Elongation factor 2 mutants deficient in diphthamide formation show temperature-sensitive cell growth.
Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains an unusual modified histidine residue, termed diphthamide. Diphthamide has been shown to be a site of ADP-ribosylation by bacterial toxins, but its function remains obscure. We expressed mutant genes of EF-2 with substitutions of 19 other amino acids for His-699, which is modified to diphthamide, in yeast cells and found that they can be classified into three groups. In the first group (Group 1), replacement of His-699 by the basic amino acid Arg or Lys showed not only loss of EF-2 activity but also inhibitory effects on the growth of cells co-expressing wild-type EF-2. In the second group (Group 2), replacement with Gly, Pro, Ser, or Asp resulted in nonfunctional EF-2, but it did not affect the growth of cells co-expressing wild-type EF-2. In the third group (Group 3), replacement by one of the other 13 amino acids resulted in a functional EF-2. In the Group 3 mutants, EF-2 was not ribosylated by diphtheria toxin, indicating that the mutant EF-2s did not form diphthamide. However, the viable cells grew more slowly than cells expressing wild-type EF-2 and showed temperature sensitivities. This result suggests that diphthamide may confer heat resistance on EF-2, although it still may be active without diphthamide at a normal temperature.
Kimata
Y
Y
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Kohno
K
K
D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D002455:Cell Division; D004274:DNA, Recombinant; D005796:Genes; D006639:Histidine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006639:Histidine; C027527:diphthamide