Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice (Budweis), Czech Republic.
Faculty of Science, Charles University, Prague, Czech Republic.
PLoS One. 2018 Apr 26;13(4):e0196474. doi: 10.1371/journal.pone.0196474. eCollection 2018.
Upon their translocation into the mitochondrial matrix, the N-terminal pre-sequence of nuclear-encoded proteins undergoes cleavage by mitochondrial processing peptidases. Some proteins require more than a single processing step, which involves several peptidases. Down-regulation of the putative Trypanosoma brucei mitochondrial intermediate peptidase (MIP) homolog by RNAi renders the cells unable to grow after 48 hours of induction. Ablation of MIP results in the accumulation of the precursor of the trypanosomatid-specific trCOIV protein, the largest nuclear-encoded subunit of the cytochrome c oxidase complex in this flagellate. However, the trCOIV precursor of the same size accumulates also in trypanosomes in which either alpha or beta subunits of the mitochondrial processing peptidase (MPP) have been depleted. Using a chimeric protein that consists of the N-terminal sequence of a putative subunit of respiratory complex I fused to a yellow fluorescent protein, we assessed the accumulation of the precursor protein in trypanosomes, in which RNAi was induced against the alpha or beta subunits of MPP or MIP. The observed accumulation of precursors indicates MIP depletion affects the activity of the cannonical MPP, or at least one of its subunits.
在被转运到线粒体基质中后，核编码蛋白的 N 端前导序列会被线粒体加工肽酶切割。一些蛋白质需要不止一个加工步骤，这涉及到几种肽酶。通过 RNAi 下调假定的布氏锥虫线粒体中间肽酶（MIP）同源物，会导致细胞在诱导后 48 小时内无法生长。MIP 的缺失会导致 trCOIV 蛋白前体的积累，该蛋白是这种鞭毛原生动物细胞色素 c 氧化酶复合物中最大的核编码亚基。然而，在α或β亚基的线粒体加工肽酶（MPP）被耗尽的锥虫中，也会积累相同大小的 trCOIV 前体。我们使用一种嵌合蛋白进行评估，该蛋白由呼吸复合物 I 的假定亚基的 N 端序列与黄色荧光蛋白融合而成，我们评估了针对 MPP 或 MIP 的α或β亚基的 RNAi 诱导后锥虫中前体蛋白的积累。观察到的前体积累表明，MIP 缺失会影响经典 MPP 的活性，或者至少影响其一个亚基的活性。
