Vitamin K2 Is a Mitochondrial Electron Carrier That Rescues Pink1 Deficiency

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Science  08 Jun 2012:
Vol. 336, Issue 6086, pp. 1306-1310
DOI: 10.1126/science.1218632

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  1. Fig. 1

    Identification of heix as modifier of pink1B9. Enhancement of pink1B9 phenotypes by heterozygosity for heix (pink) compared with pink1RV controls heterozygous for heix (black) and rescue with UAS-heix (k11403/+;da>heix); flight (n > 50) (A), ATP levels (n = 10 assays) (B), and Ψm measurements using the potentiometric dye JC-1. Red aggregate/green monomeric fluorescence ratio measured at third instar neuromuscular boutons; aggregates accumulate at negative Ψm (n = 20 synapses) (C). Suppression of pink1B9 and park1/Δ21 phenotypes by overexpression of Heix (da>heix); flight (n > 50) (D), ATP levels (n = 10 assays) (E), and JC-1 red/green fluorescence ratio (n = 20 synapses) (F). Data are percentage [(A) and (D)] and mean normalized to control [(B), (C), (E), and (F)]. Error bars indicate SEM. Analysis of variance (ANOVA)/Dunnett: *P < 0.05; **P < 0.01; ***P < 0.001.

  2. Fig. 2

    Heix is involved in vitamin K2 production. (A) Improved survival of first instar heix mutants placed on MK-4, on ubiquinone (Q10), or on menadione (black) shown as the difference in the percentage of control larvae that survive on control medium (n = 10 experiments, each with 10 larvae); that is, about 20% more heix mutant larvae survive with vitamin K2 compared with heix mutants on control medium. Error bars, SEM. ANOVA Dunnett: *P < 0.05; **P < 0.01. ns, not significant. (B) Schematic of the biochemical conversion of menadione to vitamin K2.

  3. Fig. 3

    Vitamin K2 rescues pink1B9 mutant phenotypes. Flying ability of pink1RV (A) and pink1B9 (B) and ATP levels in pink1RV and pink1B9 flies (C) that were placed on control medium (black), on medium with E. coli that produce vitamin K2 (MK-8, yellow), or on medium with menA mutant E. coli that do not produce vitamin K2 (green) or with menA mutant E. coli transformed with wild-type menA that produce vitamin K2 (blue) (fig. S4). Flying ability (D and E), ATP levels (F), Ψm at neuromuscular boutons determined by using JC-1 red/green ratio (G), and mitochondrial morphology in third instar larval muscles (H to J) in control pink1RV and in pink1B9 on MK-4 containing medium (gray) or control medium (black) [(D) to (I)] or upon overexpression of Heix (da>heix) (J). The amount of rounded mitochondria is quantified (H). Arrows are normal mitochondrial structure in (I) and (J). Error bars, SEM. n > 50 flies, and data are percentages in (A), (B), (D), and (E); 10 assays in (C) and (F); 20 synapses or muscles in (G) to (J); and mean normalized to control in (C) and (F) to (H). ANOVA/Dunnett: *P < 0.05; **P < 0.01; ***P < 0.001.

  4. Fig. 4

    Vitamin K2 acts as a mitochondrial electron carrier. Immunolabeling of S2 Drosophila cells (A) and Western blot of fractionations (B) transfected with Heix-His and labeled with anti-His and anti–ATP synthase β (A). (C) ATP levels of heix1/Df and heix/Df;da>heix (n = 8). (D) Time-dependent ATP production in mitochondria isolated from heix1/Df mutant larvae (n = 8). (E) Measurements of complex II activity [normalized to citrate synthase activity (CS)] using a dose-response of MK-4 as electron carrier (n = 4). (F) Measurements of complex II activity using ubiquinone (0.13 mM; Q-10, light gray) or MK-4 (0.35 mM, gray) as electron carrier (n = 4). (G) Time-dependent ATP production in mitochondria isolated from pink1B9 mutant flies. Reactions are either not supplemented with MK-4 (black) or supplemented with 0.15 mM (dark gray) or 0.35 mM MK-4 (light gray) (n = 8). (H) Adenosine diphosphate–stimulated complex I–driven respiration rate (oxygen consumption) in mitochondria isolated from pink1RV controls and pink1B9 mutants placed for 72 hours on MK-4 medium or on control medium. Mitochondria from vitamin K2-fed mutant flies consume oxygen faster. Data are mean normalized to controls in (C) and (H) or mean in (D) to (G). Error bars, SEM. ANOVA/Dunnett: *P < 0.05, **P < 0.01, ***P < 0.001.