Modulation of diphthamide synthesis by 5′-deoxy-5′-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of an aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5′-deoxy-5′-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase

The inactivation of elongation factor 2 (EF-2) by diphtheria toxin requires the presence of a post-translationally modified histidine residue in EF-2. This residue, diphthamide, has the structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. The present work was undertaken to study the pathway of diphthamide biosynthesis using diphtheria toxin-resistant yeast mutants (Chen. J.-Y., Bodley, J. W., and Livingston, D. M. (1985) Mol. Cell. Biol. 5, 3357-3360) which are defective in diphthamide formation. We demonstrate here that one of these mutants (dph5) contains a toxin-resistant form of EF-2 which can be converted in vitro to a toxin-sensitive form through the action of an enzyme present in other yeast strains. Both this toxin-resistant EF-2 and its modifying enzyme have been partially purified and evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase. In vitro complementation to diphtheria toxin sensitivity required S-adenosylmethionine, and when partially purified components were incubated with [methyl-3H]S-adenosylmethionine, label was incorporated specifically into EF-2. Hydrolysis of labeled EF-2 yielded diphthine (the unamidated form of diphthamide) and a single chromatographically separable labeling intermediate. We conclude that the S-adenosylmethionine:EF-2 methyltransferase adds at least the last two of the three methyl groups present in diphthine and that this modification is sufficient to create diphtheria toxin sensitivity. Evidence is also presented for the existence of an ATP-dependent amidating enzyme which catalyzes the final step in the biosynthesis of diphthamide in EF-2.     

ADP-ribosyltransferase from beef liver which ADP-ribosylates elongation factor-2

Fragment A of diphtheria toxin and Pseudomonas toxin A intoxicate cells by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) resulting in an inhibition of protein synthesis [1-3]. A cellular enzyme from polyoma virus transformed baby hamster kidney (pyBHK) cells ADP-ribosylates EF-2 in an identical manner [4]. Here we describe a similar cellular enzyme from beef liver which transfers [adenosine-14C]ADP-ribose from NAD to EF-2. The 14C-label can be removed from the EF-2 by snake venom phosphodiesterase as a soluble product which comigrates with AMP on TLC plates, indicating the 14C-label is present on EF-2 as monomeric units of ADP-ribose. Furthermore, the forward transferase reaction catalyzed by the beef liver ADP-ribosyltransferase is reversible by excess diphtheria toxin fragment A, with the formation of 14C-labeled NAD, indicating that both transferases ADP-ribosylate the same site on the diphthamide residue of EF-2. Thus, beef liver and pyBHK mono(ADP-ribosyl)transferases both modify the diphthamide residue of EF-2, in a manner identical to diphtheria toxin fragment A and Pseudomonas toxin A. These results suggest the cellular enzyme is probably ubiquitous among eukaryotic cells.     

Gene for the diphtheria toxin-susceptible elongation factor 2 from Methanococcus vannielii.

Protein synthesis elongation factor 2 (EF-2) from all archaebacteria so far analysed, is susceptible to inactivation by diphtheria toxin, a property which it shares with EF-2 from the eukaryotic 8OS translation system. To resolve the structural basis of diphtheria toxin susceptibility, the structural gene for the EF-2 from an archaebacterium, Methanococcus vannielii, was cloned and its nucleotide sequence determined. It was found that (i) this gene is closely linked to that coding for elongation factor 1 alpha-(EF-1 alpha), (ii) the size of the gene product, as derived from the nucleotide sequence, lies between those for EF-2 from eukaryotes and eubacteria, (iii) it displays a higher sequence similarity to eukaryotic EF-2 than to eubacterial homologues, and (iv) the histidine residue which is modified to diphthamide and then ADP-ribosylated by diphtheria toxin is present in a sequence context similar to that of eukaryotic EF-2 but it is not conserved in eubacterial EF-G. The EF-2 gene from Methanococcus is expressed in transformed Saccharomyces cerevisiae but is not ADP-ribosylated by diphtheria toxin. This indicates that the Saccharomyces enzyme system is unable to post-translationally convert the respective histidine residue from the Methanococcus EF-2 into diphthamide.     

Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin

Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains a conserved post-translationally modified histidine residue known as diphthamide. Diphthamide is a unique site of ADP-ribosylation by diphtheria toxin (DT), which is responsible for cell killing. In this report, we describe the construction of DT-resistant HeLa cell lines by engineering the toxin-resistant form of its specific substrate, protein elongation factor-2. Using site-specific mutagenesis of the histidine precursor of diphthamide, the histidine residue of codon 715 in human EF-2 cDNA was substituted with one of four amino acid residue codons: leucine, methionine, asparagine or glutamine. Mutant EF-2s were subcloned into a pCMVexSVneo expression vector, transfected into HeLa cells, and DT-resistant cell clones were isolated. The protective effect of mutant EF-2s against cell killing by DT, after exposing all four mutant strains derived from HeLa cells to different concentrations of the toxin (5-20 ng/mL) was demonstrated by: (1) the normal morphological appearance of the cells; (2) their unaffected or slightly slower growth rates; (3) their undisturbed electrophoretic DNA profiles whose integrity was virtually preserved. Mutant cell strains showed also considerable levels of resistance to very high concentrations of DT, in that they maintained slower but consistent rates of cell growth. It was hence concluded that despite its strict conservation and unique modification, the diphthamide histidine appears not to be essential to the function of human EF-2 in protein synthesis. In addition, DT-resistant HeLa cell clones should prove valuable hosts for various DT gene-containing vectors that express the toxin intracellularly.     

The histidine residue of codon 715 is essential for function of elongation factor 2

Several mutant cDNAs of elongation factor 2 (EF-2) were constructed by site-directed mutagenesis and their products expressed in mouse cells were investigated. Amino acid substitution for the histidine residue of codon 715, which is modified post-translationally to diphthamide, resulted in non-functional EF-2 and this substitution did not render EF-2 resistant to Pseudomonas aeruginosa exotoxin A, which inactivates EF-2 transferring ADP-ribose to the diphthamide residue. These non-functional EF-2s with replacements of the histidine-715 residue showed various extents of inhibition of protein synthesis by competing with functional EF-2 in vivo. These results suggest that histidine-715 is essential for the translocase activity of EF-2 and that the region around diphthamide functions in recognition of, and/or binding to ribosomes. Substitution of proline for the alanine-713 residue and substitution of glutamine for the glycine-717 residue converted EF-2 to partially toxin-resistant forms. Two-dimensional gel analysis with fragment A of diphtheria toxin of these toxin-resistant EF-2s revealed that their ADP-ribosylations by toxin were much less than that of wild-type EF-2.     

The post-translational trimethylation of diphthamide studied in vitro

The amino acid diphthamide is a complex post-translational derivative of histidine that exists in eukaryotic and Archaebacterial elongation factor 2 (EF-2). Diphtheria toxin and Pseudomonas exotoxin A catalyze the transfer of an ADP-ribose residue from NAD to diphthamide, causing the inactivation of EF-2. We have used cytosolic extracts of mutant CHO-K1 cells to study the biosynthesis of diphthamide in vitro. We have identified chromatographically a precursor form of diphthamide that exists in one complementation group of mutant cells and have documented the addition of 3 methyl residues from S-adenosylmethionine to this precursor. We have identified the presence of methyltransferase capable of carrying out this reaction in vitro in cells of 15 diverse eukaryotic species.     

Diphtheria toxin- and Pseudomonas A toxin-mediated apoptosis. ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha.

We have reported that diphtheria toxin (DTX) mediates target cell lysis and intranucleosomal DNA fragmentation (apoptosis) and also synergizes with TNF-alpha. In this paper, we examined which step in the pathway of DTX-mediated inhibition of protein synthesis was important for induction of cytolytic activity and for synergy. Using a DTX-sensitive tumor cell line, we first examined the activity of the mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2). CRM 197 was not cytolytic for target cells and did not mediate intranucleosomal DNA fragmentation of viable cells. The failure of CRM 197 to mediate target cell lysis suggested that the catalytic activity of DTX is prerequisite for target cell lysis. This was corroborated by demonstrating that MeSAdo, which blocks the biosynthesis of diphthamide, inhibited DTX-mediated protein synthesis inhibition and also blocked target cell lysis. Furthermore, the addition of nicotinamide, which competes with NAD+ on the DTX action site of EF-2, also blocked DTX-mediated lysis. These findings suggest that ADP-ribosylation of EF-2 may be a necessary step in the pathway leading to target cell lysis. In contrast to the sensitive line, the SKOV-3 tumor cell line is sensitive to protein synthesis inhibition by DTX but is not susceptible to cytolysis and apoptosis by DTX. Thus, protein synthesis inhibition by DTX is not sufficient to mediate target cell lysis. The synergy in cytotoxicity obtained with the combination of DTX and TNF-alpha was examined in order to determine the pathway mediated by DTX in synergy. Like the direct lysis by DTX, synergy was significantly reduced by MeSAdo and by nicotinamide. Furthermore, synergy was not observed with combination of CRM 197 and TNF-alpha. These results demonstrate that, in synergy, DTX may utilize the same pathway required for its cytolytic activity. Pseudomonas aeruginosa exotoxin shared most the properties shown for DTX. Altogether, these findings demonstrate that DTX-mediated apoptosis is initiated at a step beyond the ADP ribosylation of EF-2.     

