NADH-ubiquinone oxidoreductase（complex I）是粒線體呼吸鏈中最大且最複雜的酵素複合體。哺乳動物的complex I共含45個次單元，其中7個次單元是由mitochondrial DNA（mtDNA）所表現，其他的38個次單元則是由nuclear DNA（nDNA）表現後再經粒線體標的訊號(mitochondrial targeting signal, MTS)的帶領運送至粒線體。NADH dehydrogenase ubiquinone flavoprotein 2，簡稱NDUFV2，則為complex I中由nDNA所表現的一個次單元，其含有一個[2Fe-2S]鐵硫中心，並於演化上具高度保留的特性。臨床上發現NDUFV2的缺失與一些神經退化性疾病有關，例如：帕金森氏症、阿茲海默症、躁鬱症及精神分裂症等。本實驗主要以T-REx293細胞為研究模式，針對NDUFV2的功能及其MTS進行系統性的研究。首先，利用核醣核酸干擾技術（RNAi）來抑制NDUFV2基因的表現，單純化探討NDUFV2在人類細胞中的功能。結果發現NDUFV2基因表現的下降會造成細胞有生長遲緩、耗氧能力下降、粒線體膜電位下降及自由基上升等表型，但NDUFV2的缺失並不影響complex I的組裝能力。由這些結果顯示NDUFV2對粒線體能量生成上扮演很重要的角色。另外，我們還定義了NDUFV2的MTS位於蛋白質N端及其被剪輯的切位位於第32個胺基酸，其中只需含有前22個胺基酸的片段即具有運送enhanced green fluorescent protein (EGFP) 至粒線體的能力。隨後利用定點突變的方法探討MTS的序列訊號，結果發現鹼性及疏水性胺基酸對NDUFV2 MTS的運送功能很重要。實驗中我們還利用T-REx293細胞模擬早發性心肌腫大與腦病在臨床上所發現的NDUFV2基因剪輯異常的情形(IVS2+5_+8delGTAA)，並從分子層次來研究NDUFV2基因缺失與此疾病的關係。結果發現上述突變正好破壞了NDUFV2的粒線體標的訊號，進而導致蛋白無法正確的被運送至粒線體執行其功能。本篇研究證實NDUFV2在粒線體能量生成的過程中扮演很重要的角色並定義了粒線體標的訊號所座落的位置及其重要性。

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.

1. Brown GC: Control of respiration and ATP synthesis in mammalian mitochondria and cells. Biochem J 1992, 284 ( Pt 1):1-13.
2. Hatefi Y: The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem 1985, 54:1015-1069.
3. Taanman JW: The mitochondrial genome: structure, transcription, translation and replication. Biochim Biophys Acta 1999, 1410(2):103-123.
4. Wallace DC: The mitochondrial genome in human adaptive radiation and disease: on the road to therapeutics and performance enhancement. Gene 2005, 354:169-180.
5. Balaban RS, Nemoto S, Finkel T: Mitochondria, oxidants, and aging. Cell 2005, 120(4):483-495.
6. Huang H, Manton KG: The role of oxidative damage in mitochondria during aging: a review. Front Biosci 2004, 9:1100-1117.
7. Lynn S, Wardell T, Johnson MA, Chinnery PF, Daly ME, Walker M, Turnbull DM: Mitochondrial diabetes: investigation and identification of a novel mutation. Diabetes 1998, 47(11):1800-1802.
8. Wallace DC: A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 2005, 39:359-407.
9. Wallace DC: Why do we still have a maternally inherited mitochondrial DNA? Insights from evolutionary medicine. Annu Rev Biochem 2007, 76:781-821.
10. Bolender N, Sickmann A, Wagner R, Meisinger C, Pfanner N: Multiple pathways for sorting mitochondrial precursor proteins. EMBO Rep 2008, 9(1):42-49.
11. Horwich AL, Kalousek F, Mellman I, Rosenberg LE: A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. EMBO J 1985, 4(5):1129-1135.
12. von Heijne G: A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 1986, 14(11):4683-4690.
13. Gavel Y, von Heijne G: Cleavage-site motifs in mitochondrial targeting peptides. Protein Eng 1990, 4(1):33-37.
14. Lenaz G, Fato R, Baracca A, Genova ML: Mitochondrial quinone reductases: complex I. Methods Enzymol 2004, 382:3-20.
15. Bai Y, Hu P, Park JS, Deng JH, Song X, Chomyn A, Yagi T, Attardi G: Genetic and functional analysis of mitochondrial DNA-encoded complex I genes. Ann N Y Acad Sci 2004, 1011:272-283.
