Báo cáo khoa học: Unique modifications of translation elongation factors pot

REVIEW ARTICLE Unique modifications of translation elongation factors Eva Greganova*, Michael Altmann and Peter Bu ¨ tikofer Institute for Biochemistry and Molecular Medicine, University of Berne, Switzerland Keywords diphthamide; eEF1A; eEF2; eIF5A; ethanolamine phosphoglycerol; hypusine; protein modifcation; translation elongation Correspondence P. Bu ¨ tikofer, Institute of Biochemistry and Molecular Medicine, University of Bern, Bu ¨ hlstrasse 28, 3012 Bern, Switzerland Fax: +41 31 631 3737 Tel: +41 31 631 4113 E-mail: peter.buetikofer@mci.unibe.ch M. Altmann, Institute of Biochemistry and Molecular Medicine, University of Bern, Bu ¨ hlstrasse 28, 3012 Bern, Switzerland Fax: +41 31 631 3737 Tel: +41 31 631 4127 E-mail: michael.altmann@mci.unibe.ch *Present address Swiss Tropical and Public Health Institute Socinstrasse 57, 4002 Basel, Switzerland (Received 7 April 2011, revised 12 May 2011, accepted 26 May 2011) doi:10.1111/j.1742-4658.2011.08199.x Covalent modifications of proteins often modulate their biological func- tions or change their subcellular location. Among the many known protein modifications, three are exceptional in that they only occur on single pro- teins: ethanolamine phosphoglycerol, diphthamide and hypusine. Remark- ably, the corresponding proteins carrying these modifications, elongation factor 1A, elongation factor 2 and initiation factor 5A, are all involved in elongation steps of translation. For diphthamide and, in part, hypusine, functional essentiality has been demonstrated, whereas no functional role has been reported so far for ethanolamine phosphoglycerol. We review the biosynthesis, attachment and physiological roles of these unique protein modifications and discuss common and separate features of the target proteins, which represent essential proteins in all organisms. Introduction Several hundred protein modifications are known today, making proteomes far more complex than could be predicted by the encoding genomes. Covalent modi- fications modulate the biological functions or change the subcellular location of proteins and affect interac- tions of proteins with a variety of molecules, such as nucleic acids, lipids or other proteins [1–3]. Particular modifications are usually present on many proteins and often proteins carry several modifications at multi- ple amino acid residues [4]. The synthesis and attach- ment of protein modifications often involves multiple gene products and sets of metabolites, making these events costly for a cell in terms of substrate and energy requirements. On the other hand, modifications may generate additional functions for proteins or allow novel pathways of regulation, providing a cell with Abbreviations DHS, deoxyhypusine synthase; DOOH, deoxyhypusine hydroxylase; e(a)EF1A, eukaryotic (archaeal) elongation factor 1A; e(a)EF2, eukaryotic (archaeal) elongation factor 2; e(a)IF5A, eukaryotic (archaeal) initiation factor 5A; EF-G, bacterial elongation factor 2; EF-P, bacterial elongation factor P; EF-Tu, bacterial elongation factor 1A; EPG, ethanolamine phosphoglycerol; PE, phosphatidylethanolamine. FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2613 extra means to diversify and develop. While some modifications are transient and thus depend on rapid attachment and removal of molecules from target pro- teins, others are stable and attached to proteins shortly after their synthesis or before degradation [4]. Among many protein modifications, three are excep- tional in that they only occur on single proteins: etha- nolamine phosphoglycerol (EPG), diphthamide and hypusine. Remarkably, the corresponding proteins carrying these modifications, eukaryotic elongation factor 1A (eEF1A), eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 5A (eIF5A) respectively, are all involved in the elongation steps of translation. Elongation of polypeptide chains during translation is a conserved process among prokaryotes and eukary- otes. Single steps of elongation consist of (a) binding of aminoacyl-tRNAs to the A(minoacyl)-site of the ribo- some, (b) peptide bond formation with the adjacent peptide-tRNA at the P(eptidyl)-site and (c) transloca- tion of the extended peptide-tRNA from the A-site to the P-site and of the previously loaded tRNA from the P-site to the E(xit)-site. These steps are well conserved between organisms and the enzymatic involvement of ribosomal RNA at the transpeptidation center is nowa- days generally accepted. Accordingly, homologs of most factors involved in elongation can be found across bacterial, archaeal and eukaryotic genomes. eEF1A, eEF2 and eIF5A are phylogenetically among the most highly conserved proteins. Their bio- logical roles during elongation of translation are as fol- lows: eEF1A (called EF-Tu in bacteria and aEF1A in archaea), one of the most abundant cytosolic proteins, catalyzes binding of aminoacyl-tRNAs to the A-site of the ribosome. In addition, it has been reported to par- ticipate in a variety of other functions (so called moon- lighting functions; see below). In contrast, eEF2 (called EF-G in bacteria and aEF2 in archaea) is involved in translocation of the peptide-tRNA complex from the A- to the P-site, while eIF5A (called EF-P in bacteria and aIF5A in archaea) directly stimulates protein elon- gation, yet its precise mode of action on the ribosome is unclear [5]. Bacterial EF-P facilitates the proper positioning of the initiator-tRNA-methionine complex at the P-site [6]. Both eEF1A and eEF2 are GTP-binding proteins, i.e. their enzymatic activity requires the hydrolysis of GTP