Evaluating Cytochrome C Diffusion in the Intermembrane Spaces of Mitochondria During Cytochrome C Release

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Evaluating Cytochrome C Diffusion in the Intermembrane Spaces of Mitochondria During Cytochrome C Release 618 Research Article Evaluating cytochrome c diffusion in the intermembrane spaces of mitochondria during cytochrome c release Kieran Gillick and Martin Crompton* Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK *Author for correspondence (e-mail: [email protected]) Accepted 19 November 2007 Journal of Cell Science 121, 618-626 Published by The Company of Biologists 2008 doi:10.1242/jcs.021303 Summary Truncated Bid (tBid) releases cytochrome c from mitochondria was attributable to the permeability transition. Basal by inducing Bak (and Bax) pore formation in the outer cytochrome c diffusibility in the intermembrane spaces in the membrane. An important issue is whether a second tBid action, absence of tBid was determined to be approximately 0.2 independent of Bak and Bax, is also required to enhance minute–1, which is sufficient to support cytochrome c release cytochrome c mobility in the intermembrane spaces. To with a half-time of 3.4 minutes. It is concluded that tBid has a investigate this, we developed a kinetic analysis enabling changes monofunctional action at low concentrations and, more in the diffusibility of cytochrome c in the intermembrane generally, that the basal cytochrome c diffusibility in the spaces of isolated mitochondria to be differentiated from intermembrane spaces is adequate for rapid and complete changes resulting from Bak activation. Cytochrome c cytochrome c release irrespective of the mode of outer diffusibility in the intermembrane spaces was unaffected by membrane permeabilisation. changes in [tBid] over the range 0.5-19.0 pmol per mg of mitochondrial protein, when tBid-dependent Bak activation was increased several-thousand fold. However, high [tBid] (100 pmol Key words: Bid, Bak, Cytochrome c, Diffusion, Mitochondria, mg–1) did increase diffusibility by approximately twofold. This Apoptosis Introduction Bcl-XL and Mcl-l (Willis et al., 2005). The function of BH3-only Journal of Cell Science Mitochondria play a key role in apoptosis by releasing cytochrome Bid in this respect is greatly enhanced by its caspase-8 cleavage to c and other apoptogenic proteins into the cytosol [including form truncated Bid (tBid), in which the BH3 domain is fully exposed apoptosis-inducing factor (AIF), endonuclease G, Omi (Htra2) and and free to interact with Bax, Bak and anti-apoptotic proteins. Thus, Smac (Diablo)]. These proteins are normally retained in the death-receptor-activation of caspase-8 can lead, via tBid and Bax mitochondrial intermembrane spaces, but pass into the cytosol upon or Bak, to cytochrome c release from mitochondria, and both Bid outer membrane permeabilisation (reviewed in Martinou and Green, (Yin et al., 1999) and Bax or Bak (Zong et al., 2001) are necessary. 2001). In the cytosol, cytochrome c forms a complex (apoptosome) In addition, tBid was reported to alter the organisation of the with Apaf1 and procaspase-9 leading to caspase-9 activation and mitochondrial inner membrane independently of its BH3 domain the subsequent processing of other caspases. The release of and of Bax and Bak (Kim et al., 2004; Scorrano et al., 2002). The cytochrome c and other intermembrane space proteins is controlled restructuring is believed to enable cytochrome c release by opening by members of the Bcl2 family (reviewed in Danial and Korsmeyer, the junctions between the intracristal and intermembrane spaces, 2004). Some of these are anti-apoptotic, inhibiting permeabilisation and allowing cytochrome c to diffuse from within the intracristal of the mitochondrial outer membrane. These contain three or four spaces to outer membrane Bak and Bax pores. These changes have Bcl2 homology (BH) domains (e.g. Bcl2, Bcl-XL, Mcl-l). Other Bcl2 been attributed to opening of the inner membrane permeability relatives bring about outer membrane permeabilisation and are pro- transition (PT) pore (Alirol et al., 2006; Scorrano et al., 2002). apoptotic. The pro-apoptotics comprise proteins with multiple BH Consistent with this, release of cytochrome c from isolated domains (i.e. Bax, Bak) and proteins with only a single BH domain mitochondria was attenuated by cyclosporin A (CSA) (Karpinich (BH3) (e.g. Bid, Bad, Bik, Bim). The functional interactions et al., 2006; Scorrano et al., 2002; Zhao et al., 2003), which inhibits between single- and multi-BH-domain proteins are complex, but cyclophilin D, a component of the PT pore. In other studies, are becoming clearer. Bax and Bak oligomerise to form pores in however, cytochrome c release was unaffected by cyclophilin D the mitochondrial outer membrane large enough for the egress of inhibition (Brustovetsky et al., 2005; Eskes et al., 1998) or by cytochrome c and other intermembrane space proteins (Dejean et cyclophilin D knockout (Baines et al., 2005; Nakagawa et al., 2005). al., 2005; Korsmeyer et al., 2000; Kuwana et al., 2002). In order It is