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. 2009 May 7;459(7243):118-21.
doi: 10.1038/nature08034.

Hypusine-containing protein eIF5A promotes translation elongation

Preeti Saini  1 , Daniel E Eyler, Rachel Green, Thomas E Dever
Affiliations

Hypusine-containing protein eIF5A promotes translation elongation

Preeti Saini et al. Nature. .

Abstract

Translation elongation factors facilitate protein synthesis by the ribosome. Previous studies identified two universally conserved translation elongation factors, EF-Tu in bacteria (known as eEF1A in eukaryotes) and EF-G (eEF2), which deliver aminoacyl-tRNAs to the ribosome and promote ribosomal translocation, respectively. The factor eIF5A (encoded by HYP2 and ANB1 in Saccharomyces cerevisiae), the sole protein in eukaryotes and archaea to contain the unusual amino acid hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine), was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis-a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation. Hypusine is required for eIF5A to associate with ribosomes and to stimulate methionyl-puromycin synthesis. Because eIF5A did not stimulate earlier steps of translation initiation, and depletion of eIF5A in yeast only modestly impaired protein synthesis, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular messenger RNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay, including the nonsense-mediated mRNA decay pathway, and to nucleocytoplasmic transport. Here we use molecular genetic and biochemical studies to show that eIF5A promotes translation elongation. Depletion or inactivation of eIF5A in the yeast S. cerevisiae resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, enhanced the rate of tripeptide synthesis in vitro. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function together with eEF2 to promote ribosomal translocation. Because eIF5A is a structural homologue of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor.

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Figures

Figure 1
Figure 1. eIF5A depletion impairs yeast cell growth and protein synthesis, and causes retention of polysomes
(a) Isogenic WT (J714) and tif51a-td mutant strains (J702) were serially diluted, spotted on permissive (SCRaf + Cu2+) or non-permissive (SCGal) medium, and incubated 5 days at 25 °C. Total protein synthesis (b) and polysome profiles (c) were analyzed in tif51a-td (J702), tif32-td (YAJ22), and their isogenic WT strains following growth under non-permissive conditions. Incorporation (dpm) of [35S]Met is expressed per A600 unit, and results are representative of triplicate experiments. WT* and tif32-td* strains are isogenic. (c) Cells were treated (+) or untreated (-) with 50 μg/ml cycloheximide (CHX) prior to harvesting, and WCEs were separated on sucrose gradients and fractionated to visualize the indicated ribosomal species. P/M ratios were calculated by comparing areas under the 80S and polysome peaks; ↓polysomes indicates reduced amount of polysomes.
Figure 2
Figure 2. Translation elongation defect in temperature-sensitive tif51a-D63V mutant
(a) Isogenic WT (J697) and tif51a-D63V mutant (D63V; J698) strains were serially diluted, spotted on SC medium, and incubated 3 days at 25, 37, or 38 °C. (b) WT (left panel) and tif51a-D63V (right) mutant strains were shifted to 36 °C for 2 h, and then labeled with [35S]Met. Fitting lines of total (filled symbols) and completed (open symbols) protein synthesis were obtained by linear regression and used to determine the half-average (t1/2) and average (T) transit time. Data are the average of three independent experiments. (c) In vitro translation activity of heat-treated extracts from WT and tif51a-D63V strains following addition of eIF5A or eIF5AK51R. Results are representative of six independent experiments.
Figure 3
Figure 3. eIF5A stimulates elongation and termination in vitro
Rate constants for (a) tripeptide synthesis and (b) dipeptide release in the presence of eIF5A, eIF5AK51R, or no added factors. Error bars are the standard error from three independent experiments.
Figure 4
Figure 4. Functional connection between eIF5A and eEF2
(a) Isogenic WT (J697) and tif51a-D63V (D63V; J698) strains were serially diluted, spotted on SC medium lacking or containing 200 ng/ml sordarin, and incubated 3 days at 25 or 32 °C. (b,c) Cultures of WT or tif51-td strains grown under non-permissive conditions were cross-linked with 1% formaldehyde. WCEs were separated on sucrose gradients, and gradient fractions were subjected to Western analysis using antibodies against the indicated proteins (PUB2 = 60S subunit protein RPL39).
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References

    1. Merrick WC, Nyborg J. In: Translational Control of Gene Expression. Sonenberg N, Hershey JWB, Mathews MB, editors. Cold Spring Harbor Laboratory Press; 2000. pp. 89–125.
    1. Wolff EC, Kang KR, Kim YS, Park MH. Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification. Amino Acids. 2007;33:341–350. - PMC - PubMed
    1. Kemper WM, Berry KW, Merrick WC. Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Bα and M2Bβ. J Biol Chem. 1976;251:5551–5557. - PubMed
    1. Schreier MH, Erni B, Staehelin T. Initiation of mammalian protein synthesis: purification and characterization of seven initiation factors. J Mol Biol. 1977;116:727–753. - PubMed
    1. Jao DL, Chen KY. Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex. J Cell Biochem. 2006;97:583–598. - PubMed

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