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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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Co-immune precipitations reveal eEF2 interactions with Dph6 and Dph5.(A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His)6-tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7; B: dph1, dph6 and dph7) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His)6-tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His)6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His)6-marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His)6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).
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pgen-1003334-g005: Co-immune precipitations reveal eEF2 interactions with Dph6 and Dph5.(A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His)6-tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7; B: dph1, dph6 and dph7) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His)6-tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His)6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His)6-marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His)6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).

Mentions: Although Dph6 and Dph7 appear to function within the same step of the diphthamide synthesis pathway, using co-immune precipitation they were not found to interact either with one another or with Dph2 and Dph5, players involved in the two earlier pathway steps (Figure S4; Figure S5 and data not shown). However, in support of our evidence that Dph6 is a diphthamide biosynthetic factor, we observed by co-immune precipitation that Dph6-HA bound to a fraction of (His)6-tagged eEF2 (Figure 5A). Intriguingly, this interaction was independent of Dph7 (Figure 5A), suggesting Dph7 may not mediate interaction between Dph6 and the translation factor. Dph7 is also unlikely to play an indirect role through regulation of DPH6 gene expression because Dph6 protein levels were unaltered in the DPH7 deletion strain (Figure 5A).


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)

Co-immune precipitations reveal eEF2 interactions with Dph6 and Dph5.(A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His)6-tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7; B: dph1, dph6 and dph7) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His)6-tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His)6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His)6-marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His)6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).
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pgen-1003334-g005: Co-immune precipitations reveal eEF2 interactions with Dph6 and Dph5.(A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His)6-tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7; B: dph1, dph6 and dph7) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His)6-tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His)6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His)6-marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His)6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).
Mentions: Although Dph6 and Dph7 appear to function within the same step of the diphthamide synthesis pathway, using co-immune precipitation they were not found to interact either with one another or with Dph2 and Dph5, players involved in the two earlier pathway steps (Figure S4; Figure S5 and data not shown). However, in support of our evidence that Dph6 is a diphthamide biosynthetic factor, we observed by co-immune precipitation that Dph6-HA bound to a fraction of (His)6-tagged eEF2 (Figure 5A). Intriguingly, this interaction was independent of Dph7 (Figure 5A), suggesting Dph7 may not mediate interaction between Dph6 and the translation factor. Dph7 is also unlikely to play an indirect role through regulation of DPH6 gene expression because Dph6 protein levels were unaltered in the DPH7 deletion strain (Figure 5A).

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