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Elongation factor 2 mutants deficient in diphthamide formation show temperature-sensitive cell growth.

1994; Elsevier BV; Volume: 269; Issue: 18 Linguagem: Inglês

10.1016/s0021-9258(17)36859-x

1083-351X

Yukio Kimata, Kenji Kohno,

Fungal Infections and Studies

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.W e 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 l), 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 21, 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.Eukaryotic elongation factor 2 (EF-2)' is an essential factor that catalyzes the GTP hydrolysis-dependent translocation of peptidyl-tRNA from the aminoacyl site to the peptidyl site on the ribosome during protein synthesis.Diphtheria toxin (DT) or Pseudomonas exotoxin A specifically inactivates EF-2 by transferring the ADP-ribose moiety of NAD+ to the histidine N1-imidazole ring of diphthamide, which is a unique amino acid and is the site of the action of the toxin that results in lethal blockage of protein synthesis (1-5).Diphthamide is synthesized in cells by a multistep post-translational modification of the His residue, encoded at codon 715 in mammalian EF-2 and at codon 699 in budding yeast Saccharomyces cerevisiae EF-2, to 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine (5-9).Archaebacteria harbor EF-2 containing diphthamide or diphthine, a deamidated form of diphthamide, that is also recognized by DT (10,11).