The diphthamide modification on elongation factor-2 renders mammalian cells resistant to ricin

Diphthamide is a post-translational derivative of histidine in protein synthesis elongation factor-2 (eEF-2) that is present in all eukaryotes with no known normal physiological role. Five proteins Dph1–Dph5 are required for the biosynthesis of diphthamide. Chinese hamster ovary (CHO) cells mutated in the biosynthetic genes lack diphthamide and are resistant to bacterial toxins such as diphtheria toxin. We found that diphthamide-deficient cultured cells were threefold more sensitive than their parental cells towards ricin, a r ibosome- i nactivating p rotein (RIP). RIPs bind to ribosomes at the same site as eEF-2 and cleave the large ribosomal RNA, inhibiting translation and causing cell death. We hypothesized that one role of diphthamide may be to protect ribosomes, and therefore all eukaryotic life forms, from RIPs, which are widely distributed in nature. A protective role of diphthamide against ricin was further demonstrated by complementation where dph mutant CHO cells transfected with the corresponding DPH gene acquired increased resistance to ricin in comparison with the control-transfected cells, and resembled the parental CHO cells in their response to the toxin. These data show that the presence of diphthamide in eEF-2 provides protection against ricin and suggest the hypothesis that diphthamide may have evolved to provide protection against RIPs.     

Characterization of the endogenous ADP-ribosylation of wild-type and mutant elongation factor 2 in eukaryotic cells.

Anti-[ADP-ribosylated elongation factor 2 (EF-2)] antiserum has been used to immunoprecipitate the modified form of EF-2 from polyoma-virus-transformed baby hamster kidney (pyBHK) cells [Fendrick, J. L. & Iglewski, W. J. (1989) Proc. Natl Acad. Sci. USA 86, 554-557]. This antiserum also immunoprecipitates a 32P-labelled protein of similar size to EF-2 from a variety of primary and continuous cell lines derived from many species of animals. One of these cell lines, chinese hamster ovary CHO-K1 cells was further characterized. The time course of labelling of ADP-ribosylated EF-2 with [32P]orthophosphate was similar in pyBHK cells and in CHO-K1 cells. The kinetics of labelling were more rapid for cells cultured in 2% serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively. EF-2 mutants of pyBHK and CHO-K1 cells resistant to diphtheria-toxin-catalyzed ADP-ribosylation of EF-2 remain sensitive to cellular ADP-ribosylation of EF-2. The 32P-labelled moiety of ADP-ribosylated EF-2 was digested by snake venom phosphodiesterase and the product was identified as AMP. The same 32P-labelled tryptic peptide was modified by toxin in wild-type EF-2 and by the cellular transferase in mutant EF-2. When purified EF-2 from pyBHK cells was incubated with [carbonyl-14C]nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP-ribosylation reaction, [14C]NAD was generated. The results suggest that cellular ADP-ribosylated EF-2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP-ribosylation of EF-2, except in diphthamide mutant cells. Studies with the mutant cell lines indicate that the toxin and the cellular transferase, however, recognize different determinants at the ADP-ribose acceptor site in EF-2. The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF-2 for the transfer of ADP-ribose to the ring. Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudomonas exotoxin A.     

OVCA1: tumor suppressor gene

OVCA1, also known as DPH2L1, is a tumor suppressor gene associated with ovarian carcinoma and other tumors. Ovca1 homozygous mutant mice die at birth with developmental delay and cell-autonomous proliferation defects. Ovca1 heterozygous mutant mice are tumor-prone but rarely develop ovarian tumors. OVCA1 appears to be the homolog of yeast DPH2, which participates in the first biosynthetic step of diphthamide, by modification of histidine on translation elongation factor 2 (EF-2). Yeast dph2 mutants are resistant to diphtheria toxin, which catalyses ADP ribosylation of EF-2 at diphthamide. Thus, there appears to be growing evidence implicating alterations in protein translation with tumorigenesis.     

