Genetic Analysis of Protein and Trna Modifications Required for Growth Inhibition of Saccharomyces Cerevisiae by a Fungal Ribotoxin

Toxicity of the zymocin complex from Kluyveromyces lactis, which causes cell death of Saccharomyces cerevisiae, relies on a specific tRNA anticodon modification in sensitive yeast cells. Primarily tRNAGlu with a highly modified uridine base in the anticodon wobble position (U34) serves as recognition and cleavage site for zymocin, which functions as a tRNA endoribonuclease. Cleavage results in depletion of essential tRNAs, translational breakdown and ultimately cell death. KTI11, URM1 and SIT4 are genes which confer zymocin sensitivity. Interestingly, deletion of each of these genes protects against zymocin due to U34 modification defects. Here, using genetic analysis, data was generated to further our understanding about how KTI11, URM1 and SIT4 function in the process of tRNA modification and zymocin dependent tRNA-cleavage. This work shows that Kti11 associates with two different protein complexes involved in translationally relevant modification pathways. Kti11 partners with the multifunctional Elongator complex and this association is crucial for Elongator functions in tRNA modification, explaining why kti11 mutants phenocopy Elongator minus cells. Furthermore, evidence is provide that Kti11 is subunit of a trimeric complex, Dph1•Dph2•Kti11, which is required to form a unique diphthamide modification on eEF2. Moreover, based on the zymocin resistance of urm1Δ cells and since Urm1 harbours features of prokaryotic sulphur carriers, it was hypothesised that Urm1 may be involved in U34 thio-modification. Data are presented suggesting that Urm1-Uba4 function as a sulphur relay system responsible for thiolation of tRNAs. Finally, this study focussed on the investigation of protein phosphatase Sit4. It is shown that Sit4 and Rrd1 form a functional phosphatase complex supporting Sit4 functions in the Target Of Rapamycin (TOR) pathway. Finally, the data suggest that SIT4, URM1 and KTI11 dependent tRNA anticodon modification has a signalling function and is implicated in the Nitrogen Catabolite Repression (NCR) branch of the TOR pathway.