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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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Genome-wide gene interaction databases identify additional diphthamide related candidate genes: YLR143w/DPH6 and YBR246w/DPH7.(A) SGA database (DRYGIN). Genetic interaction profiles among DPH1, DPH2, DPH4, DPH5, YBR246w and YLR143w query gene deletion strains and 3885 or 4457 array ORF mutants were extracted from data for a total of ∼1700 query strains deposited at DRYGIN (for full details, see excel spread sheet in Table S1). Ranking of top interactors for each query ORF was according to PCC (Pearson correlation coefficient) determination. For simplicity, array ORFs DPH1, DPH2, DPH4, DPH5, EFT1, EFT2 (shown in bold) as well as potentially diphthamide related candidate loci YLR143w and YBR246w (red circles) are listed that score repeatedly as significantly high interactors of the query ORFs. (B) Yeast Fitness database (FitDB). Genes whose deletions phenocluster with the six query ORFs above were extracted from FitDB, which is based on genome-scale co-fitness defect analysis of homozygous yeast deletion mutants in response to greater than 1144 different conditions. For simplicity, the top ten interactors for three of the six query genes (DPH5, YLR143w and YBR246w: pale blue central nodes) above are depicted. (C) Representation of the tightly clustered and expanded DPH1-DPH7 gene network where nodes (pale blue) correspond to individual DPH gene family members and edges connect gene pairs by PCC>0.14. Enhanced gene interaction strength is proportional to PCC stringency. Enriched GO process likelihoods in the diphthamide modification pathway are listed as P-values for the identified candidates DPH6/YLR143w and DPH7/YBR246w.
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pgen-1003334-g002: Genome-wide gene interaction databases identify additional diphthamide related candidate genes: YLR143w/DPH6 and YBR246w/DPH7.(A) SGA database (DRYGIN). Genetic interaction profiles among DPH1, DPH2, DPH4, DPH5, YBR246w and YLR143w query gene deletion strains and 3885 or 4457 array ORF mutants were extracted from data for a total of ∼1700 query strains deposited at DRYGIN (for full details, see excel spread sheet in Table S1). Ranking of top interactors for each query ORF was according to PCC (Pearson correlation coefficient) determination. For simplicity, array ORFs DPH1, DPH2, DPH4, DPH5, EFT1, EFT2 (shown in bold) as well as potentially diphthamide related candidate loci YLR143w and YBR246w (red circles) are listed that score repeatedly as significantly high interactors of the query ORFs. (B) Yeast Fitness database (FitDB). Genes whose deletions phenocluster with the six query ORFs above were extracted from FitDB, which is based on genome-scale co-fitness defect analysis of homozygous yeast deletion mutants in response to greater than 1144 different conditions. For simplicity, the top ten interactors for three of the six query genes (DPH5, YLR143w and YBR246w: pale blue central nodes) above are depicted. (C) Representation of the tightly clustered and expanded DPH1-DPH7 gene network where nodes (pale blue) correspond to individual DPH gene family members and edges connect gene pairs by PCC>0.14. Enhanced gene interaction strength is proportional to PCC stringency. Enriched GO process likelihoods in the diphthamide modification pathway are listed as P-values for the identified candidates DPH6/YLR143w and DPH7/YBR246w.

Mentions: Since our preliminary examination of the yeast genetic interaction landscape placed two uncharacterized yeast ORFs, YLR143w and YBR246w, within the diphthamide gene network [13], we next examined this network in more detail by mining the SGA DRYGIN database for quantitative S. cerevisiae genetic interactions [44], [50]. We compared DPH1, DPH2, DPH4, DPH5, YLR143w and YBR246w gene interactions with every array ORF represented in the SGA network and deposited at DRYGIN, ranking the similarity between all possible pairwise profiles according to their Pearson correlation coefficient (PCC; see Table S1 for full details). As expected, the other known DPH genes scored significantly highly among the correlation profiles generated for each specific DPH query gene, consistently being ranked among the top ten genetic interactors (Figure 2A). Strikingly, YLR143w and YBR246w were among the top interactors of DPH1, DPH2, DPH4 and DPH5, while the most correlated interactors for YLR143w and YBR246w included each other and several bone fide DPH genes (Figure 2A). Such highly correlated interaction patterns suggest that YLR143w and YBR246w are both functionally interrelated and qualify as candidate ORFs of the pathway for eEF2 modification by diphthamide. In line with this notion, the two eEF2 encoding gene copies, EFT1 and EFT2, also ranked among the top ten interactors of DPH1, DPH2 and DPH5 (Figure 2A).


