Evidence for aerobic metabolism in retinal rod outer segment disks

The International Journal of Biochemistry & Cell Biology 41 (2009) 2555–2565

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Evidence for aerobic metabolism in retinal rod outer segment disks Isabella Panfoli a,∗ , Daniela Calzia a , Paolo Bianchini b , Silvia Ravera a , Alberto Diaspro c , Giovanni Candiano d , Angela Bachi e , Massimiliano Monticone f , Maria Grazia Aluigi a , Stefano Barabino g , Giovanni Calabria g , Maurizio Rolando g , Carlo Tacchetti h , Alessandro Morelli a , Isidoro M. Pepe a a

DIBIO, University of Genoa, Viale Benedetto XV, 5, 16132 Genova, Italy LAMBS-MicroSCoBiO Research Center, University of Genoa, Via Dodecaneso 33, 16146 Genova, Italy c Nanophysics, IIT- Italian Institute of Technology, IIT, Via Morego, 30, Genova, Italy d Laboratory on Pathophysiology of Uraemia, G. Gaslini Institute Genova, Via V Maggio 39, 16148 Genova, Italy e Dibit-San Raffaele Scientific Institute,Via Olgettina 60, 20132 Milano, Italy f ABC - Advanced Biotechnology Centre, Largo Rosanna Benzi 10, 16132 Genova, Italy g DINOG, University of Genoa, Via de Toni, 5, 16132 Genova, Italy h IFOM-Center for Cell Oncology and Ultrastructure, DIMES, University of Genoa, Via de Toni 14, 16132 Genova, Italy b

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Article history: Received 21 May 2009 Received in revised form 11 August 2009 Accepted 20 August 2009 Available online 26 August 2009 Keywords: Aerobic metabolism F1 F0 -ATP synthase Bioenergetics Oxidative phosphorylation Respiratory chain complexes

a b s t r a c t The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site of visual transduction, a very energy demanding process. In a previous proteomic study we reported the expression of the respiratory chain complexes I–IV and the oxidative phosphorylation Complex V (F1 F0 ATP synthase) in disks. In the present study, the functional localization of these proteins in disks was investigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laser scanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. An oxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A and KCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescence quenching kinetics showed the existence of a proton potential difference across the disk membranes. Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesis by disks (0.7 ␮mol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, would largely account for the rod ATP need in the light. Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria, thank to the presence of ectopically located mitochondrial proteins. These findings may provide important new insight into energy production in outer segments via aerobic metabolism and additional information about protein components in OS disk membranes. © 2009 Elsevier Ltd. All rights reserved.

Abbreviations: ANT, adenosine nucleotide translocase; CLSM, Confocal Laser Scanning Microscopy; CS, citrate synthase; GA, glutaraldehyde; MTP, mitochondrial permeability transition pore; M.W., molecular weight; O2 , oxygen; OXPHOS, oxidative phosphorylation; PBS, phosphate buffered saline; RH-123, Rhodamine 123; R.O.D., Relative Optical Density; TIM, mitochondrial import inner membrane translocase; TOM, mitochondrial import outer membrane translocase; WB, Western Blot. ∗ Corresponding author at: Università di Genova, Dipartimento di Biologia, V.le Benedetto XV, 3, 16132 Genova, Italy. Tel.: +39 010 353 7397; fax: +39 010 353 8153. E-mail addresses: [email protected], [email protected] (I. Panfoli), [email protected] (D. Calzia), bianchini@fisica.unige.it (P. Bianchini), [email protected] (S. Ravera), [email protected] (A. Diaspro), [email protected] (G. Candiano), [email protected] (A. Bachi), [email protected] (M. Monticone), [email protected] (M.G. Aluigi), [email protected] (S. Barabino), [email protected] (G. Calabria), [email protected] (M. Rolando), [email protected] (C. Tacchetti), [email protected] (A. Morelli), [email protected] (I.M. Pepe). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.08.013

1. Introduction Light captured by visual pigment Rhodopsin (Rh) in rod Outer Segments (OS) of the vertebrate retina triggers phototransduction (Stryer, 1996; Pepe, 2001; Ridge et al., 2003; Lamb and Pugh, 2006). Rods, associated with scotopic vision, have reached the outmost sensitivity to light (Fain et al., 2001), being triggered by a single photon (Lamb and Pugh, 2006). A unique property of OS is the presence of a stack of floating membranous disks surrounded by plasma membrane, housing the integral and peripheral membrane proteins that perform photon capture and visual transduction (Ridge et al., 2003). Disks are synthesized disks at the base of the rod and move toward the tip, where the upper disks are eventually phagocytized by the pigment epithelium (Bok, 1985; Finnemann et al., 1997). This localization is corre-

