Ae. Yermovsky-kammerer et Sl. Hajduk, In vitro import of a nuclearly encoded tRNA into the mitochondrion of Trypanosoma brucei, MOL CELL B, 19(9), 1999, pp. 6253-6259
All of the mitochondrial tRNAs of Trypanosoma brucei have been shown to be
encoded in the nucleus and must be imported into the mitochondrion, The imp
ort of nuclearly encoded tRNAs into the mitochondrion has been demonstrated
in a variety of organisms and is essential for proper function in the mito
chondrion. An in vitro import assay has been developed to study the pathway
of tRNA import in T, brucei. The in vitro system utilizes crude isolated t
rypanosome mitochondria and synthetic RNAs transcribed from a cloned nucleu
s-encoded tRNA gene cluster, The substrate, composed of tRNA(Ser) and tRNA(
Leu), is transcribed in tandem with a 59-nucleotide intergenic region, The
tandem tRNA substrate is imported rapidly, while the mature-size tRNA(Leu)
fails to be imported in this system. These results suggest that the preferr
ed substrate for tRNA import into trypanosome mitochondria is a precursor m
olecule composed of tandemly linked tRNAs. Import of the tandem tRNA substr
ate requires (i) a protein component that is associated with the surface of
the mitochondrion, (ii) ATP pools both outside and within the mitochondrio
n, and (iii) a membrane potential. Dissipation of the proton gradient acros
s the inner mitochondrial membrane by treatment with an uncoupling agent in
hibits import of the tandem tRNA substrate. Characterization of the import
requirements indicates that mitochondrial RNA import proceeds by a pathway
including a protein component associated with the outer mitochondrial membr
ane, ATP-dependent steps, and a mitochondrial membrane potential.