The eukaryotic translation elongation factor eIF5A is the only protein known to contain the unusual amino acid hypusine which is essential for its biological activity. This post-translational modification is achieved by the sequential action of the enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The crucial molecular function of eIF5A during translation has been recently elucidated in yeast and it is expected to be fully conserved in every eukaryotic cell, however the functional description of this pathway in plants is still sparse. The genetic approaches with transgenic plants for either eIF5A overexpression or antisense have revealed some activities related to the control of cell death processes but the molecular details remain to be characterized. One important aspect of fully understanding this pathway is the biochemical description of the hypusine modification system. Here we have used recombinant eIF5A proteins either modified by hypusination or non-modified to establish a bi-dimensional electrophoresis (2D-E) profile for the three eIF5A protein isoforms and their hypusinated or unmodified proteoforms present in Arabidopsis thaliana. The combined use of the recombinant 2D-E profile together with 2D-E/western blot analysis from whole plant extracts has provided a quantitative approach to measure the hypusination status of eIF5A. We have used this information to demonstrate that treatment with the hormone abscisic acid produces an alteration of the hypusine modification system in Arabidopsis thaliana. Overall this study presents the first biochemical description of the post-translational modification of eIF5A by hypusination which will be functionally relevant for future studies related to the characterization of this pathway in Arabidopsis thaliana.Belda Palazón, B.; Nohales Zafra, MA.; Rambla Nebot, JL.; Aceña, JL.; Delgado, O.; Fustero Lardies, S.; Martínez, MC.... (2014). Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation. Frontiers in Plant Science. 5:202-1-202-11. doi:10.3389/fpls.2014.00202S202-1202-115Belda-Palazón, B., Ruiz, L., Martí, E., Tárraga, S., Tiburcio, A. F., Culiáñez, F., … Ferrando, A. (2012). Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells. PLoS ONE, 7(10), e46907. doi:10.1371/journal.pone.0046907Bergeron, R. J., Weimar, W. R., Müller, R., Zimmerman, C. O., McCosar, B. H., Yao, H., & Smith, R. E. (1998). Synthesis of Reagents for the Construction of Hypusine and Deoxyhypusine Peptides and Their Application as Peptidic Antigens. Journal of Medicinal Chemistry, 41(20), 3888-3900. doi:10.1021/jm980389pChattopadhyay, M. K., Park, M. H., & Tabor, H. (2008). Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine. Proceedings of the National Academy of Sciences, 105(18), 6554-6559. doi:10.1073/pnas.0710970105Chevallet, M., Luche, S., & Rabilloud, T. (2006). Silver staining of proteins in polyacrylamide gels. Nature Protocols, 1(4), 1852-1858. doi:10.1038/nprot.2006.288Cuevas, J. C., López-Cobollo, R., Alcázar, R., Zarza, X., Koncz, C., Altabella, T., … Ferrando, A. (2008). Putrescine Is Involved in Arabidopsis Freezing Tolerance and Cold Acclimation by Regulating Abscisic Acid Levels in Response to Low Temperature. Plant Physiology, 148(2), 1094-1105. doi:10.1104/pp.108.122945Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K., & Scheible, W.-R. (2005). Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis. Plant Physiology, 139(1), 5-17. doi:10.1104/pp.105.063743Dias, C. A. O., Garcia, W., Zanelli, C. F., & Valentini, S. R. (2012). eIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer. Amino Acids, 44(2), 631-644. doi:10.1007/s00726-012-1387-7Doerfel, L. K., Wohlgemuth, I., Kothe, C., Peske, F., Urlaub, H., & Rodnina, M. V. (2012). EF-P Is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues. Science, 339(6115), 85-88. doi:10.1126/science.1229017Duguay, J., Jamal, S., Liu, Z., Wang, T.