cAMP response element-binding protein (CREB) is imported into mitochondria and promotes protein synthesis Domenico De Rasmo 1 , Anna Signorile 1 , Emilio Roca 1 and Sergio Papa 1,2 1 Department of Medical Biochemistry, Biology and Physics (DIBIFIM), University of Bari, Italy 2 Institute of Biomembranes and Bioenergetics (IBBE), Consiglio Nazionale delle Ricerche, Bari, Italy Introduction The cAMP response element-binding protein (CREB) is a ubiquitous transcription factor in the higher eukaryotes that recognizes the DNA consensus sequence TGACGTCA, the cAMP response element (CRE) in gene promoters [1,2]. Phosphorylation of CREB by cAMP-dependent pro- tein kinase (protein kinase A; PKA), as well as by Ca 2+ -dependent and other protein kinases, in response to different cellular signals, promotes tran- scription of CRE-regulated genes [1–4]. Activation of the expression of nuclear CRE-regulated genes has been shown to be involved in a variety of cellular pro- cesses, including apoptosis [5,6], oxidative stress [7], neuronal growth, and plasticity [5,8]. In yeast, cAMP was found to reverse the glucose repression of mito- chondriogenesis [9] and to activate the expression of mitochondrial genes [10,11] and nuclear genes [12,13] of respiratory chain proteins. In Saccharomyces cerere- visiae, where the RAS ⁄ cAMP ⁄ PKA system appears to be involved in regulation of the biogenesis of the oxidative phosphorylation system [13], a probable cis- regulatory element on mtDNA, responsible for cAMP- mediated transcription, was identified [11]. In yeast and mammalian cells, the cAMP cascade is involved in the regulation of mitochondrial dynamics [14] and bioenergetics [15–17]. In 1999, findings were presented [18] indicating that CREB is localized in the inner mitochondrial compart- ment as well as in the nucleus. These observations, based on the use of CREB and phospho-CREB anti- Keywords cAMP cascade; complex I; CREB; mitochondrial protein synthesis; PKA Correspondence S. Papa, Department of Medical Biochemistry, Biology and Physics, University of Bari, Policlinico, P.zza G. Cesare, 70124 Bari, Italy Fax: +39 080 5448538 Tel: +39 080 5448540 E-mail: papabchm@cimedoc.uniba.it (Received 9 March 2009, revised 26 May 2009, accepted 4 June 2009) doi:10.1111/j.1742-4658.2009.07133.x The cAMP response element-binding protein (CREB) is a ubiquitous transcription factor in the higher eukaryotes that, once phosphorylated, promotes transcription of cAMP response element-regulated genes. We have studied the mitochondrial import of CREB and its effect on the expression of mtDNA-encoded proteins. [ 35 S]Methionine-labelled CREB, synthesized in vitro in the Rabbit Reticulocyte Lysate system using a con- struct of the human cDNA, was imported into the matrix of isolated rat liver mitochondria by a membrane potential and TOM complex-dependent process. The imported CREB caused cAMP-dependent promotion of the synthesis of mitochondrially encoded subunits of oxidative phosphorylation enzyme complexes. Thus, CREB moves from the cytosol to mitochondria, in addition to the nucleus, and, when phosphorylated by cAMP-dependent protein kinase, promotes the expression of mitochondrial genes. Abbreviations ADU, arbitrary densitometric units; AKAP, A kinase anchoring protein; CAP, chloramphenicol; cPKA, catalytic subunit of cAMP dependent protein kinase; CRE, cAMP response element; CREB, cAMP response element-binding protein; db-cAMP, dibutyryl cAMP; IBMX, isobutylmethylxanthine; PKA, cAMP-dependent protein kinase (protein kinase A); RRL, Rabbit Reticulocyte Lysate. FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4325 bodies, as well as on mtDNA mobility shift assays, were confirmed by Lee et al. [19] and Ryu et al. [20]. Lee et al. also showed that binding of CREB to CRE sequences in the D-loop of mtDNA increased the tran- script levels of the ND2, ND5 and ND6 mitochondrial genes of complex I of the respiratory chain. Ryu et al. [20] found that activation by the antioxidant iron che- lator deferoxamine of PKA, localized in the mitochon- drial matrix [21], promoted CREB binding to the mtDNA D-loop. The presence in mitochondria of the CREB factor and, in particular, the use of phospho- CREB antibody to detect this transcription factor in mitochondria were, however, questioned by Platenik et al. [22]. These authors showed that commercially available antibodies for phospho-CREB crossreact with the E1 a-subunit of mitochondrial pyruvate dehy- drogenase. Because