Figure 1
From: The porin VDAC2 is the mitochondrial platform for Bax retrotranslocation
Bax retrotranslocates from isolated mitochondria.
(A) Retrotranslocation of endogenous Bax from isolated HCT116 mitochondria is not accompanied by the movement of the intermembrane space proteins cytochrome c (cyt c) and Smac or the OMM-associated and OMM-integral proteins GAPDH, Tom20 and VDAC into the supernatant (right). The mitochondrial sample prior retrotranslocation served as control (CM). The retrotranslocated Bax pool in the supernatant after 60 min (CS) is compared to mitochondria. VDAC serves as fractionation control. n = 4. (B) Retrotranslocation of endogenous Bax. Remaining Bax (●) or VDAC (◯) on isolated HCT116 mitochondria relative to protein levels at 0 min. (C) Level of endogenous Bax (●) and GFP-Bax (◯) retrotranslocated from isolated mitochondria into the supernatant shown in Figure S1. Data is presented relative to the shuttled Bax pool at 60 min. (D) Endogenous Bax retrotranslocation with (top) and without (bottom) the apoptotic stimulus staurosporine (STS, 1 μM). Supernatants (left) and corresponding mitochondria (right) for indicated time points (top) with STS are compared to samples after 0 min. (C0) and 60 min. (C60) of samples without STS and vice versa. Mitochondria (CM) prior retrotranslocation served as control for the supernatant and supernatant after 60 min (CS) is compared with the mitochondria. VDAC serves as fractionation control. n = 3. (E) GFP-Bax retrotranslocation determined by fluorescence spectrometry in the presence (◯, red) or the absence (●, black) of STS. Bax retrotranslocation is displayed relative to the Bax shuttled in the absence of STS after 60 min. Data represent normalized averages ± SEM. n = 3. (F) OMM permeabilization of mitochondria isolated from HCT116 Bax/Bak DKO cells with or without endogenous Bax retrotranslocated from mitochondria (HCT116 WT cells) with or without tBid. VDAC serves as loading control. n = 3. (G) Cross-linking of endogenous Bax present on isolated mitochondria after incubation for up to 60 min at 37 °C in the presence of 0.5 mM DSG with and without tBid. In the absence of tBid mitochondrial Bax (Bax (M)) including a larger protein complex (C) is diminished and retrotranslocated Bax (Bax (S)) increased, while tBid induced a Bax complex (probably Bax dimers, D, roughly the size of 2x Bax monomers, M). Tom20 serves as loading control. n = 4.
