Saccharomyces cerevisiae elongation factor 2. Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin.
Protein synthesis elongation factor 2 (EF-2) is the target of the ADP-ribosylating activity of diphtheria toxin which is responsible for cell killing. Diphthamide, an unique post-translationally modified histidine residue, is both required for and the site of this ADP-ribosylation. Although present in the EF-2 of all eukaryotes and archaebacteria, the function of diphthamide is unknown. Here we describe the site-specific mutagenesis of the histidine precursor of diphthamide, histidine 699, in yeast EF-2. Plasmid-borne EFT was randomly mutagenized at the histidine 699 codon, and the technique of plasmid shuffling was utilized to select strains that were maintained by the mutant EFT. These mutants were screened for diphtheria toxin resistance. Sequence analysis of the EFT in 49 toxin-resistant isolates showed that histidine 699 had been replaced by 1 of 4 amino acids: asparagine, glutamine, leucine, or methionine. All 11 of the possible codons corresponding to these 4 amino acids were found. The growth rates of cells sustained by the mutant forms of EF-2 were slightly slower than those of isogenic wild-type cells. We conclude that despite its strict conservation and universal post-translational modification, the histidine precursor of diphthamide is not essential to the function of yeast EF-2 in protein synthesis.
Phan
L D
LD
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Perentesis
J P
JP
Bodley
J W
JW
D001483:Base Sequence; D004271:DNA, Fungal; D004167:Diphtheria Toxin / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D005656:Fungal Proteins / Q000235:genetics; D006639:Histidine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*
D004271:DNA, Fungal; D004167:Diphtheria Toxin; D005656:Fungal Proteins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006639:Histidine; C027527:diphthamide