Ionic strength of the intermembrane space of intact mitochondria as estimated with fluorescein-BSA delivered by low pH fusion

The electrostatic interactions of cytochrome c with its redox partners and membrane lipids, as well as other protein interactions and biochemical reactions, may be modulated by the ionic strength of the intermembrane space of the mitochondrion. FITC-BSA was used to determine the relative value of the mitochondrial intermembrane ionic strength with respect to bulk medium external to the mitochondrial outer membrane. FITC-BSA exhibited an ionic strength-dependent fluorescence change with an affinity in the mM range as opposed to its pH sensitivity in the microM range. A controlled, low pH-induced membrane fusion procedure was developed to transfer FITC-BSA encapsulated in asolectin liposomes, to the intermembrane space of intact mitochondria. The fusion procedure did not significantly affect mitochondrial ultrastructure, electron transport, or respiratory control ratios. The extent of fusion of liposomes with the mitochondrial outer membrane was monitored by fluorescence dequenching assays using a membrane fluorescent probe (octadecylrhodamine B) and the soluble FITC-BSA fluorescent probe, which report membrane and contents mixing, respectively. Assays were consistent with a rapid, low pH-induced vesicle-outer membrane fusion and delivery of FITC-BSA into the intermembrane space. Similar affinities for the ionic strength-dependent change in fluorescence were found for bulk medium, soluble (9.8 +/- 0.8 mM) and intermembrane space-entrapped FITC-BSA (10.2 +/- 0.6 mM). FITC-BSA consistently reported an ionic strength in the intermembrane space of the functionally and structurally intact mitochondria within +/- 20% of the external bulk solution. These findings reveal that the intermembrane ionic strength changes as does the external ionic strength and suggest that cytochrome c interactions, as well as other protein interactions and biochemical reactions, proceed in the intermembrane space of mitochondria in the intact cell at physiological ionic strength, i.e., 100-150 mM.     

Deleterious role of superoxide dismutase in the mitochondrial intermembrane space

This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1(-/-)) did not. Also, lymphocytes isolated from SOD1(-/-) mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.     

Equilibration of adenylates in the mitochondrial intermembrane space maintains respiration and regulates cytosolic metabolism

Adenylate kinase (AK) uses one each of Mg-complexed and free adenylates as substrates in both directions of its reaction. It is very active in the mitochondrial intermembrane space (IMS), but is absent from the mitochondrial matrix where low [ADP] upon intensive respiration limits the respiratory rate. AK activity in the IMS is linked to ATP/ADP exchange across the inner mitochondrial membrane by using ATP (imported from the matrix) and AMP as substrates, the latter provided by apyrase and other AMP-generating reactions. The ADP formed by AK is exported to the matrix (in exchange for ATP), providing a mechanism for regeneration of ADP during respiration. From the AK equilibrium, and taking pH values characteristic of subcellular compartments, [Mg2+] in the IMS is calculated as 0.4-0.5 mM and in the cytosol as 0.2-0.3 mM, whereas the MgATP:MgADP ratio in the IMS and cytosol is 6-9 and 10-15, respectively. These represent optimal conditions for transport of adenylates (via the maintenance of an ATPfree:ADPfree ratio close to 1) and mitochondrial respiratory rates (via the maintenance of submillimolar [ADPfree] in the IMS). This, in turn, has important consequences for mitochondrial and cytosolic metabolism, including regulation of the protein phosphorylation rate (via changes in the MgATP:AMPfree ratio) and allosteric regulation of mitochondrial and cytosolic enzymes. Metabolomic consequences are discussed in connection with the calculation of metabolic fluxes from subcompartmental distributions of total adenylates and Mg2+.     

Reconstitution and partial purification of the Na(+)-selective Na+/H+ antiporter of beef heart mitochondria.

