Eukaryotic elongation factor 2 (eEF2) mediates translocation in protein synthesis. The molecular mimicry model proposes that the tip of domain IV mimics the anticodon loop of tRNA. His-699 in this region is post-translationally modified to diphthamide, the target for Corynebacterium diphtheriae and Pseudomonas aeruginosa toxins. ADP-ribosylation by these toxins inhibits eEF2 function causing cell death. Mutagenesis of the tip of domain IV was used to assess both functions. A H694A mutant strain was non-functional, whereas D696A, I698A, and H699N strains conferred conditional growth defects, sensitivity to translation inhibitors, and decreased total translation in vivo. These mutant strains and those lacking diphthamide modification enzymes showed increased ؊1 frameshifting. The effects are not due to reduced protein levels, ribosome binding, or GTP hydrolysis. Functional eEF2 forms substituted in domain IV confer dominant diphtheria toxin resistance, which correlates with an in vivo effect on translation-linked phenotypes. These results provide a new mechanism in which the translational machinery maintains the accurate production of proteins, establishes a role for the diphthamide modification, and provides evidence of the ability to suppress the lethal effect of a toxin targeted to eEF2.The eukaryotic translation elongation factor 2 (eEF2) 2 and its bacterial homolog elongation factor G (EF-G) are members of the G-protein superfamily. These two proteins catalyze the translocation step of translation elongation after peptide bond formation occurs. The tRNAs located in the A-and P-sites are translocated to the P-and E-sites followed by the advancement of three bases of the mRNA to allow another round of translation elongation (reviewed in Ref. 1). In the yeast Saccharomyces cerevisiae, eEF2 is encoded by two genes, EFT1 and EFT2. The encoded proteins are identical, and one must be present for viability (2).Even though work on EF-G has proven to be invaluable in our understanding of the function of eEF2 on protein synthesis, marked differences are evident between the two homologous proteins. The most pronounced are the post-translation modifications that occur on eEF2. These modifications are the phosphorylation of Thr-57 and the diphthamide modification of His-699 in yeast and His-715 in mammals. S. cerevisiae eEF2 is phosphorylated by the Rck2p kinase (3), a Ser/Thr protein kinase homologous to the mammalian calmodulin kinases, which requires phosphorylation for activation (4, 5). In mammalian cells, eEF2 is phosphorylated on Thr-57 by the eEF2 kinase, a Ca 2ϩ /calmodulin-dependent protein kinase (6). The unique diphthamide modification is the result of a multistep conversion requiring several enzymatic activities performed by the DPH gene products in yeast (7). This modification is located at the tip of domain IV of the protein (8), a region proposed to mimic the tRNA anticodon loop (reviewed in Ref. 9). Although phosphorylation reduces the affinity for GTP, but not GDP, and decreases ribosome binding (10), a r...

Eukaryotic translation elongation factor 3 (eEF3) is a fungalspecific ATPase proposed to catalyze the release of deacylatedtRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775-980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperaturesensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.The protein synthetic machinery is characterized by the interplay of different soluble factors in conjunction with ribosomes to translate the mRNA into the correct sequence of amino acids. The three phases of translation, initiation, elongation, and termination, are driven by factors that are highly conserved between yeast and metazoans (1). However, a major difference in elongation is the indispensability of eukaryotic elongation factor 3 (eEF3) 3 with yeast ribosomes (2, 3). eEF3 catalyzes an essential step in each elongation cycle by virtue of its ATPase activity. It has been proposed to act as an Exit-site (E-site) factor, facilitating the release of deacylated-tRNA and simultaneously impacting on the delivery of aminoacyl-tRNA (aa-tRNA) at the aminoacyl site (A-site) (4). Metazoan ribosomes have been reported to possess a compensatory intrinsic ATPase activity, although they differ kinetically from the fungal eEF3 (5). Escherichia coli, on the other hand, expresses the 911 amino acid RbbA protein that exhibits ATPase activity and is tightly associated with ribosomes (6, 7). Both pathogenic and non-pathogenic fungi have been reported to contain eEF3 (8 -10). In Saccharomyces cerevisiae, eEF3 is encoded by a single copy essential YEF3 gene. A paralog of the YEF3 gene, designated HEF3 or YEF3B, encodes an 84% identical protein but is not expressed during vegetative growth (11). However, expression of the HEF3 coding sequence under the YEF3 promoter produces a protein that has similar ATPase activity and ribosome binding properties to YEF3-encoded eEF3.eEF3 is a class 1 member of the ATP binding cassette (ABC) family of proteins. eEF3 possesses distinct motifs including the HEAT repeats on the N terminus, two nucleotide binding domains with tandemly arranged bipartite (ABC) cassettes in the middle, a conserved insertion in the intervening region of the Walker A and B motifs of ABC2, and a highly basic C terminus. HEAT (Huntington elongation factor 3, ...

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