to GDP. Interestingly, GTPases involved in translation elongation show a remarkable structural similarity pointing at a common ancestral GTPase (reviewed by [7]). Its presumed function was to trans- port aminoacyl-tRNAs to an ancestral membrane- bound self-folding RNA, which catalyzed peptide bond formation and constituted the original peptidyltrans- ferase center that evolved later into the corresponding domain of the ribosomal large subunit. Co-evolution of translational GTPases with ribosomal structures may have occurred to allow interaction of GTPases with ribosomal structures by addition of new structural elements [7]. In accordance with the concept of co-evo- lution between proteins and RNA structures, elonga- tion (and termination) factors of translation show a remarkable molecular mimicry between proteins and tRNAs. For example, the crystal structure of EF-G from Thermus thermophilus perfectly fits the structure of the ternary prokaryotic EF-Tu-GDPNP-Phe- tRNA Phe complex [8]. In addition, the crystal structure of EF-P from Escherichia coli with its post-transla- tional lysine modification resembling the covalently bound amino acid lysine charged to the 3¢ end of a tRNA (see below) mimics the structure of a charged tRNA [9]. The unique modifications attached to eEF1A, eEF2 and eIF5A have been known for decades. In addition, their biosynthetic precursors and pathways for produc- tion and attachment to protein have been partially established (see below). Surprisingly, their biological functions have remained elusive despite the fact that EPG, diphthamide and hypusine are attached to essen- tial proteins involved in a highly conserved process, i.e. elongation of protein translation, and that species- specific variants of the three proteins have been crys- tallized and their 3D structures solved. The aim of this review is to describe common and separate features of EPG, diphthamide and hypusine attachment to their respective acceptor proteins. Inter- estingly, despite the fact that not only the function but also the 3D structures of e(a)EF1A ⁄ EF-Tu, e(a)EF2 ⁄ EF-G and e(a)IF5A ⁄ EF-P proteins have been conserved during evolution (Fig. 1), the presence of EPG, diphthamide and hypusine shows striking differ- ences: whereas hypusine (or lysine) attachment to e(a)IF5A ⁄ EF-P proteins has been demonstrated in all three domains of life, diphthamide modification has only been found in e(a)EF2 of eukarya and archaea but not in EF-G of bacteria, while EPG has so far only been reported in eEF1A of eukarya (Fig. 1). Eukaryotic elongation factors and their unique modifications eEF1A and EPG eEF1A represents an essential protein involved in pep- tide chain elongation in all eukaryotic cells. It interacts in its GTP-bound form with an aminoacylated tRNA Unique modifications of translation elongation factors E. Greganova et al. 2614 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS to mediate binding to the acceptor site of a ribosome via codon–anticodon interaction. Following ribosome- dependent hydrolysis of GTP, eEF1A dissociates from the ribosome in its GDP-bound form and interacts with nucleotide exchange factor eEF1B (called EF-Ts in bacteria) that replaces GDP by GTP to reacti- vate eEF1A (reviewed in [10,11]). Crystal structures of eEF1A in complex with subunits of eEF1B show that eEF1A from Saccharomyces cerevisiae consists of three distinct structural domains [12,13]. The N-terminal domain I contains the binding site for guanine nucleo- tides whereas binding of aminoacyl-tRNAs occurs in domain II [12,14–17]. In addition, domains I and II share the recognition site for the a-subunit of eEF1B [12,13]. In S. cerevisiae, domain III has been shown to harbor the binding site for the fungal-specific elonga- tion factor 3 [18,19]. Beside its canonical role in pro- tein synthesis, eEF1A has been shown to also bind to cytoskeletal proteins and mediate their interactions [20–22]. This function, which has been localized to Fig. 1. 3D structure of translation elongation factors. The 3D structure of representative examples of e(a)IF5A ⁄ EF-P (top row), e(a)EF2 ⁄ EF-G (middle row) and e(a)EF1A ⁄ EF-Tu (bottom row) proteins is drawn to demonstrate the structural similarity between eukarya, archaea and bac- teria. The position of the unique modifications hypusine (Hyp), diphthamide (Dph) and ethanolamine phosphoglycerol (EPG) attached to con- served amino acids (numbered) is indicated by arrows. Structures represent eIF5A from Homo sapiens (UniProt, Q6IS14), aIF5A from Sulfolobus acidocaldarius (GenBank, CAA44842) and EF-P from E. coli (GenBank, AP_004648), eEF2 from S. cerevisiae (UniProt, P32324), aEF2 from H. salinarum (UniProt, Q9HM85) and EF-G from T. thermophilus (UniProt, Q5SHN5), and eEF1A from Mus musculus (GenBank NP_034236), aEF1A from H. salinarum (GenBank, NP_281202) and EF-Tu from E. coli (GenBank, YP_001465471), and are drawn using the PYMOL program [99]. E. Greganova et al. Unique modifications of translation elongation factors FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2615 domains II and III, seems not to be connected to its role during polypeptide elongation [21,22]. In addition, eEF1A was reported to be involved in signal transduc- tion processes [23], nuclear export of proteins [24] and import of tRNAs into mitochondria [25]. Based on the high conservation of the primary sequence of eEF1A among eukaryotes (Fig. S1) and its highly conserved role during protein synthesis, it can be speculated that many interactions with its binding partners are con- served among other eukaryotic organisms. The activity of eEF1A during peptide synthesis has been