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.
Kimata
Y
Y
Institute for Molecular and Cellular Biology, Faculty of Engineering, Osaka University, Japan.
Harashima
S
S
Kohno
K
K
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004167:Diphtheria Toxin / Q000633:toxicity*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D013172:Spores, Fungal
D004167:Diphtheria Toxin; D009838:Oligodeoxyribonucleotides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D000246:Adenosine Diphosphate Ribose