Eukaryotic initiation factor 5A dephosphorylation is required for translational arrest in stationary phase cells

Abstract

The protein known as eIF5A (eukaryotic initiation factor 5A) has an elusive role in translation. It has a unique and essential hypusine modification at a conserved lysine residue in most eukaryotes. In addition, this protein is modified by phosphorylation with unknown functions. In the present study we show that a phosphorylated state of eIF5A predominates in exponentially growing Trypanosoma cruzi cells, and extensive dephosphorylation occurs in cells in stationary phase. Phosphorylation occurs mainly at Ser(2), as shown in yeast eIF5A. In addition, a novel phosphorylation site was identified at Tyr(21). In exponential cells, T. cruzi eIF5A is partially associated with polysomes, compatible with a proposed function as an elongation factor, and becomes relatively enriched in polysomal fractions in stationary phase. Overexpression of the wild-type eIF5A, or eIF5A with Ser(2) replaced by an aspartate residue, but not by alanine, increases the rate of cell proliferation and protein synthesis. However, the presence of an aspartate residue instead of Ser(2) is toxic for cells reaching the stationary phase, which show a less-pronounced protein synthesis arrest and a decreased amount of eIF5A in dense fractions of sucrose gradients. We conclude that eIF5A phosphorylation and dephosphorylation cycles regulate translation according to the growth conditions.

Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

“…Another highly conserved and essential protein involved in translation is eukaryotic translation initiation factor 5A (eIF5A). Its role in translation has been studied in many organisms and its expression is induced in proliferating cells, while it is repressed under carbohydrate starvation6465. Although eIF5A is conserved across eukaryotes, the N-terminus of the protein was shown to differ when comparing plants and mammals.…”

“…The transcript levels of rice ( Oryza sativa ) OseIF5A1 and OseIF5A1 in suspension cell culture are strongly affected by carbohydrate starvation and the amount of mRNA is reduced when sugars are not available65. In exponentially growing protozoan parasite Trypanosoma cruzi TceIF5A is highly phosphorylated at the same N-terminal Ser 2 as in Arabidopsis64, indicating that the phosphorylation has a significant role regulating protein synthesis and growth. Moreover, depletion of eIF5A in yeast ( Saccharomyces cerevisiae ) rapidly affects the protein translation rate impairing it by 2–3-fold73.…”

Characterization of two different Asf1 histone chaperones with distinct cellular localizations and functions in Trypanosoma brucei

“…Specific antibodies to Asf1A were eluted from the column with 0.1 M triethylamine (pH 11.5), and the eluted fractions were neutralized with 1 M Tris–HCl (pH 7.4) and kept at 4°C in the presence of 2 mg/ml BSA. Anti- Trypanosoma cruzi β-tubulin antibodies were prepared as described previously (39). Anti-histone H4 was obtained from Abcam and anti- T .…”

Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

“…For HS experiments, cells were maintained at 40°C for 2 h. After each treatment, cells were collected and centrifuged at 2,000 ϫ g for 2 min, and the resulting pellet was washed with phosphate-buffered saline (PBS) and used for Western blotting or immunofluorescence. The following antibodies were used: affinity-purified anti-TcRNA Pol II antibody (21), anti-T. cruzi ␤-tubulin C-terminal sequence antibody, anti-eukaryotic initiation factor 5A (eIF5A) antibody (34), and anti-nonacetylated histone H4 N-terminal sequence antibody (35).…”