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lated to a decrease in membrane cholesterol content in parallel with the acquisition of functionality (Albert and Boesze-Battaglia, 2005). The excitation phase of the vertebrate rod photoresponse leads to a light-stimulated enzymatic cascade culminating in the hydrolysis of guanosine 3 -5 cyclic monophosphate (cGMP), generating the neuronal signal. The recovery of photoresponse is due to a guanylyl cyclase stimulated by the lowered Ca2+ levels following photoexcitation (Koch and Stryer, 1988; Ridge et al., 2003; Lamb and Pugh, 2006). Current hypotheses for continual GTP supply in OS include its production via glycolysis or its diffusion from the Inner Segment (IS) (Carretta and Cavaggioni, 1976; Ames et al., 1986; Hsu and Molday, 1994; Pepe, 2001). Although anaerobic glycolysis may provide enough ATP, i.e. GTP thank to the conversion of ATP to GTP by the activity of a nucleoside diphosphate kinase (NDP) (Orlov et al., 1996), for cGMP turnover in dark adapted rods, illumination increases cGMP turnover by 5-folds, exceeding the glycolytic capacity of rods to produce ATP (Biernbaum and Bownds, 1985; Ames et al., 1986; Pepe, 2001). There is a mismatch between the energy that can be produced by glycolysis to supply processes in the OS (Hsu and Molday, 1994; Pepe, 2001) which sets up the requirement for another energy source. Our recent proteomic studies have identified several subunits of the four redox complexes and of F1 F0 -ATPase in enriched outer segment disk membrane preparations (Panfoli et al., 2008). A proteomic analysis of rod OS was recently reported (Kwok et al., 2008) focused on proteins implicated in vesicle trafficking and membrane fusion. In the present paper, we show that purified disks aerobically synthesize ATP at a rate which largely accounts for the need of energy during phototransduction, by employing a transmembrane proton (H+ ) potential (H+ ). 2. Materials and methods 2.1. Materials Ficoll, protease inhibitor cocktail, Salts, leupeptin, ampicillin, rotenone, antimycin A, Nigericin, Valinomycin, (DCCD), carbonylcyanide-4N,N -dicyclohexylcarbodiimide (trifluoromethoxy)-phenylhydrazone (FCCP), Oligomycin and all other chemicals (of analytical grade) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Protein Molecular Weight (M.W.) markers were from Fermentas Life Sciences (Hanover, MD, USA). Ultrapure water (Milli-Q; Millipore, Billerica, MA, USA) was used throughout. Safety precautions were taken for chemical hazards in carrying out the experiments. Ampicillin (100 ␮g/ml) was used in all the solutions and sterile experimental conditions were employed throughout where appropriate. 2.2. Sample preparations 2.2.1. Purified bovine rod OS preparations Purified bovine rod OS were prepared under dim red light from 20 retinas (from freshly enucleated bovine eyes, obtained from a local slaughterhouse) at 4 ◦ C, by sucrose/Ficoll continuous gradient centrifugation (Schnetkamp and Daemen, 1982, Panfoli et al., 2008) in the presence of protease inhibitor cocktail (Sigma–Aldrich, S. Louis, MO) and ampicillin (100 ␮g/ml). 2.2.2. Osmotically intact OS disk preparations Osmotically intact disks were prepared by Ficoll flotation (Smith and Litman, 1982) from purified OS. OS were burst for 3 h in distilled water with 70 ␮g/ml leupeptin, and ampicillin (100 ␮g/ml), suspension layered onto 5% Ficoll solution in distilled water with 70 ␮g/ml leupeptin, and ampicillin (100 ␮g/ml) and centrifuged

for 2 h at 25,000 rpm in a Beckman FW-27 rotor. Disks were collected at the interface between water and Ficoll under sterile conditions. In the osmotically intact disk preparation A280 /A500 ratio was 1.8 ± 0.1 (average ± SD) and Rh concentration was 0.8 mM (Panfoli et al., 2008). OS and disks were prepared in the absence of Cyclosporin A (Crompton et al., 1988) and 2-aminoethoxydiphenyl borate (Chinopoulos et al., 2003), inhibitors of the mitochondrial permeability transition pore (MTP) (Berman et al., 2000). Inhibitors of the MTP were not employed as they proved not necessary, in fact respiration and ATP synthesis were observed in their absence. Furthermore, these conditions promoted the MTP formation in contaminant mitochondria, if any, during the disk isolation procedure, so that these would not be functional. 2.2.3. Isolation of retinal mitochondria-enriched preparations All steps were performed at 4 ◦ C. Bovine retinal mitochondria-enriched fractions were isolated by standard differential centrifugation techniques from residual retinae after OS preparation as previously reported (Panfoli et al., 2008). Pellet was retained as a mitochondria-enriched preparation, stored on ice and used within 4 h of isolation. Respiratory control ratio values of these preparations were 4.7 ± 0.2 with pyruvate + malate as substrates. To quantitatively prove that the measured aerobic metabolic activity arises from OS disks and not from putative contaminating fractured mitochondrial membranes, aliquots of mitochondria were “prepared as disks” i.e. subjected to the treatment that OS undergo for preparation of disks (Smith and Litman, 1982): diluted in distilled water plus 70 ␮g/ml leupeptin, and ampicillin (100 ␮g/ml) to a final protein concentration of 1 mg/ml (identical to that of OS pellets) for 1 h. 2.3. Electrophoresis, semiquantitive Western Blot (WB) and quantification Denaturing electrophoresis (SDS-PAGE) was performed using a Laemmli (Laemmli, 1970) protocol on gradient 8–12% gels. Following SDS-PAGE, the polypeptides were transferred electrophoretically onto 0.2 ␮M nitrocellulose membranes and probed with primary antibodies (Abs ): mouse monoclonal anti bovine Rh (C-terminus, last 9 amino acids, Chemicon Int., Temecula, CA); mouse monoclonal Ab against ␣1 rabbit Na/K-ATPase; goat polyclonal Ab against TIM subunit 23; goat polyclonal antibody against a peptide mapping at the C-terminus of mitochondrial import outer membrane translocase (TOM20) of human origin (sc-11021); rabbit polyclonal antibody against amino acids 3–97 of NADH-ubiquinone oxidoreductase subunit ND4L of human origin (sc-20665); mouse monoclonal antibody raised against mitochondrial Succinate Dehydrogenase of cow origin (sc-59687); mouse monoclonal antibody raised against UQCRC1 Cytochrome c reductase (sc-65238); goat polyclonal anti Cytochrome c oxydase II (sc-23984); goat polyclonal antibody raised against a peptide mapping at the C-terminus of citrate synthase (CS) of human origin (sc-30538); and rabbit polyclonal anti-ANT (Santa Cruz Biotecnhnology Inc., Santa Cruz, CA), diluted in Phosphate Buffered Saline (PBS) (1:500 for antiRh; 1:1000 for anti-Na/K-ATPase, -Succinate Dehydrogenase and -Cyt c reductase; 1:200 for anti-TIM, -TOM, -ANT, -ND4L, -CS and -Cytochrome c oxydase). Secondary HPR-conjugated Abs were diluted in PBS (1:5000 for anti-mouse IgG; 1:7500 for anti-rabbit and anti-goat IgG). Bands were visualized with Lumi-Light Western Blotting Substrate (Roche Diagnostics Corp., Indianapolis, IN) and acquired using Kodak Developer films (Sigma–Aldrich St. Louis, MO). Quantitative densitometry was performed using the ImageJ 1.31v software (http://rsb.info.nih.gov/ij/). Each band was converted into a densitometric trace allowing calculations of intensity