unclear, therefore, whether tBid has a secondary action in to oligomerise, Bax and Bak undergo conformational change enabling cytochrome c diffusion within mitochondria and whether brought about by BH3-only proteins (Desagher et al., 1999; Ruffolo any such action is PT-mediated. and Shore, 2003). For Bak, an integral outer membrane protein, To address this question, we need a means of measuring this first involves its displacement from inhibitory interactions with changes in the diffusibility of cytochrome c in the intracristal/ Cytochrome c diffusibility 619 intermembrane spaces during its release from mitochondria. Current A techniques do not allow this (see Discussion). Here, we describe a 1 kinetic analysis of tBid-induced cytochrome c release from isolated 15 mitochondria that differentiates basic steps in the process, including 4 8 31 cytochrome c mobility in the intermembrane spaces. The analyses 0.5 reveal that, although cytochrome c mobility is facilitated by the PT at high tBid concentrations, a substantial mobility exists even in the absence of tBid. This basal mobility is sufficient for rapid and Fraction of Cytc released 0 complete cytochrome c release at low concentrations of tBid, at 010203040 which it has no effect on internal cytochrome c mobility. [tBid] (nM) Results B 25 tBid-induced release of cytochrome c in isolated mitochondria 20 (nM) from B50 cells 15 50 Cytochrome c release was measured routinely by quantification of 10 secondary-antibody fluorescence in western blots. Preliminary 5 experiments established that the fluorescence signal was linear with tBid EC 0 cytochrome c concentration over the range used (0-60 ␮g 0102030 mitochondrial protein) and that incubation for up to 120 minutes Mitochondrial protein (μg) without tBid or Bid caused no detectable cytochrome c release. We also confirmed that cytochrome c released from the mitochondria Fig. 2. The dependence of tBid-induced cytochrome c release on mitochondrial concentration. (Top) tBid (15 nM)-induced cytochrome c was quantitatively recovered in the incubation medium. Fig. 1 release was measured with either 4, 8, 15 or 31 ␮g of mitochondrial protein compares the ability of tBid to release cytochrome c with respect to per reaction volume (20 ␮l), as indicated. Arrows show the [tBid] giving half- full-length Bid. After 30 minutes, half-maximal release was obtained maximal cytochrome c release (EC50 values). (Bottom) The EC50 values are with 10 nM tBid or 140 nM Bid. Thus, tBid was about 14-fold more plotted against mitochondrial protein per reaction. potent than Bid under these conditions, which is similar to the degree of activation on Bid cleavage reported for other cell types (generally occur exclusively via these pores (Desagher et al., 1999; Korsmeyer 10- to 20-fold) (Yin, 2006). The [tBid] giving half-maximal release et al., 2000; Kuwana et al., 2002; Ruffolo and Shore, 2003; Zong (EC50 value) depended on the amount of mitochondria. For example, et al., 2001). Whereas Bax is cytosolic and migrates to mitochondria the EC50 value increased from 3.7 nM to 22 nM as mitochondrial only during apoptosis (Capano and Crompton, 2002; Wolter et al., protein in the reaction volume (20 ␮l) was increased from 4 ␮g to 1997), Bak is constitutively mitochondrial (Griffiths et al., 1999). 31 ␮g (Fig. 2A), yielding a direct proportionality between the EC50 In agreement, isolated B50 cell mitochondria contained Bak, but not for tBid and mitochondrial protein (Fig. 2B). This indicates that Bax (Fig. 3A). Moreover, cytochrome c release was completely essentially all of the added tBid was bound to mitochondria prevented by anti-Bak antibody G-23 (Fig. 3B), which blocks Bak Journal of Cell Science (otherwise, increasing the amount of mitochondria would result in interaction with tBid and prevents Bak-mediated cytochrome c efflux a less-than-proportional increase in EC50). (Scorrano et al., 2002; Wei et al., 2000). Accordingly, kinetic analyses tBid triggers the formation of outer membrane pores from both (discussed below) of tBid-induced cytochrome c release were Bak and Bax, and tBid-induced cytochrome c efflux is believed to formulated on the basis of tBid-induced formation of Bak pores. tBid-induced change in Bak conformation Cytc Bak conformational change from the ‘closed’ to the ‘open’ form (Fig. 4) was monitored by tryptic cleavage (Ruffolo and Shore, 2003). [tBid] (nM) 0 2 4 7 10 14 20 27 50 Whereas the ‘closed’ conformation resists tryptic cleavage, a trypsin Cytc cleavage site is exposed by the conformation change, so that the [Bid] (nM) 0 63 75 90 108 130 155 187 223 268 322 387 ‘open’ Bak is digested to fragments that are unreactive with the antibody used. The technique is demonstrated in Fig. 5: mitochondria 1 were incubated with or without tBid for 30 minutes and then treated 0.8 tBid Bid with varying [trypsin] on ice for a further 10 minutes. Only tBid- 0.6 incubated samples were digested. About 50-100 ␮g trypsin ml–1 was 0.4 needed for maximal digestion; under standard conditions, 80 ␮g ml–1 0.2 Fraction of Fraction Cytc released was used.
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