The quantitation of ADP diffusion gradients across the outer membrane of heart mitochondria in the presence of macromolecules

We have previously provided evidence that diffusion of metabolites across the porin pores of mitochondrial outer membrane is hindered. A functional consequence of this diffusion limitation is the dynamic compartmentation of ADP in the intermembrane space. These earlier studies were done on isolated mitochondria suspended in isotonic media without macromolecules, in which intermembrane space of mitochondria is enlarged. The present study was undertaken to assess the diffusion limitation of outer membrane in the presence of 10% (w/v) dextran M20, in order to mimic the action of cytosolic macromolecules on mitochondria. Under these conditions, mitochondria have a more native, condensed configuration.Flux-dependent concentration gradients of ADP were estimated by measuring the ADP diffusion fluxes across the porin pores of isolated rat heart mitochondria incubated together with pyruvate kinase (PK), both of which compete for ADP regenerated by mitochondrial creatine kinase (mtCK) within the intermembrane space or by yeast hexokinase (HK) extramitochondrially. From diffusion fluxes and bulk phase concentrations of ADP, its concentrations in the intermembrane space were calculated using Fick's law of diffusion. Flux-dependent gradients up to 23 microM ADP (for a diffusion rate of J(Dif)=1.9 micromol ADP/min/mg mitochondrial protein) were observed. These gradients are about twice those estimated in the absence of dextran and in the same order of magnitude as the cytosolic ADP concentration (30 microM), but they are negligibly low for cytosolic ATP (5 mM). Therefore, it is concluded that the dynamic ADP compartmentation is of biological importance for intact heart cells.If mtCK generates ADP within the intermembrane space, the local ADP concentration can be clearly higher than in the cytosol resulting in higher extramitochondrial phosphorylation potentials. In this way, mtCK contributes to ensure optimal kinetic conditions for ATP-splitting reactions in the extramitochondrial compartment.     

Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells

Dextran M20 was added to isolated rat liver mitochondria to mimic cytosolic macromolecules. Under these conditions, the morphological changes in the mitochondrial periphery that occur upon isolation of the organelle are restored, i.e. the volume of the intermembrane space decreases and the contact site frequency increases. The ADP routing from mitochondrial kinases at various locations was investigated by using the activities of oxidative phosphorylation and externally added pyruvate kinase as sensors for ADP transport into the matrix and extramitochondrial compartment, respectively. The studies reveal that a significant fraction of the ADP generated by either adenylate kinase in the intermembrane space or by outer membrane bound hexokinase isozyme I, is not accessible to extramitochondrial pyruvate kinase. Quantitative information on the ADP compartmentation in rat liver mitochondria was obtained by comparing the ADP supply from mitochondrial kinases to oxidative phosphorylation with that of non-bound, extramitochondrially located kinases. This approach allowed us to estimate the ADP diffusion gradients which were present across the outer membrane and between the compartment formed by bound hexokinase and the extramitochondrial compartment. In the presence of 10% dextran M20 these ADP gradients amounted to approximately 12 µM. The possible role of mitochondrial kinases in ADP transport into mitochondria in vivo is discussed. (Mol Cell Biochem 174: 43–51, 1997)     

The cation-selective substate of the mitochondrial outer membrane pore: Single-channel conductance and influence on intermembrane and peripheral kinases

The polyanion-induced substate of the outer mitochondrial membrane was studiedin vivo andin vitro. Study of the substate in artificial bilayers showed that it is highly cation selective. The induction of the substate in intact mitochondria leads to a complete inhibition of the intermembrane kinases, such as creatine kinase and adenylate kinase, which were excluded from the external ATP pool. Peripheral kinases, such as hexokinase, were blocked when they utilized internal ATP. The results with intact mitochondria suggested the existence of two regions of the outer membrane containing channels of different states, which may be involved in the regulation of intermembrane and peripheral kinases.     

The influence of the cytosolic oncotic pressure on the permeability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the intermembrane space

Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.     

Early ischemia-induced alterations of the outer mitochondrial membrane and the intermembrane space: A potential cause for altered energy transfer in cardiac muscle?