of the relatively high abundance in mitochondria of pyruvate dehydrogenase, this crossre- activity might overwhelm the reaction of the antibody with phospho-CREB and produce false-positive results. The general physiological relevance of a possi- ble role of CREB and PKA in providing a regulatory mechanism in the expression of mitochondrially encoded proteins of the respiratory chain prompted us to investigate the mitochondrial import of the CREB protein, synthesized in vitro, and its effect on the expression of mitochondrial genes. The results unequivocally show that exogenous CREB is imported into isolated rat liver mitochondria and causes a marked, cAMP-dependent, stimulation of the expres- sion of the mitochondrially encoded subunits of oxida- tive phosphorylation complexes. Results [ 35 S]Methionine-labelled CREB is imported into isolated mitochondria Mitochondrial import of CREB was investigated by incubating freshly isolated, intact rat liver mitochon- dria with the protein synthesized in the Rabbit Reti- culocyte Lysate (RRL) system with [ 35 S]methionine. Figure 1A,B shows time-dependent mitochondrial uptake of the radioactive CREB, which was largely resistant to trypsin digestion unless mitochondria were dissolved by Triton X-100 (Fig. 1C, lane 3). The amount of mitochondrial proteins and mitochondrial integrity were checked by immunodetection of the 39 kDa subunit of the inner membrane complex I, in the absence and in the presence of trypsin. Mitochon- drial uptake of CREB was promoted by the mitochon- drial membrane potential, as shown by its inhibition by valinomycin (Fig. 1C, lane 4). The residual radio- active CREB detected in the mitochondrial pellet in the presence of valinomycin represents the amount of protein bound at the mitochondrial outer surface, as shown by its complete digestion by trypsin (Fig. 1C, lane 5). When mitochondria were pretreated with A B C 400 300 200 100 0 0 10203040506070 Fig. 1. Import into isolated mitochondria of [ 35 S]methionine-labelled CREB. [ 35 S]Methionine-labelled CREB, synthesized in the RRL translation system, was added to isolated rat liver mitochondria. (A, C) Autoradiograms of SDS ⁄ PAGE slabs of the mitochondrial pellet. The RRL gel slab on the left of (A) is an autoradiogram of an amount of the radioactive CREB synthesis mixture corresponding to half of the amount added to mitochondria for the import assay. No precipitable aggregate of radioactive CREB was detectable after centrifugation of the RRL CREB synthesis translation mixture in the absence of added mitochondria. Where indicated, mitochondria, after completion of the import incubation, were treated, before pelletting, with trypsin (1 lg per 50 lg of mitochondrial protein) for 35 min at 0 °C. (C) Import incubation for 60 min: in lane 3, mito- chondria were treated with trypsin in the presence of 0.2% Triton X-100; in lanes 4 and 5, valinomycin (Val) (0.1 lg per mg of mitochondrial protein) was present during the import incubation. (B) Mean values in arbitrary densitometric units (ADU) (three sepa- rate experiments) of the trypsin-resistant [ 35 S]methionine-labelled CREB radioactivity, detected in the mitochondrial pellet, plotted as a function of the import incubation time. The SDS ⁄ PAGE slabs were also blotted with an antibody against the 39 kDa subunit of complex I. See Experimental procedures and [24] for further details. CREB and mitochondrial protein synthesis D. De Rasmo et al. 4326 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS trypsin, before the import assay of radioactive CREB (Fig. 2A, lanes 3 and 4), the amount of imported CREB, resistant to trypsin treatment after completion of the uptake, and that of externally bound CREB, digested by trypsin, were both reduced. This indicates that mitochondrial binding and import of CREB involve surface components of the outer membrane import complex. Addition to the mitochondrial import mixture of an antibody against Tom20, an outer membrane receptor of the mitochondrial import sys- tem [23], reduced the binding of [ 35 S]methionine- labelled CREB to the mitochondrial surface (amount digested by trypsin) and the accumulation in mito- chondria (trypsin-resistant amount) (Fig. 2B, lanes 5 and 6). No significant effect was exerted on CREB binding and import by an antibody against Tom70 (Fig. 2B, lanes 3 and 4). The dependence of CREB uptake on Tom20 and membrane potential indicates that the protein reaches the inner mitochondrial