Na+/H+ antiporters play important physiological roles in most biological membranes. Although they were first discovered in mitochondria (Mitchell, P., and Moyle, J. (1969) Eur. J. Biochem. 9, 149-155), the mitochondrial Na+/H+ antiporter has not yet been reconstituted nor has the protein responsible for its activity been identified. We used detergents to extract proteins from beef heart mitochondria and reconstituted these proteins into lipid vesicles loaded with the fluorescent probe, sodium-binding benzofuran isophthalate. The vesicles exhibited spontaneous, electroneutral Na+ transport that was inhibited by Li+ and Mn2+ with appropriate kinetic constants. These protocols were then used to follow fractionation of the solubilized proteins with DEAE-cellulose columns. We obtained a fraction that catalyzed Na+/H+ antiport with Vmax values of 75-120 mumol/mg protein/min, 500-700 times faster than observed in intact mitochondria. Na+ transport was inhibited by Li+ with I50 values of 0.5-1.0 mM and by Mn2+ with I50 value of 0.5 mM. The Km for Na+ was 31 mM. These values correspond to those found in intact mitochondria, and we conclude that the solubilized mitochondrial Na+/H+ antiporter has been partially purified in a reconstitutively active state.     

Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.     

In vitro import of cytochrome c peroxidase into the intermembrane space: release of the processed form by intact mitochondria

Cytochrome c peroxidase (CCP) is a nuclearly encoded hemoprotein located in the intermembrane space (IMS) of Saccharomyces cerevisiae mitochondria. Wild-type preCCP synthesized in rabbit reticulocyte lysates, however, was inefficiently translocated into isolated mitochondria and was inherently resistant to externally added proteases. To test whether premature heme addition to the apoprecursor was responsible for the protease resistance and the inability to import preCCP, site-directed mutagenesis was used to replace the axial heme ligand (His175) involved in forming a pseudo-covalent link between the heme iron and CCP. Mutant proteins containing Leu, Arg, Met, or Pro at residue 175 of mature CCP were sensitive to proteolysis and were imported into isolated mitochondria as judged by proteolytic processing of the precursor. The inhibition of wild-type CCP translocation across the outer membrane may result from the inability of the heme-containing protein to unfold during the translocation process. Although the protease responsible for cleaving preCCP to its mature form is believed to be located in the IMS, most of the processed CCP was located in the supernatant rather than the mitochondrial pellet. Since the outer membranes were shown to be intact, the anomalous localization indicated that preCCP may not have been completely translocated into the IMS before proteolytic processing or that conformationally labile proteins may not be retained by the outer membrane. Proteolytic maturation of preCCP also occurred in the presence of valinomycin, suggesting that the precursor may be completely or partially translocated across the outer mitochondrial membrane independent of a potential across the inner mitochondrial membrane.     

The Erv1-Mia40 disulfide relay system in the intermembrane space of mitochondria

The compartment between the outer and the inner membranes of mitochondria, the intermembrane space (IMS), harbours a variety of proteins that contain disulfide bonds. Many of these proteins possess a conserved twin Cx(3)C motif or twin Cx(9)C motif. Recently, a disulfide relay system in the IMS has been identified which consists of two essential components, the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40/Tim40. The disulfide relay system drives the import of these cysteine-rich proteins into the IMS of mitochondria by an oxidative folding mechanism. In order to enable Mia40 to perform the oxidation of substrate proteins, the sulfhydryl oxidase Erv1 mediates the oxidation of Mia40 in a disulfide transfer reaction. To recycle Erv1 into its oxidized form, electrons are transferred to cytochrome c connecting the disulfide relay system to the electron transport chain of mitochondria. Despite the lack of homology of the components, the disulfide relay system in the IMS resembles the oxidation system in the periplasm of bacteria presumably reflecting the evolutionary origin of the IMS from the bacterial periplasm.     

Mammalian polynucleotide phosphorylase is an intermembrane space RNase that maintains mitochondrial homeostasis

We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-beta-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in FoF1-ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.     

