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. 2018 Oct 18;13(10):e0205870.
doi: 10.1371/journal.pone.0205870. eCollection 2018.

Importance of Diphthamide Modified EF2 for Translational Accuracy and Competitive Cell Growth in Yeast

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Free PMC article

Importance of Diphthamide Modified EF2 for Translational Accuracy and Competitive Cell Growth in Yeast

Harmen Hawer et al. PLoS One. .
Free PMC article

Abstract

In eukaryotes, the modification of an invariant histidine (His-699 in yeast) residue in translation elongation factor 2 (EF2) with diphthamide involves a conserved pathway encoded by the DPH1-DPH7 gene network. Diphthamide is the target for diphtheria toxin and related lethal ADP ribosylases, which collectively kill cells by inactivating the essential translocase function of EF2 during mRNA translation and protein biosynthesis. Although this notion emphasizes the pathological importance of diphthamide, precisely why cells including our own require EF2 to carry it, is unclear. Mining the synthetic genetic array (SGA) landscape from the budding yeast Saccharomyces cerevisiae has revealed negative interactions between EF2 (EFT1-EFT2) and diphthamide (DPH1-DPH7) gene deletions. In line with these correlations, we confirm in here that loss of diphthamide modification (dphΔ) on EF2 combined with EF2 undersupply (eft2Δ) causes synthetic growth phenotypes in the composite mutant (dphΔ eft2Δ). These reflect negative interference with cell performance under standard as well as thermal and/or chemical stress conditions, cell growth rates and doubling times, competitive fitness, cell viability in the presence of TOR inhibitors (rapamycin, caffeine) and translation indicator drugs (hygromycin, anisomycin). Together with significantly suppressed tolerance towards EF2 inhibition by cytotoxic DPH5 overexpression and increased ribosomal -1 frame-shift errors in mutants lacking modifiable pools of EF2 (dphΔ, dphΔ eft2Δ), our data indicate that diphthamide is important for the fidelity of the EF2 translocation function during mRNA translation.

