Fig. 2: Diphthamide modification of eEF2 and translational reading frame accuracy depend on DPH1 that localizes to the cytosol.

a Representative confocal laser scanning microscopic image of a root tip of an 8-day-old dph1-1 pDPH1:DPH1-GFP (line Y22-10) seedling stained with propidium iodide (PI, red). Scale bars, 50 μm. b Immunoblot probed with antibodies specific for detection of eEF2 lacking the diphthamide modification (unmodified), recognizing all forms of eEF2 (global), or serving as a loading control (anti-β-Actin for MCF-7, anti-plant ACTIN for A. thaliana), respectively. Wild-type (WT) and DPH1^−/− mutant human cell line MCF-7 served as controls (left). c MS/MS spectra of eEF2 peptide 684-GICFEVCDVVLHSDAIHR-701 from WT (top) and dph1-1 mutant (bottom), with diphthamide (_D) or without diphthamide (_N) modification on H700 respectively (_C: carbamidomethylation). The selected monoisotopic precursor m/z (z = +4) is given in each diagram; all detected y fragment ions are shown in blue. Red/pink lines visualize the consistent m/z difference of 71.055 between equivalent y_n²⁺ ions of the two genotypes. Tissues were shoots of 16-day-old 0.5× MS-grown seedlings (b, c). d Rates of ribosomal −1 frameshifting error. We normalized the ratio of firefly/renilla luciferase activity for a test −1 frameshift reporter construct to the ratio for an in-frame control reporter construct, as measured in transiently transfected Arabidopsis leaf mesophyll protoplasts prepared from 4-week-old soil-grown dph1 mutants and wild-type plants. Mean ± s.d., n = 3 independently transfected replicate aliquots of protoplasts, shown normalized to the wild-type (see Supplementary Fig. 4b-d). Significant differences from WT: **P < 0.01 (one-way ANOVA, Tukey’s test).