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. 2015 Aug 21;290(34):20815-20826.
doi: 10.1074/jbc.M115.641498. Epub 2015 Jun 10.

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)

Mahmoud M Elguindy  1 , Eiko Nakamaru-Ogiso  2
Affiliations

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)

Mahmoud M Elguindy et al. J Biol Chem. .

Abstract

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O₂ activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC₅₀ = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O₂ activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O₂ activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells.

Keywords: apoptosis; apoptosis-inducing factor; complex I; mitochondrial metabolism; mitochondrial respiratory chain complex; nicotinamide adenine dinucleotide (NADH); rotenone; ubiquinone.

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Figures

FIGURE 1.
FIGURE 1.
A, phylogenic tree of AIF/AMID proteins and type 2 NADH:quinone oxidoreductases. (The sequences used are as follow: Arabidopsis thaliana (A.t.), AMID (NP_563783); C. elegans (C.e.), Wah-1 (NP_499564); Drosophila melanogaster (D.m.), AIF (NP_001259907); E. coli (E.c.), Ndh (NP_415627); Homo sapiens (H.s.), AIF (NP_001124318); H. sapiens, AMID (NP_001185625); Mus musculus (M.m.), AIF (NP_001277293); M. musculus, AMID (NP_001034283); S. cerevisiae (S.c.), Ndi1 (NP_013586); S. cerevisiae, Nde1 (NP_013865); S. cerevisiae, AIF (NP_014472); and Y. lipolytica (Y.l.), Nde1 (XP_504691). B, NADH-O2 activities of wild-type, ΔnuoB, Δndh, and Ndi1- or AMID-overexpressed membranes in Δndh in the presence (solid bars) or absence (open bars) of piericidin A. 5 μl of membrane vesicles were used for the assay. Values are shown as means ± S.D. (n = 3).
FIGURE 2.
FIGURE 2.
A, SDS-PAGE analysis of purified Ndi1, AMID, and AIF proteins. Ndi1 (20 μg, lane 1), AMID (15 μg, lane 2), C-terminally tagged AIFΔ1–101 and AIFΔ1–53 (20 μg, lane 3; 15 μg, lane 4), and N-terminally tagged AIF Δ1–101 and Δ1–53 (10 μg, lane 5; 10 μg, lane 6) were loaded onto a 10% Laemmli SDS-polyacrylamide gel. B, UV-visible absorption spectra of purified Ndi1, AMID, C-terminally tagged AIFΔ1–53 and AIFΔ1–101, and N-terminally tagged AIFΔ1–53 and AIFΔ1–101. The UV-visible spectra were taken in 50 mm Tris-HCl (pH 8.0) containing 10% glycerol and 200 μm EDTA. All the data were shown normalized at 1 mg/ml (black, Ndi1; red, AMID; thick blue, C-terminally tagged AIFΔ1–53; thin blue, C-terminally tagged AIFΔ1–101; broken blue, N-terminally tagged AIFΔ1–53; dotted blue, N-terminally tagged AIFΔ1–101).
FIGURE 3.
FIGURE 3.
A, NADH-O2 activities of Ndi1, AMID, and AIF proteins reconstituted in double knock-out (DKO) membranes. 5 μl of reconstituted membranes were used for the assay. Values are means ± S.D. (n = 3). B, proton pumping activities in the DKO membrane vesicles reconstituted with isolated Ndi1 (panel a), AMID (panel b), C-terminally tagged (panel c), and N-terminally tagged (panel d) AIF proteins. Generation of a proton gradient across the membranes was monitored by the quenching of ACMA fluorescence. At the time indicated by arrowheads, 200 μm NADH or 1 μm gramicidin was added to the assay mixture. Line 1, DKO membrane alone; line 2, Ndi1-reconstituted; line 3, AMID-D285N; line 4, AMID-D285E; line 5, AMID; line 6, C-terminally tagged AIFΔ1–53; line 7, C-terminally tagged AIFΔ1–101; line 8, N-terminally tagged AIFΔ1–53; line 9, N-terminally tagged AIFΔ1–101; line 10, N-terminally tagged AIFΔ1–53-ΔR201; line 11, N-terminally tagged AIFΔ1–53-D444N; line 12, DKO membrane.
FIGURE 4.
FIGURE 4.