-W., & Thompson, J. E. (2007). Leaf-specific suppression of deoxyhypusine synthase in Arabidopsis thaliana enhances growth without negative pleiotropic effects. Journal of Plant Physiology, 164(4), 408-420. doi:10.1016/j.jplph.2006.02.001Feng, H., Chen, Q., Feng, J., Zhang, J., Yang, X., & Zuo, J. (2007). Functional Characterization of the Arabidopsis Eukaryotic Translation Initiation Factor 5A-2 That Plays a Crucial Role in Plant Growth and Development by Regulating Cell Division, Cell Growth, and Cell Death. Plant Physiology, 144(3), 1531-1545. doi:10.1104/pp.107.098079Gregio, A. P. B., Cano, V. P. S., Avaca, J. S., Valentini, S. R., & Zanelli, C. F. (2009). eIF5A has a function in the elongation step of translation in yeast. Biochemical and Biophysical Research Communications, 380(4), 785-790. doi:10.1016/j.bbrc.2009.01.148Guo, J., Wang, S., Valerius, O., Hall, H., Zeng, Q., Li, J.-F., … Chen, J.-G. (2010). Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid. Plant Physiology, 155(1), 370-383. doi:10.1104/pp.110.160663Gutierrez, E., Shin, B.-S., Woolstenhulme, C. J., Kim, J.-R., Saini, P., Buskirk, A. R., & Dever, T. E. (2013). eIF5A Promotes Translation of Polyproline Motifs. Molecular Cell, 51(1), 35-45. doi:10.1016/j.molcel.2013.04.021Hamasaki-Katagiri, N., Tabor, C. W., & Tabor, H. (1997). Spermidine biosynthesis in Saccharomyces cerevisiae: Polyaminerequirement of a null mutant of the SPE3 gene (spermidine synthase). Gene, 187(1), 35-43. doi:10.1016/s0378-1119(96)00660-9Imai, A., Matsuyama, T., Hanzawa, Y., Akiyama, T., Tamaoki, M., Saji, H., … Takahashi, T. (2004). Spermidine Synthase Genes Are Essential for Survival of Arabidopsis. Plant Physiology, 135(3), 1565-1573. doi:10.1104/pp.104.041699Ishfaq, M., Maeta, K., Maeda, S., Natsume, T., Ito, A., & Yoshida, M. (2012). Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A). FEBS Letters, 586(19), 3236-3241. doi:10.1016/j.febslet.2012.06.042Jin, B.-F., He, K., Wang, H.-X., Wang, J., Zhou, T., Lan, Y., … Zhang, X.-M. (2003). Proteomic analysis of ubiquitin-proteasome effects: insight into the function of eukaryotic initiation factor 5A. Oncogene, 22(31), 4819-4830. doi:10.1038/sj.onc.1206738Kang, K. R., & Chung, S. I. (2003). Protein kinase CK2 phosphorylates and interacts with deoxyhypusine synthase in HeLa cells. Experimental & Molecular Medicine, 35(6), 556-564. doi:10.1038/emm.2003.73Klier, H., Csonga, R., Joao, H. C., Eckerskorn, C., Auer, M., Lottspeich, F., & Eder, J. (1995). Isolation and Structural Characterization of Different Isoforms of the Hypusine-Containing Protein eIF-5A from HeLa Cells. Biochemistry, 34(45), 14693-14702. doi:10.1021/bi00045a010Łebska, M., Ciesielski, A., Szymona, L., Godecka, L., Lewandowska-Gnatowska, E., Szczegielniak, J., & Muszyńska, G. (2009). Phosphorylation of Maize Eukaryotic Translation Initiation Factor 5A (eIF5A) by Casein Kinase 2. Journal of Biological Chemistry, 285(9), 6217-6226. doi:10.1074/jbc.m109.018770Lee, S. B., Park, J. H., Kaevel, J., Sramkova, M., Weigert, R., & Park, M. H. (2009). The effect of hypusine modification on the intracellular localization of eIF5A. Biochemical and Biophysical Research Communications, 383(4), 497-502. doi:10.1016/j.bbrc.2009.04.049Li, C. H., Ohn, T., Ivanov, P., Tisdale, S., & Anderson, P. (2010). eIF5A Promotes Translation Elongation, Polysome Disassembly and Stress Granule Assembly. PLoS ONE, 5(4), e9942. doi:10.1371/journal.pone.0009942Liu, Z., Duguay, J., Ma, F., Wang, T.-W., Tshin, R., Hopkins, M. T., … Thompson, J. E. (2008). Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana. Journal of Experimental Botany, 59(4), 939-950. doi:10.1093/jxb/ern017MA, F., LIU, Z., WANG, T.-W., HOPKINS, M. T., PETERSON, C. A., & THOMPSON, J. E. (2010). Arabidopsis eIF5A3 influences growth and the response to osmotic and nutrient stress. Plant, Cell & Environment, 33(10), 1682-1696. doi:10.1111/j.1365-3040.2010.02173.xMaier, B., Ogihara, T., Trace, A. P., Tersey, S. A., Robbins, R. D., Chakrabarti, S. K., … Mirmira, R. G. (2010). The unique hypusine modification of eIF5A promotes islet β cell inflammation and dysfunction in mice. Journal of Clinical Investigation, 120(6), 2156-2170. doi:10.1172/jci38924Mandal, S., Mandal, A., Johansson, H. E., Orjalo, A. V., & Park, M. H. (2013). Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells. Proceedings of the National Academy of Sciences, 110(6), 2169-2174. doi:10.1073/pnas.1219002110Moreno-Romero, J., Carme Espunya, M., Platara, M., Ariño, J., & Carmen Martínez, M. (2008). A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutant. The Plant Journal, 55(1), 118-130. doi:10.1111/j.1365-313x.2008.03494.xNishimura, K., Lee, S. B., Park, J. H., & Park, M. H. (2011). Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development. Amino Acids, 42(2-3), 703-710. doi:10.1007/s00726-011-0986-zNISHIMURA, K., OHKI, Y., FUKUCHI-SHIMOGORI, T., SAKATA, K., SAIGA, K., BEPPU, T., … IGARASHI, K. (2002). Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin. Biochemical Journal, 363(3), 761. doi:10.1042/0264-6021:3630761Pagnussat, G. C. (2005). Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis. Development, 132(3), 603-614. doi:10.1242/dev.01595Park, J. H., Dias, C. A. O., Lee, S. B., Valentini, S. R., Sokabe, M., Fraser, C. S., & Park, M. H. (2010). Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes. Protein Engineering Design and Selection, 24(3), 301-309. doi:10.1093/protein/gzq110Park, M. H. (2006). The Post-Translational Synthesis of a Polyamine-Derived Amino Acid, Hypusine, in the Eukaryotic Translation Initiation Factor 5A (eIF5A). The Journal of Biochemistry, 139(2), 161-169. doi:10.1093/jb/mvj034Park, M. H., Lee, Y. B., & Joe, Y. A. (1997). Hypusine Is Essential for Eukaryotic Cell Proliferation. Neurosignals, 6(3), 115-123. doi:10.1159/000109117Patel, P. H., Costa-Mattioli, M., Schulze, K. L., & Bellen, H. J. (2009). The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy. The Journal of Cell Biology, 185(7), 1181-1194. doi:10.1083/jcb.200904161Ren, B., Chen, Q., Hong, S., Zhao, W., Feng, J., Feng, H., & Zuo, J. (2013). The Arabidopsis Eukaryotic Translation Initiation Factor eIF5A-2 Regulates Root Protoxylem Development by Modulating Cytokinin Signaling. The Plant Cell, 25(10), 3841-3857. doi:10.1105/tpc.113.116236Saez, A., Robert, N., Maktabi, M. H., Schroeder, J. I., Serrano, R., & Rodriguez, P. L. (2006). Enhancement of Abscisic Acid Sensitivity and Reduction of Water Consumption in Arabidopsis by Combined Inactivation of the Protein Phosphatases Type 2C ABI1 and HAB1. Plant Physiology, 141(4), 1389-1399. doi:10.1104/pp.106.081018Saini, P., Eyler, D. E., Green, R., & Dever, T. E. (2009). Hypusine-containing protein eIF5A promotes translation elongation. Nature, 459(7243), 118-121. doi:10.1038/nature08034Scheich, C., Kummel, D., Soumailakakis, D., Heinemann, U., & Bussow, K. (2007). Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Research, 35(6), e43-e43. doi:10.1093/nar/gkm067Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V., Sagliocco, F., Wilm, M., Vorm, O., … Mann, M. (1996). Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels. Proceedings of the National Academy of Sciences, 93(25), 14440-14445. doi:10.1073/pnas.93.25.14440Strohalm, M., Hassman, M., Košata, B., & Kodíček, M. (2008). mMass data miner: an open source alternative for mass spectrometric data analysis. Rapid Communications in Mass Spectrometry, 22(6), 905-908. doi:10.1002/rcm.3444Vizcaíno, J. A., Côté, R. G., Csordas, A., Dianes, J. A., Fabregat, A., Foster, J. M., … Hermjakob, H. (2012). The Proteomics Identifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Research, 41(D1), D1063-D1069. doi:10.1093/nar/gks1262Wang, L., Xu, C., Wang, C., & Wang, Y. (2012). Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance. BMC Plant Biology, 12(1), 118. doi:10.1186/1471-2229-12-118Wolff, E. C., Kang, K. R., Kim, Y. S., & Park, M. H. (2007). Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification. Amino Acids, 33(2), 341-350. doi:10.1007/s00726-007-0525-0Xu, A., & Chen, K. Y. (2000). Hypusine Is Required for a Sequence-specific Interaction of Eukaryotic Initiation Factor 5A with Postsystematic Evolution of Ligands by Exponential Enrichment RNA. Journal of Biological Chemistry, 276(4), 2555-2561. doi:10.1074/jbc.m008982200XU, A., JAO, D. L.-E., & CHEN, K. Y. (2004). Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display. Biochemical Journal, 384(3), 585-590. doi:10.1042/bj20041232Xu, J., Zhang, B., Jiang, C., & Ming, F. (2010). RceIF5A, encoding an eukaryotic translation initiation factor 5A in Rosa chinensis, can enhance thermotolerance, oxidative and osmotic stress resistance of Arabidopsis thaliana. Plant Molecular Biology, 75(1-2), 167-178. doi:10.1007/s11103-010-9716-2Zanor, M. I., Rambla, J.-L., Chaïb, J., Steppa, A., Medina, A., Granell, A., … Causse, M. (2009). Metabolic characterization of loci affecting sensory attributes in tomato allows an assessment of the influence of the levels of primary metabolites and volatile organic contents. Journal of Experimental Botany, 60(7), 2139-2154. doi:10.1093/jxb/erp08
