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PubMed · 14722084

Development and characterization of a conditional mitochondrial complex I assembly system.

Nagendra Yadava·Toby Houchens·Prasanth Potluri·Immo E Scheffler
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Abstract

We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line, CCL16-B2, that does not express the NDUFA1 gene (encoding the MWFE protein). In this mutant, a hemagglutinin (HA) epitope-tagged MWFE protein was expressed from a doxycycline-inducible promoter. The expression of the protein was absolutely dependent on the presence of doxycycline, and the gene could be turned off completely by removal of doxycycline. These experiments demonstrated a key role of MWFE in the pathway of complex I assembly. Upon induction the MWFE.HA protein reached steady-state levels within 24 h, but the appearance of fully active complex I was delayed by another approximately 24 h. The MWFE appeared in a precomplex that probably includes one or more subunits encoded by mtDNA. The fate of MWFE and the stability of complex I were themselves very tightly linked to the activity of mitochondrial protein synthesis and to the assembly of subunits encoded by mtDNA (ND1-6 and ND4L). This novel conditional system can shed light not only on the mechanism of complex I assembly but emphasizes the role of subunits previously thought of as "accessory." It promises to have broader applications in the study of cellular energy metabolism and production of reactive oxygen species and related processes.

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AnimalsBiochemical PhenomenaBiochemistryBlotting, NorthernBlotting, WesternCell LineChloramphenicolCricetinaeDNA, MitochondrialDose-Response Relationship, DrugDoxycyclineElectron TransportElectron Transport Complex IElectrophoresis, Polyacrylamide GelEpitopesFibroblastsGene DeletionHemagglutininsKineticsMembrane ProteinsMitochondriaMutationPlasmidsPromoter Regions, GeneticProtein BindingProtein Structure, TertiaryRNA, MessengerReactive Oxygen SpeciesTime FactorsJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

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Nagendra Yadava, Toby Houchens, Prasanth Potluri, Immo E Scheffler. 2004-01-13. Development and characterization of a conditional mitochondrial complex I assembly system.. https://doi.org/10.1074/jbc.m313588200

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