Bax releases cytochrome c preferentially from a complex between porin and adenine nucleotide translocator. Hexokinase activity suppresses this effect.

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1 Bax releases cytochrome c preferentially from a complex between porin and adenine nucleotide translocator. Hexokinase activity suppresses this effect. Mikhail, Y. Vyssokikh 1, Ljubava Zorova 1, Dmitry Zorov 1, Gerd Heimlich 2, Juliane, M. Jürgensmeier 2 and Dieter Brdiczka 3 1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University 2 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne 3 Department of Biology, University of Konstanz D Konstanz Keywords: mitochondria, structure, apoptosis, growth factor, protein kinase B/Akt Correspondence: Dieter.Brdiczka@uni-konstanz.de Abstract The mechanism by which external Bax releases cytochrome c is still controversial and may also depend on the type of mitochondria and the actual localisation of cytochrome c. Outer membrane porin acquires high binding affinity for hexokinase by interacting with the adenine nucleotide translocator (ANT) in the contact sites. (I) The hexokinase protein was thus used as a tool to isolate the contact site forming complex between outer membrane porin and inner membrane ANT from a TritonX100 extract of brain membranes. (II) A significant amount of cytochrome c was co-purified with the isolated hexokinase porin ANT complexes that were reconstituted in phospholipid vesicles. Bax-DC released the endogenous cytochrome c from the vesicles without forming unspecific pores. This was shown by loading the vesicles with malate that was not liberated by Bax-DC. (III) The Bax-DC effect was dependent on a specific association of cytochrome c with the porin ANT complex, as dissociation of the complex by bongkrekate abolished the Bax dependent cytochrome c liberation. (IV) The Bax-DC effect was as well suppressed by hexokinase phosphorylating glucose. Introduction In studies of Bax dependent cytochrome c release so far, cytochrome c compartmentation in mitochondria has not been considered. Based on the new mitochondrial model (Mannella et al. 1997), we suggest that different fractions of cytochrome c associated to different membrane areas of the inner membrane have to be regarded such as cristae membranes (CM), inner boundary membrane (IBM) and contact sites in addition (Fig 3 appendix). Energy transferring contact sites are formed by interaction between porin and the ANT (Vyssokikh et al. 2001). Porin being in a complex with ANT, exerts higher affinity for hexokinase (Wicker et al. 1993) compared to porin beyond the contact sites. Hexokinase remains attached to porin and ANT in a Triton extract of mitochondrial membranes from rat brain (Beutner et al. 1996). The enzyme could be used as an instrument to isolate the porin ANT complexes from the Triton extract by anion exchange chromatography. The complexes were reconstituted in phospholipid vesicles. Hexokinase was functionally coupled to the ANT, phosphorylating external glucose by utilising internal ATP (Beutner et al. 1996). It was observed that cytochrome c was co-purified with the isolated complexes. Recent investigations showed that Bax and hexokinase compete for the same binding sites in hepatocyte mitochondria (Pastorino et al. 2002). Furthermore, protein kinase B linked suppression of cytochrome c release and apoptosis was found to depend on the activity of mitochondrial bound hexokinase (Gottlob et al. 2001). In the light of these results we studied whether the complex bound cytochrome c could be the cytochrome c pool preferentially targeted by Bax. Methods Isolation of hexokinase porin ANT complexes from brain membranes: The method was performed essentially as described recently (Beutner et al. 1996) and is schematically shown in Fig 3 appendix.