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(Jarskog and Gilmore, 2000). Chemiluminescent bands were compared with the signal of whole protein patterns as reported (Ravera et al., 2007b). Results were expressed as Relative Optical Density (R.O.D.). 2.4. ATP synthesis assays 2.4.1. Spectrophotometric determination ATP formation from ADP and inorganic phosphate (Pi ) in disks was studied by an upgrading of the experimental protocol previously reported (Panfoli et al., 2008), according to Mangiullo et al. (2008). Disks (0.03 mg protein/ml) were incubated for 5 min at 37 ◦ C in 50 mM Tris/HCl (pH 7.4), 5 mM KCl, 1 mM EGTA, 5 mM MgCl2, 0.6 mM ouabain, 0.4 mM of the adenylate kinase inhibitor di(adenosine-5 ) penta-phosphate (Ap5 A), and ampicillin (25 ␮g/ml). ATP synthesis was then induced by adding 5 mM KH2 PO4 , 20 mM succinate, 0.35 mM NADH, and 0.1 mM ADP (or 0.3 mM ADP for time course analyses) at the same pH of the mixture. After stopping the reaction with 7% perchloric acid, neutralized and clarified supernatant was added to a mixture containing 2 mM MgCl2 , 0.5 mM NADP, 5 mM Glucose, 100 mM Tris/HCl pH 7.4 and 7 U/ml of a mix of hexokinase and glucose-6-phosphate dehydrogenase (Roche Diagnostics Corp., Indianapolis, IN). NADP reduction was followed in a dual-beam spectrophotometer (UNICAM UV2, Analytical S.n.c., Italy). 2.4.2. Bioluminescent luciferase ATP assay ATP synthesis in bovine retinal mitochondria was conducted first incubating samples (0.03 mg protein/ml) for 5 min at 37 ◦ C in 10 mM Tris/HCl (pH 7.4), 100 mM KCl, 1 mM EGTA, 2.5 mM EDTA,  and 5 mM MgCl2 with 0.2 mM di(adenosine-5 ) penta-phosphate, 0.6 mM ouabain, 50 nM Cylcosporin A and ampicillin (25 ␮g/ml). ATP synthesis was then induced adding 5 mM KH2 PO4 , 20 mM succinate, 5 mM pyruvate/2.5 mM malate and 0.1 mM ADP at the same pH of the mixture, to the sample. When necessary, incubation contained either 0.5 mM DCCD, 10 ␮M Nigericin plus 4 ␮M Valinomycin, 1 mM FCCP or 10 ␮M Olygomycin (Turina et al., 1991). After stopping the reaction with 7% perchloric acid, the ATP concentration in each sample was measured in a luminometer (Lumi-Scint, Bioscan) by the luciferin/luciferase chemiluminescent method with ATP standard solutions between 10−9 and 10−7 M for calibration (Roche Diagnostics Corp., Indianapolis, IN). 2.5. Spectrofluorimetric measurements of disk membrane potential H+ of isolated disk was assayed using Rhodamine 123 (RH-123) (Emaus, 1986) (Excitation 503 nm-Emission 527 nm). Fluorescence was detected at 25 ◦ C with a thermostatically controlled PerkinElmer (MA, USA) LS 50B spectrofluorometer, under continuous stirring. 0.020 mg bovine rod disks were added to buffer (2 ml final volume) containing: 10 mM HEPES pH 7.2, 5 mM MgCl2 , 5 mM NaH2 PO4 , 0.1 mM ouabain, 50 mM KCl, 0.2 mM Ap5 A, and an ADPregenerating system (10 mM glucose and 2.5 U hexokinase), and incubated for 15 min. Then 50 nM RH-123 was added, dissolved in ethanol (kept below 0.4%). 2.6. Oxygraphic measurements Respiration rates (0.04 mg protein/1.7 ml) were measured at 22 ◦ C in a closed chamber according to (Aicardi and Solaini, 1982), using a thermostatically controlled oxygraph apparatus equipped with an amperometric electrode (Unisense–Microrespiration, Unisense A/S, Denmark) that determines the ␮M of O2 of any solution. Oxymeter was connected to a PC running dedicated proprietary datalogger software (MicOx, Unisense) converting data in

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Excel files. An electromagnetic stirrer bar was used to mix the contents of the chamber. Additions were conducted by Hamilton syringes, through a rubber cup in a volume of no more than 0.05 ml. Electrode was equilibrated with the appropriate medium before each experiment, until the oxygen consumption remained constant. For disks the incubation medium was: 50 mM HEPES, 120 mM KCl, 2 mM MgCl2 , 5 mM KH2 PO4 , 25 ␮g/ml ampicillin, and 0.25 mM Ap5 A, pH 7.3 (Sgobbo et al., 2007). For retinal mitochondria-enriched-fractions standard medium was: 75 mM sucrose, 30 mM Tris–HCl, 40 mM KCl, 2 mM MgCl2 , 5 mM KH2 PO4 , 0.5 mM EDTA, 25 ␮g/ml ampicillin, and 0.2 mM Ap5 A, pH 7.4 (Sgobbo et al., 2007). In mitochondria to stimulate the Complex I + III + IV pathway, 5 mM pyruvate and 2.5 mM malate were employed as oxidative substrates. The respiratory control ratios (RCR) of mitochondria-enriched fractions, calculated as the ratio between state 3 respiration and state 4 respiration, were above the value of 5. To observe the ADP-stimulated respiration rats 0.19 mM ADP was added. Respiratory rates are expressed in ␮MO2 /min/mg. For uncoupled measurements, Nigericin (5 ␮M) and Valinomycin (10 ␮M) were added to the suspension after the membrane additions. 2.7. Spectrophotometric assay of disk redox complexes Complex I (NADH-ubiquinone oxidoreductase) activity was measured on 50 ␮g of protein following the reduction of ferricyanide at 420 nm (Sottocasa et al., 1967). Complex II (Succinic dehydrogenase) was assayed at 600 nm, in 2 mM EDTA, 0.2 mM ATP, 20 mM succinate 0.5 mM cyanide, 80 ␮M dicloroindophenol (DCIP), 50 ␮M decylubiquinone, 40 ␮M antimycin A and 10 ␮M rotenone and 10 mM Phosphate buffer, pH 7.2, with 50 ␮g of protein (Janssen et al., 2007). Complex III (Cytochrome c reductase) activity was measured following the reduction of oxidized Cytochrome c at 550 nm with 50 ␮g of protein (Sottocasa et al., 1967). Complex IV (Cytochrome c oxidase) activity was measured following the oxidation of Ascorbate-reduced Cytochrome c at 550 nm with 5 ␮g of protein (Baracca et al., 2003). 2.8. Confocal Laser Scanning Microscopy of disks Treatments on disks (60 ␮g protein/50 ␮l) were conducted in solution, as reported (Ravera et al., 2007a). 2 ␮g/ml of anti-Rh, Na/K-ATPase, -TIM, or -ANT were used as primary Abs . Secondary Abs were: Cy3-labeled anti-mouse, or -rabbit IgG; Alexa Fluor® 488-labeled anti-goat IgG (Molecular Probes, Invitrogen Corporation). CLSM imaging was performed as reported (Panfoli et al., 2008) on an inverted LEICA TCS SP5 AOBS confocal laser scanning microscope (Leica Microsystems CMS, Mannheim, Germany). In controls, disks were treated with secondary Abs only, and yielded negligible immunoreactivity (data not shown). 2.9. TEM microscopy Disks were fixed in 3% paraformaldehyde and glutaraldehyde (GA) 0.2% included in gelatine and freezed in liquid nitrogen. Ultrathin sections (20 nm thick) obtained with a microtome were put onto classical copper grids (1 mm × 1 mm). Disk sections were labeled with anti-Rh (1:200) as primary Ab. Primary Ab was recognized by rabbit anti-mouse secondary Ab and protein-A bound to colloidal gold (15 nm) (Amersham Biosciences, Piscataway, NJ). 2.10. DNA extraction from retinas and disk fractions DNA from small aliquots of fresh whole bovine retinas and purified disks (16 ␮g of total protein) was obtained according to standard procedure (Maniatis et al., 1982). The final pellet was

