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The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network.

Uthman S, Bär C, Scheidt V, Liu S, ten Have S, Giorgini F, Stark MJ, Schaffrath R - PLoS Genet. (2013)

Bottom Line: The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7.Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal -1 frameshifting and altered responses to translation inhibitors.Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
Diphthamide is a highly modified histidine residue in eukaryal translation elongation factor 2 (eEF2) that is the target for irreversible ADP ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step-amidation of the intermediate diphthine to diphthamide-is ill-defined. Here we mine the genetic interaction landscapes of DPH1-DPH5 to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants maintain eEF2 forms that evade inhibition by DT and sordarin, a diphthamide-dependent antifungal. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Consistent with an expected requirement for ATP in diphthine amidation, Dph6 contains an essential adenine nucleotide hydrolase domain and binds to eEF2. Dph6 is therefore a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal -1 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation.

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dph mutants show sensitivity to elevated diphthine synthase levels and confer reduced translational accuracy.(A) DPH5 overexpression in dph1-dph4 and dph7 mutants causes cytotoxicity and a severe cell growth defect. Cells of yeast strains with the indicated genetic backgrounds and maintaining plasmid pGAL-DPH5 for galactose inducible overexpression of diphthine synthase Dph5 were serially diluted and replica spotted onto glucose (2% glc) and galactose (2% gal) media to assay their response to DPH5 overexpression. Growth was for 3 days at 30°C. Unaltered (T), slightly weakened tolerance (∼T) and sensitive (S) responses are indicated. Note that dph1-dph4 and dph7 mutants are extremely sensitive to DPH5 overexpression. (B) Ribosomal frameshift analysis reveals erroneous translation in dph1-dph7 mutants. Strains with the indicated genetic backgrounds were transformed with control (pJD240.0) or lacZ −1 frameshift (pJD240.−1) plasmids [59] to monitor lacZ expression through β-galactosidase (β-Gal) production using O-nitrophenol-D- galactopyranoside assays and to score translation efficiency (pJD240.0) and fidelity (pJD240.−1). Ribosomal −1 frameshifts are expressed relative to the level of overall translation efficiency with statistical significance determined by one-way ANOVA followed by Dunnett's multiple comparison. With the exception of dph4 and dph7, post-hoc comparison found that all other mutant backgrounds showed a significant increase in ribosomal −1 frameshifting relative to wild-type (wt) yeast cells (* = P<0.05; *** = P<0.001; ns. = not significant).
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pgen-1003334-g006: dph mutants show sensitivity to elevated diphthine synthase levels and confer reduced translational accuracy.(A) DPH5 overexpression in dph1-dph4 and dph7 mutants causes cytotoxicity and a severe cell growth defect. Cells of yeast strains with the indicated genetic backgrounds and maintaining plasmid pGAL-DPH5 for galactose inducible overexpression of diphthine synthase Dph5 were serially diluted and replica spotted onto glucose (2% glc) and galactose (2% gal) media to assay their response to DPH5 overexpression. Growth was for 3 days at 30°C. Unaltered (T), slightly weakened tolerance (∼T) and sensitive (S) responses are indicated. Note that dph1-dph4 and dph7 mutants are extremely sensitive to DPH5 overexpression. (B) Ribosomal frameshift analysis reveals erroneous translation in dph1-dph7 mutants. Strains with the indicated genetic backgrounds were transformed with control (pJD240.0) or lacZ −1 frameshift (pJD240.−1) plasmids [59] to monitor lacZ expression through β-galactosidase (β-Gal) production using O-nitrophenol-D- galactopyranoside assays and to score translation efficiency (pJD240.0) and fidelity (pJD240.−1). Ribosomal −1 frameshifts are expressed relative to the level of overall translation efficiency with statistical significance determined by one-way ANOVA followed by Dunnett's multiple comparison. With the exception of dph4 and dph7, post-hoc comparison found that all other mutant backgrounds showed a significant increase in ribosomal −1 frameshifting relative to wild-type (wt) yeast cells (* = P<0.05; *** = P<0.001; ns. = not significant).

