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. 2009 Jun 12;383(4):497-502.
doi: 10.1016/j.bbrc.2009.04.049. Epub 2009 Apr 18.

The effect of hypusine modification on the intracellular localization of eIF5A

Seung Bum Lee  1 , Jong Hwan Park, Jörn Kaevel, Monika Sramkova, Roberto Weigert, Myung Hee Park
Affiliations

The effect of hypusine modification on the intracellular localization of eIF5A

Seung Bum Lee et al. Biochem Biophys Res Commun. .

Abstract

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential for eukaryotic cell proliferation and is the only protein containing hypusine, [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine. eIF5A also undergoes an acetylation at specific Lys residue(s). In this study, we have investigated the effect of hypusine modification and acetylation on the subcellular localization of eIF5A. Immunocytochemical analyses showed differences in the distribution of non-hypusinated eIF5A precursor and the hypusine-containing mature eIF5A. While the precursor is found in both cytoplasm and nucleus, the hypusinated eIF5A is primarily localized in cytoplasm. eIF5A mutant proteins, defective in hypusine modification (K50A, K50R) were localized in a similar manner to the eIF5A precursor, whereas hypusine-modified mutant proteins (K47A, K47R, K68A) were localized mainly in the cytoplasm. These findings provide strong evidence that the hypusine modification of eIF5A dictates its localization in the cytoplasmic compartment where it is required for protein synthesis.

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Figures

Fig. 1
Fig. 1. Exogenous expression of (A) FLAG-eIF5A and (B) GFP-eIF5A precursors or their hypusine-modified forms
HeLa cells were transfected with 3XFLAG-CMV-7.1, 3XFLAG-CMV-7.1/heIF5A-1, pCEFL, or pCEFL/GFP-heIF5A-1 with or without pCEFL/hDHS and pCEFL/hDOHH as indicated. Transfection was performed in parallel in two sets, one set of cells (6 well dishes) were used for western blotting using an eIF5A antibody (BD Bioscience) that detects eIF5A(Lys), eIF5A(Dhp) and eIF5A(Hpu) equally well. The other set (60 mm dishes) was used for labeling with [3H]spermidine (5 μCi/ml). Cells were harvested after 42 h of transfection.
Fig. 2
Fig. 2. Identification of the major acetylation site of eIF5A as Lys47
HeLa cells were transfected as indicated and harvested after 48 h of transfection. 10 μg of proteins was used for western blotting using either AcLys antibody (Santa Cruze Biotechnology), eIF5A antibody (BD Bioscience), or DHS and DOHH rabbit polyclonal antibodies.
Fig. 3
Fig. 3. Comparison of subcellular distribution of endogenous eIF5A(Hpu), exogenous eIF5A(Lys) and exogenous eIF5A(Hpu)
(A) HeLa cells were fixed after 48 h of transfection, incubated with primary antibodies, eIF5A antibody (NIH353, rabbit polyclonal) or anti FLAG antibody (mouse monoclonal from Sigma) followed by the secondary antibodies, Alexa Fluor 488-conjugated secondary anti-rabbit antibodies (green, Invitrogen) or Alexa Fluor 594-conjugated secondary anti-mouse antibodies (red, Invitrogen) for detection of endogenous eIF5A and exogenous FLAG-eIF5A, respectively. (B) HeLa cells, transfected with pCEFL/GFP-eIF5A without or with vectors encoding DHS and DOHH, were fixed and GFP-eIF5A precursor and GFP-eIF5A(Hpu) were directly visualized. (C) Fluorescence of GFP or GFP-eIF5A formed in HeLa cells transfected as in (B) was visualized by a confocal microscope.
Fig. 4
Fig. 4. Comparison of the cellular distribution of GFP-eIF5A mutant proteins
(A) direct visualization of GFP fluorescence after 48h of transfection. HeLa cells were transfected with vectors encoding GFP-eIF5A wt and mutants, without (upper panels) or with (lower panels) vectors encoding DHS and DOHH. (B) western blots of the above samples using AcLys and eIF5A antibodies (BD Bioscience).
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References

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