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resuspended in PCR-grade water. A NanoDrop® (NanoDrop® ND1000 Spectro-photometer, NanoDrop Technologies) was used to measure UV absorbance spectra, 260/280 absorbance ratios and concentrations of DNA extracted from disks bovine and retinas. Stock DNAs were stored at −20 ◦ C. 2.11. Primers and PCR amplification A primer pair, to specifically detect bovine mitochondrial DNA, was designed by the on-line Primer3 software program (Rozen and Skaletsky, 2000). The sequences for primers were forward 5 -GCTAAGACCCAAACTGGGATT-3 , reverse 5 AGCCCATTTCTTCCCATTTC-3 . Amplification reactions were carried out in a total volume of 20 ␮l, and reaction mixtures containing 5× PCR-Buffer, 2 mM MgCl2 , 1 pmol of each primer, 0.2 mM deoxynucleoside triphosphates, and 1 U of AmpliTaq DNA polymerase (PerkinElmer, Emeryville, Calif.). Starting from an equal amount of DNA (0.3 ␮g), the two samples were subjected to a PCR program consisted of 1 cycle at 95 ◦ C for 2 min, then 12–27 cycles at 95 ◦ C for 30 s for denaturing, 56 ◦ C for 45 s for annealing, and 72 ◦ C for 30 s for extension, and finally 1 cycle at 72 ◦ C for 7 min. PCR products were separated on 1% agarose gels in Tris-acetate-EDTA buffer and stained with ethidium bromide (0.4 mg/ml). 2.12. Citrate synthase assay Citrate synthase (CS, EC 4.1.3.7) activity was assayed in disks, rod OS, mitochondria and mitochondria prepared as disks by the reduction of acetyl-CoA to CoA with 5-5 -dithio-bis-2-nitrobenzoic acid (DTNB); thionitrobenzoic acid (TNB) formation was monitored at 412 nm. The reaction mixture was composed by 100 mM Tris–HCl pH 8.0, 0.1 mM DTNB, 0.4 mm acetyl-CoA and 10 mM of oxaloacetate. In order to allow the accessibility of the substrate to the enzyme, for mitochondria and mitochondria prepared as disks 0.1% Triton X-100 was added to the reaction mixture while disks and ROS were subjected to 15 passages through a needle (25 G). Reaction was started by addition of samples (20 ␮g of protein). 2.13. Standard procedures Protein concentration was determined by the BCA protein assay from Pierce Biotechnology, Inc. 3. Results 3.1. Characterization of the disk fractions The osmotically intact disk fractions were collected at the interface between a 5% Ficoll solution and water, after ultracentrifugation (Smith and Litman, 1982). The sediment contained the cellular material that bursts upon 5 h permanence in hyposmotic solution. Disks swell but do not burst by hypotonic shock. Their osmotic resistance is due to the phospholipid disk environment (Lamba et al., 1994). Purity of the disk preparations was evaluated by Transmission Electron Microscopy (TEM) imaging. Results are shown in Fig. 1. Panel A, is a TEM image of the disk fraction, representative of three experiments on different disk preparations. Flattened disks and sealed vesicles were observed. Careful, examination of the 12 × 3 shots taken in all the experiments showed absence of RIS organelles, such as mitochondria, endoplasmic reticulum, or melanosomes. Vesicles of other nature or resealed pieces of plasma membranes may be present, however these would not float up (Smith and Litman, 1982). Fig. 1(panel B) is a immunogold TEM image of disks labeled with anti bovine Rh Ab. Panel C is an overview

Fig. 1. Electron microscopy of intact disks. (A) Disks observed by TEM. The length of the profiles of the vesicles ranged around 1 ␮m. (B) Immunogold analysis of disks, with a primary Ab against bovine Rh and a secondary Ab coupled to colloidal gold (15 nm). (C) Magnification (1.3-folds) of (B) showing a single labeled disk. Scale bar is 501 nm. Figures are representative of 12 different fields.

of a single labeled disk. Panels B and C show that the visual pigment is distributed on disks. 3.2. ATP synthesis by rod disks Fig. 2 shows the extra vesicular ATP formation determined after incubation of disks (0.03 mg of protein/sample) in the presence of ADP and inorganic phosphate (Pi ). A maximal activity of 0.7 ± 0.1 ␮mol/min/mg of protein was detected in the presence of 0.35 mM NADH, 20 mM succinate and 0.1 mM ADP. Inset shows that the time course of the reaction was linear within 2 min. ATP synthesis was inhibited by the mitochondrial F1 F0 -ATPase/H+ -pump inhibitor oligomycin (75%). The oligomycinsensitive extravesicular ATP production was depressed by DCCD, inhibitor of passive H+ conduction (66%), the oxidative phosphorylation uncoupler FCCP (95%), and by the H+ –K+ /iononophores Nigericin/Valinomycin (97%). Additions of medium in which some

