DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

Mattheakis, Larry; Shen, W H; Collier, R J

doi:10.1128/mcb.12.9.4026

Cited by 68 publications

(72 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A truncated version of DT has been used to select strains that are deficient in diphthamide biosynthesis and thus resistant to DT (17). This method allowed the identification of DPH1-5 (9).…”

Section: Resultsmentioning

confidence: 99%

Chemogenomic approach identified yeast YLR143W as diphthamide synthetase

Su¹,

Lin²,

Chen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Many genes are of unknown functions in any sequenced genome. A combination of chemical and genetic perturbations has been used to investigate gene functions. Here we present a case that such "chemogenomics" information can be effectively used to identify missing genes in a defined biological pathway. In particular, we identified the previously unknown enzyme diphthamide synthetase for the last step of diphthamide biosynthesis. We found that yeast protein YLR143W is the diphthamide synthetase catalyzing the last amidation step using ammonium and ATP. Diphthamide synthetase is evolutionarily conserved in eukaryotes. The previously uncharacterized human gene ATPBD4 is the ortholog of yeast YLR143W and fully rescues the deletion of YLR143W in yeast.

show abstract

Section: Resultsmentioning

confidence: 99%

Chemogenomic approach identified yeast YLR143W as diphthamide synthetase

Su¹,

Lin²,

Chen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…ϩ/ϩ , dph3 ϩ/Ϫ , and dph3 Ϫ/Ϫ E10.5 embryos were hybridized to oligo(dT) [12][13][14][15][16][17][18] . The first-strand cDNAs were synthesized by adding 1 l (200 units) SuperScript reverse transcriptase (RT) (Invitrogen), 1 l dNTP mix (10 mM each), 2 l dithiothreitol (0.1 M) in 20-l reaction mixtures in accordance with the product manuals.…”

Section: Gene Targeting and Genotypingmentioning

confidence: 99%

“…Recent work has identified all the proteins required for diphthamide biosynthesis (10,11,13,14), with the exception of an unidentified amidating enzyme required at the last step.…”

mentioning

confidence: 99%

Dph3, a Small Protein Required for Diphthamide Biosynthesis, Is Essential in Mouse Development

Liu

Wiggins

Sreenath

et al. 2006

Molecular and Cellular Biology

View full text Add to dashboard Cite

The translation elongation factor 2 in eukaryotes (eEF-2) contains a unique posttranslationally modified histidine residue, termed diphthamide, which serves as the only target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Diphthamide biosynthesis is carried out by five highly conserved proteins, Dph1 to Dph5, and an as-yet-unidentified amidating enzyme. The evolutionary conservation of the complex diphthamide biosynthesis pathway throughout eukaryotes implies a key role for diphthamide in normal cellular physiology. Of the proteins required for diphthamide synthesis, Dph3 is the smallest, containing only 82 residues. In addition to having a role in diphthamide biosynthesis, Dph3 is also involved in modulating the functions of the Elongator complex in yeast. To explore the physiological roles of Dph3 and to begin to investigate the function of diphthamide, we generated dph3 knockout mice and showed that dph3 ؉/؊ mice are phenotypically normal, whereas dph3 ؊/؊ mice, which lack the diphthamide modification on eEF-2, are embryonic lethal. Loss of both dph3 alleles causes a general delay in embryonic development accompanied by lack of allantois fusion to the chorion and increased degeneration and necrosis in neural tubes and is not compatible with life beyond embryonic day 11.5. The dph3 ؊/؊ placentas also developed abnormally, showing a thinner labyrinth lacking embryonic erythrocytes and blood vessels. These results attest to the physiological importance of Dph3 in development. The biological roles of Dph3 are also discussed.

show abstract

“…Because the diphthamide modification is required for the action of the ADP-ribosylating toxins, the complex diphthamide biosynthesis pathway is amenable to genetic analysis, and mutants defective in diphthamide biosynthesis have been isolated in both Chinese hamster ovary (CHO) cells and yeast (Saccharomyces cerevisiae) by selection for resistance to DT or an engineered ADP-ribosylating toxin − anthrax protective antigen (PA) + fusion protein 59 (FP59) (5)(6)(7)(8)(9)(10). PA is the cellular binding moiety of anthrax toxin, which specifically binds to either capillary morphogenesis protein 2 (CMG2, major toxin receptor) or tumor endothelium marker 8 (TEM8, minor toxin receptor) on host cells (11)(12)(13)(14) causing the endocytosis and translocation of anthrax lethal factor (LF), edema factor, or FP59 into the cytosol of host cells.…”

mentioning

confidence: 99%

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Liu

Bachran

Gupta

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly 717 Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly 717 -to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1 −/− mice, eEF2 G717R/G717R MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2 G717R/G717R mice showed milder phenotypes than OVCA1 −/− mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2 G717R/G717R /OVCA1 −/− double mutant mice displayed the milder phenotypes of the eEF2 G717R/G717R mice, suggesting that the embryonic lethality of OVCA1 −/− mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent −1 frameshifting during translation and show that the Gly 717 -to-Arg mutation cannot rescue this defect. E ukaryotic elongation factor 2 (eEF2) is a member of the GTPbinding translation elongation factor family, and an essential factor for protein synthesis and cell survival. eEF2 drives the GTP-dependent translocation of the nascent polypeptide chain from the A site to the P site of the ribosome and advances mRNA by three bases during the elongation cycle of protein synthesis (1). eEF2 is highly homologous in all eukaryotes. In fact, eEF2 of humans, rats, mice, hamsters, and other mammals have exactly the same amino acid sequence. Intriguingly, all eukaryotic eEF2 proteins contain a unique posttranslationally modified histidine residue termed diphthamide (2, 3). Diphthamide modification occurs after eEF2 is translated and is irreversible, marking the completion of the biosynthesis of eEF2.Although the physiological role of the diphthamide modification on eEF2 remains elusive, diphthamide is the well-known target for the adenosine diphosphate (ADP)-ribosylating toxins from bacterial pathogens, such as diphtheria toxin (DT) from Corynebacterium diphtheriae, Pseudomonas exotoxin A (ETA) from Pseudomonas aeruginosa, and the recently identified cholix toxin (CT) from Vibrio cholerae (4). As virulence factors, these ADP-ribosylating toxins catalyze transfer of the ADP ribose from nicotinamide adenine dinucleotide (NAD + ) to diphthamide on eEF2 (Fig. S1), thus inactivating eEF2, halting cellular protein synthesis, and causing cell death.Because the diphthamide modification is required for the action of the ADP-ribosylating toxins, the complex diphthamide biosynthesis pathway is amenable to genetic analysis, and mutants defective in diphthamide biosynthesis have been isolated in both Chinese hamster ovary (CHO) cells and yeast (Saccharomyces cerevisiae) by selection for resistance to DT or an engineered ADP-ribosylating toxin − anthrax protective a...

show abstract

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

Cited by 68 publications

References 20 publications

Chemogenomic approach identified yeast YLR143W as diphthamide synthetase

Chemogenomic approach identified yeast YLR143W as diphthamide synthetase

Dph3, a Small Protein Required for Diphthamide Biosynthesis, Is Essential in Mouse Development

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Contact Info

Product

Resources

About