Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania

Abstract

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

FIG. 1.

Homology modeling of LeishIF4E isoforms. A computerized prediction of the tertiary structures was performed using PDBView (A to H), based on the mouse eIF4E crystal structure (Protein Data Bank no. 1EJ1a) (I and J). The side residues that comprise the ligand binding pocket are shown in the left panels. The ligand (m⁷GDP) is marked in black.

FIG. 2.

Affinity purification of LeishIF4E. LeishIF4E isoforms were expressed in E. coli, the cells were lysed by sonication, and the supernatants were loaded on an m⁷GTP-Sepharose column (S represents 1% of the total input protein). After collection of the flowthrough fractions (FT), the column was washed with equilibrium buffer (wash). The proteins were eluted with the same buffer containing 600 mM NaCl (elution). Samples of equal volume from the different fractions were analyzed on Western blots using specific antibodies.

FIG. 3.

Results of fluorescence binding assays of LeishIF4E isoforms to cap analogues. (A) Comparison of the titration curves for complexes of Leishmania eIF4E isoforms with m⁷GTP. The insert shows the changes of protein fluorescence (F_t) in response to binding of m⁷GTP. These were obtained by subtracting the fluorescence of the free cap analogues (F_c), which were calculated from the obtained K_as. (B) Titration curves for binding of LeishIF4E-2 to cap 4 (•), m⁷GTP (□), and GTP (▾). Protein fluorescence was excited at 295 nm, and emission was observed at 320 nm. Fluorescence is presented as relative values. The increasing fluorescence intensity observed at the higher ligand concentrations originates from emission of the free cap analogues in solution. au, arbitrary units.

FIG. 4.

LeishIF4E-1 and LeishIF4E-4 interact with h4EBP1. GST or GST-4EBP1 was immobilized on glutathione-agarose beads. The beads were incubated with supernatants of bacteria that express the different LeishIF4E isoforms or mouse eIF4E (Sup represents 1% of the input for the pull-down assay and indicates that equimolar amounts of the recombinant protein were used). After extensive washes (W), the protein was eluted (E) with Laemmli's sample buffer and analyzed with specific antibodies against the different eIF4E isoforms.

FIG. 5.

None of the LeishIF4E isoforms complements for yeast eIF4E. p301-TRP1-eIF4E constructs were transformed into S. cerevisiae strain CWO4#368. Upon transformation, yeast cells were replicated on minimal medium containing SGal or SGal plus 0.5% 5′-FOA. After 3 days of growth, none of the four Leishmania eIF4E gene constructs was able to grow on plates containing 5′-FOA, and only p301-DmeIF4E-1 (Drosophila eIF4E-1) and p301-eIF4E (expressing yeast eIF4E [yeIF4E], clones 1 and 2) could complement for the loss of endogenous yeast eIF4E. Expression in yeast of the foreign genes tested is shown at the bottom, using extracts of cells freshly transformed and grown in SGal (in the absence of 5′-FOA). The top row shows expression of yeast eIF4E from the pVTURA3-4E plasmid, and the bottom row shows expression of the exogenous genes LeishIF4E-1 to LeishIF4E-4 and deIF4E (the yeast gene expressed from pVTURA3 cannot be distinguished from that expressed from p301).

FIG. 6.

Differential expression of the LeishIF4E isoforms in promastigotes and axenic amastigotes. (A) Sequence alignment of the 450-nucleotide amastin element of Leishmania infantum (GenBank accession no. AF195531) with the 3′ UTR of LeishIF4E-1 from L. major (AAZ09911) performed with ClustalX. (B) L. amazonensis promastigotes were grown at 26°C and transferred overnight to 33°C. Cells were harvested, extracted, and analyzed on Western blots with antibodies against the different eIF4E isoforms, with anti-Hsp70 and antitubulin antibodies as controls.

FIG. 7.

Localization by Western analysis of fractionated cells. Extracts were prepared from whole cells (T) and from subcellular fractions containing the cytoplasm (S) and nucleocytoskeletal (P) fractions and separated over 15% SDS-PAGE. Western blots were reacted with antibodies against LeishIF4E isoforms. Control blots containing equal loads of the same fractions were reacted with monoclonal antibodies against Hsp70 and α-tubulin, representing the cytoplasmic and nonsoluble nucleocytoskeletal fractions, respectively.

FIG. 8.

Polysome distribution of the different LeishIF4E isoforms. L. major cells were lysed, and the extracts were loaded on 10 to 50% sucrose gradients. (A) Fractions were collected from the top, and the optical density at 260 nm (OD₂₆₀) was monitored. The graphs depict patterns of migration in sucrose gradients of untreated polysomes (continuous line), EDTA-treated polysome extracts (dots), and polysomes treated with micrococcal nuclease (broken line). Proteins from untreated extracts (B) and from EDTA-treated (C) and micrococcal nuclease-treated (D) proteins were recovered by trichloroacetic acid precipitation and further immunoblotted with antibodies that were raised against the different LeishIF4E isoforms.