The biosynthesis and biological function of diphthamide

Abstract

Eukaryotic and archaeal elongation factor 2 contains a unique post-translationally modified histidine residue, named diphthamide. Genetic and biochemical studies have revealed that diphthamide biosynthesis involves a multi-step pathway that is evolutionally conserved among lower and higher eukaryotes. During certain bacterial infections, diphthamide is specifically recognized by bacterial toxins, including diphtheria toxin, Pseudomonas exotoxin A and cholix toxin. Although the pathological relevance is well studied, the physiological function of diphthamide is still poorly understood. Recently, many new interesting developments in understanding the biosynthesis have been reported. Here, we review the current understanding of the biosynthesis and biological function of diphthamide.     

Dph3, a small protein required for diphthamide biosynthesis, is essential in mouse development

The translation elongation factor 2 in eukaryotes (eEF-2) contains a unique posttranslationally modified histidine residue, termed diphthamide, which serves as the only target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Diphthamide biosynthesis is carried out by five highly conserved proteins, Dph1 to Dph5, and an as-yet-unidentified amidating enzyme. The evolutionary conservation of the complex diphthamide biosynthesis pathway throughout eukaryotes implies a key role for diphthamide in normal cellular physiology. Of the proteins required for diphthamide synthesis, Dph3 is the smallest, containing only 82 residues. In addition to having a role in diphthamide biosynthesis, Dph3 is also involved in modulating the functions of the Elongator complex in yeast. To explore the physiological roles of Dph3 and to begin to investigate the function of diphthamide, we generated dph3 knockout mice and showed that dph3+/- mice are phenotypically normal, whereas dph3-/- mice, which lack the diphthamide modification on eEF-2, are embryonic lethal. Loss of both dph3 alleles causes a general delay in embryonic development accompanied by lack of allantois fusion to the chorion and increased degeneration and necrosis in neural tubes and is not compatible with life beyond embryonic day 11.5. The dph3-/- placentas also developed abnormally, showing a thinner labyrinth lacking embryonic erythrocytes and blood vessels. These results attest to the physiological importance of Dph3 in development. The biological roles of Dph3 are also discussed.     

A dominant-negative approach that prevents diphthamide formation confers resistance to Pseudomonas exotoxin A and diphtheria toxin

Diphtheria toxin (DT), Pseudomonas aeruginosa Exotoxin A (ETA) and cholix toxin from Vibrio cholerae share the same mechanism of toxicity; these enzymes ADP-rybosylate elongation factor-2 (EF-2) on a modified histidine residue called diphthamide, leading to a block in protein synthesis. Mutant Chinese hamster ovary cells that are defective in the formation of diphthamide have no distinct phenotype except their resistance to DT and ETA. These observations led us to predict that a strategy that prevents the formation of diphthamide to confer DT and ETA resistance is likely to be safe. It is well documented that Dph1 and Dph2 are involved in the first biochemical step of diphthamide formation and that these two proteins interact with each other. We hypothesized that we could block diphthamide formation with a dominant negative mutant of either Dph1 or Dph2. We report in this study the first cellular-targeted strategy that protects against DT and ETA toxicity. We have generated Dph2(C-), a dominant-negative mutant of Dph2, that could block very efficiently the formation of diphthamide. Cells expressing Dph2(C-) were 1000-fold more resistant to DT than parental cells, and a similar protection against Pseudomonas exotoxin A was also obtained. The targeting of a cellular component with this approach should have a reduced risk of generating resistance as it is commonly seen with antibiotic treatments.     

1-N6-Etheno-ADP-ribosylation of elongation factor-2 by diphtheria toxin

Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (epsilon NAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the epsilon ADP-ribose, to the EF-2. As a consequence of the transfer of the epsilon ADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The epsilon ADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed.     

Diphtheria toxin. Site and configuration of ADP-ribosylation of diphthamide in elongation factor 2

Diphtheria toxin inactivates protein synthesis elongation factor 2 by catalyzing the ADP-ribosylation of a novel derivative of histidine, diphthamide, in the protein (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10710-10716). In this report, we describe experiments involving nuclear Overhauser enhancement NMR spectroscopy which were undertaken to elucidate the site of ADP-ribosylation of diphthamide and the configuration of the glycosidic bond formed by the toxin. The essential result of these experiments is that, in ribosyl-diphthamide obtained by enzymatic digestion of ADP-ribosyl-elongation factor-2, the H-5 imidazole proton is near the R-4 proton of ribose. This result and others are consistent with the interpretation that diphtheria toxin covalently attaches ADP-ribose to the imidazole N-1 of diphthamide via an alpha-glycosidic linkage.     