Mentions: Inactivation of WDR85, the mammalian homolog of Dph7, was recently shown to dramatically enhance association of diphthine synthase Dph5 with eEF2 [41]. We therefore examined whether Dph7 impacts on the interaction between Dph5 and eEF2 in budding yeast. We found that a much higher level of affinity tagged eEF2 could be co-immune precipitated with HA-tagged Dph5 from extracts of the dph7 mutant in comparison to wild-type extracts (Figure 5B). A smaller increase was also seen with the dph6 mutant (Figure 5B). This strongly suggests a conserved role for Dph7/WDR85 as a regulator of the Dph5•eEF2 interaction. Remarkably, we also found similarly enhanced binding of Dph5 to eEF2 in the dph1 mutant, which has a defect in the first step of the diphthamide pathway and therefore lacks the ACP modification that is the immediate substrate of diphthine synthase (Figure 5B). Strikingly, DPH5 overproduction from a galactose-inducible promoter was found to be highly detrimental to cells deleted for DPH7 and to all mutants blocked at the first step of the pathway, but had little effect on the dph6 mutant and no effect on wild-type or dph5 cells (Figure 6A). Intriguingly, this cytotoxicity goes hand in hand with the enhanced Dph5•eEF2 interaction profiles we observed in dph1, dph6 and dph7 cells under conditions of wild-type DPH5 copy number and normal Dph5 expression levels (Figure 5B). Taken together, our results suggest that binding of Dph5 to incompletely modified eEF2 may be inhibitory to the function of the translation factor. Our data also indicate that both unmodified eEF2, and diphthine-modified eEF2 in the absence of Dph7, show strongly enhanced binding to Dph5. Furthermore, since we failed to detect association between Dph5 and Dph6 despite demonstrating interaction of each with eEF2, it is likely that Dph5 and Dph6 do not bind concurrently to eEF2 and that their binding may therefore be mutually exclusive.


The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network.

Uthman S, Bär C, Scheidt V, Liu S, ten Have S, Giorgini F, Stark MJ, Schaffrath R - PLoS Genet. (2013)

