Due to a complete lack of the tRNA genes in the mitochondrial genome of Trypanosoma brucei, all tRNAs needed for mitochondrial translation have to be imported into the organelle from the cytosol. A previous study showed that the modified nucleotide s(2)U could act as a negative determinant for mitochondrial tRNA import in another kinetoplastid, Leishmania tarentolae. We have investigated whether the same type of cytosolic control for tRNA retention exists in T. brucei. Based on Northern analysis with subcellular RNA fractions and in vitro import assays, we demonstrate that silencing of the cysteine desulfurase, TbNfs (TbIscS), the key enzyme in tRNA thiolation (s(2)U) and Fe-S cluster formation in vivo, has no effect on tRNA partitioning. This observation is especially surprising in light of a recent report suggesting that in L. tropica the Rieske Fe-S protein is an essential component of the RNA import complex (RIC). In line with the above observation, we also show that down-regulation of the Rieske protein by RNA interference, similar to the TbNfs knockdowns, has no effect on import. The data presented here supports the view that in T. brucei: (1) s(2)U is not a negative determinant for tRNA import; (2) the Rieske protein is not an essential component of the import machinery, and (3) since the Rieske protein is essential for respiration and maintenance of inner mitochondrial membrane potential, neither process plays a critical role in tRNA import. We therefore suggest that the T. brucei import machinery differs substantially from what has been described in Leishmania.

Biology Centre Institute of Parasitology Czech Academy of Sciences 37005 Ceské Budejovice Czech Republic

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Bhattacharyya SN, Adhya S. tRNA-triggered ATP hydrolysis and generation of membrane potential by the Leishmania mitochondrial tRNA import complex. J Biol Chem. 2004;279:11259–11263. PubMed

Bouzaidi-Tiali N, Aeby E, Charriere F, Pusnik M, Schneider A. Elongation factor 1a mediates the specificity of mitochondrial tRNA import in T. brucei. EMBO J. 2007;26:4302–4312. PubMed PMC

Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159. PubMed

Crain PF, Alfonzo JD, Rozenski J, Kapushoc ST, McCloskey JA, Simpson L. Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency. RNA. 2002;8:752–761. PubMed PMC

Entelis N, Brandina I, Kamenski P, Krasheninnikov IA, Martin RP, Tarassov I. A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae. Genes & Dev. 2006;20:1609–1620. PubMed PMC

Esseiva AC, Naguleswaran A, Hemphill A, Schneider A. Mitochondrial tRNA import in Toxoplasma gondii. J Biol Chem. 2004;279:42363–42368. PubMed

Haanstra JR, van Tuijl A, Kessler P, Reijnders W, Michels PA, Westerhoff HV, Parsons M, Bakker BM. Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes. Proc Natl Acad Sci. 2008;105:17718–17723. PubMed PMC

Hancock K, LeBlanc AJ, Donze D, Hajduk SL. Identification of nuclear encoded precursor tRNAs within the mitochondrion of Trypanosoma brucei. J Biol Chem. 1992;267:23963–23971. PubMed

Horváth A, Horáková E, Dunajčíková P, Verner Z, Pravdová E, Šlapetová I, Cuninková L, Lukeš J. Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei. Mol Microbiol. 2005;58:116–130. PubMed

Ikeuchi Y, Shigi N, Kato J, Nishimura A, Suzuki T. Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol Cell. 2006;21:97–108. PubMed

Kambampati R, Lauhon CT. Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. J Biol Chem. 2000;275:10727–10730. PubMed

Kambampati R, Lauhon CT. MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli. Biochemistry. 2003;42:1109–1117. PubMed

Kaneko T, Suzuki T, Kapushoc ST, Rubio MA, Ghazvini J, Watanabe K, Simpson L. Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: Implication for tRNA sorting mechanism. EMBO J. 2003;22:657–667. PubMed PMC

Kapushoc ST, Alfonzo JD, Rubio MA, Simpson L. End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae. J Biol Chem. 2000;275:37907–37914. PubMed

Lill R, Mühlenhoff U. Maturation of iron–sulfur proteins in eukaryotes: Mechanisms, connected processes, and diseases. Annu Rev Biochem. 2008;77:669–700. PubMed

Lima BD, Simpson L. Sequence-dependent in vivo importation of tRNAs into the mitochondrion of Leishmania tarentolae. RNA. 1996;2:429–440. PubMed PMC

Mahapatra S, Ghosh T, Adhya S. Import of small RNAs into Leishmania mitochondria in vitro. Nucleic Acids Res. 1994;22:3381–3386. PubMed PMC

Mahapatra S, Ghosh S, Bera SK, Ghosh T, Das A, Adhya S. The D arm of tRNATyr is necessary and sufficient for import into Leishmania mitochondria in vitro. Nucleic Acids Res. 1998;26:2037–2041. PubMed PMC

Marechal-Drouard L, Weil JH, Guillemaut P. Import of several tRNAs from the cytoplasm into the mitochondria in bean Phaseolus vulgaris. Nucleic Acids Res. 1988;16:4777–4788. PubMed PMC

