Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis

Abstract

Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1. Ribosomal −1 frameshifting-error rates are increased in Arabidopsis dph1 mutants, similar to yeast and mice. Compared to the wild type, shorter roots and smaller rosettes of dph1 mutants result from fewer formed cells. TARGET OF RAPAMYCIN (TOR) kinase activity is attenuated, and autophagy is activated, in dph1 mutants. Under abiotic stress diphthamide-unmodified eEF2 accumulates in wild-type seedlings, most strongly upon heavy metal excess, which is conserved in human cells. In summary, our results suggest that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth.

DPH1 and DPH2 variants that confer susceptibility to diphthamide deficiency syndrome in human cells and yeast models

“…Diphthamide deficiency affects development and manifests with distinct clinical phenotypes in humans, and developmental issues in mice and even plants ( Chen and Behringer, 2004 ; Liu et al, 2006 , 2012 ; Shankar et al, 2022 ; Zhang et al, 2022 ). Compromised alleles affect diphthamide synthesis on eEF2 and thus can interfere with translational fidelity and protein homeostasis ( Dever et al, 2018 ; Liu et al, 2012 ; Shin et al, 2023 ).…”

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines

“…Both antibodies were originally raised to detect human eEF2. As the diphthamide acceptor contexts in eEF2 from human and yeast cells are identical, anti-EF2(no diphthamide) can also differentiate the modification states of eEF2 in S. cerevisiae [32] and in plants, as recently shown in [33]. Unmodified eEF2 signals relative to dph1∆ or dph2∆ were quantified using densitometry with ImageJ version 1.50i.…”

“…Next, we analyzed the Cys-to-Ser variants using Western blots with anti-eEF2(no diphthamide) antibodies that specifically recognize unmodified eEF2 [31][32][33] (Figure 3A). Thus, in support of our phenotypic assays above (Figure 2B), such immune blots can provide further insights into the relevance of each cysteine replaced in our Dph1 or Dph2 substitution variants and confirm that modified eEF2 samples from WT cells with active Dph1•Dph2 enzymes will not respond towards this diagnostic antibody [31][32][33]. While our WT control contained next to no unmodified eEF2, the dph1∆ and dph2∆ deletion strains accumulated substantial pools of eEF2 not modified by diphthamide (Figure 3A).…”

What, where, and how: Regulation of translation and the translational landscape in plants

“…During translation elongation, eukaryotic Elongation Factor 2 (eEF2) regulates the transfer of peptidyl tRNA from the ribosomal P-site to the A-site. A post-translational modification on a histidine residue of eEF2, termed diphthamide (a target of diphtheria toxin in humans), is conserved in Arabidopsis ( Zhang et al 2022 ). Arabidopsis mutants of Dph1, which catalyzes the first step of diphthamide biosynthesis, lose the modification on the H700 residue and increase −1 frameshift errors, as shown by a reporter assay.…”