Use of a Yeast tRNase Killer Toxin to Diagnose Kti12 Motifs Required for tRNA Modification by Elongator.

Constance Mehlgarten,Sebastian Glatt,Wael Abdel-Fattah,Heike Prochaska,Sang Jun,Karin Breunig,Alexander Hammermeister,Melanie Wagner,Rościsław Krutyhołowa,Raffael Schaffrath,Gyung-Tae Kim,Michael Stark

doi:10.3390/toxins9090272

Abstract

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin’s action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g., nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them.

Highlights

A composite chitinase and tRNA anticodon nuclease ribotoxin produced from the dairy yeast Kluyveromyces lactis, kills other yeast species including Saccharomyces cerevisiae [1,2]
The zymocin tRNA anticodon nuclease (tRNase) ribotoxin can be exploited as a molecular diagnostic tool to assign function to motifs conserved in two Elongator partner proteins, Kti12 and plant ELO4
These motifs are potentially involved in cofactor binding and when mutated, abolish Elongator interaction and trigger phenotypes typical of tRNA modification defects in known

Summary

Introduction

A composite chitinase and tRNA anticodon nuclease (tRNase) ribotoxin produced from the dairy yeast Kluyveromyces lactis, kills other yeast species including Saccharomyces cerevisiae [1,2]. zymocin is a trimeric complex (αβγ), expression of its γ-subunit (i.e., γ-toxin tRNase) aloneToxins 2017, 9, 272; doi:10.3390/toxins9090272 www.mdpi.com/journal/toxinsToxins 2017, 9, 272 is lethal in S. cerevisiae, suggesting that the α/β-subunits facilitate zymocin docking and γ-toxin delivery [2,3]. A composite chitinase and tRNA anticodon nuclease (tRNase) ribotoxin produced from the dairy yeast Kluyveromyces lactis, kills other yeast species including Saccharomyces cerevisiae [1,2]. Class I genes operate in the synthesis of cell wall and membrane components (chitin, sphingolipids, H+ pump Pma1) required for zymocin binding and γ-toxin entry, and class II loci identified a toxin–target effector role for the Elongator complex (Elp1–Elp6) [2,6]. The latter had been co-purified with elongating RNA polymerase II, its name [7]. Loss of U34 modification in Elongator mutants protects efficiently against the toxin’s attack [6,15,16]

Methods

Results

Conclusion