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Fig. 2. ATP synthesis in purified OS disks. Hystogram shows ATP formation over 1 min, at 37 ◦ C, at pH 7.3 by disks (0.06 mg/ml) (bar 1). Addition of 10 ␮M oligomycin (bar 2), 0.5 mM DDCD (bar 3), 5 ␮M Nigericin/10 ␮M Valinomycin (bar 4) or 1 mM FCCP (bar 5) inhibited ATP production by 75%, 66%, 97% and 95% respectively. Inset shows the time course of the reaction, in 2 min. Each point, representative of four separate experiments, is the mean ± S.D; paired Student’s t test was used. *p < 0.01.

inhibitors are dissolved (ethanol) elicited no effects (data not shown). 3.3. Assay of proton gradient on disks by Rhodamine 123 fluorescence quenching Data suggests that the putative disk surface ATP synthase employs a transmembrane electrochemical H+ , whose existence has been previously envisaged (Kaupp et al., 1981; McConnell et al., 1968; Uhl et al., 1979; Uhl and Desel, 1989). The actual presence of a H+ across the disk membrane was assessed monitoring fluorescence quenching of RH-123 (Baracca et al., 2003). In isolated organelles, RH-123 electrophoretically accumulates where a steep H+ is present (Uckermann et al., 2004; Poot et al., 1996; Kahlert et al., 2008) with a fluorescence quenching, due to aggregation of the dye (Emaus, 1986), proportional to H+ . Fig. 3 shows the effect of a series of additions on the dye fluorescence. 50 nM RH-123 was added to the medium containing 0.02 mg/ml disks and an ADP-regenerating system. No fluorescence due to disks alone was detected. Addition of ADP induced an enhancement of steady-state fluorescence, indicating a decrease of H+ . Rotenone, a specific inhibitor of NADH dehydrogenase, caused a further fluorescence increase, likely due to the membrane potential dissipation. Disk membrane potential could be recovered by addition of succinate, which induced the quenching of the dye. Inhibition of ATP synthase by oligomycin induced a further increase of the membrane potential, causing decrease of fluorescence quenching. Addition of antimycin A, an inhibitor of Complex

Fig. 3. Rhodamine-123 fluorescence measurements. Representative trace of the time course of RH-123 fluorescence in disks. Additions were: 0.15 mM ADP, 0.08 mM rotenone, 20 mM succinate, 0.0002 mM oligomycin and 0.04 mM antimycin A.

Fig. 4. Measurements of respiration rates in disks. (A) Representative amperometric recording trace of Nigericin/Valinomycin-uncoupled respiration rates in disks. Additions were: NADH (0.35 mM); ADP (0.19 mM); rotenone (0.1 mM); succinate (20 mM); antimycin A (0.05 mM); ascorbate (10 mM); KCN (0.5 mM); (B) as (A) but with coupled disk in the presence of oxidative substrates (NADH and succinate) and ADP. Insets report the mean activity of the classical states of respiration, expressed as mean ␮MO2 /s ± S.E. from five independent experiments. In both cases 0.04 mg of total disk proteins were used, in 1.7 ml volume. Rotenone, antimycin A inhibited oxygen consumption, while the addition of medium (ethanol) alone had no effect (not shown).

III of the respiratory chain, allowed a recovery of fluorescence, likely as a consequence of H+ disappearance. Dynamic RH-123 redistribution across the disk membrane is likely a consequence of its potential changes. 3.4. Intact disks consume oxygen The rate of oxygen (O2 ) consumption by disks was measured at 23 ◦ C with an amperometric electrode (Unisense A/S, Denmark). Fig. 4(panel A) reports a typical amperographic tracing of Nigericin/Valinomycin-uncoupled respiration rate of purified intact disks (0.024 mg/ml). Following Nigericin/Valinomycin incubation, the maximal respiratory fluxes by NADH (Complex I + III + IV), succinate (Complex II + III + IV) and ascorbate (Complex IV) were measured as rates sensitive to inhibition by rotenone, antimycin A and cyanide, respectively. Fig. 4(panel B) reports a typical amperometric recording of ADP-stimulated respiration rates in disks. NADH and succinate were added as respiratory substrates followed by subsaturating concentrations of ADP (0.19 mM) in the case of NADH, which induced a transient stimulation of the O2 consumption rate. Respiration rates decreased by addition of rotenone and antimycin A, respectively. Inset of Fig. 4 reports the mean O2 consumption expressed as ␮M O2 /min/mg of protein for the different classical states of respiration. A series of analogous experiments performed separately allowed to couple disk respiration to ATP production oxidative

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Fig. 5. Assay of the four respiratory complexes in disks. Activity of Complex I (NADH-ubiquinone oxidoreductase) is expressed as IU/mg of total protein (␮mol of reduced ferricyanide/min/mg). In the presence of 30 ␮M rotenone, activity decreased by about 72% (A). Activity of Complex II (B) is expressed as IU/mg (␮mol of reduced DCPI/min/mg of total protein). Activity of Complex III (Cytochrome c reductase) is expressed as IU/mg (␮mol of reduced Cytochrome c/min/mg of total proteins). In the presence of 50 ␮M antimycin A, activity decreased by about 75% (C). Activity of Complex IV (Cytochrome c oxidase) is expressed as IU/mg (␮mol of oxidized Cytochrome c/min/mg of total proteins). In the presence of 0.5 mM KCN, activity decreased by about 99% (D). Panels are representative of at least 10 identical experiments.

phosphorylation efficiency (P/O ratio). The P/O ratio was calculated as the ratio between the nanomoles of ADP consumed and the nanomoles of O consumed during the ADP-stimulated respiration (conditions analogue to mitochondrial state 3). P/O ratio with NADH was 2.8 ± 0.15; and with succinate was 1.7 ± 0.11. Addition of ethanol alone elicited no effects (data not shown). 3.5. Respiratory complex assays Fig. 5 shows the time course of the activities of the redox complexes in disks, in the absence or presence of specific inhibitors (rotenone for NADH-ubiquinone oxidoreductase, 72% inhibition, panel A; antimycin A for Cytochrome c reductase, 75% inhibition, panel C; cyanide for Cytochrome c oxidase, 99% inhibition, panel D). Succinic dehydrogenase activity is in panel B. Activity of each complex in disks was comparable to retinal mitochondriaenriched fractions in the same experimental conditions (data not shown). 3.6. Evaluation of the results in terms of the entity of mitochondrial contamination The extent of contamination of the disk sample by IS organella was evaluated by immunofluorescence CLSM imaging of disks, using a new technique that we have developed (Ravera et al., 2007a). By this technique, we showed that ATP synthase ␣/␤-chains are distributed on the surface of disks and enriched in disks with respect to OS and mitochondria (Panfoli et al., 2008). Fig. 6 shows the monitoring with specific Abs of the presence on disks of: (i) Rh; (ii) Na+ /K+ -ATPase pump, an example of an IS plasma membrane protein; (iii) mitochondrial import inner membrane translocase (TIM) and adenosine nucleotide translocase (ANT), as examples of inner mitochondrial membrane proteins; (iv) mitochondrial import outer membrane translocase (TOM), as example of outer mitochondrial membrane protein. Panel A1 shows the fluorescence of a Cy3-labeled secondary Ab bound to the same Ab anti-Rh used in Fig. 1. Rh was present only in OS, that appear as spherical objects of a mean diameter of 1.2 ␮m (Ravera et al.,

