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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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MS/MS spectra of diphthamide-, ACP-, and diphthine-modified EF2 peptide 686-VNILDVTLHADAIHR-700 from wild-type and mutant yeast strains.Spectra are shown for (A) diphthamide-modified peptide from the wild-type yeast strain; (B) ACP-modified peptide from the dph5Δ mutant; (C) diphthine-modified peptide in the dph7Δ strain; (D) diphthine-modified peptide in the dph6Δ strain; (E) diphthine-modified peptide in the dph6Δ strain with loss of the trimethylamino group before analysis in the mass spectrometer indicated by the parent ion m/z. In each case the parent ion m/z and charge state is indicated. In (A), (C) and (D), * indicates neutral loss of trimethylamino during MS/MS. The inset in (C) shows greater detail for the more crowded part of the MS/MS spectrum. Figure S2A indicates how the B and Y ions are derived from the peptide sequence.
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pgen-1003334-g004: MS/MS spectra of diphthamide-, ACP-, and diphthine-modified EF2 peptide 686-VNILDVTLHADAIHR-700 from wild-type and mutant yeast strains.Spectra are shown for (A) diphthamide-modified peptide from the wild-type yeast strain; (B) ACP-modified peptide from the dph5Δ mutant; (C) diphthine-modified peptide in the dph7Δ strain; (D) diphthine-modified peptide in the dph6Δ strain; (E) diphthine-modified peptide in the dph6Δ strain with loss of the trimethylamino group before analysis in the mass spectrometer indicated by the parent ion m/z. In each case the parent ion m/z and charge state is indicated. In (A), (C) and (D), * indicates neutral loss of trimethylamino during MS/MS. The inset in (C) shows greater detail for the more crowded part of the MS/MS spectrum. Figure S2A indicates how the B and Y ions are derived from the peptide sequence.

Mentions: The modified histidine in eEF2 (His699) is located in the tryptic peptide 686-VNILDVTLHADAIHR-700 and, as expected, unmodified versions of this peptide were readily detected in eEF2 prepared from the dph1 mutant (Figure S1C). Unmodified peptide was also found in eEF2 prepared from dph5, dph6 and dph7 deletion strains as well as from wild-type cells (Figures S1 and S2). Thus even in wild-type cells not all of the eEF2 is modified by diphthamide. In addition to the unmodified peptide, we readily detected diphthamide-modified peptide in eEF2 prepared from the wild-type strain (Figure 4A), but failed to detect this in any of the mutants. Instead, ACP-modified peptide was found in eEF2 prepared from the dph5 mutant (Figure 4B), as expected given its known role in generating diphthine [32] from the ACP intermediate in the pathway.


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)

MS/MS spectra of diphthamide-, ACP-, and diphthine-modified EF2 peptide 686-VNILDVTLHADAIHR-700 from wild-type and mutant yeast strains.Spectra are shown for (A) diphthamide-modified peptide from the wild-type yeast strain; (B) ACP-modified peptide from the dph5Δ mutant; (C) diphthine-modified peptide in the dph7Δ strain; (D) diphthine-modified peptide in the dph6Δ strain; (E) diphthine-modified peptide in the dph6Δ strain with loss of the trimethylamino group before analysis in the mass spectrometer indicated by the parent ion m/z. In each case the parent ion m/z and charge state is indicated. In (A), (C) and (D), * indicates neutral loss of trimethylamino during MS/MS. The inset in (C) shows greater detail for the more crowded part of the MS/MS spectrum. Figure S2A indicates how the B and Y ions are derived from the peptide sequence.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585130&req=5

pgen-1003334-g004: MS/MS spectra of diphthamide-, ACP-, and diphthine-modified EF2 peptide 686-VNILDVTLHADAIHR-700 from wild-type and mutant yeast strains.Spectra are shown for (A) diphthamide-modified peptide from the wild-type yeast strain; (B) ACP-modified peptide from the dph5Δ mutant; (C) diphthine-modified peptide in the dph7Δ strain; (D) diphthine-modified peptide in the dph6Δ strain; (E) diphthine-modified peptide in the dph6Δ strain with loss of the trimethylamino group before analysis in the mass spectrometer indicated by the parent ion m/z. In each case the parent ion m/z and charge state is indicated. In (A), (C) and (D), * indicates neutral loss of trimethylamino during MS/MS. The inset in (C) shows greater detail for the more crowded part of the MS/MS spectrum. Figure S2A indicates how the B and Y ions are derived from the peptide sequence.
Mentions: The modified histidine in eEF2 (His699) is located in the tryptic peptide 686-VNILDVTLHADAIHR-700 and, as expected, unmodified versions of this peptide were readily detected in eEF2 prepared from the dph1 mutant (Figure S1C). Unmodified peptide was also found in eEF2 prepared from dph5, dph6 and dph7 deletion strains as well as from wild-type cells (Figures S1 and S2). Thus even in wild-type cells not all of the eEF2 is modified by diphthamide. In addition to the unmodified peptide, we readily detected diphthamide-modified peptide in eEF2 prepared from the wild-type strain (Figure 4A), but failed to detect this in any of the mutants. Instead, ACP-modified peptide was found in eEF2 prepared from the dph5 mutant (Figure 4B), as expected given its known role in generating diphthine [32] from the ACP intermediate in the pathway.

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