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First published online 5 February 2008
doi: 10.1242/jcs.021303

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Evaluating cytochrome c diffusion in the intermembrane spaces of mitochondria during cytochrome c release

Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (11K): [in this window] [in a new window]   Fig. 1. Bid- and tBid-induced cytochrome c release from B50 cell mitochondria. Mitochondria were incubated for 30 minutes with either Bid or tBid, as indicated, and then sedimented. The mitochondrial pellets were analysed for cytochrome c in western blots using fluorescent secondary antibodies as shown. (Graph) Bands were quantified by fluorescence imaging and fractional cytochrome c release calculated (where 0-1 indicates 0-100% release). Cytc, cytochrome c.

View larger version (7K): [in this window] [in a new window]   Fig. 2. The dependence of tBid-induced cytochrome c release on mitochondrial concentration. (Top) tBid (15 nM)-induced cytochrome c release was measured with either 4, 8, 15 or 31 µg of mitochondrial protein per reaction volume (20 µl), as indicated. Arrows show the [tBid] giving half-maximal cytochrome c release (EC50 values). (Bottom) The EC50 values are plotted against mitochondrial protein per reaction.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Isolated B50 cell mitochondria contain Bak, but not Bax; inhibition of tBid-induced cytochrome c release by anti-Bak antibodies. (A) Mitochondria and whole B50 cells, each containing 20 µg protein, were analysed in western blots developed with anti-Bak and anti-Bax antibodies. (B) Mitochondria (150 µg protein) were pre-incubated with or without anti-Bak G-23 antibodies (1 µg) for 30 minutes. tBid was added and mitochondrial cytochrome c determined at the times indicated (graph).

View larger version (3K): [in this window] [in a new window]   Fig. 4. Scheme of tBid-induced Bak-mediated cytochrome c release. In the inactive state, Bak assumes a closed conformation, designated Bak. tBid induces a change in conformation to an open conformer, Bak*. Open conformers self-assemble into an outer membrane pore [Bak*]n through which cytochrome c permeates. Bak is also activated autocatalytically by Bak*. The rate constants for tBid-induced and autocatalytic Bak* formation are c (minute–1) and f (minute–1), respectively. The rate constant for cytochrome c permeation from the intracristal/intermembrane spaces to the external medium is a (minute–1).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Rat mitochondrial Bak is digested by trypsin after tBid treatment, but mitochondrial cytochrome c is resistant. (A) Mitochondria were incubated with or without 5 nM tBid for 30 minutes and then with the indicated concentration of trypsin at 0°C for 10 minutes. Western blots were probed with anti-Bak antibody that only recognises uncleaved Bak. (B) The Bak signal was used to calculate the level of undigested Bak. (C) Mitochondria were incubated with or without 5 nM tBid for 9 minutes (yielding partial release of cytochrome c) and then with trypsin, as indicated, for 10 minutes at 0°C. Mitochondria were sedimented and their cytochrome c analysed in immunoblots.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Kinetic analyses of Bak activation and cytochrome c efflux at two [tBid]. (A) The immunoblots show mitochondrial cytochrome c and trypsin-resistant Bak following the addition of either 15 or 0.5 nM tBid to mitochondria at zero time. (B) The blots were used to determine the time courses of Bak conformation change (black symbols) and cytochrome c release (white symbols). Circles, 15 nM tBid; triangles, 0.5 nM tBid. The Bak data were best-fitted according to equation 1 (see Materials and Methods) by the curves shown, yielding the c and f values given (C; f values constrained equal). These values were then used to derive the best fits for the cytochrome c data according to equation 2 (see Materials and Methods) to yield the curves shown and the a values given.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Saline washing of mitochondria selectively suppresses autocatalysis of Bak conformation change. (A) Conventional (designated `unwashed') and saline-washed (`washed') mitochondria were prepared from the same cell homogenate. tBid-induced Bak conformation change (black symbols) and cytochrome c release (white symbols) were measured as in Fig. 6. The Bak data were best-fitted according to equation 1 (see Materials and Methods) to yield the f and c values given. These values were then used to obtain the best fits for the cytochrome c data according to equation 2 (see Materials and Methods) to give the curves shown and the a values given. (B) SDS-PAGE gels (Coomassie stained) and Bak immunoblots of the washed and unwashed mitochondrial preparations. Arrows show bands that were reduced in the washed preparation.

View larger version (8K): [in this window] [in a new window]   Fig. 8. The dependence of cytochrome c diffusibility and Bak conformation change on [tBid]. (A,C) The rate constants a and c at varying [tBid] were evaluated in a single mitochondrial preparation by the two-step procedure used in Fig. 6, but with 1 µM Ca2+ buffer (5 mM EGTA/4 mM Ca2+). (B) The basal values of the cytochrome c diffusibility constant a were determined in five mitochondrial preparations by extrapolation of lines (as in A) to zero [tBid]; mean ± s.e.m.

View larger version (12K): [in this window] [in a new window]   Fig. 9. The PT pore increases diffusibility of intermembrane space proteins but is not involved in the action of tBid at low concentrations. CyP-D(+) mitochondria were used throughout. (A) Calcein-loaded mitochondria were incubated in SRM containing either 400 µM Ca2+ (400 Ca) with/without 1 µM CSA, or 1 µM buffered free Ca2+ (1 Ca) with/without 20 nM tBid. The Ca2+ buffer comprised 5 mM EGTA and 4 mM CaCl2. After incubation for the time specified, mitochondria were sedimented and calcein in the supernatant was determined fluorometrically. (B) Calcein-loaded mitochondria were incubated in SRM containing 400 µM Ca2+, 1 µM free Ca2+ and 20 nM tBid, as indicated. Cytochrome c release was measured at the times specified. The data of A and B are representative of three such experiments. (C) The two-step procedure was used to determine the diffusibility rate constants for cytochrome c (white columns) and Smac (black columns) in the presence of 20 nM tBid. Other additions were 400 µM Ca2+, 1 µM buffered free Ca2+ and 1 µM CSA, as indicated. Means ± s.e.m. (four experiments).

View larger version (9K): [in this window] [in a new window]   Fig. 10. High concentrations of tBid induce the PT and increase cytochrome c diffusibility in the intermembrane spaces. CyP-D(+) mitochondria were used and were saline-washed. All experiments were conducted with 1 µM buffered free Ca2+. Additions: 20 nM tBid, 100 nM tBid, 200 nM tBid and 1 µM CSA, as indicated. (A) Calcein-loaded mitochondria were incubated in SRM; other details as in Fig. 9A. Data are representative of three such experiments. (B) The two-step procedure was used to determine the diffusibility constant for cytochrome c. Means ± s.e.m. (four experiments).

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