reported to be modulated by post-translational modifications such as phosphorylation [26,27], lysine methylation (reviewed in [28,29]) and C-terminal methyl-esterification [30]. The precise role of these modifications is unclear (reviewed in [31]). In contrast, no studies have been reported on the role of EPG that is attached to conserved glutamate residues in eEF1A of several eukaryotes (Fig. S1). Chemical and mass spectrometric analyses demonstrated that murine [32], rabbit [33] and carrot [34] eEF1A contain two EPG modification sites, located in domains II and III. In contrast, although both glutamates are conserved in eEF1A of the protozoan parasite Trypanosoma brucei (Fig. S1), trypanosome eEF1A is modified only by a single EPG moiety attached to Glu362 in domain III [35] (Fig. 2A). Amino acid point mutations of the modification site in T. brucei eEF1A were found to prevent attachment of EPG, even when glutamate was replaced by aspartate [36], demonstrating that EPG attachment is strictly specific for glutamate. Interest- ingly, S. cerevisiae represents the only eukaryote so far reported where eEF1A is not modified with EPG [28], although the glutamate residue in domain III is con- served among yeast and other eukaryotes (Fig. S1). Amino acid sequence comparisons between eEF1A and EF-Tu show that eukaryotic EPG modification sites are not strictly conserved in bacteria (Figs S2 and S3). For E. coli, the lack of EPG modification has been proven experimentally [32]. Recent analyses of aEF1A from Halobacterium salinarum and Haloquad- ratum walsbyi showed no evidence for the presence of EPG (E. Greganova, R. Vitale, A. Corcelli, M. Heller &P.Bu ¨ tikofer, unpublished results) suggesting that EPG is absent in archaea. Interestingly, despite the high amino acid sequence identity between eEF1A proteins from different eukary- otes, the residues around the EPG modification sites are less well conserved (Fig. S1) suggesting that they may not be essential for EPG attachment [36]. Additionally, when expressing eEF1A deletion mutants or chimeric proteins consisting of domain III of T. brucei eEF1A fused to soluble reporter proteins, a peptide consisting of 80 amino acids of domain III of eEF1A was found to be sufficient for EPG attachment to occur, indicating that EPG attachment is dependent on the three-dimen- sional structure of domain III rather than the sequence of amino acids around the attachment site [36]. Fig. 2. Attachment of EPG to eEF1A. (A) Predicted 3D structure of eEF1A from T. brucei (TriTrypDB Tb927.10.2100) showing three distinct structural domains (I–III) and the EPG attachment site (Glu362). (B) Proposed pathway for attachment of EPG to eEF1A: PE is attached to Glu362 and subsequently deacylated to EPG. Unique modifications of translation elongation factors E. Greganova et al. 2616 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS The biosynthetic pathway for EPG attachment has not been firmly established. Although an early study proposed that binding of free ethanolamine to eEF1A may represent the first reaction towards a stepwise assembly of EPG [37], the chemical structure of EPG (Fig. 2B) suggests that the entire EPG moiety may derive from phosphatidylethanolamine (PE). Studies using T. brucei parasites defective in PE biosynthesis showed that, indeed, PE is a direct precursor of EPG in T. brucei eEF1A [35]. Based on these findings, we propose a model in which eEF1A is first modified by PE and then becomes deacylated to EPG (Fig. 2B). If correct, such a model would predict that a PE- linked eEF1A intermediate might transiently bind to membranes. Surprisingly, although the covalent attachment of EPG to eEF1A was described more than 20 years ago, nothing is known about its biological function. eEF2 and diphthamide The GTPase eEF2 catalyzes the coordinated move- ment of peptide-tRNA, unloaded tRNA and mRNA, and induces conformational changes in the ribosome (reviewed in [38]). Bacterial EF-G, archaeal aEF2 and eukaryotic eEF2 clearly show structural and functional homologies (Fig. 1). They all consist of six structural domains (I–V and G¢; Fig. 3A) with the binding pocket for GDP ⁄ GTP being located in domain I [39]. It has been shown that, upon binding of the antifun- gal inhibitor sordarin, yeast eEF2 can undergo dra- matic conformational changes involving rotations of up to 75° of domains IV–V relative to the amino-ter- minal domains I–II and G¢ through a switch in domain III [40] that may be decisive for its transloca- tion activity. eEF2 was reported to be negatively regu- lated by phosphorylation by eEF2-kinase leading to a complete arrest of translation elongation (reviewed in [41]). The unique diphthamide [2-(3-carboxyamido-3-(trim- ethylammonio)propyl)-histidine] modification [42] is conserved from archaea to human but is absent in bac- teria (Figs 1 and S4). Diphthamide serves as cellular target for diphtheria toxin from Corynebacterium diph- theriae (reviewed in [43,44]), exotoxin A from Pseudo- monas aeruginosa [45,46] and cholix toxin from Vibrio cholerae [47,48]. These toxins catalyze the trans- fer of ADP-ribose from NAD + to eEF2-bound diph- thamide resulting in irreversible inactivation of eEF2 and cell death. Enzymatic mono-ADP ribosylation is a phylogeneti- cally ancient mechanism to modulate protein function in prokaryotes, eukaryotes and viruses [49–51]. Exo- toxin A mimics part of the 80S ribosomal structure and interacts with diphthamide-modified eEF2 leading to its ADP ribosylation [52]. Fig. 3. Attachment of diphthamide to eEF2. (A) 3D structure of eEF2 from S. cerevisiae (PDB, 2P8Z) showing six distinct structural domains (I–V and G¢) and diphthamide attachment to His699. (B) Pathway for diphthamide synthesis: histidine is modified by a reaction sequence involving five separate enzymes (Dph1–5) to diphthine followed by conversion to diphthamide. E. Greganova et al. Unique modifications of translation elongation factors FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2617 The biosynthesis of diphthamide involves the step- wise addition of different functional groups to the side chain of a distinct histidine residue in eEF2 (His715 in mammals and His699 in S. cerevisiae) by a coordi- nated action of the conserved enzymes Dph1–Dph5 and a yet unknown amidase (Fig. 3B) [53–58]. The diphthamide modification is located at the tip of domain IV of eEF2 (Fig. 3A) that is supposed to mimic the tRNA anticodon loop [59]. To determine the amino acid requirements of eEF2 for recognition by diphthamide biosynthetic enzymes, site-directed mutagenesis was performed on several residues within the diphthamide-containing loop (Leu693–Gly703) of yeast eEF2. Upon replacement of six residues by ala- nine, mutated eEF2 proteins were lacking the diphtha- mide moiety [46]. Similarly, replacement of Gly717 or Gly719 in mammalian eEF2 led to diphtheria toxin- resistant cells [60,61]. Despite the fact that this modification was first described more than 30 years ago [42], its role in normal cellular function has remained largely elusive. System- atic mutagenesis of yeast eEF2-His699 showed that the resulting eEF2 proteins were lacking diphthamide and, consequently, were not ADP-ribosylated by diphtheria toxin [62]. Interestingly, the various yeast eEF2 mutants were either lethal indicating a key role of His699 for eEF2 function or led to temperature-sensitive growth of yeast indicating that diphthamide attachment to eEF2 is not strictly required for cell growth [62,63]. The dispens- ability of diphthamide for eEF2 function was later confirmed by mutagenesis of eEF2-His715 in mammals [64]. Moreover, yeast mutants lacking Dph1, Dph2, Dph4 or Dph5 genes showed no growth phenotypes compared with wild-type cells [58]. The non-essentiality of diphthamide and the Dph enzymes raises the question why such a complex post- translational modification has been maintained in archaea and eukarya. It has been postulated that essential functions of diphthamide may only become apparent under certain circumstances, e.g. in the con- text of a multi-cellular organism or during stress con- ditions [65]. In mouse and human, Dph1 has been identified as a tumor suppressor gene [66–68]. In mice, knockout of one Dph1 allele lead to increased tumor development whereas loss of both Dph1 alleles resulted in death at an early age [69]. Similarly, Dph3 knockout mice showed embryonic lethality [70]. These observa- tions indicate a potential role for diphthamide in the control of tumorigenesis, cell growth and embryonic development. However, the effects caused by loss of dph genes in mammals may be related to other func- tions of the gene products such as tRNA modification by Dph3 [71]. As mentioned, the importance of diphthamide in eEF2 function may become apparent during stress con- ditions [65]. For instance, yeast strains expressing H699N eEF2 or lacking Dph2 or Dph5 are viable but reveal increased frequency in ())1 ribosomal frame shifting [59]. Furthermore, diphthamide has been pro- posed to protect ribosomes from ribosome-inactivating proteins by showing that cultured Chinese hamster ovary cells lacking the diphthamide biosynthetic enzymes Dph2, Dph3 or Dph5 were threefold more sensitive towards ricin than wild-type cells [65]. After complementation with the corresponding dph genes, the mutant cells gained resistance to ricin. Alternatively, diphthamide may serve as a regulatory modification site of eEF2. It has been previously pos- tulated that ADP ribosylation by diphtheria toxin may represent a normal cellular control mechanism (reviewed in [72]). In mammalian cells, an endogenous ADP-ribosyltransferase activity specific for eEF2 has been described [73–75] that may function in controlling protein synthesis. eIF5A and hypusine For many years, eIF5A was assumed to be involved in translation initiation [76–78]. Only recently, studies in yeast demonstrated that eIF5A promotes translation elongation rather than translation initiation [5,14,79]. eIF5A stimulates translation directly and functions as a general translation elongation factor in a manner determined by its hypusine modification [5]. The unique hypusine [N e-(4-amino-2-hydroxybutyl)- lysine] modification [80] attached to domain I of eIF5A has been found in all eukaryotes examined so far (reviewed in [81,82]) (Fig. 4A). In addition, it also occurs in certain archaea [83] but has not been detected in bacteria. However, in E. coli the conserved lysine residue in domain I of EF-P (Fig. S5) is modi- fied by lysine by a paralog of lysyl-tRNA synthetase. Interestingly, the structure of EF-P mimics that of L-shaped tRNA and its lysylation site (Lys34) corre- sponds to the tRNA 3¢ end [9]. Domains I and II are highly conserved among all organisms; however, eIF5A and aIF5A lack a carboxyterminal domain III found in bacterial EF-P (see Fig. 1). While the amino- terminal domain I is located close to the aminoacyl acceptor stem of initiator tRNA bound to the P-site of the 70S ribosome, the carboxyterminal domain III of bacterial EF-P is positioned close to the anticodon stem-loop [6]. Hypusine is formed by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOOH) (Fig. 4B). Unique modifications of translation elongation factors E. Greganova et al. 2618 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS Both enzymes are highly conserved among eukaryotes and display similar structural requirements for their substrates, eIF5A-lysine and eIF5A-deoxyhypusine [84–86]. While neither DHS nor DOOH are found in bacteria, a gene homolog for DHS has been identified in archaea. However, it is not clear how hypusinated aIF5A is generated in archaea [87]. Mutations at the hypusine attachment site Lys50 in human eIF5A (Fig. 4A) completely blocked deoxyhypusine synthesis whereas substitutions in its vicinity resulted in reduced efficiency of deoxyhypusine synthesis or inhibition of the hydroxylation reaction catalyzed by DOOH [88]. A truncated peptide consisting of 80 residues of human eIF5A (amino acids 10–90; expressed in E. coli) was nearly as good a substrate as the full-length protein for hypusine attachment [85,86]. Disruption of the eIF5A [89,90] or DHS [91,92] gene results in a lethal phenotype. In contrast, the DOOH gene does not appear to be essential in S. cerevisiae since growth of a DOOH null mutant strain was only slightly reduced compared with the parental strain [93]. However, in multi-cellular organisms such as Caenor- habditis elegans or Drosophila melanogaster inactiva- tion of the DOOH gene was found to be recessively lethal [94,95]. Thus, although in single cell eukaryotes deoxyhypusinated eIF5A is sufficient to perform its essential cellular functions, multi-cellular eukaryotes require hypusinated eIF5A. In addition to the above- mentioned phenotypes, hypusine is necessary for homodimerization of eIF5A and affects its subcellular localization [96,97]. However, the precise mode of eIF5A action and how hypusine modulates eIF5A function remain to be answered. It is possible that eIF5A fulfills the same function as its bacterial ortho- log EF-P, which has been shown to catalyze the forma- tion of the first peptide bond in protein synthesis (reviewed in [98]). The recent resolution of its crystal structure [6] has provided new insights into the function of EF-P, indicating that it allows proper positioning of initiator met-tRNA at the P-site of the ribosome in a situation where the E-site of the ribosome is not occu- pied by unloaded tRNA. Conclusions We have reviewed the unusual post-translational modi- fications of three different translation elongation fac- tors that are present in all cells and participate in a conserved mechanistic pathway among eukaryotes and prokaryotes. Though not essential in all organisms (Fig. 1), EPG, diphthamide and hypusine are impor- tant to maintain the activity (and probably also the Fig. 4. Attachment of hypusine to eIF5A. (A) Predicted 3D structure of human eIF5A (PDB, 1FH4) showing two distinct structural domains (I, II) and the hypusine attachment site (Lys50). (B) Pathway for hypusine synthesis: spermidine is attached to lysine and subsequently modi- fied to hypusine. E. Greganova et al. Unique modifications of translation elongation factors FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2619 proper structure) of acceptor proteins. The biological significance of these modifications may only become evident in vivo or under certain stress or competition conditions, which so far have not been mimicked in the laboratory. In all eukaryotes studied, the function of eIF1A, eEF2 and eIF5A is essential for cell survival. To our knowledge, cross-complementation experiments with paralog prokaryotic and eukaryotic factors have so far not been reported. One possible reason why such experiments may not work would be due to co-evolu- tion of these proteins with their interacting partners which might have given rise to subtle differences that do not allow for cross-complementation of single para- logs in different organisms. Whether EPG, diphthamide and hypusine play a role in protein–protein interactions is unknown. The availability of efficient knockout ⁄ knockin and knock- down techniques using mono- and multi-cellular organisms may allow our knowledge about the impor- tance of these modifications to be extended in the near future. Why are the three modifications EPG, diphthamide and hypusine restricted to single proteins and why are the three modified proteins all involved in elonga- tion of translation? We propose that the modifica- tions are remnants of an evolutionary process that might have been more common in an ancient world, i.e. that multiple proteins were modified by EPG, diphthamide and hypusine. During the course of evo- lution, however, these modifications may have mostly disappeared, except for the translation elongation proteins e(a)EF1A ⁄ EF-Tu, e(a)EF2 ⁄ EF-G and e(a)IF5A ⁄ EF-P, which are highly conserved between organisms and for which EPG, diphthamide and hypusine may fulfill important functions to enhance accuracy or catalytic activity of enzymes interacting with translating ribosomes. For diphthamide, and in part hypusine, functional essentiality has been demon- strated. In contrast, no functional role has so far been reported for EPG. Acknowledgements We thank U. Baumann (University of Ko ¨ ln) and G. Hernandez (McGill University, Montreal) for advice during preparation of the manuscript. E.G. thanks P. Ma ¨ ser (Swiss Tropical and Public Health Institute, Basel) for support. P.B. thanks G. Moore for stimula- tion and input and O. Bu ¨ tikofer for support. Research in our laboratories is supported by Swiss National Science Foundation grants 31003A-130815 to P.B. and 31003A-119996 to M.A. References 1 Walsh CT, Garneau-Tsodikova S & Gatto GJ Jr (2005) Protein posttranslational modifications: the chemistry of proteome diversifications. Angew Chem Int Ed Engl 44, 7342–7372. 