Selective importation of RNA into isolated mitochondria from Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleu s and imported from the cytosol. Incubation of two in vitro-transcribed tRN As, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leis hmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nucleas e protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane pot ential is not required for in vitro importation. A variety of small synthet ic RNAs were also found to be efficiently imported in vitro. The data sugge st that there is a structural requirement for importation of RNAs greater t han similar to 17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determin ed, but the specificity of the process was illustrated by the higher satura tion level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA( Gln). Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA( Gln) resulted in a reversal of the in vitro importation behavior and this c ould also be interpreted in terms of tertiary structure specificity.