Our aim was to carefully analyse the time-dependent changes that affect the mitochondrial function of myocardial cells during and after an ischemic episode. To this end, variables characterizing mitochondrial function have been evaluated on myocardial samples from isolated rat hearts subjected to different conditions of ischemia. The technique of permeabilized fibers was used in order to evaluate the mitochondrial function whilst retaining intracellular structure.The earliest alteration that could be detected was a decrease in the stimulatory effect of creatine on mitochondrial respiration. This alteration became more pronounced as the severity (or duration) of the ischemia increased. Afterwards, a significant decrease in the apparent K_m of mitochondrial respiration for ADP also appeared, followed by a diminution of the maximal respiration rate which was partly restored by adding cytochrome c. Finally, for the most severe conditions of ischemia, the basal respiratory rate also increased. These observations are indicative of a sequence of alterations affecting first the intermembrane space, then the outer mitochondrial membrane, and finally the inner membrane. The discussion is focused on the very early alterations, that could not be detected using the conventional techniques of isolated mitochondria. We postulate that these alterations to the intermembrane space and outer mitochondrial membrane can induce disturbances both in the channelling of energy from the mitochondria, and on the signalling towards the mitochondria. The potential consequences on the regulation of the production of energy (ATP, PC) by the mitochondria are evoked.     

The role of contact sites between inner and outer mitochondrial membrane in energy transfer

Three functions have been suggested to be localized in contact sites between the inner and the outer membrane of mitochondria from mammalian cells: (i) transfer of energy from matrix to cytosol through the action of peripheral kinases; (ii) import of mitochondrial precursor proteins; and (iii) transfer of lipids between outer and inner membrane. In the contact site-related energy transfer a number of kinases localized in the periphery of the mitochondrion play a crucial role. Two examples of such kinases are relevant here: (i) hexokinase isoenzyme I which is capable of binding to the outer aspect of the outer membrane; and (ii) the mitochondrial isoenzyme of creatine kinase which is localized in the intermembrane space. Recently, evidence was presented that both hexokinase and creatine kinase are preferentially localized in contact sites (Adams, V. et al. (1989) Biochim. Biophys. Acta 981, 213-225). The aim of the present experiments was two-fold. First, to establish methods which enable the bioenergetic aspects of energy transfer mediated by kinases in contact sites to be measured. In these experiments emphasis was on hexokinase, while 31P-NMR was the major experimental technique. Second, we wanted to develop methods which can give insight into factors playing a role in the formation of contact sites involved in energy transfer. In the latter approach, mitochondrial creatine kinase was studied using monolayer techniques.     

Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.     

Dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space of rat-heart mitochondria

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.     

Alterations of the bioenergetics systems of the cell in acute and chronic myocardial ischemia

The aim of the works presented here is to analyze the alterations induced by acute ischemia-reperfusion and chronic ischemia on mitochondrial function, in relation to alterations on heart function. Parameters of mitochondrial function were assessed on skinned fibers coming from isolated perfused rat hearts. The effects of chronic ischemia were studied on a rat model of left descending coronary artery stenosis. Two key events observed after acute ischemia-reperfusion and chronic ischemia are the decrease (or the loss) of the stimulatory effect of creatine and the alteration of outer mitochondrial permeability to cytochrome c and ADP. Taken together, these effects indicate the alteration of the intermembrane space architecture leading to the loss of intracellular adenine nucleotides compartmentation and possibly of functional coupling of mitochondrial creatine kinase and adenine nucleotide translocase. These alterations result in the impairment of intracellular energy transfer (channeling) from mitochondria to ATP-utilizing sites located in the cytosol. This may play a significant role in ischemic injury and alterations in heart function. We show that these effects were prevented by effective cardioprotective strategies like ischemic preconditioning or pharmacological preconditioning by perfusion of mitochondrial ATP-sensitive potassium channel openers. We hypothesize that an open mitochondrial ATP-sensitive potassium channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP.     

Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane-matrix preparation

To define more clearly the interactions between mitochondrial creatine kinase and the adenine nucleotide translocase, the outer membrane of rat heart mitochondria was removed by digitonin, producing an inner membrane-matrix (mitoplast) preparation. This mitoplast fracton was well-coupled and contained a high specific activity of mitochondrial creatine kinase. Outer membrane permeabilization was documented by the loss of adenylate kinase, a soluble intermembrane enzyme, and by direct antibody inhibition of mitochondrial creatine kinase activity. With this preparation, we documented four important aspects of functional coupling. Kinetic studies showed that oxidative phosphorylation decreased the value of the ternary enzyme-substrate complex dissociation constant for MgATP from 140 to 16 microM. Two approaches were used to document the adenine nucleotide translocase specificity for ADP generated by mitochondrial creatine kinase. Exogenous pyruvate kinase (20 IU/ml) could not readily phosphorylate ADP produced by creatine kinase, since added pyruvate kinase did not markedly inhibit creatine + ATP-stimulated respiration. Additionally, when ADP was produced by mitochondrial creatine kinase, the inhibition of the translocase required 2 nmol of atractyloside/mg of mitoplast protein, while only 1 nmol/mg was necessary when exogenous ADP was added. Finally, the mass action ratio of the mitochondrial creatine kinase reaction exceeded the apparent equilibrium constant when ATP was supplied to the creatine kinase reaction by oxidative phosphorylation. Overall, these results are consistent with much data from intact rat heart mitochondria, and suggest that the outer membrane plays a minor role in the compartmentation of adenine nucleotides. Furthermore, since the removal of the outer membrane does not alter the unique coupling between oxidative phosphorylation and mitochondrial creatine kinase, we suggest that this cooperation is the result of protein-protein proximity at the inner membrane surface.     

Heart mitochondrial creatine kinase revisited: the outer mitochondrial membrane is not important for coupling of phosphocreatine production to oxidative phosphorylation

The state of mitochondrial creatine kinase (CKmi-mi) in intact dog heart mitochondria and mitoplasts and the mechanism of its functional coupling with the oxidative phosphorylation system have been reinvestigated under different osmotic conditions and ionic compositions of the medium. It has been established that in a medium which mimics the cardiac cell cytoplasma, dissociation of CKmi-mi from the membrane of mitoplasts increases when the mitoplasts are swollen due to hypoosmotic treatment. It was shown by EPR that hypoosmotic treatment results in the enhancement of the mobility of phospholipids in the membrane bilayer. It has been also shown that when CKmi-mi is detached from the inner membrane in intact mitochondria in isotonic KCl solution, the effects of the coupling between CKmi-mi and oxidative phosphorylation via ATP/ADP translocase disappear in spite of the presence of CKmi-mi in the intermembrane space and intactness of the outer mitochondrial membrane. Therefore, this coupling cannot be explained by the "compartmented coupling" mechanism or "dynamic adenine nucleotide compartmentation" in the intermembrane space due to diffusion limitation for adenine nucleotides through the outer mitochondrial membrane, as has been supposed by several authors (F.N. Gellerich et al. (1987) Biochim. Biophys. Acta 890, 117-126; S.P.J. Brooks and C.H. Suelter (1987) Arch. Biochem. Biophys. 253, 122-132). The data obtained show that the displacement of the enzyme from the membrane results in significantly increased sensitivity of the coupled processes of aerobic phosphocreatine synthesis to inhibition by the product, phosphocreatine. Thus, all results show that under physiological osmotic and ionic conditions CKmi-mi remains firmly attached to the inner mitochondrial membrane and effectively coupled with ATP/ADP translocase due to intimate dynamic interaction between those proteins.     

Compartmented coupling of chicken heart mitochondrial creatine kinase to the nucleotide translocase requires the outer mitochondrial membrane

The kinetic coupling of mitochondrial creatine kinase (MiMi-CK) to ADP/ATP translocase in chicken heart mitochondrial preparations is demonstrated. Measuring the MiMi-CK apparent Km value for MgATP2- (at saturating creatine) gives a value of 36 microM when MiMi-CK is coupled to oxidative phosphorylation. This Km value is threefold lower than the Km for enzyme bound to mitoplasts or free in solution. The nucleotide translocase Km value for ADP decreases from 20 to 10 microM in the presence of 50 mM creatine only with intact mitochondria. Similar experiments with mitoplasts do not give decreased Km values. The observed Km differences can be used to calculate the concentration of ATP and ADP under steady-state conditions showing that the observed differences in the kinetic constants accurately reflect the enzyme activities of MiMi-CK under the different conditions. The behavior of the Km values provides evidence for what we term compartmented coupling. Therefore, like the rabbit heart system (S. Erickson-Viitanen, P. Viitanen, P. J. Geiger, W. C. T. Yang, and S. P. Bessman (1982) J. Biol. Chem. 257, 14395-14404) compartmented coupling requires an intact outer mitochondrial membrane. The apparent Km values for normal or compartmentally coupled systems can be used to calculate steady-state values of ATP and ADP by coupling enzyme theory. Hence, the overall kinetic parameters accurately reflect the behavior of the enzymes whether free in solution or in the intermembrane space.     

