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. 2008 Jan;275(1):44-58.
doi: 10.1111/j.1742-4658.2007.06172.x. Epub 2007 Dec 6.

Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification

Veridiana S P Cano  1 , Geoung A Jeon, Hans E Johansson, C Allen Henderson, Jong-Hwan Park, Sandro R Valentini, John W B Hershey, Myung Hee Park
Affiliations

Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification

Veridiana S P Cano et al. FEBS J. 2008 Jan.

Abstract

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.

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Figures

Fig. 1
Fig. 1. Growth analysis of S. cerevisiae strains expressing human eIF5A wild type and mutant proteins
(A) The haploid strain HHY13a was transformed with recombinant p414GAL1 plasmid encoding human eIF5A-1 wild type or mutant proteins with single amino acid substitutions, indicated on the left side. Trp+ transformants were selected on minimal galactose plates (SGal,-His, -Leu, -Trp, -Ura). Four individual transformant colonies (HHY212d) were resuspended in water and spotted in parallel on the same selection plates (left panels) and on the 5-FOA containing plates (SGal, -His,- Leu, -Trp, plus 5-FOA) (middle and right panels) to derive HHY212s that lost pBM-TIF51A. The plates were photographed after incubation at 30 C for 2 days without 5-FOA (left panels), or 3 and 5 days with 5-FOA (middle and right panels). (B), Growth curves of HHY212s strains harboring only the human eIF5A proteins. The experiments were repeated two to three times with virtually the same results: a typical experiment is shown.
Fig. 2
Fig. 2. Expression and stability of human eIF5A wild type and mutant proteins in S. cerevisiae strains
Western blot analyses of proteins of HHY212d strains harboring both recombinant plasmids, pBM-TIF51A and p414GAL1-heIF5A (A) and of HHY212s strains expressing only the human eIF5A proteins (B). All the strains in A were cultured in SGal,-His,-Leu,-Trp,-Ura and those in B, cultured in SGal,-His,-Leu,-Trp. The experiments were repeated two times with virtually the same results: a typical experiment is shown.
Fig. 3
Fig. 3. Human eIF5A mutant proteins as substrates for deoxyhypusine synthase and deoxyhypusine hydroxylase in vitro
Human recombinant eIF5A proteins were expressed in E. coli, BL21(DE3) and cell lysates were used as substrates for DHS and DOHH in a combined assay. Coomassie-Blue staining of BL21(DE3) lysates expressing human mutant proteins (A, top panel) and fluorogram of the SDS gel of DHS/DOHH reaction mixture showing labeling of several mutant proteins (A, bottom panel). Portions of DHS/DOHH reaction mixtures were analyzed for radioactive deoxyhypusine and hypusine content in the products by ion exchange chromatographic separation (B) as described previously (49). The experiments were repeated two to three times with virtually the same results: a typical experiment is shown.
Fig. 4
Fig. 4. Growth analysis of S. cerevisiae strains expressing truncated human eIF5A and expression and stability of truncated proteins
Growth analysis was performed as described in Fig. 1 (A) and western blots of proteins of HHY212d strains harboring both recombinant plasmids, pBM-TIF51A and p414GAL1-heIF5A (B) are shown. The strains were cultured in minimal galactose medium (SGal,-His,-Leu,-Trp,-Ura). The experiments were repeated two to three times with virtually the same results: a typical experiment is shown.
Fig. 5
Fig. 5. The effects of human wild type and mutant eIF5A expression on growth and protein synthesis in UBHY-R
Exponential cultures (in YPGal medium) of W303-1A, UBHY-R, and UBHY-R strains transformed with p414GAL1 vectors encoding human eIF5A-1 wild type or mutant proteins K47A, K47R, and K50R were washed with water, resuspended in YPD medium at the density of ~0.125 (OD 600 nm) and growth was followed for 5 h (A). At 0, 1, 3, and 5 h after shift to the glucose medium, aliquots of cells were used to measure protein synthesis as described under Experimental Procedures and the rate of protein synthesis was calculated for each sample as dpm/μg/20 min (B). The levels of human and yeast eIF5A and yeast UBR5A proteins were determined by western blot analysis (C). The experiments were repeated two to three times with virtually the same results: a typical experiment is shown.
Scheme 1
Scheme 1. Model structure of human eIF5A with critical amino acid residues
The structure of human eIF5A-1 is based on the model (PDB 1FH4) constructed by Facchiano et al. (36). Both the N- and C-terminal domains (in blue and aqua-blue) consist of β-sheet core structures and are connected by a hinge at Asn83-Ile84. The hypusine modification site (Lys 50, in red) is located at an exposed loop (hypusine loop aa46–54) in the basic N-terminal domain. No growth is observed upon Ala substitution of the red and orange colored residues (Lys50, Gly49, Gly52 and Lys55) and slow growth upon Ala substitution of the green colored residues (Lys47, His55, Pro74, Leu91 and Leu101).
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