Yeast gene KTI13 (alias DPH8) operates in the initiation step of diphthamide synthesis on elongation factor 2

Abstract

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four biosynthetic steps encoded by the DPH1-DPH7 network and an ill-defined KTI13 function. On further examining the latter gene in yeast, we found that kti13Δ null-mutants maintain unmodified EF2 able to escape ADP-ribosylation by DT and to survive EF2 inhibition by sordarin, a diphthamide-dependent antifungal. Consistently, mass spectrometry shows kti13Δ cells are blocked in proper formation of amino-carboxyl-propyl-EF2, the first diphthamide pathway intermediate. Thus, apart from their common function in tRNA modification, both Kti11/Dph3 and Kti13 share roles in the initiation step of EF2 modification. We suggest an alias KTI13/DPH8 nomenclature indicating dual-functionality analogous to KTI11/DPH3.

Keywords: EF2 diphthamide modification; budding yeast; diphtheria toxin; elongator; tRNA modification; tRNase zymocin.

Figure 1. FIGURE 1: Potential role of yeast KTI13 in diphthamide modification.

(A) Simplified pathway overview [24,25]. Diphthamide synthesis initiates with modifcation of EF2 at His-699 by ACP involving proteins Dph1-Dph4. Subsequential reactions to convert ACP into end product diphthamide entail Dph5-Dph7. Potential Kti13 involvement in the synthesis steps is indicated (‘?’). Diphthamide can be hijacked by diphtheria toxin (DT) for ADP-ribosylation in an NAD⁺ fashion and induces cell death by EF2 inactivation (skull-crossbones). (B) KTI13 and DPH1 gene deletion strains resist against DT cytotoxicity. Yeast strains carrying pGAL-DT [39], a plasmid for galactose-inducible expression of the lethal ADP-ribosylase domain from DT (see A) were spotted onto medium containing 0.5-1% (w/v) galactose (gal) or 2% (w/v) glucose (glc). Following DT induction, growth inhibition of diphthamide-proficient wild-type is distinguishable from DT resistance of diphthamide-deficient dph1Δ and kti13Δ mutants (green arrows).

Figure 2. FIGURE 2: Among genes involved in Elongator regulation and tRNA modification, KTI11 and KTI13 also function in EF2 modification.

(A) Growth assays in response to zymocin (0.02% [v/v]) or sordarin (9 µg/mL) and diagnostic for tRNA or diphthamide modifciation defects, respectively. Dilutions of cells with indicated genotypes were incubated at 30°C for 3 days. Note, that while all ktiΔ and sit4Δ mutants resist growth inhibition by Elongator-dependent tRNase zymocin, only kti11/dph3Δ and kti13Δ cells are protected (green arrows) against diphthamide-dependent EF inhibitor sordarin. (B) Western blot analysis of total cell extracts from strains with genotypes as in A in order to profile their amounts of total EF2 and unmodified EF2 using anti-EF2(pan) (left panel) and anti-EF2(no diphthamide) antibodies (right panel), respectively. Black asterisks (left & right panels) denote EF2 degradation products, the red asterisk indicates full-length unmodified EF2 (right panel). The anti-Cdc19 antibody (bottom panel) was used as loading control. Note the anti-EF2(no diphthamide) Western blot (right panel) detects unmodified EF2 pools for kti11/dph3Δ and kti13Δ cells indicative for diphthamide defects.

Figure 3. 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.

Figure 4. FIGURE 4: Kti11•Kti13 dimer (alias Dph3•Dph8), dual modification regulator.

The dimer is located upstream of two radical SAM (RS) enzyme complexes (Dph1•Dph2; Elongator: Elp1•Elp6). Its dual regulator roles ensure proper synthesis of diphthamide on EF2 and modification of tRNA anticodon wobble uridine (U34) bases in order to support accurate mRNA translation and de novo protein synthesis [27,50].