The tRNA binding protein Kti12 and its role in the regulation of Elongator complex

[Summary] Kti12 is a conserved eukaryotic protein showing sequence homology to O-phosphoseryltRNA kinase (Pstk), which is required for the synthesis of selenocysteine tRNA. Saccharomyces cerevisiae Kti12 was originally discovered in a genetic screening for resistance against zymocin, a protein toxin produced by Kluyveromyces lactis. The cytotoxicity of zymocin lies in its anticodon nuclease activity, causing tRNA depletion and cell death in sensitive yeast cells. It recognizes and cleaves a particular wobble uridine (U34) modification in tRNAGlu. The biosynthesis of nearly all U34 modifications requires a pivotal substitution at the U34, which is achieved by the Elongator complex and its co-factors, including Kti12, Hrr25 and Sit4. Unlike the other co-factors, Kti12 shows a unique behavior as it affects Elongator activity when deleted or overexpressed, causing zymocin resistance. It was shown, that a variation of Kti12 levels affects the balanced phosphorylation state of the largest Elongator subunit Elp1 by influencing the function of both Hrr25 kinase and Sit4 phosphatase complex. Cells lacking functional KTI12 display a hypophosphorylated Elp1 isoform, as Hrr25 is unable to interact and phosphorylate Elp1. An opposing effect is observable when Kti12 is overexpressed. In this case, Elp1 shows a hyperphosphorylated form resembling a situation, in which Sit4 is unable to mediate Elp1 dephosphorylation, because it is either deleted or inhibited by overexpression of its regulatory subunit Sap155. However, it is not fully understood how elevated expression of Kti12 affects Elongator activity. In this work, the overexpression effect of Kti12 and Sap155 on Elongators tRNA modification function was compared, using zymocin sensitivity assays to monitor the activity state of the complex. It was investigated, which genetic requirements are necessary for these effects. Overexpressed Sap155 exhibited a toxin resistance effect similar to Δkti12 mutants. On the opposite, high-copy expression of Kti12 provided an intermediate protection against zymocin, which was absent in hypersensitive Δsap155 cells. Furthermore, protein interaction studies showed that overexpression of Kti12 results in an enhanced co-precipitation with Sit4, independent of Sap155. The high dosage of Kti12 simultaneously increased Hrr25 and decreased Sit4 interaction with Elp1. Interestingly, the Kti12 presence on Elp1 remained unaffected. These results suggest that the overexpression effect of Kti12 on Elongator activity is likely due to a direct misregulation of Hrr25 accompanied by a reduction in Elongator specific Sit4 function. Additionally, as Kti12 shares a high sequence similarity with Pstk, it could further be shown that Kti12 binds any tRNA predominantly through its C-terminal domain in a Pstklike fashion. Furthermore, a tRNA bound Kti12 form shows a reduced binding ability with Elp1, which is in line with the observation that only the Elongator-free Kti12 pool is UV crosslinkable to nucleic acids.