Nutritional stress affects an atypical cap-binding protein in Leishmania

Abstract

Many eukaryotes encode multiple isoforms of the cap-binding translation initiation factor (eIF4E). Leishmanias and other trypanosomatids encode four paralogs of this protein, but none can complement the eIF4E function in a yeast mutant. A low conservation is observed between the four paralogs, suggesting they assist these organisms survive a multitude of conditions encountered throughout the life cycle. Earlier attempts to decipher their function led to identification of LeishIF4E-4 as the canonical translation initiation factor. LeishIF4E-1 appears to function during thermal stress, via a mechanism not yet understood. LeishIF4E-3 hardly binds cap-4 and is, therefore, less likely to serve as a typical initiation factor. Although it interacts with an eIF4G homolog, LeishIF4G-4, the two polypeptides do not co-migrate on sucrose gradients. While LeishIF4E-3 enters large particles that increase in size during nutritional stress, LeishIF4G-4 is found only in the top fractions. Confocal microscopy localized LeishIF4E-3 (but not LeishIF4G-4) within nutritional stress-induced granules. Accordingly, interaction between the two proteins reduced upon starvation. We therefore propose that under normal conditions, LeishIF4G-4 sequesters LeishIF4E-3 in the cytoplasm. During a nutritional stress, LeishIF4E-3 is modified and released from LeishIF4G-4 to enter stress granules, where inactive mRNAs are stored. Binding of LeishIF4G-4 to LeishIF4E-3 requires a short peptide within the LeishIF4G-4 N-terminus, which bears no similarity to the consensus 4E-binding peptide, YXXXXLΦ. Mutational analysis combined with structure prediction indicates that this interaction is based on an obligatory, conserved α helix in LeishIF4G-4. These features further highlight the uniqueness of LeishIF4E-3 and how it interacts with its binding partners.

Keywords: :

• Leishmania
• eIF4E
• eIF4G
• 4E-binding peptide
• RNA granules

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by the Israel Science Foundation, (ISF, grant No 395/09) and by the US-Israel Binational Foundation (BSF, grant number 2007287). We are grateful to Charles Jaffe from the Hebrew University of Jerusalem and Joachim Clos from the Bernhard Nocht Institute for Tropical Diseases, Hamburg, Germany for providing specific antibodies against GP46 and Hsp100, respectively. We acknowledge Steve Beverley from Washington University, St. Louis, MO for providing the pX-GFP expression vector.