Abstract
ND4L, the smallest subunit of the hydrophobic membrane segment of complex I, is encoded by the mitochondrial genome. Defects in this subunit have been associated with Leber Hereditary Optic Neuropathy (LHON) diseases. In spite of its importance, no effective remedies have been established for ND4L deficiencies. To provide a way for investigating the functional and pathophysiological role of the ND4L subunit, we adopted the allotopic expression strategy and successfully constructed and synthesized the recoded ND4L genes with the conjugation of P1, S9 and SOD2(16A) or SOD2(16V) mitochondrial targeting sequence (MTS). In addition, we also generated several derivatives with the SOD2 3’ untranslated region (SOD23’UTR) from the aforementioned constructs to facilitate mitochondrial import. The results demonstrated that these newly designed MTSs were doing their jobs in helping EGFP protein in the process of mitochondrial import. However, the precursor proteins of our targeted ND4L were not completely import competent. The addition of SOD23’UTR in the transgenes only had a very limited success in contranlational mitochondrial import. Interestingly, some TREx-293 cells expressed ND4L with P1MTS or S9MTS addition were partially associated with vimentin. In contrast, some expressed ND4L proteins with SOD2MTS were stuck on the surface of nuclei. Analyses of the contents of ND4L amino acid sequence suggested that the highly hydrophobic characteristics of this protein may hamper its correct mitochondrial import. Finally, we suggested that mitochondrial targeting sequences from Chlamydomonas reinhardtii ND4L which is nDNA-encoded may help us to achieve this challenging task.