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In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells
Published on: September 2, 2019
Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development
Tom R Webb1, Sally H Cross, Lisa McKie
1MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, UK.
Journal of Cell Science
|September 4, 2008
Summary
The DPH4 gene is essential for diphthamide modification of elongation factor 2 (eEF2), crucial for eukaryotic translation. Mouse mutants lacking DPH4 exhibit developmental defects and resistance to diphtheria toxin.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Diphthamide is a unique histidine modification on eukaryotic translation elongation factor 2 (eEF2).
- This modification is essential for protein synthesis and is conserved across eukaryotes.
- Five genes (DPH1-DPH5) are known to be involved in diphthamide biosynthesis in yeast, with homologues found in higher eukaryotes.
Purpose of the Study:
- To investigate the function of the DPH4 gene in diphthamide biosynthesis in mammals.
- To characterize the phenotypic consequences of Dph4 deficiency in mice.
Main Methods:
- Identification and characterization of a mouse mutant lacking functional Dph4.
- Analysis of diphthamide modification status in eEF2 from mutant cells.
- Assessment of diphtheria toxin sensitivity in Dph4-deficient cells.
- Localization studies of DPH4 protein using reporter tags.
- Phenotypic analysis of homozygous Dph4 mutant mice.
Main Results:
- Homozygous Dph4 mutant mouse cells completely lacked diphthamide modification on eEF2.
- These cells were resistant to diphtheria toxin-induced cell death.
- DPH4 protein localized to the cytoskeleton, distinct from other DPH proteins.
- Dph4 mutant mice displayed growth retardation, developmental defects, and often died perinatally.
- Surviving mutant mice exhibited preaxial polydactyly, a digit abnormality.
Conclusions:
- DPH4 is essential for diphthamide biosynthesis in mammals.
- The absence of diphthamide modification in eEF2 leads to specific developmental abnormalities, possibly due to translational defects of certain proteins.
- DPH4 functions independently of the DPH1/DPH2/DPH3 complex in diphthamide synthesis.
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