title: Subunit-specific incorporation efficiency and kinetics in mitochondrial complex I homeostasis.
abstract: 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.
authors.affiliation: Department of Biochemistry, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands.
authors.forename: Cindy E J
authors.initials: CE
authors.lastname: Dieteren
authors.forename: Werner J H
authors.initials: WJ
authors.lastname: Koopman
authors.forename: Herman G
authors.initials: HG
authors.lastname: Swarts
authors.forename: Janny G P
authors.initials: JG
authors.lastname: Peters
authors.forename: Piotr
authors.initials: P
authors.lastname: Maczuga
authors.forename: Jasper J
authors.initials: JJ
authors.lastname: van Gemst
authors.forename: Rosalinde
authors.initials: R
authors.lastname: Masereeuw
authors.forename: Jan A M
authors.initials: JA
authors.lastname: Smeitink
authors.forename: Leo G J
authors.initials: LG
authors.lastname: Nijtmans
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
mesh_terms: D000818:Animals; D006224:Cricetinae; D003412:Cricetulus; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*
chemical_list: D024101:Mitochondrial Proteins; D021122:Protein Subunits; D042967:Electron Transport Complex I