title: Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I.
abstract: Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, and ARC Centre of Excellence for Coherent X-ray Science, La Trobe University, Melbourne 3086, Australia.
authors.forename: David A
authors.initials: DA
authors.lastname: Stroud
authors.forename: Luke E
authors.initials: LE
authors.lastname: Formosa
authors.forename: Xiaonan W
authors.initials: XW
authors.lastname: Wijeyeratne
authors.forename: Thanh N
authors.initials: TN
authors.lastname: Nguyen
authors.forename: Michael T
authors.initials: MT
authors.lastname: Ryan
mesh_terms: D001483:Base Sequence; D003851:Deoxyribonucleases / Q000378:metabolism*; D042967:Electron Transport Complex I / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005091:Exons; D015870:Gene Expression; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D051336:Mitochondrial Membranes / Q000737:chemistry* / Q000201:enzymology; D008969:Molecular Sequence Data; D015533:Transcriptional Activation
chemical_list: D003851:Deoxyribonucleases; D042967:Electron Transport Complex I; C571164:NDUFA9 protein, human