2007a; Nickell et al., 2007). Indirect fluorescence of Na+ /K+ -ATPase, TIM, ANT and TOM are in panels B1, C1, D1, and E1, respectively. Na+ /K+ -ATPase was only present in mitochondria-enriched fractions, probably due to a contamination from plasma membrane. TIM, TOM and ANT were only detectable in mitochondria. Immunofluorescence was specific, as shown by spectral analysis of fluorochromes (data not shown). Specificity of the Abs was assayed by Semiquantitative WB. The chemiluminescent WB signal of Rh, Na+ /K+ -ATPase, TIM ANT, and TOM in OS, disks and retinal mitochondria-enriched fractions, used as a control are in panels A2, B2, C2, D2, and E2, respectively. IS organella contamination in disk fractions seems negligible. Relative quantification (panel G) of chemiluminescent band signal was calculated as the ratio of densitometric value of the band to that of total protein in each lane of the SDS-PAGE gel (as stained with Colloidal Blue Coomassie, shown in panel F). The presence of total DNA, and of mitochondrial DNA was assessed in disk samples, in comparison to bovine retinal aliquots, taken as a control. Data NanoDrop® analysis of the samples show a great difference in the concentration of the DNA extraced, being 1645.5 and 51.4 ng/␮l in retinas and disks, respectively. The ratio between the absorption at 260 vs 280 nm was 1.8 ± 0.02 for retinal DNA and 1.67 ± 0.02 for disks. The smooth absorbance spectrum from retina (green) showing an absorbance maximum at 260 nm demonstrates the high quality of the extracted DNA (Fig. 1S). The flat absorbance spectrum of disks (red in Fig. 1S), without any evident absorbance maximum, likely depends on the sensibly lower starting material and suggests the presence of nucleic acid degradation. The presence of mitochondrial DNA in disks, was assessed with a primer pair specific for bovine mitochondrial DNA (see Section 2). Semiquantitative PCR assays conducted on an equal amount of DNA of the samples showed that while the amplification product of retinal mitochondrial DNA was visualized after 15 cycles and augmented with the increase in cycle number, disk mitochondrial DNA was detectable only after 18 cycles. Overall, the amplification of disk DNA confirmed the poor quality of DNA extracted from disks (Fig. 2S).

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Fig. 6. Evaluation of disk contamination by IS organella. (A1) CLSM image of Cy3 indirect fluorescence of Rh; (B1, C1, and D1) are CLSM images of indirect fluorescence of Na+ /K+ -ATPase, TIM, and ANT on disks, merged to the transmission image of the disks in the field. E1 is CLSM image of indirect fluorescence of TOM on disks. WB, conducted with the same primary Abs on disks (D), rod OS (R) and mitochondria (M). Chemiluminescence of Rh, Na+ /K+ -ATPase, TIM, ANT and TOM is in (A2), (B2), (C2), (D2) and (E2) respectively. (F) Reports the semiquantitative densitometric analysis of these signals. The protein pattern is in (G). Panels are representative of at least five experiments.

3.7. Comparison among mitochondria and “mitochondria prepared as disks” Fig. 7, shows that coupled mitochondria prepared and assayed in the presence of 50 nM Cyclosporin A conduct ATP synthesis (1.2 ± 0.2 ␮mol/min/mg of protein) sensitive to inhibition by oligomycin (95%), DCCD (93%), and FCCP (97%) (Fig. 7). Conversely, “mitochondria prepared as disks” (i.e.: diluted in Milli-Q water in the absence of nutrients and of Cyclosporin A, for 1 h (see Section 2)), synthesize a negligible amount of ATP (bar 5). Table 1 reports the mean values of ATP production expressed as ␮mol/min/mg of protein.

Table 1 ATP synthesis in mitochondria, and “mitochondria prepared as disks”. Fig. 7. ATP synthesis in retinal mitochondria-enriched fractions and in “mitochondria prepared as disks”. Histogram shows ATP formation in mitochondria with 50 nM Cyclosporin A over 1 min, at 37 ◦ C, at pH 7.4 in Controls (bar 1) (0.03 mg/ml). Addition of 10 ␮M oligomycin (bar 2), 0.5 mM DCCD (bar 3), or 1 mM FCCP (bar 4) inhibited ATP production by 95%, 93%, and 96%, respectively. Lane 5 shows ATP synthase activity by “mitochondria prepared as disks” (see Section 2). Each solid bar, representative of four separate experiments, represents mean ± S.D; paired Student’s t test was used. *p < 0.01.

␮mol ATP synthesized/min/mg protein Mitochondria-enriched fraction “Mitochondria prepared as disks”

1.2 ± 0.2 0.009 ± 0.002

Table shows ATP production from ADP and Pi at 37 ◦ C by coupled mitochondria, or “mitochondria prepared as disks”. Data are the mean of four separate experiments ± S.D.

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Fig. 8. Respiration rates in retinal mitochondria-enriched fraction and in “mitochondria prepared as disks”. (A) Respiration rates in retinal coupled mitochondria (0.04 mg/1.7 ml) prepared and assayed in the presence of 50 nM Cyclosporin A. (B) Respiration rates in coupled “mitochondria prepared as disks” (0.04 mg/1.7 ml) (see Section 2). Additions: pyruvate (2.5 mM); malate (5 mM); ADP (0.19 mM); rotenone (0.1 mM); succinate (5 mM); antimycin A (0.05 mM); ascorbate (10 mM); KCN, potassium-cyanide (0.5 mM).

