| Summary: | 碩士 === 國立清華大學 === 分子醫學研究所 === 97 === Human NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7), one of the most conserved core subunits of mitochondrial complex I, plays an important role in the oxidative phosphorylation system (OXPHOS). This protein binds one iron-sulfur cluster N2 (tetranuclear) which is the terminal redox center in the electron transport process of complex I. Three types of mutations in this subunit have been associated with Leigh syndrome (LS). NDUFS7 protein is encoded by nuclear genome and incorporated in the peripheral segment of complex I facing the mitochondrial matrix. The NDUFS7 was suppressed in human T-REx-293 cells using the RNA interference (RNAi) technology. The reduction in the NDUFS7 expression caused a slow growth rate in galactose containing medium and increased H2O2 generation. These results indicated that NDUFS7 may play an important role in cell energy production and survival. Most mitochondrial matrix proteins are synthesized in the cytoplasm and imported into mitochondria by TIM/TOM complexes recognizing the mitochondrial targeting sequences (MTSs). Using predication softwares, the N-terminal fragment was suggested to be the MTS of NDUFS7. In this study, we fused NDUFS7 containing N-terminal deletions, C-terminal deletions or different portions of the protein to enhanced green fluorescent protein (EGFP), and used these obtained constructs to study their intracellular localization in T-REx-293 cells. We found that the chimeric NDUFS71-60-EGFP was colocalized with mitochondria, demonstrating that this N-terminal fragment contained an effective MTS. We then used site-directed mutagenesis analyses to study the role of basic and hydrophobic residues in the first 60 amino acids of NDUFS7. These results suggested that both two types of amino acids are important for the mitochondrial import of the MTS in NDUFS7. In addition, we also demonstrated that there is a nuclear localization signal (NLS) and a nuclear export signal (NES) located in the C-terminus of NDUFS7. These results suggested that NDUFS7 has a dual distribution both in mitochondria and nuclei.
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