Mechanisms by which opening the mitochondrial ATP- sensitive K(+) channel protects the ischemic heart

Diazoxide opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoK(ATP) channel opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoK(ATP) channel inhibitor 5-hydroxydecanoate. We hypothesize that an open mitoK(ATP) channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced by either osmotic swelling or diazoxide, increased the half-saturation constant for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion.     

Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane

The diffusion of metabolites across the outer mitochondrial membrane is essential for coupled cellular respiration. The outer membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-x(L) restores metabolite exchange across the outer membrane without inducing the loss of cytochrome c from the intermembrane space. Restoration of outer membrane permeability to anionic metabolites does not occur directly through Bcl-x(L) ion channels. Instead, recombinant Bcl-x(L) maintains the outer mitochondrial membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous beta-NADH, recombinant Bcl-x(L) can sustain outer mitochondrial membrane permeability to ADP. beta-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the outer mitochondrial membrane by inhibiting VDAC closure.     

Multiple pathways of cytochrome c release from mitochondria in apoptosis

Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating permeabilization of the outer mitochondrial membrane and the release of intermembrane space proteins remain controversial. Here, we discuss possible scenarios of the outer membrane permeabilization. The mechanisms by which the intermembrane space proteins are released from mitochondria depend presumably on cell type and on the nature of the apoptotic stimulus. The variety of mechanisms that can lead to outer membrane permeabilization might explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex

Import of the ADP/ATP carrier (AAC) into mitochondria requires the soluble TIM10 complex to cross the intermembrane space. We report here that Tim9 and Tim10 purified from Escherichia coli can form a complex of the same size as the endogenous complex from yeast mitochondria. This shows that no other mitochondrial protein is required for the formation of the TIM10 complex. Co-expression of both proteins rendered Tim9 more soluble and allowed purification of the reconstituted complex in a single step. Urea/EDTA treatment of recombinant Tim10 allowed its import into tim10-ts mitochondria that lack endogenous Tim10 and cannot import AAC. In this way, we were able to (i) reconstitute the TIM10 complex in the intermembrane space and (ii) restore import of AAC to almost wild-type levels. The reconstituted TIM10 complex not only facilitated passage of AAC across the outer membrane but also ensured its accurate membrane insertion. We conclude that the TIM10 complex can be formed exclusively from Tim9 and Tim10 and that the reconstituted complex efficiently restores AAC import in a strain lacking the TIM10 complex.     

Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice

The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.     

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.     

Mitochondrial import of the ADP/ATP carrier: the essential TIM complex of the intermembrane space is required for precursor release from the TOM complex

The mitochondrial intermembrane space contains a protein complex essential for cell viability, the Tim9-Tim10 complex. This complex is required for the import of hydrophobic membrane proteins, such as the ADP/ATP carrier (AAC), into the inner membrane. Different views exist about the role played by the Tim9-Tim10 complex in translocation of the AAC precursor across the outer membrane. For this report we have generated a new tim10 yeast mutant that leads to a strong defect in AAC import into mitochondria. Thereby, for the first time, authentic AAC is stably arrested in the translocase complex of the outer membrane (TOM), as shown by antibody shift blue native electrophoresis. Surprisingly, AAC is still associated with the receptors Tom70 and Tom20 when the function of Tim10 is impaired. The nonessential Tim8-Tim13 complex of the intermembrane space is not involved in the transfer of AAC across the outer membrane. These results define a two-step mechanism for translocation of AAC across the outer membrane. The initial insertion of AAC into the import channel is independent of the function of Tim9-Tim10; however, completion of translocation across the outer membrane, including release from the TOM complex, requires a functional Tim9-Tim10 complex.