Y. Kimata et K. Kohno, ELONGATION-FACTOR-2 MUTANTS DEFICIENT IN DIPHTHAMIDE FORMATION SHOW TEMPERATURE-SENSITIVE CELL-GROWTH, The Journal of biological chemistry, 269(18), 1994, pp. 13497-13501
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 wi
th substitutions of 19 other amino acids for His-699, which is modifie
d to diphthamide, in yeast cells and found that they can be classified
into three groups. In the first group (Group 1), replacement of His-6
99 by the basic amino acid Arg or Lys showed not only loss of EF 2 act
ivity 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 dipht
hamide. However, the viable cells grew more slowly than cells expressi
ng wild-type EF-2 and showed temperature sensitivities. This result su
ggests that diphthamide may confer heat resistance on EF-2, although i
t still may be active without diphthamide at a normal temperature.