Expression of the mitochondrial ADP/ATP carrier in Escherichia coli. Renaturation, reconstitution, and the effect of mutations on 10 positive residues

Previously, the role of residues in the ADP/ATP carrier (AAC) from Saccharomyces cerevisiae has been studied by mutagenesis, but the dependence of mitochondrial biogenesis on functional AAC impedes segregation of the mutational effects on transport and biogenesis. Unlike other mitochondrial carriers, expression of the AAC from yeast or mammalians in Escherichia coli encountered difficulties because of disparate codon usage. Here we introduce the AAC from Neurospora crassa in E. coli, where it is accumulated in inclusion bodies and establish the reconstitution conditions. AAC expressed with heat shock vector gave higher activity than with pET-3a. Transport activity was absolutely dependent on cardiolipin. The 10 single mutations of intrahelical positive residues and of the matrix repeat (+X+) motif resulted in lower activity, except of R245A. R143A had decreased sensitivity toward carboxyatractylate. The ATP-linked exchange is generally more affected than ADP exchange. This reflects a charge network that propagates positive charge defects to ATP(4-) more strongly than to ADP(3-) transport. Comparison to the homologous mutants of yeast AAC2 permits attribution of the roles of these residues more to ADP/ATP transport or to AAC import into mitochondria.     

Import of ADP/ATP carrier into mitochondria: two receptors act in parallel

We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.     

ADP/ATP carrier is required for mitochondrial outer membrane permeabilization and cytochrome c release in yeast apoptosis

Adenine nucleotide translocator (ANT) is a mitochondrial inner membrane protein involved in the ADP/ATP exchange and is a component of the mitochondrial permeability transition pore (PTP). In mammalian apoptosis, the PTP can mediate mitochondrial outer membrane permeabilization (MOMP), which is suspected to be responsible for the release of apoptogenic factors, including cytochrome c. Although release of cytochrome c in yeast apoptosis has previously been reported, it is not known how it occurs. Herein we used yeast genetics to investigate whether depletion of proteins putatively involved in MOMP and cytochrome c release affects these processes in yeast. While deletion of POR1 (yeast voltage-dependent anion channel) enhances apoptosis triggered by acetic acid, H(2)O(2) and diamide, CPR3 (mitochondrial cyclophilin) deletion had no effect. Absence of ADP/ATP carrier (AAC) proteins, yeast orthologues of ANT, protects cells exposed to acetic acid and diamide but not to H(2)O(2). Expression of a mutated form of Aac2p (op1) exhibiting very low ADP/ATP translocase activity indicates that AAC's pro-death role does not require translocase activity. Absence of AAC proteins impairs MOMP and release of cytochrome c, which, together with other mitochondrial inner membrane proteins, is degraded. Our findings point to a crucial role of AAC in yeast apoptosis.     

Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa. One gene codes for both the cytosolic and mitochondrial forms

Cyclophilin (cyclosporin A-binding protein) has a dual localization in the mitochondria and in the cytosol of Neurospora crassa. The two forms are encoded by a single gene which is transcribed into mRNAs having different lengths and 5' termini (approximately 1 and 0.8 kilobases). The shorter mRNA specifies the cytosolic protein consisting of 179 amino acids. The longer mRNA is translated into a precursor polypeptide with an amino-terminal extension of 44 amino acids which is cleaved in two steps upon entry into the mitochondrial matrix. Neurospora cyclophilin shows about 60% sequence homology to human and bovine cyclophilins.     

Acyl carrier protein is present in the mitochondria of plants and eucaryotic micro-organisms

Proteins antigenically similar to the acyl carrier protein (ACP) found in the mitochondria of Neurospora crassa were detected by immunoblotting and radioimmunoassay techniques in mitochondria isolated from yeast, potatoes, and pea leaves. These mitochondrial proteins were similar to Neurospora ACP both in their electrophoretic mobility and in their unusual decrease in mobility upon reduction. Authentic ACP(s) show this type of change upon conversion of the acylated to the unacylated form. Purified ACP from both spinach chloroplasts and Escherichia coli cells cross-reacted with antibodies raised against Neurospora ACP. Purified ACP from Neurospora cross-reacted with antibodies raised against spinach chloroplast ACP and E. coli ACP. Mitochondria isolated from beef heart and rat brain were tested extensively and exhibited no cross-reaction with any of the three anti-ACP preparations. The discovery of ACP in the mitochondria of other organisms raises questions concerning the possible relationship between ACP and beta-oxidation in mitochondria, the involvement of ACP in de novo biosynthesis of some of the acyl chains in mitochondria and the subcellular locations of fatty acid biosynthesis in plants and eucaryotic micro-organisms.     