dph mutants show sensitivity to elevated diphthine synthase levels and confer reduced translational accuracy.(A) DPH5 overexpression in dph1-dph4 and dph7 mutants causes cytotoxicity and a severe cell growth defect. Cells of yeast strains with the indicated genetic backgrounds and maintaining plasmid pGAL-DPH5 for galactose inducible overexpression of diphthine synthase Dph5 were serially diluted and replica spotted onto glucose (2% glc) and galactose (2% gal) media to assay their response to DPH5 overexpression. Growth was for 3 days at 30°C. Unaltered (T), slightly weakened tolerance (∼T) and sensitive (S) responses are indicated. Note that dph1-dph4 and dph7 mutants are extremely sensitive to DPH5 overexpression. (B) Ribosomal frameshift analysis reveals erroneous translation in dph1-dph7 mutants. Strains with the indicated genetic backgrounds were transformed with control (pJD240.0) or lacZ −1 frameshift (pJD240.−1) plasmids [59] to monitor lacZ expression through β-galactosidase (β-Gal) production using O-nitrophenol-D- galactopyranoside assays and to score translation efficiency (pJD240.0) and fidelity (pJD240.−1). Ribosomal −1 frameshifts are expressed relative to the level of overall translation efficiency with statistical significance determined by one-way ANOVA followed by Dunnett's multiple comparison. With the exception of dph4 and dph7, post-hoc comparison found that all other mutant backgrounds showed a significant increase in ribosomal −1 frameshifting relative to wild-type (wt) yeast cells (* = P<0.05; *** = P<0.001; ns. = not significant).
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pgen-1003334-g006: dph mutants show sensitivity to elevated diphthine synthase levels and confer reduced translational accuracy.(A) DPH5 overexpression in dph1-dph4 and dph7 mutants causes cytotoxicity and a severe cell growth defect. Cells of yeast strains with the indicated genetic backgrounds and maintaining plasmid pGAL-DPH5 for galactose inducible overexpression of diphthine synthase Dph5 were serially diluted and replica spotted onto glucose (2% glc) and galactose (2% gal) media to assay their response to DPH5 overexpression. Growth was for 3 days at 30°C. Unaltered (T), slightly weakened tolerance (∼T) and sensitive (S) responses are indicated. Note that dph1-dph4 and dph7 mutants are extremely sensitive to DPH5 overexpression. (B) Ribosomal frameshift analysis reveals erroneous translation in dph1-dph7 mutants. Strains with the indicated genetic backgrounds were transformed with control (pJD240.0) or lacZ −1 frameshift (pJD240.−1) plasmids [59] to monitor lacZ expression through β-galactosidase (β-Gal) production using O-nitrophenol-D- galactopyranoside assays and to score translation efficiency (pJD240.0) and fidelity (pJD240.−1). Ribosomal −1 frameshifts are expressed relative to the level of overall translation efficiency with statistical significance determined by one-way ANOVA followed by Dunnett's multiple comparison. With the exception of dph4 and dph7, post-hoc comparison found that all other mutant backgrounds showed a significant increase in ribosomal −1 frameshifting relative to wild-type (wt) yeast cells (* = P<0.05; *** = P<0.001; ns. = not significant).
Mentions: Inactivation of WDR85, the mammalian homolog of Dph7, was recently shown to dramatically enhance association of diphthine synthase Dph5 with eEF2 [41]. We therefore examined whether Dph7 impacts on the interaction between Dph5 and eEF2 in budding yeast. We found that a much higher level of affinity tagged eEF2 could be co-immune precipitated with HA-tagged Dph5 from extracts of the dph7 mutant in comparison to wild-type extracts (Figure 5B). A smaller increase was also seen with the dph6 mutant (Figure 5B). This strongly suggests a conserved role for Dph7/WDR85 as a regulator of the Dph5•eEF2 interaction. Remarkably, we also found similarly enhanced binding of Dph5 to eEF2 in the dph1 mutant, which has a defect in the first step of the diphthamide pathway and therefore lacks the ACP modification that is the immediate substrate of diphthine synthase (Figure 5B). Strikingly, DPH5 overproduction from a galactose-inducible promoter was found to be highly detrimental to cells deleted for DPH7 and to all mutants blocked at the first step of the pathway, but had little effect on the dph6 mutant and no effect on wild-type or dph5 cells (Figure 6A). Intriguingly, this cytotoxicity goes hand in hand with the enhanced Dph5•eEF2 interaction profiles we observed in dph1, dph6 and dph7 cells under conditions of wild-type DPH5 copy number and normal Dph5 expression levels (Figure 5B). Taken together, our results suggest that binding of Dph5 to incompletely modified eEF2 may be inhibitory to the function of the translation factor. Our data also indicate that both unmodified eEF2, and diphthine-modified eEF2 in the absence of Dph7, show strongly enhanced binding to Dph5. Furthermore, since we failed to detect association between Dph5 and Dph6 despite demonstrating interaction of each with eEF2, it is likely that Dph5 and Dph6 do not bind concurrently to eEF2 and that their binding may therefore be mutually exclusive.

Bottom Line: The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7.Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal -1 frameshifting and altered responses to translation inhibitors.Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, United Kingdom.

ABSTRACT
Diphthamide is a highly modified histidine residue in eukaryal translation elongation factor 2 (eEF2) that is the target for irreversible ADP ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step-amidation of the intermediate diphthine to diphthamide-is ill-defined. Here we mine the genetic interaction landscapes of DPH1-DPH5 to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants maintain eEF2 forms that evade inhibition by DT and sordarin, a diphthamide-dependent antifungal. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Consistent with an expected requirement for ATP in diphthine amidation, Dph6 contains an essential adenine nucleotide hydrolase domain and binds to eEF2. Dph6 is therefore a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal -1 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation.

Show MeSH
Related in: MedlinePlus