Fig. 9. Semiquantitative Western Blot analysis of the four respiratory complexes. WB analysis was conducted on disks (D), rod OS (R) mitochondria (M) and mitochondria prepared as disks (Md) using a primary Abs against NADH-ubiquinone oxidoreductase (A), Succinate Dehydrogenase (B), Cytochrome c reductase (C) and Cytochrome c oxidase (D). (E) Reports the semiquantitative densitometric analysis of these signals. The protein pattern of boiled samples is in (F). Panels are representative of three experiments.

Fig. 8(panel A) is a representative amperometric recording trace of respiration rates of coupled mitochondria prepared and assayed in the presence of 50 nM Cyclosporin A. “Mitochondria prepared as disks” (panel B) are unable to consume O2 likely due to the opening of the MTP (Berman et al., 2000). Table 2 shows the mean O2 consumption of mitochondria-enriched fractions and “mitochondria Table 2 Comparison of oxygen consumption by disks, mitochondria-enriched fractions and “mitochondria prepared as disks”. Samples

Oxygen consumption (␮MO2 /min/mg protein) Complex I + III + IV

Disks Mitochondria-enriched fraction “Mitochondria prepared as disks”

151 ± 12 243 ± 23 0.23 ± 0.03

Complex II + III + IV 253 ± 21 160 ± 10 0.56 ± 0.07

Respiration rates in coupled disks, mitochondria and “mitochondria prepared as disks”. To stimulate the Complex I + III + IV pathway, NADH was substituted by 5 mM pyruvate and 2,5 mM malate. Data are the mean ± SD of at least eight experiments.

prepared as disks”, in coupled conditions. Rates were calculated from the first derivative of the oxygraph traces. Substrates and inhibitors were the same used for disks.

3.8. Semiquantitative Western Blot analysis of the four respiratory complexes Fig. 9 shows the chemiluminescent WB signal of the presence on disks of: NADH-ubiquinone oxidoreductase (panel A); Succinic dehydrogenase (panel B); Cytochrome c reductase, (panel D); Cytochrome c oxidase (panel D), monitored with specific Abs in disks, OS, retinal mitochondria-enriched fractions, and mitochondria prepared as disks, the latter two used as a control. Relative quantification (panel E) of chemiluminescent band signal was calculated as the ratio of the densitometric value of each band to that of total protein in each lane of the SDS-PAGE gel (as stained with Colloidal Blue Coomassie, shown in panel F, where OS and disks proteins were boiled before the run, in order to eliminate Rh).

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Fig. 10. Assay and semiquantitative Western Blot analysis of Citrate Synthase. (A) Comparison of Citrate synthase activity in disks (D), rod OS (R), mitochondria-enriched fractions (M) and mitochondria prepared as disks (Md). Activity is expressed as IU/mg of total protein. (B) Semiquantitative WB analysis conducted with the primary Ab against C-terminus of citrate synthase of human origin, on the same samples. (C) Semiquantitative densitometric analysis of these signals. The protein pattern is the same as represented in Fig. 9. Panels are representative of three experiments.

3.9. Citrate synthase assay Citrate synthase (CS) was assayed in ROS, disks, mitochondria and mitochondria prepared as disks, as an example of a mitochondrial matrix protein. Its expression in disks, together with that of other Krebs cycle enzymes had been reported in our previous proteomic paper (Panfoli et al., 2008). The presence of a number of cytosolic proteins identified by Mass spectrometry of the 2-DE gels, among is justified by the fact that disks purified by Ficoll flotation are aggregated, entrapping some ROS cytosol (Ravera et al., 2007a). Fig. 10 shows that CS activity (panel A) is consistent, and enriched in disks with respect to OS, that is a less purified fraction, indicating that it may not be a contaminant. Mitochondria and mitochondria prepared as disks (Md) were used as a control. WB analysis (panel B) with a specific antibody confirmed the presence of the enzyme in all of the fractions. Relative quantification (panel G) of chemiluminescent band signal, calculated as the ratio of densitometric value of the band to that of total protein in the SDS-PAGE gel (similar to that in Fig. 9) where disks and OS were boiled before the run.

4. Discussion The present biochemical metabolic studies suggest that rod OS disks possess a respiratory capacity (Figs. 4 and 5) which can build up a H+ (Fig. 3) to drive ATP synthesis (Fig. 2) from ADP and Pi , accordingly to our recent proteomic studies that identified several subunits of the mitochondrial Electron Transfer Chain (ETC) complexes and of ATP synthase in enriched outer segment disk membrane preparations (Panfoli et al., 2008). ATP synthesis and O2 consumption by disks were sensitive to the classical inhibitors (Figs. 2 and 4). Biochemical assays (Fig. 5) and semiquantitative Western Blotting analyses with specific antibodies (Fig. 9) show that the ETC complexes are functionally expressed in disks. Respiration rates of disks were similar to those observed in mitochondria (compare Figs. 4 and 8), but not sensitive to the potential loss that mitochondria suffer. In fact, disks were prepared and assayed in the absence of inhibitors of the opening of the MTP (Crompton et al., 1988) (Beutner et al., 1996). The ability of disks to use NADH as a respiring substrate indicates a free access to Complex I (Figs. 2–4), suggestive of a sidedness of the ETC and of ATP synthase similar to that of submitochondrial particles, therefore devoid of substrate permeability barriers. The hypothesis that putative disk ATP synthase employs a transmembrane H+ to synthesize ATP like in mitochondria (Boyer, 1997), is supported by the redistribution of RH-123 across the disk membrane (Fig. 3) and by the inhibition of ATP synthesis by uncouplers (Fig. 2). RH-123 fluorescence quenching kinetics gives reliable and sensitive evaluation of H+ (Emaus et al., 1986, Uckermann et al., 2004). We had found that the OS selectively stains with mitochondrial vital dyes ex vivo, in a manner sensitive to pharmacological disruption of the disk H+ (Bianchini et al., 2008).

