人類粒線體酵素複合體I內NDUFV1次單元蛋白質 功能研究及其在呼吸鏈酵素複合體 組合機制中所扮演的角色

Abstract

NADH dehydrogenase (ubiquinone) flavoprotein 1 (NDUFV1) is a nuclear-encoded subunit of mitochondrial Complex I. It provides a conserved FMN binding site and is responsible for transferring electrons from NADH to FMN to facilitate the entrance of electrons into the electron transport chain (ETC). NDUFV1 also has an iron sulfur cluster [4Fe-4S] (N3) that is the first of that kind in the Complex I. The defect in NDUFV1 could cause neural disease like psychomotor retardation, and some point mutations on FMN binding site have been found in patients with Leigh syndrome. The assembly of intact OXPHOS complexes (complex I~V) is very important in mitochondrial energy production. In addition, the assembly of several respiratory complexes to form a larger supercomplex may improve the efficiency of electron transport. The supercomplex composed by complex I, complex III and complex IV has been confirmed and the defect in supercomplex assembly is associated with encephalomyopathies and neurodegenerative disorders. In present study, we applied a RNA interference system to suppress the NDUFV1 expression in human embryonic kidney cell line (HEK293), and generated three knockdown cell lines A9, B5 and E12 with 55~70% reduction in NDUFV1 protein level. We also performed oxygen consumption assay, dynamic complex I activity assay, ATP determination assay and cell growth rate measurement to evaluate the effects of NDUFV1 suppression. The results showed that the metabolic activity and growth rate were significantly decreased in NDUFV1 knockdown cell lines. In the respiratory complex assembly study, we solubilized mitochondria and applied a high resolution clear native electrophoresis (HrCNE) approach to investigate whether NDUFV1 knockdown affects individual respiratory complex assembly. The results III indicated that NDUFV1 knockdown would significantly decrease the level of complex I, III and IV. Further investigation of OXPHOS supercomplex formation also showed that while NDUFV1 was suppressed, CI/CIII2/CIVn supercomplexes were significantly reduced in cells. These finding suggests that NDUFV1 might play an important role in the assembly/stability of mitochondrial OXPHOS complexes and supercomplexes.