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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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DPH6 and DPH7 deletion strains copy traits typically related to the bona fide diphthamide mutants dph1-dph5.(A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its dph1-dph7 gene deletion derivatives (upper panels) as well an MKK-derived eft1 eft2 double deletion background maintaining plasmid pEFT2 wild-type or H699 substitution (H699 N and H699I) alleles of EFT2 (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml−1 sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from dph1, dph5, dph6 and dph7 mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast dph mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see Materials and Methods). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.
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pgen-1003334-g003: DPH6 and DPH7 deletion strains copy traits typically related to the bona fide diphthamide mutants dph1-dph5.(A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its dph1-dph7 gene deletion derivatives (upper panels) as well an MKK-derived eft1 eft2 double deletion background maintaining plasmid pEFT2 wild-type or H699 substitution (H699 N and H699I) alleles of EFT2 (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml−1 sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from dph1, dph5, dph6 and dph7 mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast dph mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see Materials and Methods). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.

Mentions: To verify the predicted roles for DPH6 and DPH7 in the diphthamide pathway, we next examined strains deleted for these ORFs for phenotypes specifically linked to defects in diphthamide formation on eEF2, namely sordarin resistance and response to DT [6], [7]. Sordarin traps eEF2 on the 80S ribosome [53], blocking mRNA translation elongation and yeast cell growth [54] in a fashion that depends on diphthamide synthesis [6], [7]. As a result, diphthamide mutants dph1-dph5 efficiently protect against sordarin inhibition [6], [7]. Like dph1-dph5, dph6 and dph7 mutants showed robust resistance towards sordarin at 10 µg/ml, a concentration inhibitory to the wild-type (Figure 3A). This resistance was comparable to that shown by eEF2 substitution mutants eft2H699I and eft2H699N (Figure 3A), which lack the His699 acceptor residue for diphthamide modification [37]. Thus DPH6 and DPH7 are novel sordarin effectors, a feature they share with the diphthamide synthesis genes DPH1-DPH5[6], [7].


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)

DPH6 and DPH7 deletion strains copy traits typically related to the bona fide diphthamide mutants dph1-dph5.(A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its dph1-dph7 gene deletion derivatives (upper panels) as well an MKK-derived eft1 eft2 double deletion background maintaining plasmid pEFT2 wild-type or H699 substitution (H699 N and H699I) alleles of EFT2 (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml−1 sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from dph1, dph5, dph6 and dph7 mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast dph mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see Materials and Methods). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.
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pgen-1003334-g003: DPH6 and DPH7 deletion strains copy traits typically related to the bona fide diphthamide mutants dph1-dph5.(A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its dph1-dph7 gene deletion derivatives (upper panels) as well an MKK-derived eft1 eft2 double deletion background maintaining plasmid pEFT2 wild-type or H699 substitution (H699 N and H699I) alleles of EFT2 (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml−1 sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from dph1, dph5, dph6 and dph7 mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast dph mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see Materials and Methods). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.
Mentions: To verify the predicted roles for DPH6 and DPH7 in the diphthamide pathway, we next examined strains deleted for these ORFs for phenotypes specifically linked to defects in diphthamide formation on eEF2, namely sordarin resistance and response to DT [6], [7]. Sordarin traps eEF2 on the 80S ribosome [53], blocking mRNA translation elongation and yeast cell growth [54] in a fashion that depends on diphthamide synthesis [6], [7]. As a result, diphthamide mutants dph1-dph5 efficiently protect against sordarin inhibition [6], [7]. Like dph1-dph5, dph6 and dph7 mutants showed robust resistance towards sordarin at 10 µg/ml, a concentration inhibitory to the wild-type (Figure 3A). This resistance was comparable to that shown by eEF2 substitution mutants eft2H699I and eft2H699N (Figure 3A), which lack the His699 acceptor residue for diphthamide modification [37]. Thus DPH6 and DPH7 are novel sordarin effectors, a feature they share with the diphthamide synthesis genes DPH1-DPH5[6], [7].

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