Steroidogenesis in the zona glomerulosa of the adrenal cortex I. Isolation and some properties of mitochondria from the zona glomerulosa of the bovine adrenal cortex

Isolation procedures for mitochondria from the zona glomerulosa of the bovine adrenal cortex are described and the properties of the mitochondria thus prepared are compared with those isolated from the zona fasciculoreticularis. The cristal membranes of mitochondria in the zona glomerulosa in situ are tubular or tubulovesicular, whereas those of mitochondria in the zona fasciculoreticularis in situ are vesicular. When mitochondria are isolated from the former zone, they invariably showed the condensed configuration regardless of isolation media, whereas those isolated from the latter zone in an ST medium showed the orthodox configuration. When Ca²⁺ was added to mitochondria isolated either from the zona glomerulosa or the zona fasciculoreticularis in an STE medium in the condensed configuration, a transition from the condensed to the orthodox configuration took place; the cristal membranes of mitochondria from the zona glomerulosa became tubular or tubulovesicular and those of mitochondria from the zona fasciculoreticularis became vesicular. Contaminations of mitrochondria of the zona glomerulosa with other cellular organelles were examined using various marker enzymes. There was no difference in cytochrome content between mitochondria of the two zones specified above. The coupling efficiency of mitochondria of the zona glomerulosa was found to be remarkably effected by temperature during the isolation procedures. Effects of various substrates, isolation media, and bovine serum albumin on the coupling efficiency of mitochondria of the glomerulosa are also described.     

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.     

Effects of VDAC isoforms on CuZn-superoxide dismutase activity in the intermembrane space of Saccharomyces cerevisiae mitochondria

Copper and zinc containing superoxide dismutase (CuZnSOD) is located primarily in the cytosol but a small amount of the enzyme has also been identified in the intermembrane space of mitochondria (termed here IMS CuZnSOD). Using Saccharomyces cerevisiae mutants depleted of either isoform of VDAC (voltage-dependent anion-selective channel), we have shown that the activity of IMS CuZnSOD coincides with the presence of a given VDAC isoform and changes in a growth phase dependent way. Moreover, the IMS CuZnSOD activity correlates with the levels of O2*- release from mitochondria and the cytosol redox state. The latter in turn seems to influence the levels of the mitochondrial outer membrane channel protein other than VDAC. Thus, we conclude that in the case of S. cerevisiae both VDAC isoforms influence the IMS CuZnSOD activity and subsequently the expression levels of some mitochondrial proteins.     

Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis

During apoptosis, the mitochondrial membrane potential (MMP) decreases, but it is not known how this relates to the apoptotic process. It was recently suggested that cytochrome c is compartmentalized in closed cristal regions and therefore, matrix remodeling is required to attain complete cytochrome c release from the mitochondria. In this work we show that, at the onset of apoptosis, changes in MMP control matrix remodeling prior to cytochrome c release. Early after growth factor withdrawal the MMP declines and the matrix condenses. Both phenomena are reversed by adding oxidizable substrates. In mitochondria isolated from healthy cells, matrix condensation can be induced by either denying oxidizable substrates or by protonophores that dissipate the membrane potential. Matrix remodeling to the condensed state results in cristal unfolding and exposes cytochrome c to the intermembrane space facilitating its release from the mitochondria during apoptosis. In contrast, when a transmembrane potential is generated due to either electron transport or a pH gradient formed by acidifying the medium, mitochondria maintain an orthodox configuration in which most cytochrome c is sequestered in the cristae and is resistant to release by agents that disrupt the mitochondrial outer membrane.     

Effect of valinomycin on mitochondrial ultrastructure and function of intact Ehrlich ascites tumor cells

Ultrastructural changes in the mitochondria of intact Ehrlich ascites tumor cells were observed after stimulation by valinomycin of the energy-dependent transport of K⁺ into mitochondria. The mitochondria in cells taken directly from the animal displayed an orthodox configuration. After repeated washings of the cells, the mitochondria were converted to the ‘condensed’ or ‘aggregated’ state. The addition of valinomycin resulted in a transformation of mitochondria from the condensed to orthodox and markedly swollen forms. Alterations in cell size, O₂ uptake, and K⁺ content accompanied the changes in mitochondrial morphology.     

Mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space

99% of all mitochondrial proteins are synthesized in the cytosol, from where they are imported into mitochondria. In contrast to matrix proteins, many proteins of the intermembrane space (IMS) lack presequences and are imported in an oxidation-driven reaction by the mitochondrial disulfide relay. Incoming polypeptides are recognized and oxidized by the IMS-located receptor Mia40. Reoxidation of Mia40 is facilitated by the sulfhydryl oxidase Erv1 and the respiratory chain. Although structurally unrelated, the mitochondrial disulfide relay functionally resembles the Dsb (disufide bond) system of the bacterial periplasm, the compartment from which the IMS was derived 2 billion years ago.     

The essential mitochondrial protein Erv1 cooperates with Mia40 in biogenesis of intermembrane space proteins

The proteins of the mitochondrial intermembrane space (IMS) are encoded by nuclear genes and synthesized on cytosolic ribosomes. While some IMS proteins are imported by the classical presequence pathway that involves the membrane potential deltapsi across the inner mitochondrial membrane and proteolytic processing to release the mature protein to the IMS, the import of numerous small IMS proteins is independent of a deltapsi and does not include proteolytic processing. The biogenesis of small IMS proteins requires an essential mitochondrial IMS import and assembly protein, termed Mia40. Here, we show that Erv1, a further essential IMS protein that has been reported to function as a sulfhydryl oxidase and participate in biogenesis of Fe/S proteins, is also required for the biogenesis of small IMS proteins. We generated a temperature-sensitive yeast mutant of Erv1 and observed a strong reduction of the levels of small IMS proteins upon shift of the cells to non-permissive temperature. Isolated erv1-2 mitochondria were selectively impaired in import of small IMS proteins while protein import pathways to other mitochondrial subcompartments were not affected. Small IMS precursor proteins remained associated with Mia40 in erv1-2 mitochondria and were not assembled into mature oligomeric complexes. Moreover, Erv1 associated with Mia40 in a reductant-sensitive manner. We conclude that two essential proteins, Mia40 and Erv1, cooperate in the assembly pathway of small proteins of the mitochondrial IMS.     

Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria

The vast majority of mitochondrial proteins are synthesized in the cytosol and transported into the organelle in a largely, if not completely, unfolded state. The proper function of mitochondria thus depends on folding of several hundreds of proteins in the various subcompartments of the organelle. Whereas folding of proteins in the mitochondrial matrix is supported by members of several chaperone families, very little is known about folding of proteins in the intermembrane space (IMS). We targeted dihydrofolate reductase (DHFR) as a model substrate to the IMS of yeast mitochondria and analyzed its folding. DHFR can fold in this compartment, and its aggregation upon heat shock can be prevented in an ATP-dependent manner. Yme1, an AAA (ATPases associated with diverse cellular activities) protease of the IMS, prevented aggregation of DHFR. Analysis of protein aggregates in mitochondria lacking Yme1 revealed the presence of a number of proteins involved in the establishment of mitochondrial ultrastructure, lipid metabolism, protein import, and respiratory growth. These findings explain the pleiotropic effects of deletion of YME1 and suggest an important role for Yme1 as a folding assistant, in addition to its proteolytic function, in the protein homeostasis of mitochondria     

Zinc-dependent intermembrane space proteins stimulate import of carrier proteins into plant mitochondria

Mitochondrial inner membrane carrier proteins are imported into mitochondria from yeast, fungi and mammals by specific machinery, some components of which are distinct from those utilized by other proteins. Import of two different carriers into plant mitochondria showed that one contains a cleavable presequence which was processed during import, while the other imported in a valinomycin-sensitive manner without processing. Mild osmotic shock of mitochondria released intermembrane space (IMS) components and impaired carrier protein import. Adding back the released IMS proteins as a concentrate in the presence of micromolar ZnCl2 stimulated carrier import into IMS-depleted mitochondria, but did not stimulate import of a non-carrier control precursor protein, the alternative oxidase. Anion-exchange separation of IMS components before addition to IMS-depleted mitochondria revealed a correlation between several 9-10 kDa proteins and stimulation of carrier import. MS/MS sequencing of these proteins identified them as plant homologues of the yeast zinc-finger carrier import components Tim9 and Tim10. Stimulation of import was dependent on either Zn2+ or Cd2+ and inhibited by both N-ethylmalamide (NEM) and a divalent cation chelator, consistent with a functional requirement for a zinc finger protein. This represents direct functional evidence for a distinct carrier import pathway in plant mitochondria, and provides a tool for determining the potential function of other IMS proteins associated with protein import.     

