Leishmania tarentolae contains distinct cytosolic and mitochondrial glutaminyl–tRNA synthetase activities

Abstract

The intracellular distribution of glutaminyltRNA synthetases and their role in mitochondrial tRNA import were evaluated in the ancient eukaryote Leishmania tarentolae. The following results were obtained: (i) Glutaminyl-tRNA synthetase was detected in leishmanial mitochondria. This was unexpected because it has been postulated that, in organelles, Gln-tRNA Gln is not formed by direct acylation of tRNA Gln but by enzymatic transamidation of misacylated Glu-tRNA Gln .(ii) Whereas the cytosolic extract is able to charge cytosolic and mitochondrial tRNAs Gln , the mitochondrial matrix extract does not aminoacylate the cytosol-specific tRNA Gln . This indicates that mitochondrial and cytosolic glutaminyl-tRNA synthetases are distinct. (iii) Seven of the 11 nucleotides that differ between the cytosolic and the mitochondrial tRNA Gln are sufficient to convert the cytosolspecific tRNA Gln into an optimal substrate for the mitochondrial enzyme. These nucleotides are arranged in three groups consisting of the nucleotides f lanking the anticodon stem, the 5 nucleotide of the anticodon, and four nucleotides within the acceptor stem. And (iv), it was shown that the identity elements for recognition by the mitochondrial glutaminyltRNA synthetase do not overlap with a previously identified sequence segment required for mitochondrial import of the tRNA Gln .

Glu-tRNA Gln amidotransferase: A novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation

“…Based on the limited sampling of a number of organisms, we predicted (4) that the transamidation route occurs only in Gram-positive eubacteria, organelles, and Archaea. While this is generally true, there are a number of exceptions: mitochondria of Leishmania tarentolae possess the direct acylation pathway (29), whereas Rhizobium shows Glu-AdT activity (30). In addition, genomics suggest the occurrence of the transamidation genes in M. catarrhalis and N. gonorrhoeae.…”

Eukaryotic-type elongator tRNA Met of Trypanosoma brucei becomes formylated after import into mitochondria

“…The unedited tRNA Trp (CCA) in the cytosol, however, as required reads the standard UGG tryptophan codon only (25). Finally, whereas organelles are known to be devoid of glutaminyl-tRNA synthetases, as their tRNAs are charged in a two-step process requiring a tRNA-dependent transamidase (9), such enzymes exist in trypanosomatid mitochondria (12). Thus, exploring the limits of adaptation of a bacterial-type translation system to eukaryotic components may help to reveal fundamental requirements of translation.…”

“…Preparation of RNA-free cytosolic and mitochondrial matrix fraction was done as described (12). Substrate tRNAs were prepared as follows.…”

Duplication and Quadruplication of Arabidopsis thaliana Cysteinyl- and Asparaginyl-tRNA Synthetase Genes of Organellar Origin

“…It is common to find two distinct aaRSs with the same amino acid specificity in eukaryotes, one mitochondrial and the other cytosolic (Kumazawa et al 1989;Nabholz et al 1997). In most of these cases, it is probable that one of the two genes encoding these proteins is derived from the original organellar gene.…”