Diphthamide Modification of eEF2 Requires a J-domain Protein and Is Essential for Normal Development

J Cell Sci. 2008 Oct 1;121(Pt 19):3140-5. doi: 10.1242/jcs.035550. Epub 2008 Sep 2.

Abstract

The intracellular target of diphtheria toxin is a modified histidine residue, diphthamide, in the translation elongation factor, eEF2 (also known as EFT1). This enigmatic modification occurs in all eukaryotes and is produced in yeast by the action of five gene products, DPH1 to DPH5. Sequence homologues of these genes are present in all sequenced eukaryotic genomes and, in higher eukaryotes, there is functional evidence for DPH1, DPH2, DPH3 and DPH5 acting in diphthamide biosynthesis. We identified a mouse that was mutant for the remaining gene, Dph4. Cells derived from homozygous mutant embryos lacked the diphthamide modification of eEF2 and were resistant to killing by diphtheria toxin. Reporter-tagged DPH4 protein localized to the cytoskeleton, in contrast to the localization of DPH1 and consistent with evidence that DPH4 is not part of a proposed complex containing DPH1, DPH2 and DPH3. Mice that were homozygous for the mutation were retarded in growth and development, and almost always die before birth. Those that survive long enough had preaxial polydactyly, a duplication of digit 1 of the hind foot. This same defect has been seen in embryos that were homozygous for mutation of DPH1, suggesting that lack of diphthamide on eEF2 could result in translational failure of specific proteins, rather than a generalized translation downregulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism*
  • Chromosomes, Mammalian / genetics
  • Crosses, Genetic
  • Cytoskeleton / drug effects
  • Cytoskeleton / metabolism
  • Diphtheria Toxin / pharmacology
  • Embryo, Mammalian / abnormalities
  • Embryo, Mammalian / drug effects
  • Embryonic Development* / drug effects
  • Eye Proteins / genetics
  • Female
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Genetic Testing
  • Genotype
  • Histidine / analogs & derivatives*
  • Histidine / metabolism
  • Homeodomain Proteins / genetics
  • Male
  • Mice
  • Mice, Mutant Strains
  • Mutation / genetics
  • NIH 3T3 Cells
  • PAX6 Transcription Factor
  • Paired Box Transcription Factors / genetics
  • Peptide Elongation Factor 2 / metabolism*
  • Protein Structure, Tertiary
  • Protein Transport / drug effects
  • RNA Splicing / drug effects
  • Repressor Proteins / genetics
  • Toes / abnormalities

Substances

  • Carrier Proteins
  • Diphtheria Toxin
  • Eye Proteins
  • Homeodomain Proteins
  • PAX6 Transcription Factor
  • PAX6 protein, human
  • Paired Box Transcription Factors
  • Pax6 protein, mouse
  • Peptide Elongation Factor 2
  • Repressor Proteins
  • Histidine
  • diphthamide