FIGURE 3: KTI13 is required for proper initiation of diphthamide synthesis on EF2. (A) kti13Δ and dph1Δ mutants in strain TKY675 resist against DT cytotoxicity. The assay was essentially performed as for BY4741 (Fig. 1B). Following galactose-inducible DT expression, wild-type growth inhibition is distinct from DT resistance (green arrows) of diphthamide-deficient mutants (kti13Δ, dph1Δ). (B) Profiling diphthamide modification states on EF2 purified from wild-type, dph1Δ, dph5Δ and kti13Δ cells via nLC-MS/MS. Amounts of modification states were normalized to amounts of unmodified EF2 in dph1Δ (EF2 peptide [%]). kti13Δ contains pools of unmodified EF2 comparable to dph1Δ and drastically reduced ACP levels (∼9%) in relation to dph5Δ (∼65%). (C) ADP-ribosylation (ADPR) assay. Cell extracts from indicated genotypes were incubated with 200 ng DT and biotin-NAD [5 µM] at 25 °C for 1 h. The transfer to EF2 of biotin-ADP-ribose (EF2-ADPR) was detected by Western blot (top panel) using an HRP-streptavidin conjugate recognizing the biotin moiety of the reaction product [26][42]. An anti-Cdc19 Western blot (bottom panel) served as control for sample loading. Note that solely diphthamide-modified EF2 from wild-type cells undergoes detectable ADPR. As has been previously detected in similar assays [29][39], there is an unspecific (n.s.) reaction product of high molecular weight.