NDI1 participates in apoptosis induced by various stimuli. (A, B) NDI1 is involved in Mn-induced apoptosis. NDI1 overexpression and deletion resulted respectively in reduced and increased viabilities under Mn stress, accompanied by changes of caspase-like activity. Yeast cells were grown on SGR-URA medium to a low cell density and treated for 12 h with 5 or 8 mM Mn. For each culture, a fraction was quantified for survival; another fraction was labeled for active caspase with FITC-VAD-fmk and analyzed by flow cytometry. (C) NDI1 is also involved in H₂O₂-induced apoptosis. Survival rates and active caspase signals of wild-type and ndi1 mutant treated with classic apoptosis inducer H₂O₂ at concentrations of 1.5 and 3 mM for 100 min. (D) NDI1 overexpression by itself results in apoptosis when grown in respiration-restricted media (glucose), also associated with an increase of caspase-like activity. (E) The increased caspase-like activity plays an insignificant role in the apoptosis resulting from Ndi1 overexpression. Left, survival rates of Ndi1 and vector-transformed yeast cells with or without preincubation of 400 μM Z-VAD-fmk for 30 min (before being cultured in SD-URA for 48 h). Dimethyl sulfoxide (DMSO) serves as the blank control. Right, the positive control to indicate that Z-VAD (100 μM) is functional, as it can increase cell viability when insulted by Mn. (F, G) NDI1-mediated apoptosis is independent of YCA1. NDI1 overexpression in yca1-deletion mutant similarly aggravated apoptosis, as shown by survival test and spotting assay. Survival data are presented as the mean ±SEM of three independent experiments.

Proapoptotic activity of Ndi1 is independent of its ubiquinone oxidoreductase activity. (A) Loss of ubiquinone oxidoreductase activity is not associated with change of Ndi1 apoptotic activity. Shown here are spotting assays of ndi1-mutant strain transformed with the vector, Ndi1, Ndi1^S83W, Ndi1^Q253A,D254A, Ndi1^{Q253A,D254A,R479A}, Ndi1^L217D,F258D, and Ndi1^L217D,F510D on SD-URA (respiration restricted), SGlycerol-URA (nonfermentable), and SGR-URA plates. Ndi1 overexpression inhibits cell growth in respiration-restricted media (glucose) but not glycerol or galactose/raffinose media. (B) Expression levels of several representative Ndi1 mutants are comparable to that of wild-type Ndi1, and these Ndi1 mutants are also associated with the mitochondrial membrane. (C, D) Ndi1 apoptotic activity is not dependent on ETC activity. (C) Viability testing of WT, ndi1, nde1, and petite yeast strains treated with 1 mM H₂O₂ indicates that loss of the ETC activity does not affect Ndi1-mediated apoptosis. (D) Various ETC mutants were tested for their effects on growth inhibition by Ndi1 overexpression. (E) Overexpression of Ndi1 mutants lacking oxidoreductase function also induced ROS when cultured in SD-URA, as shown by DHR123 staining with flow cytometry. Survival data are presented as the mean ±SEM of three independent experiments.

Ndi1 translocates from mitochondria to cytoplasm during apoptosis. Ndi1-RFP is overexpressed, whereas Ndi1-TAP is under the endogenous regulation. (A) Ndi1-RFP maintains the proapoptotic function of Ndi1. (B) Ndi1-RFP is localized in the mitochondria under normal growth conditions but moves out during apoptosis. Om45-GFP is used to indicate mitochondria. Mn is used for apoptosis induction. (C, D) Ndi1-TAP colocalizes with mitochondrial inner membrane protein CoxIV, as detected by immunofluorescence and Western blot. (E) NDI1-TAP exhibited normal NADH dehydrogenase function like wild-type NDI1. It can use glycerol well. (F) Ndi1-TAP also shows similar apoptotic activity to the wild-type Ndi1. Bar, 1 μm.

Ndi1 is cleaved in mitochondria during apoptosis. (A) Ndi1 is cleaved when treated with Mn. NDI1-TAP yeast was treated or not treated with 8 mM Mn for 12 h, and the extracts were immunoblotted and probed with a TAP antibody. (B) Ndi1 cleavage occurs in the mitochondria. Both the full-length mature form of Ndi1 and the truncated Ndi1 were observed in the mitochondrial fraction, whereas only the truncated Ndi1 was found in the postmitochondrial fraction. Pgk1 served as the cytoplasm loading control, CoxIV as the mitochondrial loading control, and Idh1 as the matrix protein control. (C, D) The cleavage patterns of Ndi1 vs. treatment time and Mn concentration. (E, F) The survival rates of stressed NDI1-TAP yeast in C and D. (G, H) Ybh3 does not affect the apoptotic activity or release of Ndi1. (G) The proapoptotic activity of Ndi1 in the ybh3-deletion mutant tested by spotting assay. (H) The release of Ndi1 in NDI1-TAP,ybh3Δ background. Representative blots from at least three independent experiments are presented.

The N-terminal likely serves normally a protective role. (A) Cytosol-NDI1 (Ndi1ΔN1-27) displays much reduced toxicity as compared with normal NDI1. (B) A Western blotting control showing that cytosol-NDI1 was expressed similarly. (C) The N-terminal 40 or so amino acids of the mature Ndi1 are protective for Ndi1 toxicity. Without the first 45 (28–72) amino acids, Ndi1 (Ndi1ΔN1-72) becomes fully toxic when expressed in the cytoplasm. (D) The C-terminal of Ndi1 is required for Ndi1 toxicity. (E) A summary of the proapoptotic activities of various Ndi1 mutants. MLS, mitochondria localization signal; TMD, transmembrane domain; +, level of proapoptotic activity; –, no proapoptotic activity. Representative blot and spotting assay results from at least three independent experiments are presented.

Model for the molecular mechanism of Ndi1’s dual actions. Ndi1 normally initiates electron transport from NADH to the electron transport chain. During stress, its normal function changes, and its N-terminal is cleaved. The cleaved Ndi1 then translocates from the mitochondria to the cytoplasm to provoke apoptosis.