2 Seet BT, Dikic I, Zhou MM & Pawson T (2006) Read- ing protein modifications with interaction domains. Nat Rev Mol Cell Biol 7, 473–483. 3 Ahrne E, Muller M & Lisacek F (2010) Unrestricted identification of modified proteins using MS ⁄ MS. Proteomics 10, 671–686. 4 Yang XJ (2005) Multisite protein modification and intramolecular signaling. Oncogene 24, 1653–1662. 5 Saini P, Eyler DE, Green R & Dever TE (2009) Hypu- sine-containing protein eIF5A promotes translation elongation. Nature 459, 118–121. 6 Blaha G, Stanley RE & Steitz TA (2009) Formation of the first peptide bond: the structure of EF-P bound to the 70S ribosome. Science 325, 966–970. 7 Hartman H & Smith TF (2010) GTPases and the origin of the ribosome. Biol Direct 5, 36. 8 Nakamura Y & Ito K (2003) Making sense of mimic in translation termination. Trends Biochem Sci 28, 99–105. 9 Yanagisawa T, Sumida T, Ishii R, Takemoto C & Yokoyama S (2010) A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P. Nat Struct Mol Biol 17, 1136–1143. 10 Andersen GR, Nissen P & Nyborg J (2003) Elongation factors in protein biosynthesis. Trends Biochem Sci 28, 434–441. 11 Le Sourd F, Boulben S, Le Bouffant R, Cormier P, Morales J, Belle R & Mulner-Lorillon O (2006) eEF1B: at the dawn of the 21st century. Biochim Biophys Acta 1759, 13–31. 12 Andersen GR, Pedersen L, Valente L, Chatterjee I, Kinzy TG, Kjeldgaard M & Nyborg J (2000) Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A:eEF1Balpha. Mol Cell 6, 1261–1266. 13 Andersen GR, Valente L, Pedersen L, Kinzy TG & Nyborg J (2001) Crystal structures of nucleotide exchange intermediates in the eEF1A-eEF1Balpha complex. Nat Struct Biol 8, 531–534. 14 Zanelli CF, Maragno AL, Gregio AP, Komili S, Pandolfi JR, Mestriner CA, Lustri WR & Valentini SR (2006) eIF5A binds to translational machinery compo- nents and affects translation in yeast. Biochem Biophys Res Commun 348, 1358–1366. 15 Carr-Schmid A, Durko N, Cavallius J, Merrick WC & Kinzy TG (1999) Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduce both transla- tional fidelity and the requirement for nucleotide exchange. J Biol Chem 274, 30297–30302. Unique modifications of translation elongation factors E. Greganova et al. 2620 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 16 Kinzy TG, Freeman JP, Johnson AE & Merrick WC (1992) A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha. J Biol Chem 267, 1623–1632. 17 Dinman JD & Kinzy TG (1997) Translational misread- ing: mutations in translation elongation factor 1alpha differentially affect programmed ribosomal frameshif- ting and drug sensitivity. RNA 3, 870–881. 18 Anand M, Chakraburtty K, Marton MJ, Hinnebusch AG & Kinzy TG (2003) Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3. J Biol Chem 278, 6985–6991. 19 Anand M, Balar B, Ulloque R, Gross SR & Kinzy TG (2006) Domain and nucleotide dependence of the inter- action between Saccharomyces cerevisiae translation elongation factors 3 and 1A. J Biol Chem 281, 32318–32326. 20 Liu G, Tang J, Edmonds BT, Murray J, Levin S & Condeelis J (1996) F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction. J Cell Biol 135, 953–963. 21 Gross SR & Kinzy TG (2005) Translation elongation factor 1A is essential for regulation of the actin cyto- skeleton and cell morphology. Nat Struct Mol Biol 12, 772–778. 22 Gross SR & Kinzy TG (2007) Improper organization of the actin cytoskeleton affects protein synthesis at initia- tion. Mol Cell Biol 27, 1974–1989. 23 Panasyuk G, Nemazanyy I, Filonenko V, Negrutskii B & El’skaya AV (2008) A2 isoform of mammalian trans- lation factor eEF1A displays increased tyrosine phos- phorylation and ability to interact with different signalling molecules. Int J Biochem Cell Biol 40, 63–71. 24 Khacho M, Mekhail K, Pilon-Larose K, Pause A, Cote J & Lee S (2008) eEF1A is a novel component of the mammalian nuclear protein export machinery. Mol Biol Cell 19, 5296–5308. 25 Bouzaidi-Tiali N, Aeby E, Charriere F, Pusnik M & Schneider A (2007) Elongation factor 1a mediates the specificity of mitochondrial tRNA import in T. brucei. EMBO J 26, 4302–4312. 26 Peters HI, Chang YW & Traugh JA (1995) Phosphory- lation of elongation factor 1 (EF-1) by protein kinase C stimulates GDP ⁄ GTP-exchange activity. Eur J Biochem 234, 550–556. 27 Chang YW & Traugh JA (1998) Insulin stimulation of phosphorylation of elongation factor 1 (eEF-1) enhances elongation activity. Eur J Biochem 251, 201–207. 28 Cavallius J, Zoll W, Chakraburtty K & Merrick WC (1993) Characterization of yeast EF-1alpha: non-conser- vation of post-translational modifications. Biochim Biophys Acta 1163, 75–80. 29 Sherman M & Sypherd PS (1989) Role of lysine methyl- ation in the activities of elongation factor 1 alpha. Arch Biochem Biophys 275, 371–378. 30 Zobel-Thropp P, Yang MC, Machado L & Clarke S (2000) A novel post-translational modification of yeast elongation factor 1A. J Biol Chem 275, 37150–37158. 31 Ejiri S (2002) Moonlighting functions of polypeptide elongation factor 1: from actin bundling to zinc finger protein R1-associated nuclear localization. Biosci Biotechnol Biochem 66, 1–21. 32 Whiteheart SW, Shenbagamurthi P, Chen L, Cotter RJ & Hart GW (1989) Murine elongation factor 1alpha is posttranslationally modified by novel amide-linked etha- nolamine-phosphoglycerol moieties. J Biol Chem 264, 14334–14341. 33 Rosenberry TL, Krall JA, Dever TE, Haas R, Louvard D & Merrick WC (1989) Biosynthetic incorporation of [3H]ethanolamine into protein synthe- sis elongation factor 1alpha reveals a new post-transla- tional protein modification. J Biol Chem 264, 7096– 7099. 