Yermovsky-Kammerer A E, Hajduk S L
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Mol Cell Biol. 1999 Sep;19(9):6253-9. doi: 10.1128/MCB.19.9.6253.
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 import of nuclearly encoded tRNAs into the mitochondrion has been demonstrated in a variety of organisms and is essential for proper function in the mitochondrion. 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 trypanosome mitochondria and synthetic RNAs transcribed from a cloned nucleus-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 preferred substrate for tRNA import into trypanosome mitochondria is a precursor molecule composed of tandemly linked tRNAs. Import of the tandem tRNA substrate requires (i) a protein component that is associated with the surface of the mitochondrion, (ii) ATP pools both outside and within the mitochondrion, and (iii) a membrane potential. Dissipation of the proton gradient across the inner mitochondrial membrane by treatment with an uncoupling agent inhibits 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 membrane, ATP-dependent steps, and a mitochondrial membrane potential.
布氏锥虫的所有线粒体tRNA已被证明是由细胞核编码的，并且必须导入线粒体。在多种生物体中都已证实细胞核编码的tRNA可导入线粒体，这对于线粒体的正常功能至关重要。已开发出一种体外导入测定法来研究布氏锥虫中tRNA的导入途径。该体外系统利用粗制的分离锥虫线粒体和从克隆的细胞核编码tRNA基因簇转录而来的合成RNA。由tRNA(Ser)和tRNA(Leu)组成的底物与一个59个核苷酸的基因间隔区串联转录。串联的tRNA底物能快速导入，而成熟大小的tRNA(Leu)在该系统中无法导入。这些结果表明，导入锥虫线粒体的tRNA的首选底物是由串联连接的tRNA组成的前体分子。串联tRNA底物的导入需要：(i) 一种与线粒体外表面相关的蛋白质成分；(ii) 线粒体外和线粒体内的ATP库；(iii) 膜电位。用解偶联剂处理以耗散跨线粒体内膜的质子梯度会抑制串联tRNA底物的导入。对导入所需条件的表征表明，线粒体RNA的导入通过一条包括与线粒体外膜相关的蛋白质成分、ATP依赖步骤和线粒体膜电位的途径进行。
