The functional and mitochondrial targeting signal studies of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) subunit in mitochondrial complexⅠ

• Main Authors: Liu, Hsin-Yu, 劉欣瑜
• Other Authors: Kao, Mou-Chieh
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/03608895926813533123

碩士 === 國立清華大學 === 分子醫學研究所 === 97 === Mammalian NADH-ubiquinone oxidoreductase (complex I) is the first, largest and most complicated respiratory complex in mitochondria. Seven subunits of complex I, including ND1-6 and ND4L, are encoded by mitochondrial DNA (mtDNA), and the other thirty-eight subunits are encoded by nuclear DNA (nDNA). NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) is one of the core nucleus-encoded subunits existing in human mitochondrial complex I. It contains one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), which may play a role in the prevention of oxidative damage. The defect of NDUFV2 subunit is associated with neurodegenerative diseases, including Parkinson disease, Alzheimer’s disease, Bipolar disorder and Schizophrenia. In this study, we applied the RNA interference (RNAi) technology in human T-REx293 cells to investigate the function of NDUFV2 subunit. We found that suppression of NDUFV2 expression in the cells would cause a slowing growth cell rate in galactose medium, decreasing oxygen consumption rate, reducing mitochondrial membrane potential (MMP) and increasing reactive oxygen species (ROS) generation, but did not affect complex I assembly. These observations provided the evidences that NDUFV2 plays an essential role for energy production in cells. In addition, we designed various truncation constructs to investigate the mitochondrial targeting mechanism of NDUFV2. We identified that the cleavage site of NDUFV2 was located around amino acid residue 32 and the first 22 residues of NDUFV2 was enough to function as a mitochondrial targeting sequence (MTS) to carry the passenger protein, enhanced green fluorescent protein (EGFP), into mitochondria successfully. Furthermore, we used the site-directed mutagenesis to study the basic, hydrophobic and hydroxylated residues in this identified N-terminal MTS. We found that the basic and hydrophobic residues were important for the MTS of NDUFV2, but the hydroxylated residues were not. In a recent study, the patients of the hypertrophic cardiomyopathy and encephalomyopathy were found to contain 4 bp deletion in the second intron of NDUFV2 (IVS2+5_+8delGTAA) to cause the exon 2 losing. To dissect the pathogenetic mechanism caused by this mutation, we established the human disease model and found that lost of this exon 2 cause NDUFV2 to lose its mitochondrial targeting ability. In this report, we proved that the NDUFV2 plays an important role for energy production in mammalian cells and identified the location of mitochondrial targeting sequence in this protein.