title: Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells.
abstract: Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells.
authors.affiliation: Department of Biochemistry, Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Cindy E J
authors.initials: CE
authors.lastname: Dieteren
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
authors.forename: Rutger O
authors.initials: RO
authors.lastname: Vogel
authors.forename: Herman G
authors.initials: HG
authors.lastname: Swarts
authors.forename: Jack
authors.initials: J
authors.lastname: Fransen
authors.forename: Ronald
authors.initials: R
authors.lastname: Roepman
authors.forename: Gijs
authors.initials: G
authors.lastname: Crienen
authors.forename: Jan A M
authors.initials: JA
authors.lastname: Smeitink
authors.forename: Leo G J
authors.initials: LG
authors.lastname: Nijtmans
authors.forename: Werner J H
authors.initials: WJ
authors.lastname: Koopman
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry; D009245:NADH Dehydrogenase / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013997:Time Factors
chemical_list: D018076:DNA, Complementary; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009247:NADH, NADPH Oxidoreductases; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I; C527690:NDUFB6 protein, human; C472512:NDUFS3 protein, human