Expression of non-ADP-ribosylatable, diphtheria toxin-resistant elongation factor 2 in Saccharomyces cerevisiae.

Eucaryotic elongation factor 2 (EF-2) contains a post-translationally modified histidine residue termed diphthamide that is specifically ADP-ribosylated by diphtheria toxin (DT) or Pseudomonas exotoxin A. To analyze the potential physiological role of ADP-ribosylation of EF-2 by cellular ADP-ribosyl transferase, we constructed DT-resistant, non-ADP-ribosylatable Saccharomyces cerevisiae EF-2 by site-directed mutagenesis and expressed the mutant EF-2 in yeast. Substitution of Arg for Gly(701) in yeast EF-2 conferred complete resistance to DT in vivo and in vitro. However, when only non-ribosylatable EF-2 was expressed in cells using genetic manipulation, the mutated EF-2 did not affect vegetative cell growth, mating, sporulation and germination of ascospores.