title: Cross kingdom functional conservation of the core universally conserved threonylcarbamoyladenosine tRNA synthesis enzymes.
abstract: Threonylcarbamoyladenosine (t(6)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(6)A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t(6)A machinery of Saccharomyces cerevisiae 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, http://dx.doi.org/10.1093/nar/gkt322). 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(6)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(6)A pathway was characterized in vitro and, for the first time, in vivo by heterologous complementation studies in Escherichia coli. The data revealed a potential for TC-AMP channeling in the t(6)A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the E. coli tsaD mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t(6)A and bring additional advancement in our understanding of the reaction mechanism.
authors.affiliation: Genetics and Genomics Graduate Program, University of Florida, Gainesville, Florida, USA University of Florida Genetics Institute, Gainesville, Florida, USA Institut de Génétique et Microbiologie, Université of Paris-Sud, Orsay, France Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Thiaville
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA.
authors.forename: Basma
authors.initials: B
authors.lastname: El Yacoubi
authors.affiliation: Institut de Génétique et Microbiologie, Université of Paris-Sud, Orsay, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Perrochia
authors.affiliation: Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, Vandoeuvre, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Hecker
authors.affiliation: Institut de Génétique et Microbiologie, Université of Paris-Sud, Orsay, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Prigent
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Thiaville
authors.affiliation: Institut de Génétique et Microbiologie, Université of Paris-Sud, Orsay, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Forterre
authors.affiliation: Institut de Génétique et Microbiologie, Université of Paris-Sud, Orsay, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Namy
authors.affiliation: Institut de Génétique et Microbiologie, Université of Paris-Sud, Orsay, France.
authors.forename: Tamara
authors.initials: T
authors.lastname: Basta
authors.affiliation: University of Florida Genetics Institute, Gainesville, Florida, USA Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA vcrecy@ufl.edu.
authors.forename: Valérie
authors.initials: V
authors.lastname: de Crécy-Lagard
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000926:Anticodon / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008928:Mitochondria / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000926:Anticodon; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C544825:Qri7 protein, S cerevisiae; C541142:SUA5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C003268:N(6)-(N-threonylcarbonyl)adenosine; D012343:RNA, Transfer; D000241:Adenosine