Preparation of highly purified mitochondria ofNeurospora crassa on a Percoll gradient

Mitochondria isolated from Neurospora crassa were purified by centrifugation in a Percoll density gradient. Enzyme activities and cytochrome differential spectra revealed a high purity of the mitochondria. As compared with a crude mitochondrial fraction the purified mitochondria exhibited a high respiratory activity and a fine ADP/O ratio. Electrophoresis of nucleic acids demonstrated the absence of cytoplasmic rRNA.     

Transfer of proteins into mitochondria. Precursor to the ADP/ATP carrier binds to receptor sites on isolated mitochondria

The precursor form of Neurospora crassa mitochondrial ADP/ATP carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. If the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. Upon reestablishment of the membrane potential, the bound precursor is imported. This occurs without dissociation of the bound precursor from the mitochondrial surface. We conclude that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway.     

Lithium desensitizes brain mitochondria to calcium, antagonizes permeability transition, and diminishes cytochrome C release

Among the numerous effects of lithium on intracellular targets, its possible action on mitochondria remains poorly explored. In the experiments with suspension of isolated brain mitochondria, replacement of KCl by LiCl suppressed mitochondrial swelling, depolarization, and a release of cytochrome c induced by a single Ca2+ bolus. Li+ robustly protected individual brain mitochondria loaded with rhodamine 123 against Ca2+-induced depolarization. In the experiments with slow calcium infusion, replacement of KCl by LiCl in the incubation medium increased resilience of synaptic and nonsynaptic brain mitochondria as well as resilience of liver and heart mitochondria to the deleterious effect of Ca2+. In LiCl medium, mitochondria accumulated larger amounts of Ca2+ before they lost the ability to sequester Ca2+. However, lithium appeared to be ineffective if mitochondria were challenged by Sr2+ instead of Ca2+. Cyclosporin A, sanglifehrin A, and Mg2+, inhibitors of the mitochondrial permeability transition (mPT), increased mitochondrial Ca2+ capacity in KCl medium but failed to do so in LiCl medium. This suggests that the mPT might be a common target for Li+ and mPT inhibitors. In addition, lithium protected mitochondria against high Ca2+ in the presence of ATP, where cyclosporin A was reported to be ineffective. SB216763 and SB415286, inhibitors of glycogen synthase kinase-3beta, which is implicated in regulating reactive oxygen species-induced mPT in cardiac mitochondria, did not increase Ca2+ capacity of brain mitochondria. Altogether, these findings suggest that Li+ desensitizes mitochondria to elevated Ca2+ and diminishes cytochrome c release from brain mitochondria by antagonizing the Ca2+-induced mPT.     

Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids.

The expression of mitochondrial and hydrogenosomal ADP/ATP carriers (AACs) from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 in Escherichia coli allows a functional integration of the recombinant proteins into the bacterial cytoplasmic membrane. For AAC1 and AAC2 from rat, apparent Km values of about 40 microm for ADP, and 105 microm or 140 microm, respectively, for ATP have been determined, similar to the data reported for isolated rat mitochondria. The apparent Km for ATP decreased up to 10-fold in the presence of the protonophore m-chlorocarbonylcyanide phenylhydrazone (CCCP). The hydrogenosomal AAC isolated from the chytrid fungus Neocallimastix spec. L2 exhibited the same characteristics, but the affinities for ADP (165 microm) and ATP (2.33 mm) were significantly lower. Notably, AAC1-3 from Arabidopsis thaliana and AAC1 from Solanum tuberosum (potato) showed significantly higher external affinities for both nucleotides (10-22 microm); they were only slightly influenced by CCCP. Studies on intact plant mitochondria confirmed these observations. Back exchange experiments with preloaded E. coli cells expressing AACs indicate a preferential export of ATP for all AACs tested. This is the first report of a functional integration of proteins belonging to the mitochondrial carrier family (MCF) into a bacterial cytoplasmic membrane. The technique described here provides a relatively simple and highly reproducible method for functional studies of individual mitochondrial-type carrier proteins from organisms that do not allow the application of sophisticated genetic techniques.     