2 Bax-DC Bax truncated of the hydrophobic C-terminal domain was used in the experiments. Reconstitution of hexokinase porin ANT complexes A mixture of phosphatidyl-cholin and 2% cholesterol was used for reconstitution. The phospholipid vesicles were mixed with 5 ml complex fraction and 0.3% n-octyl-ß-d-glucoside for 20 min at room temperature, followed by overnight dialysis at 4 o C against 125 mm sucrose and 10 mm Hepes, ph 7.4. The vesicles were loaded with 10 mm KCl and 5 mm malate by sonification and subsequent chromatography on Sephadex G 60 in 125 mm sucrose and 10 mm Hepes ph 7.4. Permeability for malate was tested. by centrifugation of the vesicles for 45 min. at 400,000 g and determination in the supernatant and sediment. Results Cytochrome c is a component of the hexokinase porin ANT complex Significant amounts of cytochrome c were co-purified during two isolation and reconstitution steps of the hexokinase porin ANT complex (Fig 3A and B appendix). As determined by spectroscopic analysis of the vesicle suspension (Fig 3C appendix), the endogenous cytochrome c was present in nm concentration. Cytochrome c release by Bax-Dc without pore formation Up to 80% of the endogenous cytochrome c in the proteoliposomes was released by Bax-DC in a concentration range between 500 and 1000 nm. At the same time malate, enclosed in the vesicles, was not liberated (Fig 1A). Thus cytochrome c was not released through pores formed by Bax-DC but was displaced from specific sites in the porin ANT complex. After dissociation of the complex by bongkrekate (BA) the Bax-DC effect was completely suppressed (Fig 1B) Figure 1 Bax dependent release of endogenous cytochrome c in the hexokinase porin ANT complex A: The vesicles containing reconstituted complexes were loaded with malate and KCl. Release of the entrapped malate and of endogenous cytochrome c was measured after incubation of the vesicles at 25 C for 30 min with increasing concentrations of Bax-DC in 125 mm sucrose, 10 mm Hepes ph 7. Mean & s.e.m. of three experiments with independent complex preparations. B: The experiment was performed as in A except that in some samples 250 µm bongkrekate was present. In the samples treated with bongkrekate cytochrome c was determined in the supernatant (BA sup) and sediment (BA sed) after centrifugation. Mean & s.e.m. of two experiments with two independent complex preparations.

3 Hexokinase activity suppresses the Bax-Dc effect Bax and hexokinase compete for the same binding sites (Pastorino et al. 2002). ADP produced by active hexokinase stabilises porin ANT complexes (Bücheler et al. 1991) and increases the enzyme binding to the mitochondrial surface. Therefore, Bax-DC was unable to displace hexokinase and to liberate cytochrome c from the reconstituted hexokinase porin ANT complex in the presence of hexokinase substrates (Fig 2A). However, when hexokinase was inhibited and detached by glucose-6-phosphate, Bax-DC released endogenous cytochrome c at lower concentrations compared to the control (Fig 2B). Figure 2 Influence of hexokinase activity on the Bax dependent release of endogenous cytochrome c A: The experiment was performed as in A but 2 mm ATP and glucose were added to the samples. B: The experiment was performed as in A without ATP and glucose. In some samples 5 mm glucose- 6- phosphate (G-6-P) was present. Cytochrome c was determined in supernatant. The results are typical for three independent experiments with three independent complex preparations. Discussion Here we show that hexokinase porin ANT complexes contain significant amounts of cytochrome c. This cytochrome c appears to be bound in the porin ANT complex because it is not detached by 200 mm KCl, that was used to elute the complex from the DE-cellulose column (Fig 3A, appendix). The binding sites for cytochrome c may be provided by cardiolipin which is tightly bound to the ANT (Beyer & Klingenberg 1988). Thus, a different arrangement of the complex associated cytochrome c was assumed explaining why it could be released by Bax without forming pores in the vesicle membranes (Fig 1A). The Bax action on endogenous cytochrome c could be suppressed in two ways: either by dissociation of the porin ANT complex by bongkrekate (Fig 1B) or induction of the complex and intensifying hexokinase binding (Fig 2A). Both effects are explained by supposing that Bax like hexokinase binds with high affinity (Pastorino et al. 2001) to a specific porin structure exposed in the porin ANT complexes. In summary, we show the existence of a cytochrome c fraction organised in mitochondrial contact sites. This cytochrome c was accessible to external Bax-DC and may be physiologically released as the first apoptotic signal without pore formation or disruption of the outer membrane. It appears plausible that cytochrome c release and apoptosis are inhibited as long as mitochondrial function can provide ATP for glucose phosphorylation and cell survival.

