title: Subcomplexes of ancestral respiratory complex I subunits rapidly turn over in vivo as productive assembly intermediates in Arabidopsis.
abstract: Subcomplexes of mitochondrial respiratory complex I (CI; EC 1.6.5.3) are shown to turn over in vivo, and we propose a role in an ancestral assembly pathway. By progressively labeling Arabidopsis cell cultures with (15)N and isolating mitochondria, we have identified CI subcomplexes through differences in (15)N incorporation into their protein subunits. The 200-kDa subcomplex, containing the ancestral γ-carbonic anhydrase (γ-CA), γ-carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CI+CIII(2). In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their specific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CI+CIII(2), indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a γ-CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occurrence of genes encoding these particular proteins broadly in eukaryotes, except for opisthokonts, provides a framework for the evolutionary conservation of these accessory subunits and evidence of their function in ancestral CI assembly.
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Western Australia, Australia.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Clark J
authors.initials: CJ
authors.lastname: Nelson
authors.forename: Chris
authors.initials: C
authors.lastname: Carrie
authors.forename: Ryan M R
authors.initials: RM
authors.lastname: Gawryluk
authors.forename: Cory
authors.initials: C
authors.lastname: Solheim
authors.forename: Michael W
authors.initials: MW
authors.lastname: Gray
authors.forename: James
authors.initials: J
authors.lastname: Whelan
authors.forename: A Harvey
authors.initials: AH
authors.lastname: Millar
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods; D002256:Carbonic Anhydrases / Q000378:metabolism; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000378:metabolism; D004734:Energy Metabolism; D018506:Gene Expression Regulation, Plant; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D018521:Plant Physiological Phenomena; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D011523:Protoplasts / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D024101:Mitochondrial Proteins; D002256:Carbonic Anhydrases; D042967:Electron Transport Complex I