16. Grigorieff N: Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I). Curr Opin Struct Biol 1999, 9(4):476-483.
17. Friedrich T, Bottcher B: The gross structure of the respiratory complex I: a Lego System. Biochim Biophys Acta 2004, 1608(1):1-9.
18. Scheffler IE, Yadava N, Potluri P: Molecular genetics of complex I-deficient Chinese hamster cell lines. Biochim Biophys Acta 2004, 1659(2-3):160-171.
19. Carroll J, Fearnley IM, Shannon RJ, Hirst J, Walker JE: Analysis of the subunit composition of complex I from bovine heart mitochondria. Mol Cell Proteomics 2003, 2(2):117-126.
20. Smeitink JA, Loeffen JL, Triepels RH, Smeets RJ, Trijbels JM, van den Heuvel LP: Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art. Hum Mol Genet 1998, 7(10):1573-1579.
21. Saraste M: Oxidative phosphorylation at the fin de siecle. Science 1999, 283(5407):1488-1493.
22. Schultz BE, Chan SI: Structures and proton-pumping strategies of mitochondrial respiratory enzymes. Annu Rev Biophys Biomol Struct 2001, 30:23-65.
23. Sled VD, Friedrich T, Leif H, Weiss H, Meinhardt SW, Fukumori Y, Calhoun MW, Gennis RB, Ohnishi T: Bacterial NADH-quinone oxidoreductases: iron-sulfur clusters and related problems. J Bioenerg Biomembr 1993, 25(4):347-356.
24. Ohnishi T: Iron-sulfur clusters/semiquinones in complex I. Biochim Biophys Acta 1998, 1364(2):186-206.
25. Yano T, Yagi T: H(+)-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans. Studies on topology and stoichiometry of the peripheral subunits. J Biol Chem 1999, 274(40):28606-28611.
26. Hinchliffe P, Sazanov LA: Organization of iron-sulfur clusters in respiratory complex I. Science 2005, 309(5735):771-774.
27. Brandt U: Energy converting NADH:quinone oxidoreductase (complex I). Annu Rev Biochem 2006, 75:69-92.
28. de Coo R, Buddiger P, Smeets H, Geurts van Kessel A, Morgan-Hughes J, Weghuis DO, Overhauser J, van Oost B: Molecular cloning and characterization of the active human mitochondrial NADH:ubiquinone oxidoreductase 24-kDa gene (NDUFV2) and its pseudogene. Genomics 1995, 26(3):461-466.
29. Hattori N: Structural organization and chromosomal localization of the human nuclear gene (NDUFV2) for the 24-kDa iron-sulfur subunit of complex I in mitochondrial respiratory chain.pdf. biochemical and biophysical research communications 1995, 216:771-777.
30. Weidner U, Geier S, Ptock A, Friedrich T, Leif H, Weiss H: The gene locus of the proton-translocating NADH: ubiquinone oxidoreductase in Escherichia coli. Organization of the 14 genes and relationship between the derived proteins and subunits of mitochondrial complex I. J Mol Biol 1993, 233(1):109-122.
31. Duborjal H, Dupuis A, Chapel A, Kieffer S, Lunardi J, Issartel JP: Immuno-purification of a dimeric subcomplex of the respiratory NADH-CoQ reductase of Rhodobacter capsulatus equivalent to the FP fraction of the mitochondrial complex I. FEBS Lett 1997, 405(3):345-350.
32. Yano T, Sled VD, Ohnishi T, Yagi T: Expression of the 25-kilodalton iron-sulfur subunit of the energy-transducing NADH-ubiquinone oxidoreductase of Paracoccus denitrificans. Biochemistry 1994, 33(2):494-499.
33. Yano T, Chu SS, Sled VD, Ohnishi T, Yagi T: The proton-translocating NADH-quinone oxidoreductase (NDH-1) of thermophilic bacterium Thermus thermophilus HB-8. Complete DNA sequence of the gene cluster and thermostable properties of the expressed NQO2 subunit. J Biol Chem 1997, 272(7):4201-4211.
34. Almeida T, Duarte M, Melo AM, Videira A: The 24-kDa iron-sulphur subunit of complex I is required for enzyme activity. Eur J Biochem 1999, 265(1):86-93.
35. Kerscher S, Benit P, Abdrakhmanova A, Zwicker K, Rais I, Karas M, Rustin P, Brandt U: Processing of the 24 kDa subunit mitochondrial import signal is not required for assembly of functional complex I in Yarrowia lipolytica. Eur J Biochem 2004, 271(17):3588-3595.
36. Velazquez I, Nakamaru-Ogiso E, Yano T, Ohnishi T, Yagi T: Amino acid residues associated with cluster N3 in the NuoF subunit of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli. FEBS Lett 2005, 579(14):3164-3168.
