Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

Abstract

Cytoplasmic tRNA(Lys)CUU is the only nuclear‐encoded tRNA of Saccharomyces cerevisiae found to be associated with mitochondria. Selective import of this tRNA into isolated organelles requires cytoplasmic factors. Here we identify two of these factors as the cytoplasmic and mitochondrial lysyl‐tRNA synthetases. The cytoplasmic enzyme is obligatory for in vitro import of the deacylated, but not of the aminoacylated tRNA. We thus infer that it is needed for aminoacylation of the tRNA, which is a prerequisite for its import. The mitochondrial synthetase, which cannot aminoacylate tRN(Lys)CUU, is required for import of both aminoacylated and deacylated forms. Its depletion leads to a total arrest of tRNA import, in vitro and in vivo. The mitochondrial lysyl‐tRNA synthetase is able to form specific and stable RNP complexes with the amino‐acylated tRNA. Furthermore, an N‐terminal truncated form of the synthetase which cannot be targeted into mitochondria is unable to direct the import of the tRNA. We therefore hypothesize that the cytosolic precursor form of the mitochondrial synthetase has a carrier function for translocation of the tRNA across the mitochondrial membranes. However, cooperation of the two synthetases is not sufficient to direct tRNA import, suggesting the need of additional factor(s).

Mitochondrial import of a yeast cytoplasmic tRNALys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

“…The precursor of MSK is also essential for the import process. Since MSK is able to form stable complexes with the aminoacylated form of tRK1, we favor the hypothesis of its carrier function for the tRNA [12].…”

“…In some experiments (see section 3), mitochondria were preincubated with various amounts of E. coli 16S and 23S rRNA (Boheringer Mannheim) at 0°C for 10 min and repelleted before the import assay. The in vitro import was directed by crude extracts (IDP) from hMOM19mv cells [18] carrying plasmid pG1 lfr6 [19] to overexpress in the cytoplasm the precursor form of MSK [12]. Immunodepletion of KRS was performed using anti-KRS antibodies (kindly provided by M. Mirande) as described previously [12].…”

“…The in vitro import was directed by crude extracts (IDP) from hMOM19mv cells [18] carrying plasmid pG1 lfr6 [19] to overexpress in the cytoplasm the precursor form of MSK [12]. Immunodepletion of KRS was performed using anti-KRS antibodies (kindly provided by M. Mirande) as described previously [12]. To obtain an in vitro tRK1 transcript, the tRK1 gene (from plasmid pY109 kindly provided by H. Feldmann) was cloned using PCR under control of T7 promoter with a BstNI site at the 3'-terminus of the coding sequence.…”

“…In addition, soluble cytoplasmic proteins are also required [7,8]. We have recently identified two of these factors as the cytoplasmic lysyl-tRNA synthetase (KRS) and the precursor of the mitochondrial lysyl-tRNA synthetase (MSK) [12]. The main role of KRS appears to be aminoacylation of tRK1, which is a prerequisite for its mitochondrial targeting.…”

“…The poor import efficiency of the deacylated transcript can be explained by its low level of aminoacylation during the incubation in import mixture. Indeed, the ratio between the KcatlKm values of aminoacylation reaction with KRS for tRK1 and for the in vitro transcript was found to be 16,7 'T7-tr.RKI', in vitro T7-transcript of the tRK1 gene; 'lys-', RNA was aminoacylated prior to the import assay, then KRS was removed by phenol treatment; IDP immuno-depleted with anti-KRS antibodies [12] were used in import assay. efficiency of 0.154).2 pmol per mg MP, which corresponds to 0.454).6% of the transcript added to the in vitro assay (i.e., 6-8% of the in vivo mitochondrial pool of tRK1).…”