Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes

<jats:title>ABSTRACT</jats:title> <jats:p> Threonylcarbamoyladenosine (t <jats:sup>6</jats:sup> A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t <jats:sup>6</jats:sup> A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent <jats:italic>in vitro</jats:italic> work suggested that the mitochondrial t <jats:sup>6</jats:sup> A machinery of <jats:named-content content-type="genus-species">Saccharomyces cerevisiae</jats:named-content> is composed of only two proteins, Sua5p and Qri7p, a member of the Kae1p/TsaD family (L. C. K. Wan et al., Nucleic Acids Res. 41:6332–6346, 2013, <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://dx.doi.org/10.1093/nar/gkt322">http://dx.doi.org/10.1093/nar/gkt322</jats:ext-link> ). 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 t <jats:sup>6</jats:sup> A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted to both the cytoplasm and the mitochondria through the use of alternative start sites. The 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. This minimal t <jats:sup>6</jats:sup> A pathway was characterized <jats:italic>in vitro</jats:italic> and, for the first time, <jats:italic>in vivo</jats:italic> by heterologous complementation studies in <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content> . The data revealed a potential for TC-AMP channeling in the t <jats:sup>6</jats:sup> A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the <jats:named-content content-type="genus-species">E. coli</jats:named-content> <jats:italic>tsaD</jats:italic> mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t <jats:sup>6</jats:sup> A and bring additional advancement in our understanding of the reaction mechanism. </jats:p>