Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes
PC Thiaville, B El Yacoubi, L Perrochia, A Hecker, M Prigent, JJ Thiaville, …
Eukaryotic Cell, EC. 00147-14

Abstract

Threonylcarbamoyladenosine (t⁶A) is a universal modification located in the anticodon stem loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t⁶A synthesis pathway was elucidated and requires Sua5p, Kae1p and four other KEOPS complex proteins. Recent in vitrowork suggested that the mitochondrial t⁶A machinery of S. cerevisiae is composed of only two proteins, Sua5p and Qri7p, a member of Kae1p/TsaD family. Sua5p catalyzes the first step leading to the threonyl-carbamoyl-AMP intermediate (TC-AMP) while Qri7 transfers the threonyl-carbamoyl moiety from TC-AMP to tRNA to form t⁶A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted both to the cytoplasm and to the mitochondria through the use of alternative start sites. Import of Sua5p into the mitochondria is required for this organelle to be functional since the TC-AMP intermediate produced by Sua5p in the cytoplasm is not transported into the mitochondria in sufficient amounts. The conservation of this minimal t⁶A pathway was characterized in vitro and, for the first time, in vivoby heterologous complementation studies in E. coli. The data revealed a potential for TC-AMP channeling in the t⁶A pathway as co-expression of Qri7p and Sua5p is required to complement the essentiality of E. coli tsaD^–. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t⁶A and bring additional advancement in our understanding of the reaction mechanism.