Suppression of apoptosis by cyclophilin D via stabilization of hexokinase II mitochondrial binding in cancer cells

The permeability transition pore is involved in the mitochondrial pathway of apoptosis. Cyclophilin D, a pore component, has catalytic activity as a peptidyl prolyl cis, trans-isomerase (PPIase), which is essential to the pore opening. It has been reported that cyclophilin D overexpression suppresses apoptosis in cancer cells. To clarify the mechanism of this effect, we generated glioma cells overexpressing wild-type or a PPIase-deficient mutant of cyclophilin D. Interestingly, we found that the PPIase-dependent apoptosis suppression by cyclophilin D correlated with the amounts of mitochondrial-bound hexokinase II, which has anti-apoptotic activity. Inactivation of endogenous cyclophilin D by small interference RNA or a cyclophilin inhibitor was found to release hexokinase II from mitochondria and to enhance Bax-mediated apoptosis. The anti-apoptotic effects of cyclophilin D were canceled out by the detachment of hexokinase II from mitochondria, demonstrating that mitochondrial binding of hexokinase II is essential to the apoptosis suppression by cyclophilin D. Furthermore, cyclophilin D dysfunction appears to abrogate hexokinase II-mediated apoptosis suppression, indicating that cyclophilin D is required for the anti-apoptotic activity of hexokinase II. Based on the above, we propose here that cyclophilin D suppresses apoptotic cell death via a mitochondrial hexokinase II-dependent mechanism in cancer cells.     

Role of the ADP/ATP-antiporter in fatty acid-induced uncoupling of Ca2+-loaded rat liver mitochondria

We show that Ca2+ loading of mitochondria substantially augments the myristate-induced decrease in the transmembrane electric potential difference (deltapsi). Such a Ca2+ action is without effect on the respiration rate and is not accompanied by the high-amplitude swelling when low concentrations of Ca2+ and myristate are used. The myristate-induced deltapsi decrease is prevented and reversed by cyclosporin A (CsA); the decrease is prevented and transiently reversed by nigericin. To explain these effects, we suggest that myristate induces opening of the mitochondrial permeability transition pore at a low-conductance state. Addition of carboxyatractylate (CAtr) after myristate induces the CsA-sensitive uncoupling, but when added after myristate and CsA, CAtr produces a decrease in deltapsi, if the interval between myristate and CsA addition is sufficiently long. The CAtr effect is completely reversed by EGTA and transiently reversed by nigericin. This suggests that the ADP/ATP-antiporter participates in the CsA-sensitive uncoupling when present as a pore complex constituent. ADP/ATP-antiporter that does not take part in the pore complex formation is involved in the CsA-insensitive uncoupling.     

Cations SkQ1 and MitoQ accumulated in mitochondria delay opening of ascorbate/FeSO4-induced nonspecific pore in the inner mitochondrial membrane

It is known that an addition of FeSO4 in the presence of ascorbic acid to cells or mitochondria can injure energy coupling and some other functions in mitochondria. The present study demonstrates that decrease in ascorbate concentration from 4 to 0.2 mM in the presence of the same low concentrations of FeSO4 accelerates the nonspecific pore opening, while cyclosporin A prevents and under some conditions reverses the pore opening. Hydrophobic cations SkQ1 and MitoQ (structural analogs of plastoquinone and coenzyme Q(10), respectively) delay pore opening, SkQ1 being more efficient. It is known that an increase in matrix ADP concentration delays pore opening, while an addition of carboxyatractylate to mitochondria accelerates the beginning of pore opening. Preliminary addition of SkQ1 into a mitochondrial suspension increased the effect of ADP and decreased the effect of carboxyatractylate. These results suggest that under the conditions used SkQ1 protects mitochondria from oxidative damage as an antioxidant when added at extremely low concentrations.     