4 References: Beutner, G., Rück, A., Riede, B., Welte, W., Brdiczka D. (1996) Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. FEBS Lett 396: Beyer, K., Klingenberg, M. (1985) ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by 31P nuclear magnetic resonance Biochemistry 24, Bücheler, K., Adams, V., Brdiczka, D. (1991) Localization of the ATP/ADP translocator in the inner membrane and regulation of contact sites between mitochondrial envelope membranes by ADP. A study on freeze fractured isolated liver mitochondria Biochim Biophys Acta 1061, Gottlob, K., Majewski, N., Kennedy, S., Kandel, E., Robey, RB., Hay, N. (2001) Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase Genes Dev. 15, Mannella, C. Marko, M. Buttle, K. (1997) Reconsidering mitochondrial structure: new views of an old organelle. TIBS 22, Pastorino JG, Shulga N, Hoek JB (2002) Mitochondrial Binding of Hexokinase II Inhibits Bax-induced Cytochrome c Release and Apoptosis J Biol. Chem 277, Vyssokikh, MY., Katz, A., Rück, A., Wünsch, C., Dörner, A., Zorov, DB. Brdiczka, D. (2001) Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilind. Biochem J (2001) 358, Wicker, U., Bücheler, K., Gellerich, FN., Wagner, M., Kapischke, M., Brdiczka, D. (1993) Effect of macromolecules on the structure of the mitochondrial inter-membrane space and the regulation of hexokinase Biochim Biophys Acta 1142,

5 Appendix Methods: Cytochrome c determination Cytochrome c was determined by difference spectroscopy according to fundamental work of Keilin and Margoliash. We used the a band of the spectrum at 550 nm after reduction of the cytochrome by Na-dithionite. Bax-DC preparations The Bax truncated of the hydrophobic C-terminal domain (Bax-DC) used in the experiments shown in Fig 1 A,B was a gift of Bruno Antonsson, Serono Geneva. It was purified as described by Antonsson et al The purified protein was stored at 80 C in 25 mm Hepes, 0.2 mm DTT, 30% glycerol (v/v), ph 7.5. Recombinant Bax-DC protein used in the experiments shown in Fig. 2 A,B was prepared according to Xie et al with slight modifications. Figure 3 Isolation and reconstitution of a complex between hexokinase, porin, and adenine nucleotide translocator (ANT) Rat brain membranes were extracted as described in Methods with 1% Triton X-100. Not dissolved material was removed by centrifugation and the supernatant was bound to DE- Cellulose. A: Hexokinase was eluted from the DE-Cellulose column between 100 and 300 mm KCl together with significant amount of cytochrome c. B: The porin ANT complex bound to hexokinase activity was reconstituted in phosphatidyl-cholin cholesterol vesicles that were loaded with 5 mm malate. The vesicles were run through a Sephadex G 60 column. C: Spectroscopic identification of reduced cytochrome c by the a band at 550 nm in the vesicle fraction containing the reconstituted complex. Results Preparation of a hexokinase porin ANT complex containing cytochrome c About 15% of the total cytochrome c in brain and kidney mitochondria are found in the contact sites. At these high affinity binding sites, hexokinase forms tetramers (Beutner et al. 1997) It can thus be used as an instrument to isolate the ANT porin complex from a Triton extract of brain membranes (Beutner et al. 1996). Hexokinase in the Triton extract was bound to an anion exchanger and could be eluted by a KCl gradient. Figure 3A shows the elution profile of hexokinase