Short-form OPA1 is a molecular chaperone in mitochondrial intermembrane space

Mitochondria, double-membrane organelles, are known to participate in a variety of metabolic and signal transduction pathways. The intermembrane space (IMS) of mitochondria is proposed to subject to multiple damages emanating from the respiratory chain. The optic atrophy 1 (OPA1), an important protein for mitochondrial fusion, is cleaved into soluble short-form (S-OPA1) under stresses. Here we report that S-OPA1 could function as a molecular chaperone in IMS. We purified the S-OPA1 (amino acid sequence after OPA1 isoform 5 S1 site) protein and showed it protected substrate proteins from thermally and chemically induced aggregation and strengthened the thermotolerance of Escherichia coli (E. coli). We also showed that S-OPA1 conferred thermotolerance on IMS proteins, e.g., neurolysin. The chaperone activity of S-OPA1 may be required for maintaining IMS homeostasis in mitochondria.

     

Topology of beef heart cytochrome c oxidase from studies on reconstituted membranes

The orientation of purified beef heart cytochrome c oxidase, incorporated into vesicles by the cholate dialysis procedure [Carroll, R.C., & Racker, E. (1977) J. Biol. Chem. 252, 6981], has been investigated by functional and structural approaches. The level of heme reduction obtained by using cytochrome c along with the membrane-impermeant electron donor ascorbate was 78 +/- 2% of that obtained with cytochrome c and the membrane-permeant reagent N,N,N',N'-tetramethyl-p-phenylenediamine. Electron transfer from cytochrome c is known to occur exclusively from the outer surface of the mitochondrial inner membrane (C side), implying that at least 78% of the oxidase molecules are oriented in the same way in these vesicles as in the intact mitochondria. Trypsin, which cleaves subunit IV near its N terminus, modifies only 5-7% of this subunit in intact vesicles. This removal of the N-terminal residues has been shown to occur only in mitochondrial membranes with their inner side (M side) exposed. Diazobenzene [35S]sulfonate [( 35S]DABS) likewise modifies subunit IV only in submitochondrial particles. Labeling of intact membranes with [35S]DABS resulted in incorporation of only 4-8% of the total counts that could be incorporated into this subunit in membranes made leaky to the reagent by addition of 2% Triton X-100. Therefore, both the functional and structural data show that at least 80% and probably more of the cytochrome c oxidase molecules are oriented with their C domain outermost and M domains in the lumen of vesicles prepared by the cholate dialysis method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Import of small Tim proteins into the mitochondrial intermembrane space

Proteins of the intermembrane space (IMS) of mitochondria are typically synthesized without presequences. Little is known about their topogenesis. We used Tim13, a member of the 'small Tim protein' family, as model protein to investigate the mechanism of translocation into the IMS. Tim13 contains four conserved cysteine residues that bind a zinc ion as cofactor. Import of Tim13 did not depend on the membrane potential or ATP hydrolysis. Upon import into mitochondria Tim13 adopted a stably folded conformation in the IMS. Mutagenesis of the cysteine residues or pretreatment with metal chelators interfered with folding of Tim13 in vitro and impaired its import into mitochondria. Upon depletion of metal ions or modification of cysteine residues, imported Tim13 diffused back out of the IMS. We propose an import pathway in which (1) Tim13 can pass through the TOM complex into and out of the IMS in an unfolded conformation, and (2) cofactor acquisition stabilizes folding on the trans side of the outer membrane and traps Tim13 in the IMS, and drives unidirectional movement of the protein across the outer membrane of mitochondria.