34 Ransom WD, Lao P-C, Gage DA & Boss WF (1998) Phosphoglycerylethanolamine posttranslational modifi- cation of plant eukaryotic elongation factor 1alpha. Plant Physiol 117, 949–960. 35 Signorell A, Jelk J, Rauch M & Bu ¨ tikofer P (2008) Phosphatidylethanolamine is the precursor of the ethanolamine phosphoglycerol moiety bound to eukary- otic elongation factor 1A. J Biol Chem 283, 20320– 20329. 36 Greganova E, Heller M & Bu ¨ tikofer P (2010) A struc- tural domain mediates attachment of ethanolamine phosphoglycerol to eukaryotic elongation factor 1A in Trypanosoma brucei. PLoS ONE 5, e9486. 37 Whiteheart SW & Hart GW (1994) Incorporation of [3H]ethanolamine into a single cytosolic protein in a cell free system: ethanolaminylation of EF-1 alpha in vitro. Arch Biochem Biophys 309, 387–391. 38 Jorgensen R, Merrill AR & Andersen GR (2006) The life and death of translation elongation factor 2. Biochem Soc Trans 34, 1–6. 39 Yates SP, Jorgensen R, Andersen GR & Merrill AR (2006) Stealth and mimicry by deadly bacterial toxins. Trends Biochem Sci 31, 123–133. 40 Jorgensen R, Ortiz PA, Carr-Schmid A, Nissen P, Kinzy TG & Andersen GR (2003) Two crystal struc- tures demonstrate large conformational changes in the eukaryotic ribosomal translocase. Nat Struct Biol 10, 379–385. 41 Browne GJ & Proud CG (2002) Regulation of peptide- chain elongation in mammalian cells. Eur J Biochem 269, 5360–5368. 42 Van Ness BG, Howard JB & Bodley JW (1980) ADP-ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl- amino acid and its hydrolysis products. J Biol Chem 255, 10717–10720. E. Greganova et al. Unique modifications of translation elongation factors FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2621 43 Pappenheimer AM Jr (1977) Diphtheria toxin. Annu Rev Biochem 46, 69–94. 44 Oppenheimer NJ & Bodley JW (1981) Diphtheria toxin. Site and configuration of ADP-ribosylation of diphtha- mide in elongation factor 2. J Biol Chem 256, 8579–8581. 45 Iglewski BH & Kabat D (1975) NAD-dependent inhibi- tion of protein synthesis by Pseudomonas aeruginosa toxin. Proc Natl Acad Sci USA 72, 2284–2288. 46 Zhang Y, Liu S, Lajoie G & Merrill AR (2008) The role of the diphthamide-containing loop within eukary- otic elongation factor 2 in ADP-ribosylation by Pseudo- monas aeruginosa exotoxin A. Biochem J 413, 163–174. 47 Liu S & Leppla SH (2003) Retroviral insertional muta- genesis identifies a small protein required for synthesis of diphthamide, the target of bacterial ADP-ribosylat- ing toxins. Mol Cell 12, 603–613. 48 Jorgensen R, Purdy AE, Fieldhouse RJ, Kimber MS, Bartlett DH & Merrill AR (2008) Cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae. J Biol Chem 283, 10671–10678. 49 Pallen MJ, Lam AC, Loman NJ & McBride A (2001) An abundance of bacterial ADP-ribosyltransferases – implications for the origin of exotoxins and their human homologues. Trends Microbiol 9, 302–307; discussion 308. 50 Krueger KM & Barbieri JT (1995) The family of bacte- rial ADP-ribosylating exotoxins. Clin Microbiol Rev 8, 34–47. 51 Corda D & Di Girolamo M (2003) Functional aspects of protein mono-ADP-ribosylation. EMBO J 22, 1953–1958. 52 Jorgensen R, Merrill AR, Yates SP, Marquez VE, Schwan AL, Boesen T & Andersen GR (2005) Exotoxin A-eEF2 complex structure indicates ADP ribosylation by ribosome mimicry. Nature 436, 979–984. 53 Moehring JM, Moehring TJ & Danley DE (1980) Post- translational modification of elongation factor 2 in diphtheria-toxin-resistant mutants of CHO-K1 cells. Proc Natl Acad Sci USA 77, 1010–1014. 54 Dunlop PC & Bodley JW (1983) Biosynthetic labeling of diphthamide in Saccharomyces cerevisiae. J Biol Chem 258, 4754–4758. 55 Moehring TJ, Danley DE & Moehring JM (1984) In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin. Mol Cell Biol 4, 642–650. 56 Chen JY, Bodley JW & Livingston DM (1985) Diphthe- ria toxin-resistant mutants of Saccharomyces cerevisiae. Mol Cell Biol 5, 3357–3360. 57 Chen JY & Bodley JW (1988) Biosynthesis of diphtha- mide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine: elongation factor 2 methyltransferase. J Biol Chem 263, 11692–11696. 58 Liu S, Milne GT, Kuremsky JG, Fink GR & Leppla SH (2004) Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation fac- tor 2. Mol Cell Biol 24, 9487–9497. 59 Ortiz PA, Ulloque R, Kihara GK, Zheng H & Kinzy TG (2006) Translation elongation factor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin resistance. J Biol Chem 281 , 32639–32648. 60 Foley BT, Moehring JM & Moehring TJ (1995) Mutations in the elongation factor 2 gene which confer resistance to diphtheria toxin and Pseudomonas exotoxin A. Genetic and biochemical analyses. J Biol Chem 270, 23218–23225. 61 Kohno K & Uchida T (1987) Highly frequent single amino acid substitution in mammalian elongation factor 2 (EF-2) results in expression of resistance to EF-2-ADP- ribosylating toxins. J Biol Chem 262, 12298–12305. 62 Kimata Y & Kohno K (1994) Elongation factor 2 mutants deficient in diphthamide formation show temperature-sensitive cell growth. J Biol Chem 269, 13497–13501. 63 Phan LD, Perentesis JP & Bodley JW (1993) Saccharo- myces cerevisiae elongation factor 2. Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin. J Biol Chem 268, 8665–8668. 64 Ivankovic M, Rubelj I, Matulic M, Reich E & Brdar B (2006) Site-specific mutagenesis of the histidine precur- sor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin. Mutat Res 609, 34–42. 65 Gupta PK, Liu S, Batavia MP & Leppla SH (2008) The diphthamide modification on elongation factor-2 renders mammalian cells resistant to ricin. Cell Micro- biol 10, 1687–1694. 66 Phillips NJ, Zeigler MR & Deaven LL (1996) A cDNA from the ovarian cancer critical region of deletion on chromosome 17p13.3. Cancer Lett 102, 85–90. 