Abstract
The oxidative phosphorylation system (OXPHOS) is the most important source of ATP production in eukaryotic cells, and the complex I is the first enzyme complex in this system which participates in the process of electron transfer. Defects in the mitochondrial-encoded subunits in complex I, such as ND3, have been associated with certain kinds of mitochondrial diseases including Leigh’s syndrome (LS) and lethal infantile mitochondrial disease. Unfortunately, there is currently no effective therapeutic approach to treat diseases derived from defects in the mitochondrial genome. Recently, allotopic expression has appeared to be a promising approach to overcome the blind spot of mitochondrial disease treatment. In this strategy, the mitochondrial gene must be recoded for being expressed in cytoplasm. To facilitate the importation back to mitochondria, mitochondrial targeting sequences (MTSs) should be added in front of the N-terminal region of the gene products. In this study, we examined the applicability of applying this approach for human complex I ND3 subunit. Two gene constructs, COX4MTSND3 and COX8MTSND3, were synthesized and individually inserted into the tetracycline-regulated vector for allotopic expression of ND3 by transient transfection. We showed that the recoded ND3 gene fragment could be expressed through tetracycline induction in T-REx 293 cells. Nevertheless, the two chosen MTSs, COX4MTS and COX8MTS, were not competent in importing ND3 to the correct location in the mitochondria. The unsuccessful mitochondrial import may be attributed to the high hydrophobicity of ND3 which may form barriers for the import machinery. In contrast, the nuclear-encoded gene for NDUFS7 (PSST) subunit of complex I was successfully employed in this induction system. We suggest that by adopting other suitable MTSs and addition of a 3’ untranslated region (3’-UTR) in the recoded mitochondrial gene may improve ND3 mitochondrial import. From this point of view, using a mitochondrial targeting sequence from a nuclear-encoded Chlamydomonas reinhardtii NUO3 (a ND3 homologue) may have the potential in accomplishing this challenging work.