Arabidopsis carrying only exogenous AteIFiso4E or AteIFiso4E C97S in the eifiso4e/eif4e1/apx1 triple knockout were characterized for the response to this highly oxidizing environment.