67 Bruening W, Prowse AH, Schultz DC, Holgado-Madru- ga M, Wong A & Godwin AK (1999) Expression of OVCA1, a candidate tumor suppressor, is reduced in tumors and inhibits growth of ovarian cancer cells. Cancer Res 59, 4973–4983. 68 Schultz DC, Vanderveer L, Berman DB, Hamilton TC, Wong AJ & Godwin AK (1996) Identification of two candidate tumor suppressor genes on chromosome 17p13.3. Cancer Res 56, 1997–2002. 69 Chen CM & Behringer RR (2004) Ovca1 regulates cell proliferation, embryonic development, and tumorigene- sis. Genes Dev 18, 320–332. 70 Liu S, Wiggins JF, Sreenath T, Kulkarni AB, Ward JM & Leppla SH (2006) Dph3, a small protein required for diphthamide biosynthesis, is essential in mouse develop- ment. Mol Cell Biol 26, 3835–3841. Unique modifications of translation elongation factors E. Greganova et al. 2622 FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS [...]... specificity of the hypusine modification Amino Acids 33, 341–350 85 Joe YA & Park MH (1994) Structural features of the eIF-5A precursor required for posttranslational Unique modifications of translation elongation factors 86 87 88 89 90 91 92 93 94 95 96 97 synthesis of deoxyhypusine J Biol Chem 269, 25916–25921 Kang KR, Kim YS, Wolff EC & Park MH (2007) Specificity of the deoxyhypusine hydroxylase-eukaryotic translation. .. 2011 FEBS 2623 Unique modifications of translation elongation factors E Greganova et al 98 Liljas A (2009) Leaps in translational elongation Science 326, 677–678 99 DeLano WL (2008) The PyMOL Molecular Graphics System DeLano Scientific LLC, Palo Alto, CA, USA; http://www.pymol.org Supporting information The following supplementary material is available: Fig S1 Alignment of primary sequences of eEF1A Fig... primary sequences of eEF1A Fig S2 Alignment of partial amino acid sequences of e(a)EF1A ⁄ EF-Tu Fig S3 Alignment of partial amino acid sequences of EF-Tu 2624 Fig S4 Alignment of partial amino acid sequences of e(a)EF2 ⁄ EF-G Fig S5 Alignment of partial amino acid sequences of e(a)IF5A ⁄ EF-P This supplementary material can be found in the online version of this article Please note: As a service to... comparison of native and unhypusinated forms of the protein J Biol Chem 264, 18527–18530 78 Park MH (1989) The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D) Purification of eIF-4D and its precursors and comparison of their activities J Biol Chem 264, 18531–18535 79 Gregio AP, Cano VP, Avaca JS, Valentini SR & Zanelli CF (2009) eIF5A has a function in the elongation step of. .. project: single P-element insertions mutating 25% of vital Drosophila genes Genetics 153, 135–177 Lee SB, Park JH, Kaevel J, Sramkova M, Weigert R & Park MH (2009) The effect of hypusine modification on the intracellular localization of eIF5A Biochem Biophys Res Commun 383, 497–502 Gentz PM, Blatch GL & Dorrington RA (2009) Dimerization of the yeast eukaryotic translation initiation factor 5A requires hypusine... leaves polyribosomes as a result of ADP-ribosylation FEBS Lett 176, 406–410 75 Sayhan O, Ozdemirli M, Nurten R & Bermek E (1986) On the nature of cellular ADP-ribosyltransferase from rat liver specific for elongation factor 2 Biochem Biophys Res Commun 139, 1210–1214 76 Benne R, Brown-Luedi ML & Hershey JW (1978) Purification and characterization of protein synthesis initiation factors eIF-1, eIF-4C, eIF-4D,... Saccharomyces cerevisiae Mol Cell Biol 11, 3105–3114 Wohl T, Klier H, Ammer H, Lottspeich F & Magdolen V (1993) The HYP2 gene of Saccharomyces cerevisiae is essential for aerobic growth: characterization of different isoforms of the hypusine-containing protein Hyp2p and analysis of gene disruption mutants Mol Gen Genet 241, 305–311 Sasaki K, Abid MR & Miyazaki M (1996) Deoxyhypusine synthase gene is essential... function of hypusine formation on eukaryotic initiation factor 5A Biol Signals 6, 105–109 83 Bartig D, Lemkemeier K, Frank J, Lottspeich F & Klink F (1992) The archaebacterial hypusine-containing protein Structural features suggest common ancestry with eukaryotic translation initiation factor 5A Eur J Biochem 204, 751–758 84 Wolff EC, Kang KR, Kim YS & Park MH (2007) Posttranslational synthesis of hypusine:... deoxyhypusine hydroxylase-eukaryotic translation initiation factor (eIF5A) interaction: identification of amino acid residues of the enzyme required for binding of its substrate, deoxyhypusine-containing eIF5A J Biol Chem 282, 8300–8308 Park MH, Nishimura K, Zanelli CF & Valentini SR (2010) Functional significance of eIF5A and its hypusine modification in eukaryotes Amino Acids 38, 491–500 Cano VS, Jeon GA, Johansson... 72 Collier RJ (1975) Diphtheria toxin: mode of action and structure Bacteriol Rev 39, 54–85 73 Lee H & Iglewski WJ (1984) Cellular ADP-ribosyltransferase with the same mechanism of action as diphtheria toxin and Pseudomonas toxin A Proc Natl Acad Sci USA 81, 2703–2707 74 Sitikov AS, Davydova EK, Bezlepkina TA, Ovchinnikov LP & Spirin AS (1984) Eukaryotic elongation factor 2 loses its non-specific affinity . elongation steps of translation. Elongation of polypeptide chains during translation is a conserved process among prokaryotes and eukary- otes. Single steps of elongation consist of (a) binding of aminoacyl-tRNAs. REVIEW ARTICLE Unique modifications of translation elongation factors Eva Greganova*, Michael Altmann and Peter Bu ¨ tikofer Institute for Biochemistry and Molecular Medicine, University of Berne,. al. Unique modifications of translation elongation factors FEBS Journal 278 (2011) 2613–2624 ª 2011 The Authors Journal compilation ª 2011 FEBS 2623 98 Liljas A (2009) Leaps in translational elongation. Science