title: Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts.
abstract: Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
authors.affiliation: Department of Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Federica
authors.initials: F
authors.lastname: Valsecchi
authors.forename: Claire
authors.initials: C
authors.lastname: Monge
authors.forename: Marleen
authors.initials: M
authors.lastname: Forkink
authors.forename: Ad J C
authors.initials: AJ
authors.lastname: de Groof
authors.forename: Giovanni
authors.initials: G
authors.lastname: Benard
authors.forename: Rodrigue
authors.initials: R
authors.lastname: Rossignol
authors.forename: Herman G
authors.initials: HG
authors.lastname: Swarts
authors.forename: Sjenet E
authors.initials: SE
authors.lastname: van Emst-de Vries
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Rodenburg
authors.forename: Maria A
authors.initials: MA
authors.lastname: Calvaruso
authors.forename: Leo G J
authors.initials: LG
authors.lastname: Nijtmans
authors.forename: Bavo
authors.initials: B
authors.lastname: Heeman
authors.forename: Peggy
authors.initials: P
authors.lastname: Roestenberg
authors.forename: Be
authors.initials: B
authors.lastname: Wieringa
authors.forename: Jan A M
authors.initials: JA
authors.lastname: Smeitink
authors.forename: Werner J H
authors.initials: WJ
authors.lastname: Koopman
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002461:Cell Line, Transformed; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology / Q000201:enzymology*; D004795:Enzyme Stability / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology*; D017353:Gene Deletion; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism
chemical_list: D024101:Mitochondrial Proteins; C534449:Ndufs4 protein, mouse; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D019344:Lactic Acid; D009249:NADP; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D042967:Electron Transport Complex I