compartment. Submitochon- drial localization of the imported CREB was directly verified by separation of mitochondrial subfractions (Fig. 3). After the import incubation and trypsin treat- ment of mitochondria, organelles were swollen in a hypo-osmotic medium to eliminate CREB bound at the surface. Residual mitochondria and mitoplasts, deprived of the outer membrane and of residual super- ficially bound radioactive CREB, were disrupted by sonication, and the inner membrane fraction was sepa- rated from the matrix content. The radioactive CREB of the mitoplast fraction, still resistant to trypsin in this fraction, was recovered in the matrix fraction. Mitochondrial subfractionation was checked by immunochemical detection of marker proteins of the outer membrane (porin), inner membrane (core II subunit of the cytochrome bc 1 complex), and matrix (cyclophilin D) (Fig. 3). Imported CREB promotes expression of mitochondrial genes The impact of imported CREB and PKA on the expression of mitochondrial genes was studied by testing their effect on the synthesis of [ 35 S]methionine- labelled mitochondrially encoded subunits of oxidative phosphorylation complexes in rat liver mitochondria. Addition of cAMP or dibutyryl cAMP (db-cAMP) A B Fig. 2. Inhibition of [ 35 S]methionine-labelled CREB mitochondrial import by proteolytic digestion of mitochondrial outer surface com- ponents and by an antibody against Tom20. (A) Lane 1: control import of [ 35 S]methionine-labelled CREB. Lane 2: import of the [ 35 S]methonine-labelled CREB followed by trypsin treatment. Lanes 3 and 4: CREB import in mitochondria pretreated for 35 min at 0 °C with trypsin. Where indicated, mitochondria were also treated with trypsin after completion of the import incubation. (B) Lanes 1 and 2: as in (A). Lanes 3 and 4: mitochondrial import in the presence of 3 lg of the antibody against Tom70 (Santa Cruz Biotechnology, CA, USA). Lanes 5 and 6: mitochondrial import in the presence of the antibody against Tom20 (Santa Cruz Biotechnology). Where indi- cated, mitochondria were treated with trypsin after completion of the import incubation. Aliquots of the samples were blotted with an antibody against the 39 kDa subunit of complex I. See Experi- mental procedures and [24] for further details. Fig. 3. Submitochondrial localization of imported [ 35 S]methionine- labelled CREB. Mitochondrial import of [ 35 S]methionine-labelled CREB was followed for 60 min as described in Experimental proce- dures and in the legend to Fig. 1. After import of [ 35 S]methionine- labelled CREB, followed by trypsin treatment of mitochondria (Mt), these were spun down and treated to separate mitoplasts (Mp) and inner membrane (I.M.) and matrix (M.) fractions as described in Experimental procedures. Aliquots of the samples were analysed by SDS ⁄ PAGE and autoradiography, or immunoblotted with the anti- bodies specified. Lanes 1 and 2: mitochondria isolated from the import mixture, before or after trypsin treatment, respectively. Lane 3: mitoplast fraction. Lane 4: mitoplast fraction subjected to trypsin treatment. Lane 5: inner membrane fraction. Lane 6: matrix fraction. For other details, see Experimental procedures. D. De Rasmo et al. CREB and mitochondrial protein synthesis FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4327 caused some stimulation of the overall radioactivity of the gel lane with the [ 35 S]methionine-labelled mi- tochondrially encoded proteins (Fig. 4). In particular db-cAMP increased the synthesis of ND1 by 80%, of CoxIII ⁄ ATP6 by 47%, and of ND6 by 30% (Fig. 4). The addition of the RRL-synthesized CREB alone resulted in enhancement of the synthesis of mitochondrial proteins, this effect being strongly potentiated when CREB was added together with cAMP, db-cAMP or the catalytic subunit of PKA (cPKA), respectively, in the incubation mixture (Fig. 4B, whole gel lane radioactivity of the [ 35 S]methionine-labelled mitochondrial-encoded pro- teins). In particular, the synthesis of ND1, ND6 and CoxIII ⁄ ATP6, was enhanced approximately two-fold by the combination of CREB and cAMP or cPKA A B 200 250 125 0 250 125 0 250 125 0 200 400 0 100 0 200 100 0 Fig. 4. Effect of CREB, cAMP and cPKA on mitochondrial protein synthesis. Mitochondrial protein synthesis was performed in a rat liver mitochondria suspension in the presence of [ 35 S]methionine and cycloheximide plus the RRL mixture without the addition of the cDNA CREB construct (lanes 1–4), or the RRL mixture with the cDNA