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)

Genome-wide gene interaction databases identify additional diphthamide related candidate genes: YLR143w/DPH6 and YBR246w/DPH7.(A) SGA database (DRYGIN). Genetic interaction profiles among DPH1, DPH2, DPH4, DPH5, YBR246w and YLR143w query gene deletion strains and 3885 or 4457 array ORF mutants were extracted from data for a total of ∼1700 query strains deposited at DRYGIN (for full details, see excel spread sheet in Table S1). Ranking of top interactors for each query ORF was according to PCC (Pearson correlation coefficient) determination. For simplicity, array ORFs DPH1, DPH2, DPH4, DPH5, EFT1, EFT2 (shown in bold) as well as potentially diphthamide related candidate loci YLR143w and YBR246w (red circles) are listed that score repeatedly as significantly high interactors of the query ORFs. (B) Yeast Fitness database (FitDB). Genes whose deletions phenocluster with the six query ORFs above were extracted from FitDB, which is based on genome-scale co-fitness defect analysis of homozygous yeast deletion mutants in response to greater than 1144 different conditions. For simplicity, the top ten interactors for three of the six query genes (DPH5, YLR143w and YBR246w: pale blue central nodes) above are depicted. (C) Representation of the tightly clustered and expanded DPH1-DPH7 gene network where nodes (pale blue) correspond to individual DPH gene family members and edges connect gene pairs by PCC>0.14. Enhanced gene interaction strength is proportional to PCC stringency. Enriched GO process likelihoods in the diphthamide modification pathway are listed as P-values for the identified candidates DPH6/YLR143w and DPH7/YBR246w.
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pgen-1003334-g002: Genome-wide gene interaction databases identify additional diphthamide related candidate genes: YLR143w/DPH6 and YBR246w/DPH7.(A) SGA database (DRYGIN). Genetic interaction profiles among DPH1, DPH2, DPH4, DPH5, YBR246w and YLR143w query gene deletion strains and 3885 or 4457 array ORF mutants were extracted from data for a total of ∼1700 query strains deposited at DRYGIN (for full details, see excel spread sheet in Table S1). Ranking of top interactors for each query ORF was according to PCC (Pearson correlation coefficient) determination. For simplicity, array ORFs DPH1, DPH2, DPH4, DPH5, EFT1, EFT2 (shown in bold) as well as potentially diphthamide related candidate loci YLR143w and YBR246w (red circles) are listed that score repeatedly as significantly high interactors of the query ORFs. (B) Yeast Fitness database (FitDB). Genes whose deletions phenocluster with the six query ORFs above were extracted from FitDB, which is based on genome-scale co-fitness defect analysis of homozygous yeast deletion mutants in response to greater than 1144 different conditions. For simplicity, the top ten interactors for three of the six query genes (DPH5, YLR143w and YBR246w: pale blue central nodes) above are depicted. (C) Representation of the tightly clustered and expanded DPH1-DPH7 gene network where nodes (pale blue) correspond to individual DPH gene family members and edges connect gene pairs by PCC>0.14. Enhanced gene interaction strength is proportional to PCC stringency. Enriched GO process likelihoods in the diphthamide modification pathway are listed as P-values for the identified candidates DPH6/YLR143w and DPH7/YBR246w.
Mentions: Since our preliminary examination of the yeast genetic interaction landscape placed two uncharacterized yeast ORFs, YLR143w and YBR246w, within the diphthamide gene network [13], we next examined this network in more detail by mining the SGA DRYGIN database for quantitative S. cerevisiae genetic interactions [44], [50]. We compared DPH1, DPH2, DPH4, DPH5, YLR143w and YBR246w gene interactions with every array ORF represented in the SGA network and deposited at DRYGIN, ranking the similarity between all possible pairwise profiles according to their Pearson correlation coefficient (PCC; see Table S1 for full details). As expected, the other known DPH genes scored significantly highly among the correlation profiles generated for each specific DPH query gene, consistently being ranked among the top ten genetic interactors (Figure 2A). Strikingly, YLR143w and YBR246w were among the top interactors of DPH1, DPH2, DPH4 and DPH5, while the most correlated interactors for YLR143w and YBR246w included each other and several bone fide DPH genes (Figure 2A). Such highly correlated interaction patterns suggest that YLR143w and YBR246w are both functionally interrelated and qualify as candidate ORFs of the pathway for eEF2 modification by diphthamide. In line with this notion, the two eEF2 encoding gene copies, EFT1 and EFT2, also ranked among the top ten interactors of DPH1, DPH2 and DPH5 (Figure 2A).

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