5 S rRNA and tRNA import into human mitochondria - Comparison of in vitro requirements

In vivo, human mitochondria import 5 S rRNA and do not import tRNAs from th e cytoplasm. We demonstrated previously that isolated human mitochondria ar e able to internalize a yeast tRNA(Lys) in the presence of yeast soluble fa ctors. Here, we describe an assay for specific uptake of 5 S rRNA by isolat ed human mitochondria and compare its requirements with the artificial tRNA import. The efficiency of 5 S rRNA uptake by isolated mitochondria was com parable with that found in vivo. The import was shown to depend on ATP and the transmembrane electrochemical potential and was directed by soluble pro teins. Blocking the pre-protein import channel inhibited internalization of both 5 S rRNA and tRNA, which suggests this apparatus be involved in RNA u ptake by the mitochondria. We show that human mitochondria can also selecti vely internalize several in vitro synthesized versions of yeast tRNA(Lys) a s well as a transcript of the human mitochondrial tRNA(Lys). Either yeast o r human soluble proteins can direct this import, suggesting that human cell s possess all factors needed for such an artificial translocation. On the o ther hand, the efficiency of import directed by yeast or human protein fact ors varies significantly, depending on the tRNA version. Similarly to the y east system, tRNA(Lys) import into human mitochondria depended on aminoacyl ation and on the precursor of the mitochondrial lysyl-tRNA synthetase. 5 S rRNA import was also dependent upon soluble protein(s), which were distinct from the factors providing tRNA internalization.