CREB construct and cold methionine (lanes 5–9). The mitochondrial protein synthesis control (CTRL) contained: rat liver mitochondria suspension and the RRL mixture without the addition of the cDNA CREB con- struct. (A) SDS ⁄ PAGE autoradiography of [ 35 S]methionine-labelled mitochondrial proteins. Lane 1: control. Lane 2: 50 lM cAMP plus 50 lM IBMX. Lane 3: 50 lM db-cAMP plus IBMX. Lane 4: cPKA (1 Unit per 10 lg of mitochondrial protein). Lane 5: no addition. Lane 6: cAMP plus IBMX. Lane 7: db-cAMP plus IBMX. Lane 8: cPKA. Lane 9: CAP (3 mgÆmL )1 ). (B) Histograms showing the mean ADU (as percentage of con- trol) of the whole gel lane radioactivity of the [ 35 S]methionine-labelled mitochondrial proteins and of individual protein spot radioactivity. Mean values of three separate experiments; **P < 0.01; *P < 0.05. The inset shows autoradiography of [ 35 S]methionine-labelled CREB immuno- precipitated by an antibody against phospho-CREB (Santa Cruz Biotechnology). [ 35 S]Methionine-labelled CREB was synthesized in the RRL translation system in the absence (control) or in the presence of 10 Units of cPKA. Translation products were immunoprecipitated by the phospho-CREB antibody. For other details, see Experimental procedures. CREB and mitochondrial protein synthesis D. De Rasmo et al. 4328 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS (Fig. 4B). Evidence that added cPKA catalysed the phosphorylation of CREB is provided by a control experiment showing that the radioactive CREB was immunoprecipitated by an antibody against phospho- CREB only when cPKA was added to the mixture (Fig. 4, inset). It may be noted that in the experiment presented in the Fig. 4, not all of the mitochondrially encoded subunits of the oxidative phosphorylation complexes exhibited labelling by [ 35 S]methionine under the experi- mental conditions used. The experiment presented in Fig. 5, in which a higher amount of [ 35 S]methionine was used in the mitochondrial protein synthesis and a different acrylamide gel concentration was applied for SDS ⁄ PAGE, shows more subunits of oxidative phos- phorylation complexes labelled with [ 35 S]methionine. The combined addition of CREB and cAMP or cPKA resulted also, in this case, in a marked enhancement of the overall gel lane radioactivity with the [ 35 S]methio- nine-labelled mitochondrially encoded proteins (Fig. 5B). The autoradiogram presented in Fig. 5A shows that the synthesis of individual mitochondrially encoded proteins was generally promoted by the addi- tion of CREB with cAMP or cPKA. This stimulatory effect on mitochondrially encoded subunits was com- pletely abolished by the addition of H89, a specific inhibitor of PKA. The addition to the mitochondrial protein synthesis mixture of the RRL-synthesized NDUFS4 nuclear sub- unit of complex I, used as a control, had no effect on the synthesis of mitochondrially encoded subunits of this and other oxidative phosphorylation complexes (results not shown). Evidence has been presented else- where that PKA-mediated phosphorylation of the NDUFS4 nuclear subunit of complex I [24], as well as of other nuclear-encoded mitochondrial proteins [25–27], promotes the import into mitochondria of these proteins. The presence of cPKA or cAMP in the import mixture had, however, no effect on the uptake of CREB by isolated mitochondria (results not shown). Discussion The present results show that in vitro synthesized CREB is imported into the mitochondrial matrix by a membrane potential-dependent mechanism. The CREB imported into mitochondria, and therefore resistant to digestion by trypsin unless mitochondria were dissolved by Triton, did not undergo N-terminal pro- cessing as has also been observed for other nuclear- encoded mitochondrial matrix-targeted proteins [26,28]. The inhibition of both mitochondrial surface binding and import into mitochondria of radioactive CREB by the antibody against Tom20 shows, how- ever, that the import of CREB is mediated by the TOM complex involved in the translocation of pro- teins into the matrix space [23,29–31]. The antibody against Tom70, which is an import receptor for inser- tion of hydrophobic imported proteins into the inner membrane, did not reduce the import of CREB. The CREB mitochondrial import could also be assisted by chaperones, such as the mitochondrial heat shock protein 70 [19]. A B Fig. 5. Effect of H89 on the promotion of mitochondrial protein synthesis by CREB plus cAMP or cPKA. Mitochondrial protein