6 activity from a DE-cellulose column. When cytochrome c was determined in the enzyme activity fractions, we observed that a significant amount of cytochrome c was eluted together with the hexokinase. In previous investigations it has been observed that hexokinase in these fractions was still linked to porin and the ANT (Beutner et al. 1996, Vyssokikh et al. 2001). The hexokinase porin ANT complexes were reconstituted in phospholipid vesicles. After reconstitution, the vesicles were loaded with malate and KCl by sonification (Beutner et al. 1998). The external malate and KCl was removed by size exclusion chromatography on Sephadex G 60. In the excluded fraction, cytochrome c and hexokinase activity were co-migrating (Fig 3B). As determined by spectroscopic analysis (Fig 3C), the endogenous cytochrome c in the vesicle suspension was present in nm concentration. Figure 4 Different mitochondrial fractions of cytochrome c. Considering the new mitochondrial model obtained by electron microscopic tomography (Mannella et al. 1997, Perkins et al. 1997) various fractions of cytochrome c (c) can be identified by binding to different membrane areas of the inner membrane and by different function. We understand that the cristae (CM) are connected to the inner boundary membrane (IBM) by tubules. Thus a chain of cytochrome c molecules transfers the electrons through the tubules, to cytochrome oxidase in the crista membrane (white arrow). The cytochrome c at the IBM surface accepts the electrons either from the rotenone insensitive NADH oxidase in the outer membrane (OM) through cytochrome b5 or from the rotenone sensitive NADH oxidase through the bc1 complex III (Bernardi & Azzone 1981). Bax releases the peripheral cytochrome c located at the IBM surface and in the contact sites. However, it may not directly effect cytochrome c at the CM. FeS = iron sulfur protein, SDH = succinate dehydrogenase, Mal = malate, Glut = glutamate, OxAc = oxaloacetate, KG = ketoglutarate, Suc = succinate, Fum = fumarate. Discussion Localisation and function of different cytochrome c fractions We suggest that different fractions of cytochrome c associated to different membrane areas of the inner membrane have to be regarded such as cristae membranes (CM), inner boundary membrane (IBM) and contact sites in addition (Fig 4). Specific functions can be assigned to the different cytochrome c fractions. When mitochondrial respiration is driven by Krebs Cycle substrates, it s rate largely depends on the availability of cytochrome c at the surface of the cristae forming inner membrane. When external NADH is the substrate, the respiration requires cytochrome c at the surface of the peripheral inner membrane for electron transfer from the rotenone insensitive NADH oxidase in the outer membrane to cytochrome oxidase in the cristae (Bernardi & Azzone 1981). It has been found that contact sites are involved in this electron transfer pathway (Marzulli et al.1999).

7 In many cases of apoptosis mitochondrial depolarisation, swelling and release of larger proteins (AIF, Smac ec.) occur as late events, well after cytochrome c release. In these cases, Bax-induced cytochrome c release appears to be more specific and not dependent on outer membrane disruption. In isolated mitochondria cytochrome c could be released by Bax independent of permeability transition. (Eskes et al. 1998, Jürgensmeier JM et al. 1998). Acknowledgement: The work was supported by grants of the Deutsche Forschungsgemeinschaft and Köln Fortune given to JM. Jürgensmeier, and by grants of RFBR ( , ) given to M. Vyssokikh and D. Zorov. Furthermore D. Brdiczka and D. Zorov are grateful for scientific support by the VW fondation. References Antonsson, B. Montessuit, S., Lauper, S., Eskes, R., Martinou, JC. (2000) Biochem J. 345, Bernardi, P. Azzone, GF. (1981) J Biol Chem. 256, Eskes, R., Antonsson, B., Osen-Sand, A., Montessuit, S., Richter, C., Sadoul, R., Mazzei, G., Nichols, A., Martinou, JC. (1998) J Cell Biol 143, Jürgensmeier JM., Xie, Z., Deveraux Q., Ellerby, L., Bredesen, D., Reed, JC. (1998). Proc Natl Acad Sci U S A 95, Keilin, D. (1966) The history of cell respiration and cytochromes Cambridge University Press N.Y. Perkins, G., Renken, C., Martone, ME. Young, SJ., Ellisman, M., Frey, T. (1997) J Struct Biol 119: Marzulli, D., La Piana, G., Franseve, E., Lofrumento, NE. (1999) Biochem Biophys Res Commun 259, Margoliash, E. and Lustgarten, J.(1962) J.Biol. Chem. 237: Xie, Z. Schendel, S. Matsuyama, S. Reed, JC. (1998) Biochemistry 37,