Arabidopsis thaliana mitochondrial EF-G1 functions in two different translation steps Received October 18, 2013; accepted November 4, 2013; published online November 21, 2013 Takuma Suematsu 1, *, Osamu Watanabe 1 , Kiyoshi Kita 1 , Shin-ichi Yokobori 2,y and Yoh-ichi Watanabe 1,‡ 1 Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, and 2 Department of Applied Life Sciences, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan *Present address: Takuma Suematsu, Department of Molecular and Cell Biology, Boston University, 72 East Concord Street, Boston, MA 02118, USA y Shin-ichi Yokobori, Department of Applied Life Sciences, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan. Tel: þ81-42-676-5035, Fax: þ81-42-676-7145, email: yokobori@ ls.toyaku.ac.jp ‡ Yoh-ichi Watanabe, Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel: þ81-3-5841-3528, Fax: þ81-3-5841-3444, email: ywatanab@m.u-tokyo.ac.jp Translation elongation factor G (EF-G) in bacteria catalyses the translocation of transfer RNA on ribo- somes in the elongation step as well as dissociation of post-termination state ribosomes into two subunits in the recycling step. In contrast, the dual functions of EF-G are exclusively divided into two different paralo- gues in human mitochondria, named EF-G1mt for translocation and EF-G2mt for ribosomal dissociation. Many of the two eukaryotic EF-G paralogues are phylogenetically associated with EF-G1mt and EF- G2mt groups. However, plant paralogues are associated with EF-G1mt and plastid EF-G, not with EF-G2mt. In this study, we phylogenetically and biochemically char- acterized Arabidopsis thaliana EF-G1mt (AtEF-G1mt) to clarify the factor responsible for the dissociation of ribosomes in plant mitochondria. We showed that eukaryotic EF-G1mts form one monophyletic group separated from bacterial EF-G and are classified into five sister groups. AtEF-G1mt is classified into a different group from its human counterpart. We also demonstrated that AtEF-G1mt catalyses both trans- location and ribosomal dissociation, unlike in humans. Meanwhile, AtEF-G1mt is resistant to fusidic acid, an inhibitor of bacterial EF-G. Here, we propose that the functional division is not necessarily conserved among mitochondriate eukaryotes and also that EF-G1mt in organisms lacking EF-G2mt functions in two steps, similar to conventional bacterial EF-G. Keywords: Arabidopsis thaliana/EF-G/mitochondria/ ribosome recycling factor/translation. Abbreviations: AtEF-G1mt, Arabidopsis thaliana EF-G1mt; AtRRFmt, A. thaliana RRFmt; AU, approximately unbiased; BI, Bayesian inference; BP, bootstrap proportion; cDNA, complementary DNA; DTT, dithiothreitol; EF-G, elongation factor G; FA, fusidic acid; GTP, guanosine 5 0 -triphosphate; IF-3, initiation factor 3; ML, maximum likelihood; mt, mitochondrial; PCR, polymerase chain reaction; Pi, inorganic phosphate; PP, posterior probability; RRF, ribosome recycling factor; RT-PCR, reverse tran- scription-polymerase chain reaction. Protein synthesis comprises initiation, elongation, ter- mination and recycling steps. Translation elongation factor G (EF-G) functions in two different phases. During the elongation cycle, EF-G/guanosine 5 0 -tri- phosphate (GTP) binds to a pre-translocation state ribosome with transfer RNAs (tRNAs) in the A- and P-sites and promotes translocation of the tRNAs to the P- and E-sites (1). In addition, during the recycling step of a post-termination state ribosome, EF-G/GTP cooperates with ribosome recycling factor (RRF) to dissociate the ribosome into two ribosomal subunits. Subsequently, translation initiation factor 3 (IF-3) binds to the 30S small subunit to prevent reassociation of these subunits, which are recycled back to a new round of initiation (26). Most bacterial EF-Gs are thought to be a single factor that has two distinct func- tions, although multiple copies of EF-G genes have been found in bacterial genomes (7). On the other hand, the dual functions are divided into two different EF-G paralogues in human mitochondria, which are named EF-G1mt and EF-G2mt (8). EF-G2mt func- tions exclusively in the recycling step with mitochon- drial RRF (RRFmt), whereas EF-G1mt functions in the elongation cycle. Recently, we demonstrated that the division of the dual functions into EF-G paralogues also occurs in the spirochaete Borrelia burgdorferi based on phylogenetic and biochemical analyses (9). The phylogenetic ana- lyses among bacterial EF-Gs further showed that two EF-G paralogues in the majority of Spirochaetes, Planctomycetes, Lentisphaera and some species of Deltaproteobacteria are associated with EF-G1mt and EF-G2mt groups (9, 10). Meanwhile, eukaryotic EF-G homologues are classified into three groups: EF- G1mt, EF-G2mt and plastid EF-G (9, 10). Based on these biochemical and phylogenetic data, it has been proposed that EF-G homologues in EF-G1mt and EF- G2mt groups are probably exclusive translocases and ribosome recycling factors, respectively (8, 9). Although many of the mitochondriate eukaryotic EF-G sequences are associated with the EF-G1mt and EF-G2mt groups, no EF-G paralogue in J. Biochem. 2014;155(2):107–114 doi:10.1093/jb/mvt105 ß The Authors 2013. 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