Abstract
The endosymbiotic event leads mitochondrial genes to be translocated into nuclei. However, there are still a few genes conserved in the mitochondrial genome which code for proteins involved in oxidative phosphorylation. These proteins include ND1~ND6 and ND4L subunits of complex I, cytochrome c of complex III, Cox1~Cox3 subunits of complex IV, ATP6 and ATP8 subunits of complex V. Interestingly, the mitochondrial genome in Chlamydomonas reinhardtii lacks nd3, nd4l, cox2, cox3, atp6 and atp8 genes. Previous report suggested that nd3, nd4l, cox2, cox3 and atp6 were transfered to nuclei in C. reinhardtii, and cox2 was further divided into two independent nuclear genes. In this study, allotopic and xenotopic expression strategies were adopted to evaluate the possibilities of applying these methods as treatment concepts for mtDNA diseases. When the mitochondrial targeting signal (MTS) of ND4L, ND3, ATP6, COX3, and COX2a of C. reinhardtii was fused with enhanced green fluorescent proteins (EGFP), the resultant fusion proteins were successfully imported into mitochondria of HeLa and HEK293 cells. However, when the MTSs of the ND4L and ND3 were individually fused with its human homologue, only a limited mitochondrial targeting was observed. To improve the import efficiency, xenotopic expression of several full-length C. reinhardtii nuclear-encoded mitochondrial proteins were directly applied for mitochondrial targeting studies in human cells. By this approach, a significant improvement of mitochondrial import was observed in the targeted complex I ND4L subunit. Nevertheless, when the transactivator of transcription (TAT) region from the HIV was added in front of these constructed C. reinhardtii transgenes, the new designs did not show a significant improvement on mitochondrial targeting. However, when the hydrophilic EGFP protein was added at the C-terminus of the full-length C. reinhardtii ND4L subunit, the process could decrease the hydrophobicity of the importing protein effectively and improve the ability of the mitochondrial targeting. In this report, we proved that the C. reinhardtii ND4L subunit possesses the capability of the mitochondrial targeting in human cells and the hydrophobicity of the importing proteins plays an important role for mitochondrial targeting.