syn- thesis was carried out in the presence of the RRL system supple- mented with cold synthesized CREB, as described in the legend to Fig. 4. The RRL mixture with cold synthesized CREB was present in all of the lanes, including the control. The mitochondrial protein synthesis control (CTRL) contained rat liver mitochondria suspen- sion and the RRL mixture with the addition of the cDNA CREB con- struct. (A) SDS ⁄ PAGE autoradiography of mitochondrial proteins synthesized in the presence of [ 35 S]methionine. For the experimen- tal conditions, see legend to Fig. 4. Where indicated, H89 (100 n M) was present during the import incubation. (B) Histograms showing the mean ADU (as percentage of control) of the whole gel lane radioactivity of the [ 35 S]methionine-labelled mitochondrial proteins. For other details, see Experimental procedures. D. De Rasmo et al. CREB and mitochondrial protein synthesis FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4329 Externally synthesized CREB, once imported into mitochondria, strongly stimulates the synthesis of subunits of oxidative phosphorylation encoded by mitochondrial genes; this requires, as in the case of nuclear CRE-regulated genes, CREB phosphorylation by PKA [2–4]. The synthesis of all of the mitochondri- ally encoded subunits of oxidative phosphorylation enzyme complexes was, in fact, stimulated by the combined addition of CREB and cAMP or cPKA. The stimulatory effect was particularly evident (approximately two-fold enhancement) in the case of those proteins that were more heavily labelled with [ 35 S]methionine, such as ND1, ND6, and CoxIII ⁄ ATP6 (Figs 4 and 5). Phosphorylation of CREB, which is synthesized on cytosolic ribosomes, can take place in vivo in the cytosol before its import into mitochondria, and ⁄ or after it is imported into mitochondria. PKA is, in fact, present in various subcellular regions, including the cytosol and outer and inner mitochondrial compartments [21,32,33]. Our results show that, under the experimental condi- tions used, CREB was phosphorylated by the PKA that was evidently present in the RRL system used for the in vitro synthesis of CREB and ⁄ or in the mitochondrial sample, as well as by the added catalytic subunit of PKA. In this last case, no cAMP was obviously required. The minor promoting effect on mitochondrial protein synthesis given by the addition of cAMP or cPKA (in separate samples) in the absence of added CREB results from phosphorylation of CREB present in the RRL system and ⁄ or in the mitochondrial sample [18–20]. Evidence has been produced showing the exis- tence of a pool of PKA and PKA-anchoring protein (AKAP) localized in the inner mitochondrial compart- ment [21]. Intramitochondrial PKA can be activated by cAMP generated within mitochondria by a carbon dioxide ⁄ bicarbonate-regulated soluble adenylyl cyclase [34,35]. Lee et al. [19] have shown that disruption of CREB activity, by overexpression of a mito-tagged negative dominant CREB, decreases the expression of mitochondrial genes in transfected cells. It has also been shown that activation of mitochondrial PKA by the antioxidant deferoxamine results in phosphorylation of mitochondrial CREB and its binding to the CRE sequence in the mitochondrial D-loop DNA [20]. In conclusion, the present findings provide unequiv- ocal evidence that the transcription factor CREB is imported into the mitochondrial matrix and promotes, when phosphorylated by PKA, the synthesis of mito- chondrially encoded subunits of oxidative phosphory- lation complexes. Our results also lend support, free from the uncertainties involved in immunochemical analysis [22], for the presence of CREB in the inner mitochondrial compartment, where it can also be phosphorylated by PKA present in the same compart- ment [21]. This is not a surprise, as CREB, in order to exert its effect on mitochondrial protein synthesis, has to move from the cytosol, where it is synthesized, into mitochondria, where transcription ⁄ translation of mtDNA-encoded proteins takes place. Positive modulation by CREB of the expression of nuclear [36,37] and mitochondrial genes of proteins of the oxidative phosphorylation system could represent an important regulatory mechanism for the expression of this housekeeping cellular function, thus contributing to the role of CREB in a variety of cellular processes. Experimental procedures cDNA construct and in vitro translation Full-length human CREB cDNA was generated by RT-PCR, using RNA