The related EIF4G3 and EIF4G4 initiation factors from Leishmania: dissimilar modes of action during translation revealed by a comparative proteomic approach.

BACKGROUND: The start of eukaryotic translation requires recruitment of messenger RNA (mRNA) through the action of the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is formed by joining of the eIF4G and eIF4E subunits and generally also requires the eIF4A helicase. In Leishmania infantum, five eIF4Gs form multiple eIF4F-like complexes, with those based on the related EIF4G3 and EIF4G4 being active during translation, but with likely nonredundant roles that need to be better defined. METHODS: To further investigate the roles of EIF4G3 and EIF4G4 in Leishmania infantum, we generated transgenic cell lines expressing each protein tagged with a C-terminal hemagglutinin (HA) epitope. Expression analyses were then carried out during different phases of promastigote growth, followed by gene knockout and complementation assays investigating the essentiality of the targeted eIF4Gs. The HA-tagged proteins were then used as baits in a large-scale investigation of potential protein partners, from different growth phases: early exponential, late exponential, and stationary. RESULTS: EIF4G3 and EIF4G4 were expressed as multiple isoforms during promastigote growth, with EIF4G4 isoforms changing according to the growth phase. The two HA-tagged proteins were capable of replacing the corresponding native proteins after deletion of the endogenous genes. EIF4G3-HA and EIF4G4-HA were always found with their known eIF4E partners, respectively EIF4E4 and EIF4E3. EIF4G3-HA also more consistently coprecipitated with poly(A)-binding protein 1 (PABP1), RNA-binding protein 23 (RBP23), and EIF4AI, with EIF4G4-HA having greater association with PABP3 and the HEL67 helicase. A variable number of translation factors and ribosomal proteins were found with both baits, reflecting roles in translation. Our extensive analyses, investigating also proteins with possible moonlighting roles and uncharacterized polypeptides, not only revealed new proteins bound to both baits but also identified new specific partners for EIF4G3, and possibly EIF4G4, some of those being restricted to selected growth phases. CONCLUSIONS: Overall, new and more defined binding partners were observed for EIF4G3, with EIF4G4 having an increased coprecipitation with other translation initiation factors. Newly identified partners, for both eIF4Gs, might facilitate specific mRNA recognition or function regulating translation during growth. Further studies on some of those might reveal unique and conserved aspects of the Leishmania translation and might help define targets for novel translation inhibitors.