Eukaryotic EF-2 Research Articles

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

Does phosphorylation of eukaryotic elongation factor eEF2 regulate protein synthesis in ischemic preconditioning?

Ischemia/reperfusion-associated translation inhibition in the hippocampus is attenuated significantly at reinitiation and elongation steps by ischemic preconditioning (Burda et al. [2003] Neurochem. Res. 28:1237-1243). To address potential regulation of the elongation step by changes in eukaryotic elongation factor 2 (eEF2) phosphorylation with and without acquired ischemic tolerance (IT), Wistar rats were preconditioned by 5-min sublethal ischemia and 2 days later, 30-min lethal ischemia was induced. Given the important role that oxidative stress plays in the ischemic process, eEF2 phosphorylation was also studied in a model of oxidative stress in vitro. Three blocks of our results support a lack of correlation between eEF2 phosphorylation status and protein synthesis rate. First, eEF2 was dephosphorylated significantly (activated) after transient cerebral ischemia in rats with and without IT or H2O2-treated cells; however, protein synthesis was significantly inhibited under these three conditions. Second, after 30-min reperfusion, the protein synthesis rate was maintained below control levels in cortex and hippocampus of rats without IT. Eukaryotic EF2 phosphorylated levels were notably low only in the cortex, whereas levels in the hippocampus were close to that of sham controls. In rats with IT, protein synthesis was virtually restored in both brain regions, but phosphorylated eEF2 levels were even higher than in rats without IT. Third, after 4-hr reperfusion, the protein synthesis rate in cortex and hippocampus was observed to be below sham control values in rats with and without IT. Conversely, phosphorylated eEF2 levels were below sham control in rats with IT and reached sham control values in rats without IT.

Unexpected roles of a Dictyostelium homologue of eukaryotic EF-2 in growth and differentiation.

EF-2 is believed to be indispensable for polypeptide chain elongation in protein synthesis and therefore for cell proliferation. Surprisingly, we could isolate ef2 null cells from Dictyostelium discoideum that exhibited almost normal growth and protein synthesis, which suggests that there is another molecule capable of compensating for EF-2 function. The knock-out of Dictyostelium EF-2 (Dd-EF2H; 101 kDa phosphoprotein) impairs cytokinesis, resulting in formation of multinucleate cells. The initiation of differentiation, including the acquisition of aggregation competence, was delayed in Dd-ef2 null cells compared with that in wild-type. By contrast, Dd-ef2 overexpression enhanced the progression of differentiation, thus indicating a positive involvement of Dd-EF2H in growth/differentiation transition.

Cloning and sequencing of the gene encoding thermostable elongation factor 2 in Sulfolobus solfataricus

The gene ( aEF-2) coding for the translation elongation factor 2 (aEF-2) in the thermoacidophilic archaebacterium, Sulfolobus solfataricus, has been cloned and sequenced. The deduced primary structure of aEF-2 is composed of 735 amino acids (aa), excluding the Met start residue. There are no Cys residues and the calculated M r is 81699. In the coding region of aEF-2, the high A + T content greatly influences the codon usage. From the alignment of the primary structure of aEF-2 with that of the analogous factors from the three kingdoms, aa identities were derived. The greatest identity (82%) was found with EF-2 from Sulfolobus acidocaldarius; lower values were observed with other archaebacterial EF-2 (45–47%), eukaryotic EF-2 (38–40%) and with the functional eubacterial analogue EF-G (28–31%). aEF-2 possesses the consensus sequences required for a GTP-binding protein and the four regions which are supposed to be involved in the functional regulation of EF-2/EF-G. These data should have phylogenetic implications.

Sequence analysis of the peptide‐elongation factor EF‐2 gene, downstream from those of ribosomal proteins H‐S12 and H‐S7, from the archaebacterial extreme halophile, Halobacterium halobium

The gene for the peptide-elongation factor 2 (EF-2) was cloned from the archaebacterial extreme halophile Halobacterium halobium and sequenced. The 1013 nucleotides upstream from this gene was two open reading frames similar to ribosomal proteins S12 and S7 from Escherichia coli. Sequence alignment studies showed the halobacterial elongation factor 2 to be equivalent to eukaryotic EF-2 and eubacterial EF-G. Sequence similarity to the eukaryotic elongation factor was much higher than to the eubacterial factor. Conserved sequence regions were present within the factor and are likely to constitute functionally important domains. These include the sites of GTP binding and ADP ribosylation by diphtheria toxin.

Identification and Comparison of Eighteen Archaebacteria by Means of the Diphtheria Toxin Reaction

Summary Cell-free extracts were prepared from seven extremely halophilic, six methanogenic and five thermoacidophilic archaebacteria covering nearly all known subgroups. The 150,000 X g supernatants were incubated with diphtheria toxin (DT) and NAD [adenine-14C]. TCA-precipitable radioactivity was compared with that from toxin-free controls. All eighteen archaebacterial strains gave significant, DT-dependent protein labelling, as had done all eukaryotic extracts previously assayed. No eubacterial nor mitochondrial proteins were substrates for DT. Therefore the DT reaction in this simple form provides a general method for identifying a procaryote as an archaebacterium. SDS-gel electrophoresis of the reaction products followed by autoradiography revealed in many but not all cases one predominant labelled protein band; nearly all halophilic gels and several methanogenic ones showed minor bands. Direct evidence from H. cutirubrum (Kessel and Klink, 1980) and the known specifity of DT for eukaryotic EF-2 led to the conclusion that only the analogous archaebacterial EF was ADP-ribosylated, minor bands being products of proteolysis. The apparent molecular weights showed a considerable diversity and formed three groups: The thermoacidophilic elongation factors banded between Mr 74,000 and Mr 83,000, the methanogenic ones from Mr 83,000 up to Mr 89,000, the halophilic ones between Mr 101,000 and Mr 111,000. With the same technique eukaryotic factors gave values of Mr 96,000.