Occurrence of diphthamide in archaebacteria

We examined the nature of the diphtheria toxin fragment A recognition site in the protein synthesis translocating factor present in cell-free preparations from the archaebacteria Thermoplasma acidophilum and Halobacterium halobium. In agreement with earlier work (M. Kessel and F. Klink, Nature (London) 287:250-251, 1980), we found that extracts from these organisms contain a protein factor which is a substrate for the ADP-ribosylation reaction catalyzed by diphtheria toxin fragment A. However, the rate of the reaction was approximately 1,000 times slower than that typically observed with eucaryotic elongation factor 2. We also demonstrated the presence of diphthine (the deamidated form of diphthamide, i.e., 2-[3-carboxyamide-3-(trimethylammonio)propyl]histidine) in acid hydrolysates of H. halobium protein in amounts comparable to those found in hydrolysates of similar preparations from eucaryotic cells (Saccharomyces cerevisiae and HeLa). Diphthine could not be detected in hydrolysates of protein from the eubacterium Escherichia coli. Whereas both archaebacterial and eucaryotic elongation factors contain diphthamide, they differ importantly in other respects.     

In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin.

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).     

Introduction of Additional Charges as an Aid in Protein Purification: Isolation of Elongation Factor 2 from Sulfolobus acidocaldarius by Preparative Isoelectric Focusing Before and After ADP-Ribosylation

Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor 2 (EF-2) in eukaryotes and archaebacteria. As the reaction is strictly EF-2 specific and introduces two negative charges into the molecule, the resulting shift in the isoelectric point (pI) by 0.2 pH units was used to establish a new purification method for EF-2 from Sulfolobus acidocaldarius. The cells were lysed with dithiothreitol at pH 9 and EF-2 was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-200, and three isoelectric focusing steps. The EF-2-containing fractions from the first isoelectric focusing step at pH 4-9 were refocused in a more narrow pH-gradient (pH 5-7). The EF-2 peak from the second step was eluted, collecting only the fractions above the pH region where ADP-ribosylated EF-2 would focus. The EF-2 was then ADP-ribosylated with diphtheria toxin and NAD and subjected to further isoelectric focusing (pH 5-7). The EF-2 was almost homogeneous since ADP-ribosylation had shifted it into a region of the pH gradient free of contaminating proteins. Diphtheria toxin was immobilized on CNBr-activated Sepharose to prevent a possible contamination by proteins from the diphtheria toxin preparation which might have the same pI as ADP-ribosylated EF-2. Finally, the ADP-ribosyl group was removed by equilibrium dialysis using diphtheria toxin and nicotinamide at pH 6.3. The obtained EF-2 was active in protein synthesis.     

Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling.

The elongation factor 2 (EF-2) genes of the yeast Saccharomyces cerevisiae have been cloned and characterized with the ultimate goal of gaining a better understanding of the mechanism and control of protein synthesis. Two genes (EFT1 and EFT2) were isolated by screening a bacteriophage lambda yeast genomic DNA library with an oligonucleotide probe complementary to the domain of EF-2 that contains diphthamide, the unique posttranslationally modified histidine that is specifically ADP-ribosylated by diphtheria toxin. Although EFT1 and EFT2 are located on separate chromosomes, the DNA sequences of the two genes differ at only four positions out of 2526 base pairs, and the predicted protein sequences are identical. Genetic deletion of each gene revealed that at least one functional copy of either EFT gene is required for cell viability. Messenger RNA levels of yeast EF-2 parallel cellular growth and peak in mid-log phase cultures. The EF-2 protein sequence is strikingly conserved through evolution. Yeast EF-2 is 66% identical to, and shares over 85% homology with, human EF-2. In addition, yeast and mammalian EF-2 share identical sequences at two critical functional sites: (i) the domain containing the histidine residue that is modified to diphthamide and (ii) the threonine residue that is specifically phosphorylated in vivo in mammalian cells by calmodulin-dependent protein kinase III, also known as EF-2 kinase. Furthermore, yeast EF-2 also contains the Glu-X-X-Arg-X-Ile-Thr-Ile "effector" sequence motif that is conserved among all known elongation factors, and its GTP-binding domain exhibits strong homology to the G-domain of Escherichia coli elongation factor Tu (EF-Tu) and other G-protein family members. Based upon these observations, we have modeled the G-domain of the deduced EF-2 protein sequence to the solved crystallographic structure for EF-Tu.