Observations dating since 1968 are consistent with the notion of disks as organelles able to store and release H+ and unusually impermeant to H+ (McConnell et al., 1968, Kaupp et al., 1981). A H+ uptake by disks (2.8 mol H+ /mol Rh) stimulated by ADP at acidic pH, and a H+ release inhibited by KCN at neutral pH were observed (McConnell et al., 1968, Kaupp et al., 1981). The H+ across disk membranes was studied by light scattering experiments (Uhl and Desel, 1989; Uhl et al., 1989). Signals (half life, 1 min) were accompanied by ATP hydrolysis and inhibited by oligomycin and DCCD. Caretta and Cavaggioni performed ATP turnover experiments with isolated frog OS, and reported that addition of the uncoupler 2,4-dinitrophenol caused a decrease in ATP content (Carretta and Cavaggioni, 1976). Mammalian retina consumes an elevated O2 amount: OS have a 3-fold greater O2 consumption than the inner retina (Braun et al., 1995). Anaerobic glycolysis may provide enough ATP for cGMP turnover in dark adapted rods, but illumination increases cGMP turnover by 5-folds, exceeding the glycolytic capacity of rods to produce ATP (Biernbaum and Bownds, 1985, Ames et al., 1986, Pepe, 2001). ATP need was calculated to be 127 ␮M/s in the light (Ames et al., 1986). The average diffusion time for cGMP (smaller than ATP) through the entire outer segment was estimated to be about six minutes by fluorescent probes (Olson and Pugh, 1993) while the time involved in phototransduction reactions is of the order of milliseconds. It is tempting to presume that ATP supply for phototransduction in OS be carried out mainly by oxydative phosphorylation. The aerobic ATP synthesis of 0.7 ± 0.1 ␮mol/min/mg here reported corresponds to about 1330 ␮M/s (assuming M.W. of Rh 39 kDa, and its concentration in OS 3 mM, about 85% of the disk protein), one order of magnitude in excess with respect to the photoreceptor need of 127 ␮M ATP/s in the light (Ames et al., 1986). The great membrane surface area of the OS disks would be congenial to both light and O2 capture. The presence of the ATP synthase in ectopic locations has been reviewed (Chi and Pizzo, 2006), however, most Authors ascribe to ectopic ATP synthase functions different from ATP synthesis (Arakaki et al., 2003) (Martinez et al., 2003). Besides, the membrane of a cell bears an extracellular positive charge, not favourable for the functioning of an ATP synthase with an outward facing F1 moiety (ecto-F1 -ATPase). In fact, only ATP hydrolysis by ecto-F1 -ATPase was reported in hepatocyte cultures (Fabre et al., 2006). Besides, if ATP synthase is not coupled to ETC, that carries out proton pumping and electron transfer (Wallace, 1999), its activity produces a pH unbalance. An extracellular ATP synthesis on the surface of hepathomes (Mangiullo et al., 2008) and of the human umbilical endothelial cells (HUVECs) (Arakaki et al., 2003) was reported. However, Quillen et al. (2006) did not find any ATP synthesis in HUVECs. Entity of the ectopic ATP synthesis ranged from picomoles to nanomoles. By contrast, disks synthesize micromoles of ATP. To the best of our knowledge the present is the first study reporting such a consistent ectopic ATP synthesis. It must also be noted that activities are

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referred to total protein, including Rh which accounts for about 85% (Stryer, 1996). The disk ecto-F1 would face a vesicle side bearing a negative charge, as disks form as invaginations of the plasma membrane, favouring the outward H+ flow (Capaldi et al., 1994) to the OS cytosol. Interestingly, mitochondrial disorders, a group of human diseases characterized by defects of the OXPHOS, primarily affect visual system and the CNS and PNS (Zeviani and Di Donato, 2004). The possibility that our results are due an artefact causing a fusion of mitochondrial vesicles with disks may not be ruled out. However, the quality of the disk preparations seems good (Figs. 1 and 6). Moreover, a contamination would not account for the absence of ANT, TIM, TOM and Na+/ K+ -ATPase (Fig. 6). Even if disks preparations were composed mostly of intact mitochondria, not visible by TEM analysis (Fig. 1), these would be uncoupled due to the procedure of disk isolation (Smith and Litman, 1982), as demonstrated in Figs. 7 and 8. Contaminating mitochondria, if any, would not respire after 3 h incubation in water in the absence of Cyclosporin A (Mitchell, 1961) (Boyer, 1997). Finding of ETC and ATP synthase in the purified retinal rod OS disks may mean that these be regarded as some particular mitochondria. nevertheless, the absence of assembling factors in the 2-DE gel of disks (Panfoli et al., 2008) and the observation that mitochondrial DNA is a contaminant in disks (Figs. 1S and 2S) may rule out the possibility that the assembly of complexes would proceed in the OS. Rather it may be speculated that the ETC and ATP synthase are transferred from the preformed inner mitochondrial membranes, fused to thenascent disks. In fact, the antibodies against the redox chains I to V recognized the proteins in both disks and mitochondrial membranes, which indicates a sequence homology (Fig. 9) especially considering that ND4L and COX II are subunits encoded by the mitochondrial DNA. Furthermore, as the redox complexes I, II and IV are thought to form a supercomplex in vivo (Genova et al., 2008), the assembly of the ETC may take place in mitochondria. It seems that the putative fusion would bring also some matrix proteins, considering the consistent activity of CS that we report (Fig. 10). This may mean that some proteins of the mitochondrial matrix be functionally expressed in the OS cytosol. Indeed, it is conceivable that the ETC be continually fed by Krebs cycle directly within the interdiskal space. Matrix proteins may be transferred into OS cytosol during an hypothetic fusion of mitochondria with disks in formation. On the other hand, an artefactual fusion during experiments would be ruled out by the consideration that even if a membrane embeds the respiratory complexes it may not be able to synthesize ATP, unless it is impermeant to protons, an essential prerequisite to build up a proton gradient (whose presence across disks is shown in Fig. 3), but a very unusual characteristic restricted to respiring organella. A physiological recruitment of mitochondrial proteins into disk membranes may be hypothesized. Mitochondria may then fuse with disk membranes during their formation, as recently discussed for myelin sheath in our report of ectopic ATP synthesis and O2 consumption by intact myelin vesicles (Ravera et al., 2009). In our proteomic study by ESI-MS/MS of the monodimensional gel one protein related to fusion: dynamin 1 (gi|123230374) was found. Further studies may lead to a deeper understanding of the mechanism of exportation of mitochondrial proteins on extramitochondrial membranes.

Acknowledgments We thank Pietro Calissano (University of Roma), Antonio de Flora (University of Genova) and Bruno Andrea Melandri (University of Bologna) for their invaluable contribution.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biocel.2009.08.013.

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