extracted from primary fibroblasts from skin biopsy specimens of control subjects. The CREB cDNA was cloned in the pGEM vector with the T7 promoter. Plasmid construction was confirmed by DNA sequencing. In vitro transcription ⁄ translation of CREB cDNA was performed in RRL system (Promega Biotech, Madison, WI, USA) as reported by De Rasmo et al. [24]. One microgram of CREB construct was added to 50 lLof Promega standard mixture, containing T7 RNA polymerase and a standard amino acid mixture with [ 35 S]methionine (20 lCi). Incubation was performed at 30 °C for 90 min. Rat liver mitochondria Mitochondria were isolated from rat liver as described in ref. [38]. Import assay The assay was performed as in [24]. Sixteen microlitres of the RRL translation mixture producing [ 35 S]methionine-labelled CREB was added to the import mixture containing 210 mm mannitol, 7 mm Hepes (pH 7.4), 0.35 mm MgCl 2 , 2.5 mgÆmL )1 BSA, rat liver mitochondria (500 lg of pro- tein), 3 mm ATP, 3 mm GTP, 15 mm malate and 30 mm pyruvate in a final volume 200 lL. After incubation at 30 °C for the times specified in the figures, aliquots of the mixture were transferred to ice-cooled tubes and supplemented with protease inhibitors (Sigma, St Louis, MO, USA) (1 lL per 250 lg of mitochondrial protein). Mitochondria were spun down at 4000 g for 10 min, and supernatant and mitochon- drial proteins were separated by SDS ⁄ PAGE and transferred to a nitrocellulose membrane. Radioactive protein bands were detected by personal fx at phosphorus imager (Bio-Rad, Milan, Italy) and quantified by versadoc (Bio- CREB and mitochondrial protein synthesis D. De Rasmo et al. 4330 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS Rad). The same samples were also immunoblotted with an antibody against the 39 kDa subunit of complex I of the respiratory chain (Invitrogen, Paisley, UK). Mitochondrial protein synthesis RRL translation medium with unlabelled synthesized CREB or without CREB was added to the import mixture containing a standard amino acid mixture with [ 35 S]methio- nine (20 lCi), 210 mm mannitol, 7 mm Hepes (pH 7.4), 0.35 mm MgCl 2 , 2.5 mgÆmL )1 BSA, rat liver mitochondria (125 lg of protein), 3 mm ATP, 3 mm GTP, 15 mm malate, 30 mm pyruvate and 50 ngÆmL )1 cycloheximide in a final volume of 50 lL. Incubation was performed at 30 °C for 1 h in the presence, where indicated, of cAMP, cPKA, db-cAMP, isobutylmethylxanthine (IBMX), H89, or chloramphenicol (CAP). The incubation was prolonged for 10 min after the addition of unlabelled amino acid mixture. Mitochondria were then spun down at 4000 g for 10 min, and proteins were separated by SDS ⁄ PAGE and transferred to a nitrocellulose membrane. Radioactive protein bands were detected by personal fx at phosphorus imager (Bio-Rad) and quantified by versadoc (Bio-Rad). Submitochondrial localization of imported [ 35 S]methionine-labelled CREB Mitochondrial sublocalization of imported [ 35 S]methionine- labelled CREB was performed essentially as described in ref. [39]. After mitochondrial import of [ 35 S]methionine- labelled CREB and trypsin treatment, reisolated mitochon- dria were split in two aliquots and resuspended in 250 mm sucrose, 1 mm EDTA, and 10 mm Mops ⁄ KOH (pH 7.2), or in 1 mm EDTA and 10 mm Mops ⁄ KOH (pH 7.2); the latter medium was used to obtain mitoplasts by mitochon- drial swelling. After 15 min of incubation on ice, each sample was split into two, and one of these was again subjected to trypsin treatment. Mitochondrial and mito- plasts fractions were spun down at 10 000 g for 10 min. After mitoplast sonication, the sample was centrifuged at 150 000 g for 15 min. The pellet representing the inner membrane proteins was resuspended in the SDS ⁄ PAGE loading buffer; the supernatant, representing the matrix fraction, was treated with trichloroacetic acid, and the pre- cipitate was resuspended in the SDS ⁄ PAGE loading buffer. All samples were analysed by SDS ⁄ PAGE and autoradio- graphy, or immunoblotted with antibodies against the porin, cyclophilin D and core II subunit of complex III (Invitrogen) as specified in the legend to Fig. 3. Acknowledgements This work was supported by the National Project on ‘Molecular Mechanisms, Physiology and Pathology of Membrane Bioenergetics System’, 2005, Ministero dell’Istruzione, dell’Universita ` e della Ricerca (MIUR), Italy, the University of Bari, and Research Foundation Cassa di Risparmio di Puglia. 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