Mutations in the elongation factor 2 gene which confer resistance to diphtheria toxin and Pseudomonas exotoxin A. Genetic and biochemical analyses.
Both diphtheria toxin and Pseudomonas exotoxin A inhibit eukaryotic protein synthesis by ADP-ribosylating diphthamide, a posttranslationally modified histidine residue present in the elongation factor 2 (EF-2) protein. Elongation factor 2 cannot be ADP-ribosylated by the toxins unless this histidine is modified. In this report we identify three new point mutations in toxin-resistant alleles of the Chinese hamster ovary cell elongation factor 2 gene. The mutations resulted in amino acid substitutions at positions 584 (serine to glycine), 714 (isoleucine to asparagine), and 719 (glycine to aspartic acid). All three amino acid substitutions prevented the biosynthesis of diphthamide. The amount by which the toxins reduced protein synthesis in each of these mutant cell strains suggested that all three mutations also either impaired the function of EF-2 or reduced its steady state level in the cytoplasm. Western blot analysis showed that equal amounts of EF-2 were present in each of the cell strains, indicating that the mutations impaired the catalytic function of EF-2.
Foley
B T
BT
University of Vermont, Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, Burlington 05405, USA.
Moehring
J M
JM
Moehring
T J
TJ
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001427:Bacterial Toxins / Q000633:toxicity*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004167:Diphtheria Toxin / Q000633:toxicity*; D004351:Drug Resistance / Q000235:genetics*; D005098:Exotoxins / Q000633:toxicity*; D005998:Glycine; D006639:Histidine; D006801:Humans; D007532:Isoleucine; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000378:metabolism*; D010750:Phosphoproteins; D010957:Plasmids; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014162:Transfection; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001216:Asparagine; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D005098:Exotoxins; D005998:Glycine; D006639:Histidine; D007532:Isoleucine; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D012694:Serine; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A