Martin RP, Schneller JM, Stahl AJ, Dirheimer G. Import of nuclear deoxyribonucleic acid coded lysine-accepting transfer ribonucleic acid (anticodon C-U-U) into yeast mitochondria. Biochemistry. 1979;18:4600–4605. PubMed

Mihara H, Kato S, Lacourciere GM, Stadtman TC, Kennedy RA, Kurihara T, Tokumoto U, Takahashi Y, Esaki N. The iscS gene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H. Proc Natl Acad Sci. 2002;99:6679–6683. PubMed PMC

Mottram JC, Bell SD, Nelson RG, Barry JD. tRNAs of Trypanosoma brucei. Unusual gene organization and mitochondrial importation. J Biol Chem. 1991;266:18313–18317. PubMed

Mukherjee S, Basu S, Home P, Dhar G, Adhya S. Necessary and sufficient factors for the import of transfer RNA into the kinetoplast mitochondrion. EMBO Rep. 2007;8:589–595. PubMed PMC

Nabholz CE, Horn EK, Schneider A. tRNAs and proteins are imported into mitochondria of Trypanosoma brucei by two distinct mechanisms. Mol Biol Cell. 1999;10:2547–2557. PubMed PMC

Nakai Y, Umeda N, Suzuki T, Nakai M, Hayashi H, Watanabe K, Kagamiyama H. Yeast Nfs1p is involved in thio-modification of both mitochondrial and cytoplasmic tRNAs. J Biol Chem. 2004;279:12363–12368. PubMed

Nilsson K, Lundgren HK, Hagervall TG, Bjork GR. The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium. J Bacteriol. 2002;184:6830–6835. PubMed PMC

Pusnik M, Charriere F, Maser P, Waller R, Dagley MJ, Lithgow T, Schneider A. The single mitochondrial porin of Trypanosoma brucei is the main metabolite transporter in the outer mitochondrial membrane. Mol Biol Evol. 2009;26:671–680. PubMed

Rinehart J, Krett B, Rubio MA, Alfonzo JD, Soll D. Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion. Genes & Dev. 2005;19:583–592. PubMed PMC

Rubio MA, Liu X, Yuzawa H, Alfonzo JD, Simpson L. Selective importation of RNA into isolated mitochondria from Leishmania tarentolae. RNA. 2000;6:988–1003. PubMed PMC

Rubio MA, Rinehart JJ, Krett B, Duvezin-Caubet S, Reichert AS, Soll D, Alfonzo JD. Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import. Proc Natl Acad Sci. 2008;105:9186–9191. PubMed PMC

Rusconi CP, Cech TR. The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena. Genes & Dev. 1996a;10:2870–2880. PubMed

Rusconi CP, Cech TR. Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila. EMBO J. 1996b;15:3286–3295. PubMed PMC

Salinas T, Duchene AM, Delage L, Nilsson S, Glaser E, Zaepfel M, Marechal-Drouard L. The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria. Proc Natl Acad Sci. 2006;103:18362–18367. PubMed PMC

Salinas T, Duchene AM, Marechal-Drouard L. Recent advances in tRNA mitochondrial import. Trends Biochem Sci. 2008;33:320–329. PubMed

Schmitz UK, Lonsdale DM. A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo. Plant Cell. 1989;1:783–791. PubMed PMC

Schneider A, Marechal-Drouard L. Mitochondrial tRNA import: Are there distinct mechanisms? Trends Cell Biol. 2000;10:509–513. PubMed

Simpson AM, Suyama Y, Dewes H, Campbell DA, Simpson L. Kinetoplastid mitochondria contain functional tRNAs which are encoded in nuclear DNA and also contain small minicircle and maxicircle transcripts of unknown function. Nucleic Acids Res. 1989;17:5427–5445. PubMed PMC

Smid O, Horakova E, Vilimova V, Hrdy I, Cammack R, Horvath A, Lukeš J, Tachezy J. Knock-downs of iron-sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma brucei. J Biol Chem. 2006;281:28679–28686. PubMed

Suyama Y. Two-dimensional polyacrylamide gel electrophoresis analysis of Tetrahymena mitochondrial tRNA. Curr Genet. 1986;10:411–420. PubMed

Suyama Y, Wong S, Campbell DA. Regulated tRNA import in Leishmania mitochondria. Biochim Biophys Acta. 1998;1396:138–142. PubMed

Tarassov IA, Martin RP. Mechanisms of tRNA import into yeast mitochondria: An overview. Biochimie. 1996;78:502–510. PubMed

Tarassov I, Entelis N, Martin RP. An intact protein translocating machinery is required for mitochondrial import of a yeast cytoplasmic tRNA. J Mol Biol. 1995a;245:315–323. PubMed

Tarassov I, Entelis N, Martin RP. Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases. EMBO J. 1995b;14:3461–3471. PubMed PMC

Yermovsky-Kammerer AE, Hajduk SL. In vitro import of a nuclearly encoded tRNA into the mitochondrion of Trypanosoma brucei. Mol Cell Biol. 1999;19:6253–6259. PubMed PMC

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Functions and cellular localization of cysteine desulfurase and selenocysteine lyase in Trypanosoma brucei