Subunit-specific incorporation efficiency and kinetics in mitochondrial complex I homeostasis

J Biol Chem. 2012 Dec 7;287(50):41851-60. doi: 10.1074/jbc.M112.391151. Epub 2012 Oct 4.


Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse. Here, we used inducible HEK293 cell lines stably expressing AcGFP1-tagged CI subunits and quantified the amount of tagged subunit in mitoplasts and holo-CI by non-native and native PAGE, respectively, to determine their CI incorporation efficiency. Analysis of time courses of induction revealed three subunit-specific patterns. A first pattern, represented by NDUFS1, showed overlapping time courses, indicating that imported subunits predominantly incorporate into holo-CI. A second pattern, represented by NDUFV1, consisted of parallel time courses, which were, however, not quantitatively overlapping, suggesting that imported subunits incorporate at similar rates into holo-CI and CI assembly intermediates. The third pattern, represented by NDUFS3 and NDUFA2, revealed a delayed incorporation into holo-CI, suggesting their prior appearance in CI assembly intermediates and/or as free monomers. Our analysis showed the same maximum incorporation into holo-CI for NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS4, NDUFA2, and NDUFA12 with nearly complete loss of endogenous subunit at 24 h of induction, indicative of an equimolar stoichiometry and unexpectedly rapid turnover. In conclusion, the results presented demonstrate that newly formed nDNA-encoded CI subunits rapidly incorporate into holo-CI in a subunit-specific manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cricetinae
  • Cricetulus
  • Electron Transport Complex I / genetics
  • Electron Transport Complex I / metabolism*
  • HEK293 Cells
  • Homeostasis / physiology*
  • Humans
  • Kinetics
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*


  • Mitochondrial Proteins
  • Protein Subunits
  • Electron Transport Complex I