Kinetic model for Ca2+-induced permeability transition in energized liver mitochondria discriminates between inhibitor mechanisms

Cytotoxicity associated with pathophysiological Ca(2+) overload (e.g. in stroke) appears mediated by an event termed the mitochondrial permeability transition (mPT). We built and solved a kinetic model of the mPT in populations of isolated rat liver mitochondria that quantitatively describes Ca(2+)-induced mPT as a two-step sequence of pre-swelling induction followed by Ca(2+)-driven, positive feedback, autocatalytic propagation. The model was formulated as two differential equations, each directly related to experimental parameters (Ca(2+) flux/mitochondrial swelling). These parameters were simultaneously assessed using a spectroscopic approach to monitor multiple mitochondrial properties. The derived kinetic model correctly identifies a correlation between initial Ca(2+) concentration and delay interval prior to mPT induction. Within the model's framework, Ru-360 (a ruthenium complex) and Mg(2+) were shown to compete with the Ca(2+)-stimulated initiation phase of mPT induction, consistent with known inhibition at the phenomenological level of the Ca(2+) uniporter. The model further reveals that Mg(2+), but not Ru-360, inhibits Ca(2+)-induced effects on a downstream stage of mPT induction at a site distinct from the uniporter. The analytical approach was then applied to promethazine, an FDA-approved drug previously shown to inhibit both mPT and ischemia-reperfusion injury. Kinetic analysis revealed that promethazine delayed mPT induction in a manner qualitatively distinct from that of lower concentrations of Mg(2+). In summary, we have developed a kinetic model to aid in the quantitative characterization of mPT induction. This model is consistent with/informative about the biochemistry of several mPT inhibitors, and its success suggests that this kinetic approach can aid in the classification of agents or targets that modulate mPT induction.     

High performance liquid chromatography purification and amino terminal sequence analysis of the subunits of bovine heart mitochondrial F1-ATPase

All five subunits of bovine heart mitochondrial F1-ATPase have been isolated by reverse-phase HPLC and NH2-terminal sequences determined by gas phase Edman degradations. Bovine gamma exhibits 16 identities in the first 30 residues compared with the NH2-terminus of gamma from E.coli F1. Bovine delta exhibit about 27% identity with residues 28-59 of precursor delta from N.crassa and in the first six residues is identical with delta from S.cerevisiae. Approximately half of bovine epsilon has been sequenced. Possibly significant sequence similarities exist between bovine gamma and epsilon and kinase-related gene and oncogene products. The bovine alpha subunit has a blocked NH2-terminus.     

Cyclosporin a inhibits the protonophoric uncoupling activity of laurate in liver mitochondria

The effects of cyclosporin A, carboxyatractylate, and glutamate on the protonophoric uncoupling activity of laurate in liver mitochondria have been studied. It was found that 5 μM cyclosporin A partly inhibits laurate-stimulated mitochondrial respiration, which is suggestive of its recoupling effect, i.e., the ability to suppress the protonophoric activity of this fatty acid. Under these conditions, cyclosporin A has no effect on the ability of carboxyatractylate and glutamate to inhibit the uncoupling effect of laurate. In their turn, these compounds do not influence the recoupling activity of cyclosporin A. The recoupling effects of cyclosporin A, carboxyatractylate, and glutamate are additive: acting simultaneously, they fully suppress the uncoupling activity of laurate. It is concluded that the protonophoric uncoupling activity of fatty acids in liver mitochondria is mediated not only by ADP/ATP and aspartate/glutamate antiporters, but also by a system that is sensitive to cyclosporin A, but is not related with cyclophilin D.     

Inhibition by spermine of the inner membrane permeability transition of isolated rat heart mitochondria.

