EMBO Conference on Protein Synthesis and Translational Control

TERRI KINZY
15
Speaker Abstracts
ADP-ribosylation of eukaryotic elongation factor 2 by bacterial toxins and its
effects on translation elongation in vivo and in vitro
Maria M. Mateyak 1, Pedro Ortiz 1, Eric Jan 2, Terri Kinzy 3
1 Department of Molecular Genetics, Microbiology, and Immunology, Robert Wood Johnson
Medical School, UMDNJ, Piscataway, NJ 08854, United States of America
2 Department of Biochemistry and Molecular Biology, University of British Columbia,
Vancouver, BC V6T 1Z3, United States of America
3 UMDNJ Robert Wood Johnson Medical School, United States of America
ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) by bacterial toxins leads to inhibition
of its function in translation elongation. Diphtheria toxin, produced by Corynebacterium
diphtheria, and exotoxin A, produced by Pseudomonas aeruginosa, ADP-ribosylate eEF2 on a
modified histidine residue at position 699 called diphthamide, that is highly conserved
throughout eukaryotes. However, mechanistically how ADP-ribosylation of eEF2 inhibits its
function is poorly understood. We have developed a novel Saccharomyces cerevisiae system
to directly address the effects of ADP-ribosylation on eEF2’s role in translation elongation in
vivo. This system employs eEF2 mutations identified within the anticodon mimicry domain that
display dominant resistance to galactose-induced diphtheria toxin expression in yeast. These
eEF2 mutants lack the diphthamide modification and are not a target of diphtheria toxin;
however, their expression allows growth in the presence of ADP-ribosylated wild type eEF2,
thus providing an excellent system to study the in vivo effects of eEF2 ADP-ribosylation. For
example, yeast expressing both mutant and wild type eEF2 display an increase in parmomycin
sensitivity specifically upon diphtheria toxin induction demonstrating a link between
ADP-ribosylation of eEF2 and translation defects in vivo. These effects are due to the
presence of the ADP-ribosylated form of eEF2, as determined by MS analysis. Interestingly,
the level of ADP ribosylation and the presence of a pool of eEF2 only partially modified at
histidine 699 to diphthamide and thus resistant to ADP ribosylation in vivo is not consistent
with a complete inhibition of all eEF2 activity. However, in vitro analysis indicates alterations in
the anticodon mimicry domain, which includes the location of the ADP ribosylation target
diphthamide 699, clearly affect translocation. In summary, the establishment of this novel yeast
system will provide insight into this fundamental and long-standing question of the role of the
ADP-ribosylation on eEF2