Development and characterization of a conditional mitochondrial complex I assembly system.
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.
Yadava
Nagendra
N
Section of Molecular Biology, Division of Biological Sciences, University of California-San Diego, La Jolla, CA 92093-0322, USA.
Houchens
Toby
T
Potluri
Prasanth
P
Scheffler
Immo E
IE
D000818:Animals; D001669:Biochemical Phenomena; D001671:Biochemistry; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002701:Chloramphenicol / Q000494:pharmacology; D006224:Cricetinae; D004272:DNA, Mitochondrial / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D006388:Hemagglutinins / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D013997:Time Factors
D004272:DNA, Mitochondrial; D000939:Epitopes; D006388:Hemagglutinins; D008565:Membrane Proteins; C492251:Ndufa1 protein, mammalian; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D002701:Chloramphenicol; D042967:Electron Transport Complex I; D004318:Doxycycline