In many organisms, mitochondria import nuclear DNA-encoded small RNAs. In yeast Saccharomyces cerevisiae, one out of two cytoplasmic isoacceptor tRNAs Lys is partially addressed into the organelle. Mitochondrial targeting of this tRNA was shown to depend on interaction with the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. However, preMsk1p alone was unable to direct tRNA targeting, suggesting the existence of additional protein factor(s). Here, we identify the glycolytic enzyme, enolase, as such a factor. We demonstrate that recombinant enolase and preMSK1p are sufficient to direct tRNA import in vitro and that depletion of enolase inhibits tRNA import in vivo. Enzymatic and tRNA targeting functions of enolase appear to be independent. Three newly characterized properties of the enolase can be related to its novel function: (1) specific affinity to the imported tRNA, (2) the ability to facilitate formation of the complex between preMsk1p and the imported tRNA, and (3) partial targeting toward the mitochondrial outer membrane. We propose a model suggesting that the cell exploits mitochondrial targeting of the enolase in order to address the tRNA toward peri-mitochondrially synthesized preMsk1p. Our results indicate an alternative molecular chaperone function of glycolytic enzyme enolase in tRNA mitochondrial targeting.

Although mitochondrial import of nuclear DNA-encoded RNAs is widely occurring, their functions in the organelles are not always understood. Mitochondrial function(s) of tRNA(Lys)(CUU), tRK1, targeted into Saccharomyces cerevisiae mitochondria was mysterious, since mitochondrial DNA-encoded tRNA(Lys)(UUU), tRK3, was hypothesized to decode both lysine codons, AAA and AAG. Mitochondrial targeting of tRK1 depends on the precursor of mitochondrial lysyl-tRNA synthetase, pre-Msk1p. Here we show that substitution of pre-Msk1p by its Ashbya gossypii ortholog results in a strain in which tRK3 is aminoacylated, while tRK1 is not imported. At elevated temperature, drop of tRK1 import inhibits mitochondrial translation of mRNAs containing AAG codons, which coincides with the impaired 2-thiolation of tRK3 anticodon wobble nucleotide. Restoration of tRK1 import cures the translational defect, suggesting the role of tRK1 in conditional adaptation of mitochondrial protein synthesis. In contrast with the known ways of organellar translation control, this mechanism exploits the RNA import pathway.

Mitochondrial translation is essentially bacteria-like, reflecting the bacterial endosymbiotic ancestry of the eukaryotic organelle. However, unlike the translation system of its bacterial ancestors, mitochondrial translation is limited to just a few mRNAs, mainly coding for components of the respiratory complex. The classical bacterial initiation factors (IFs) IF1, IF2 and IF3 are universal in bacteria, but only IF2 is universal in mitochondria (mIF2). We analyse the distribution of mitochondrial translation initiation factors and their sequence features, given two well-propagated claims: first, a sequence insertion in mitochondrial IF2 (mIF2) compensates for the universal lack of IF1 in mitochondria, and secondly, no homologue of mitochondrial IF3 (mIF3) is identifiable in Saccharomyces cerevisiae. Our comparative sequence analysis shows that, in fact, the mIF2 insertion is highly variable and restricted in length and primary sequence conservation to vertebrates, while phylogenetic and in vivo complementation analyses reveal that an uncharacterized S. cerevisiae mitochondrial protein currently named Aim23p is a bona fide evolutionary and functional orthologue of mIF3. Our results highlight the lineage-specific nature of mitochondrial translation and emphasise that comparative analyses among diverse taxa are essential for understanding whether generalizations from model organisms can be made across eukaryotes.