Eukaryotic initiation factor 3 (eIF3) is a large multi-protein complex that is a key component in the recruitment and assembly of the translation initiation machinery. In mammals eIF3 comprises 13 subunits, most of which are characterized by conserved structural domains. Analysis of trypanosomal genomes provided only partial annotations of the eIF3 subunits, highlighting their evolutionary divergence with respect to higher eukaryotes. In trypanosomatids, the translation machinery is tightly regulated in the different life stages of these organisms as part of their adaptation and survival in changing environments. Here, we identify the components of the Leishmania eIF3 complex by combining bioinformatics with biochemical methodologies, using affinity purification and mass spectrometry to identify the complex subunits. Using this strategy, we identified 12 subunits as part of the Leishmania eIF3 complex (LeishIF3a-l). We further addressed the mechanism by which LeishIF3 is recruited to different mRNA cap-binding complexes. Our in vitro experiments reveal a direct interaction between the fully assembled LeishIF3 complex and LeishIF4G3, the canonical scaffolding protein of the cap-binding complex in Leishmania promastigotes. We also highlight a novel and alternative interaction between LeishIF3a and LeishIF4E1, the only cap-binding protein that efficiently binds the cap structure under prolonged heat shock conditions.
