| Summary: | 碩士 === 國立清華大學 === 分子醫學研究所 === 98 === Oxidative phosphorylation system in mammalian cells contains five enzyme complexes. Among them, mitochondrial complex I is the biggest and the most complicated, with many undefined subunits and has no resolved complete structure. Mammalian mitochondrial complex I comprises of forty-five subunits, and seven of them are encoded by the mitochondrial genome. The remaining subunits are encoded by the nuclear genome and imported into mitochondria to perform their functions. NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is one of the nuclear-encoded mitochondrial core proteins of complex I. It contains two tetranuclear iron-sulfur clusters and plays an important role in electron transport. Mutations on NDUFS8 have been found to cause Leigh syndrome with mitochondrial complex I deficiency. In this study, RNA interference technique was used to knock down the NDUFS8 expression in T-REx293 cells to investigate the function of NDUFS8. Experimental results demonstrated that reducing expression of NDUFS8 would retard the cellular growth rate, slow down the oxygen consumption efficiency and increase the production of reactive oxygen species (ROS). Using high resolution clear native gel electrophoresis (HrCNE) for investigating the integrity of mitochondrial complex I revealed that knockdown of NDUFS8 would not affect the assembly of mitochondrial complex I but reduce its NADH oxidation activities. Restoration of NDUFS8 in a suppressed cell line improved the ability of oxygen consumption and NADH oxidation of complex I. In addition, various deletion and fusion constructs of NDUFS8 were generated to characterize the mitochondrial targeting sequence of this protein. The results revealed that the N-terminal fragment of 18 residues possessed the ability to import EGFP into mitochondria, which is shorter than the prediction of 34 amino acid residues proposed by MitoprotII program. Interestingly, there was also an unexpected result that all of the N-terminal deletion constructs of NDUFS8 protein were located in a specific region of nuclei. It was speculated that there is a nuclear localization signal hiding in NDUFS8 sequence. This study demonstrated that NDUFS8 play an essential role in complex I activity, and the mitochondrial targeting sequence of NDUFS8 existing or not will determine the subcellular localization in mitochondria or in nuclei.
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