37. Friedrich T: The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli. Biochim Biophys Acta 1998, 1364(2):134-146.
38. Yagi T, Dinh TM: Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificans. Biochemistry 1990, 29(23):5515-5520.
39. Yano T, Sled VD, Ohnishi T, Yagi T: Expression and characterization of the flavoprotein subcomplex composed of 50-kDa (NQO1) and 25-kDa (NQO2) subunits of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans. J Biol Chem 1996, 271(10):5907-5913.
40. Yano T, Sled VD, Ohnishi T, Yagi T: Identification of amino acid residues associated with the [2Fe-2S] cluster of the 25 kDa (NQO2) subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans. FEBS Lett 1994, 354(2):160-164.
41. Zu Y, Di Bernardo S, Yagi T, Hirst J: Redox properties of the [2Fe-2S] center in the 24 kDa (NQO2) subunit of NADH:ubiquinone oxidoreductase (complex I). Biochemistry 2002, 41(31):10056-10069.
42. Sazanov LA, Hinchliffe P: Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus. Science 2006, 311(5766):1430-1436.
43. Videira A: Complex I from the fungus Neurospora crassa. Biochim Biophys Acta 1998, 1364(2):89-100.
44. von Bahr-Lindstrom H, Galante YM, Persson M, Jornvall H: The primary structure of subunit II of NADH dehydrogenase from bovine-heart mitochondria. Eur J Biochem 1983, 134(1):145-150.
45. Pilkington SJ, Walker JE: Mitochondrial NADH-ubiquinone reductase: complementary DNA sequences of import precursors of the bovine and human 24-kDa subunit. Biochemistry 1989, 28(8):3257-3264.
46. Hattori N, Yoshino H, Tanaka M, Suzuki H, Mizuno Y: Genotype in the 24-kDa subunit gene (NDUFV2) of mitochondrial complex I and susceptibility to Parkinson disease. Genomics 1998, 49(1):52-58.
47. Kim SH, Vlkolinsky R, Cairns N, Fountoulakis M, Lubec G: The reduction of NADH ubiquinone oxidoreductase 24- and 75-kDa subunits in brains of patients with Down syndrome and Alzheimer's disease. Life Sci 2001, 68(24):2741-2750.
48. Karry R, Klein E, Ben Shachar D: Mitochondrial complex I subunits expression is altered in schizophrenia: a postmortem study. Biol Psychiatry 2004, 55(7):676-684.
49. Nakatani N, Hattori E, Ohnishi T, Dean B, Iwayama Y, Matsumoto I, Kato T, Osumi N, Higuchi T, Niwa S et al: Genome-wide expression analysis detects eight genes with robust alterations specific to bipolar I disorder: relevance to neuronal network perturbation. Hum Mol Genet 2006, 15(12):1949-1962.
50. Loeffen J, Elpeleg O, Smeitink J, Smeets R, Stockler-Ipsiroglu S, Mandel H, Sengers R, Trijbels F, van den Heuvel L: Mutations in the complex I NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy. Ann Neurol 2001, 49(2):195-201.
51. Benit P, Beugnot R, Chretien D, Giurgea I, De Lonlay-Debeney P, Issartel JP, Corral-Debrinski M, Kerscher S, Rustin P, Rotig A et al: Mutant NDUFV2 subunit of mitochondrial complex I causes early onset hypertrophic cardiomyopathy and encephalopathy. Hum Mutat 2003, 21(6):582-586.
52. Djafarzadeh R, Kerscher S, Zwicker K, Radermacher M, Lindahl M, Schagger H, Brandt U: Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica. Biochim Biophys Acta 2000, 1459(1):230-238.
53. Barrientos A: In vivo and in organello assessment of OXPHOS activities. Methods 2002, 26(4):307-316.
54. Weiss H, von Jagow G, Klingenberg M, Bucher T: Characterization of Neurospora crassa mitochondria prepared with a grind-mill. Eur J Biochem 1970, 14(1):75-82.
55. Kerscher S, Drose S, Zickermann V, Brandt U: The three families of respiratory NADH dehydrogenases. Results Probl Cell Differ 2008, 45:185-222.
56. Sabherwal N, Schneider KU, Blaschke RJ, Marchini A, Rappold G: Impairment of SHOX nuclear localization as a cause for Leri-Weill syndrome. J Cell Sci 2004, 117(Pt 14):3041-3048.
57. Claypool SM, McCaffery JM, Koehler CM: Mitochondrial mislocalization and altered assembly of a cluster of Barth syndrome mutant tafazzins. J Cell Biol 2006, 174(3):379-390.