The effect of spermine on the permeability transition of the inner mitochondrial membrane of isolated rat heart mitochondria was evaluated. The permeability transition was triggered using a series of agents (t-butyl hydroperoxide, phenylarsine oxide, carboxyatractylate, and elevated Ca2+ and inorganic phosphate concentrations), and was monitored via Ca(2+)-release, mitochondrial swelling and pyridine nucleotide oxidation. By all three criteria, spermine inhibited the transition. A C50 of 0.38 +/- 0.06 (SD) mM was measured for inhibition.     

Kinetics of electrogenic transport by the ADP/ATP carrier.

The electrogenic transport of ATP and ADP by the mitochondrial ADP/ATP carrier (AAC) was investigated by recording transient currents with two different techniques for performing concentration jump experiments: 1) the fast fluid injection method: AAC-containing proteoliposomes were adsorbed to a solid supported membrane (SSM), and the carrier was activated via ATP or ADP concentration jumps. 2) BLM (black lipid membrane) technique: proteoliposomes were adsorbed to a planar lipid bilayer, while the carrier was activated via the photolysis of caged ATP or caged ADP with a UV laser pulse. Two transport modes of the AAC were investigated, ATP(ex)-0(in) and ADP(ex)-0(in). Liposomes not loaded with nucleotides allowed half-cycles of the ADP/ATP exchange to be studied. Under these conditions the AAC transports ADP and ATP electrogenically. Mg(2+) inhibits the nucleotide transport, and the specific inhibitors carboxyatractylate (CAT) and bongkrekate (BKA) prevent the binding of the substrate. The evaluation of the transient currents yielded rate constants of 160 s(-1) for ATP and >/=400 s(-1) for ADP translocation. The function of the carrier is approximately symmetrical, i.e., the kinetic properties are similar in the inside-out and right-side-out orientations. The assumption from previous investigations, that the deprotonated nucleotides are exclusively transported by the AAC, is supported by further experimental evidence. In addition, caged ATP and caged ADP bind to the carrier with similar affinities as the free nucleotides. An inhibitory effect of anions (200-300 mM) was observed, which can be explained as a competitive effect at the binding site. The results are summarized in a transport model.     

The sensor regions of VDAC are translocated from within the membrane to the surface during the gating processes.

The motion of the sensor regions in a mitochondrial voltage-gated channel called VDAC were probed by attaching biotin at specific locations and determining its ability to bind to added streptavidin. Site-directed mutagenesis was used to introduce single cysteine residues into Neurospora crassa VDAC (naturally lacks cysteine). These were chemically biotinylated and reconstituted into planar phospholipid membranes. In the 19 sites examined, only two types of results were observed upon streptavidin addition: in type 1, channel conductance was reduced, but voltage gating could proceed; in type 2, channels were locked in a closed state. The result at type 1 sites is interpreted as streptavidin binding to sites in static regions close to the channel opening. The binding sterically interferes with ion flow. The result at type 2 sites indicates that these are located on a mobile domain and coincide with the previously identified sensor regions. The findings are consistent with closure resulting from the movement of a domain from within the transmembrane regions to the membrane surface. No single site was accessible to streptavidin from both membrane surfaces, indicating that the motion is limited. From the streptavidin-induced reduction in conductance at type 1 sites, structural information was obtained about the location of these sites.     

Projection structure of the atractyloside-inhibited mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae

ADP/ATP carriers in the inner mitochondrial membrane catalyze the exchange of cytosolic ADP for ATP synthesized in the mitochondrial matrix by ATP synthase and thereby replenish the eukaryotic cell with metabolic energy. The yeast ADP/ATP carrier (AAC3) was overexpressed, inhibited by atractyloside, purified, and reconstituted into two-dimensional crystals. Images of frozen hydrated crystals were recorded by electron microscopy, and a projection structure was calculated to 8-A resolution. The AAC3 molecule has pseudo 3-fold symmetry in agreement with the 3-fold sequence repeats that are typical of members of the mitochondrial carrier family. The density distribution is consistent with a bundle of six transmembrane alpha-helices with two or three short alpha-helical extensions closing the central pore on the matrix side. The AAC3 molecules in the crystal are arranged in symmetrical homo-dimers, but the translocation pore for adenine nucleotides lies in the center of the molecule and not along the dyad axis of the dimer.