Effect of the quinone analog inhibitor HQNO on NADH-UQ1 activities by Ndi1 (A), AMID (B), and N-terminally tagged AIFΔ1–53 (C) in the reconstituted DKO membranes. The control values (n = 3) for Ndi1, AMID, and N-terminally tagged AIFΔ1–53, were 23.4, 7.52, and 6.77 μmol/min/mg, respectively.
FIGURE 5.
FIGURE 5.
Deamino-NADH is a poor substrate for Ndi1, AMID, and N-terminally tagged AIF-mediated proton pumping activities. The DKO membrane vesicles were reconstituted with isolated Ndi1 (A), AMID (B), and AIF (C) proteins. The reaction was started with the addition of 200 μm NADH (black), 100 μm dNADH (red), or 100 μm NADPH (blue). 5, 20, and 20 μl of 1:10 reconstituted mixture of Ndi1, AMID, and N-terminally tagged AIF, respectively, were added to the reaction mixture.
FIGURE 6.
FIGURE 6.
Growth curves of E. coli DKO cells overexpressing Ndi1, AMID, or AIF proteins. A, blue, Ndi1; red, AMID; green, AMID-D285N; dashed line, empty pET16b. B, blue, Ndi1; red, N-terminally tagged AIFΔ1–53; green, C-terminally tagged AIFΔ1–53; dashed line, empty pET16b. C, red, N-terminally tagged AIFΔ1–53; green, N-terminally tagged AIFΔ53-D444N; green with open circles, N-terminally tagged AIFΔ1–53-ΔR201; dashed line, empty pET28b. D, blue, Ndi1; red, N-terminally tagged AIFΔ1–53; red with open circles, N-terminally tagged AIFΔ1–101; dashed line, empty pET28b. The arrows indicate the addition of 0.5 mm IPTG.
FIGURE 7.
FIGURE 7.
A, proton pumping activities in bovine heart SMP reconstituted with isolated Ndi1 (panel a), AMID (panel b), or N-terminally tagged AIFΔ1–53 (panel c). 10 μl of 1:10 reconstituted mixture of Ndi1, AMID, or AIF were added to the reaction mixture. At the time indicated by arrowheads, 200 μm NADH or 1 μm gramicidin were added to the assay mixture. Thick black line, SMP; thin black line, SMP + 0.1 μm rotenone; blue line, SMP + isolated proteins (panel a, Ndi1; panel b, AMID; panel c, N-terminally tagged AIFΔ1–53); red thick line, SMP + 0.1 μm rotenone + isolated proteins (panel a, Ndi1; panel b, AMID; panel c, N-terminally tagged AIFΔ1–53); red thin line, SMP + 0.5 μm rotenone + isolated proteins (panel a, Ndi1; panel b, AMID; panel c, N-terminally tagged AIFΔ1–53). B, membrane potentials generated by the addition of NADH in bovine heart SMP reconstituted with Ndi1, AMID, and N-terminally tagged AIFΔ1–53. SMP alone (panel a) SMP + Ndi1 (panel b) SMP + AMID (panel c) SMP + N-terminally tagged AIFΔ1–53 (panel d). Membrane potentials were measured in the absence (blue dotted line) or presence (red dotted line) of 0.1 μm rotenone. 10 μl of 1:10 reconstituted mixture of Ndi1, AMID, and AIF were added in the reaction mixture. At the time indicated by arrowheads, 200 μm NADH was added to the assay mixture. The representative of three measurements is shown in the figure.
FIGURE 8.
FIGURE 8.
NADH:O2 activities of Ndi1 and N-terminally tagged AIFΔ1–53 proteins reconstituted in mouse liver mitoplasts. 2.5 μl of reconstituted mitoplasts were used for the assay. Values are means ± S.D. (n = 3). NADH:O2 activities were measured without inhibitors (black bars), in the presence of 0.1 μm rotenone (Rot, open bars), or 1 mm KCN (hatched bars).
FIGURE 9.
FIGURE 9.
Effect of rotenone on NADH:O2 (A) and proton pumping activities (B) in DKO reconstituted with Ndi1 (panel a), AMID (panel b), or N-terminally tagged AIFΔ1–53 (panel c). Black (bars and lines), red, (bars and lines), and blue (bars and lines) indicated that NADH-O2 or proton pumping activities were measured in the presence of 0, 0.1, or 0.5 μm rotenone, respectively. The measurements were repeated at least three times.
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