ADP-ribosylation of translation elongation factor 2 by diphtheria toxin in yeast inhibits translation and cell separation

Abstract

Background: Diphtheria toxin inhibits translation by ADP-ribosylation of elongation factor 2. Results: Diphtheria toxin expression results in accumulation of cells that fail to separate following mitosis. Conclusion: Diphtheria toxin expression has unique effects on translocation and the production of specific proteins. Significance: Understanding how ADP-ribosylated elongation factor 2 affects translocation will increase knowledge of translation and potentially lead to new treatments for toxin exposure. 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. ADPribosylation (ADPR) 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 ADPR-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 ADPR-eEF2. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.