Conflict of interest statement

KM and UB are employed by and members of Roche Pharma Research & Early Development. Roche is interested in targeted therapies and diagnostics. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors (HH, KÜ, MA, LA and RS) have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Diphthamide synthesis on yeast EF2 involves a multi-step pathway.
In step one (1, black label), Dph1-Dph4 use SAM as amino-carboxyl-propyl (ACP) donor to modify His-699 in EF2 with ACP. Next (2, blue label), Dph5 generates methyl-diphthine using the methyl donor function of SAM. Subsequently (3, green label), this intermediate is converted to diphthine by demethylase Dph7 before finally (4, red label), Dph6 generates diphthamide from diphthine using ammonium and ATP. Diphthamide can be ADP-ribosylated by diphtheria toxin (DT) to inactivate EF2 and cause cell death. Note that prior to ACP formation, Dph5 binds to unmodified EF2 and dissociates from intermediate methyl-diphthine in a fashion licensed by Dph7 [19,50]. The model is derived and up-dated from Schaffrath et al. (2014) [11].
Fig 2
Fig 2. SGA analysis reveals strong correlation between EF2 and diphthamide gene networks.
(A) Based on Pearson correlation coefficients (PCC) of indicated stringencies, each diphthamide query gene (DPH1-DPH7) correlates to other DPH gene members and to a lesser yet significant degree to EF2 (EFT2) in a tightly clustered interaction network. (B) SGA based coefficients of query gene EFT2 identify strongest negative relations with EFT1 and weaker albeit significant ones with diphthamide network genes (DPH1-DPH7). All SGA data were retrieved from TheCellMap.org database [43] (S1 File).
Fig 3
Fig 3. Loss of diphthamide modification and EF2 downregulation lead to synthetic growth phenotypes.
(A) MS based quantification of EF2 levels from yeast mutants (dph2Δ, eft2Δ, dph2Δ eft2Δ) in relation to wild-type (wt) EF2 values (1.0). ** (P ≤ 0.01); **** (P ≤ 0.0001). (B) Western blots using antibodies for global EF2 recognition (anti-EF2(pan), top panel), specific detection of EF2 pools that lack the diphthamide modification (anti-EF2(no diphthamide), middle panel) and for internal protein expression controls (anti-β-Actin and anti-Cdc19, bottom panels). Protein extracts from homozygous (dph2ΔΔ) diphthamide-minus MCF7 [33] cell lines served as internal controls (all panels). (C) Synthetic sick growth phenotypes result from combining EF2 downregulation with loss of diphthamide. Ten-fold serial cell dilutions of yeast strains (as in A & B) were cultivated in the absence (untreated) or presence of various chemical stressors (SDS [0.2% w/v], rapamycin [15 nM] or caffeine [7.5 mM]) or at different temperatures (30°C, 37°C, 39°C or 40°C).
Fig 4
Fig 4. Competitive fitness studies in response to diphthamide defects, EF2 undersupply or both.
(A) Experimental setup. Equal amounts of wild-type (wt) and mutant (xxxΔ) cells were mixed and grown for 24 h (1 d) and successively passaged three more times for a total period of 96 h (4 d). After each passage, the relative amount (% of cells) of the two cell-types was determined. Growth experiments involving competition between wt and dph2Δ (B), eft2Δ (C) or dph2Δ eft2Δ (D) cells are shown.
Fig 5
Fig 5. Analysis of cell doubling times in diphthamide synthesis (DPH1-DPH7) and EFT2 gene deletion strains.
Doubling times of the indicated strain backgrounds were determined from biological quadruplicates. Statistical significance was determined by two-tailed t-test. * (P ≤ 0.05).
Fig 6
Fig 6. DPH5 overexpression toxicity.
(A) Model depicting that higher-than-normal Dph5 levels interact with EF2 when diphthamide synthesis is absent (dph2Δ) and can become EF2 inhibitory (red bolt) and cytotoxic upon DPH5 overexpression [13,19,24,58]. (B) Expression analysis between Dph5-HA produced from the wild-type genomic locus (DPH5-HA) and a GAL-promoter plasmid (pGAL-DPH5-HA) (in a dph5Δ strain). Protein extracts obtained after growth on glucose (glc) or galactose (gal) medium were analyzed by anti-HA (top panel) or anti-Pfk1 (bottom panel) Western blots to detect Dph5-HA or phosphofructokinase expression levels. Dph5-HA produced from pGAL-DPH5-HA carries additional tags [59] that confer a mobility-shift (*). (C) DPH5 overexpression is toxic for growth of dph2Δ and dph2Δ eft2Δ mutants. The indicated strains were cultivated for 3 days on glc (2% [w/v]) and gal (0.5, 1, 2% [w/v]) medium.
Fig 7
Fig 7. Growth response to translation indicator drugs and vulnerability to programmed -1 frame-shifting of mutants lacking diphthamide and/or proper EF2 supply (dph2Δ, eft2Δ and dph2Δ eft2Δ).
(A) Serial cell dilutions of the indicated yeast stains were cultivated on media without any drugs (untreated) or supplemented with various doses of anisomycin (10, 20 μg/ml) or hygromycin (40, 80 μg/ml) at 30°C for 2–3 days. (B) Programmed -1 frame-shift assays based on a dual luciferase reporter system (scheme) carrying a viral -1 frame-shift site: 5’-GGGTTTA-3’ [62]. Luminescence based read-outs were statistically analyzed using two-tailed t-test. * (P ≤ 0.05); n.s. (P > 0.05).

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Grant support

This study was supported by European regional development funds (EFRE—Europe Funds Saxony) for the Centre for Chemical Microscopy (ProVIS) to Lorenz Adrian; University of Kassel research unit PhosMOrg (P/1082 232) to Raffael Schaffrath; DFG Priority Program 1927 Iron-Sulfur for Life: Cooperative Function of Iron-Sulfur Centers in Assembly, Biosynthesis, Catalysis and Disease to Lorenz Adrian (AD178/7-1) and to Raffael Schaffrath (SCHA750/21-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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