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Mitochondrial structure and function are influenced by the unique phospholipid composition of its membranes. While mitochondria contain all the major classes of phospholipids, recent studies have highlighted specific roles of the non-bilayer forming phospholipids phosphatidylethanolamine (PE) and cardiolipin (CL) in the assembly and activity of mitochondrial respiratory chain (MRC) complexes. The non-bilayer phospholipids are cone-shaped molecules that introduce curvature stress in the bilayer membrane and have been shown to impact mitochondrial fusion and fission. In addition to their overlapping roles in these mitochondrial processes, each non-bilayer phospholipid also plays a unique role in mitochondrial function; for example, CL is specifically required for MRC supercomplex formation. Recent discoveries of mitochondrial PE and CL trafficking proteins and prior knowledge of their biosynthetic pathways have provided targets for precisely manipulating non-bilayer phospholipid levels in the mitochondrial membranes in vivo. Thus, the genetic mutants of these pathways could be valuable tools in illuminating molecular functions and biophysical properties of non-bilayer phospholipids in driving mitochondrial bioenergetics and dynamics.

The Saccharomyces cerevisiae MEF2 gene is a mitochondrial protein translation factor. Formerly believed to catalyze peptide elongation, evidence now suggests its involvement in ribosome recycling. This study confirms the role of the MEF2 gene for cell respiration and further uncovers a slow growth phenotype and reduced chronological lifespan. Furthermore, in comparison with cytoplasmic P0 strains, mef2? strains have a marked reduction of the inner mitochondrial membrane potential and mitochondria show a tendency to aggregate, suggesting an additional role for the MEF2 gene in maintenance of mitochondrial health, a role that may also be shared by other mitochondrial protein synthesis factors. Refereed/Peer-reviewed

The data presented here show for the first time that the protein known as “ribonuclease (RNase) inhibitor” (RI or RNH1) is present not only in the cell cytosol, but also in mitochondria, the central organelles in cell redox homeostasis. This finding directly correlates with the reported ability of RI to protect the cell from oxidative stress, with its sensitivity to oxidation and reactivity as a reactive oxygen species scavenger. While this study was carried out we also surprisingly discovered the presence of RI in the cell nucleus. We deem that these data open new views in the investigation on the cellular role(s) of the RI. Structured summary RI physically interacts with ATP synthase sub Alpha mitochondrial , Stress 70 Protein , Mitochondrial , Calcium-binding mitochondrial carrier protein Aralar 2 ADP/ATP Translocase 2 Elongation factor Tu and Cytochrome b-c1 complex Sub 2 by anti bait coimmunoprecipitation (View interaction) PARP and RI colocalize by cosedimentation (View interaction)

The tumor suppressor p53 can translocate into mitochondria and activate apoptosis. Here we studied whether p53 mitochondrial translocation and subsequent apoptosis were affected by blocking mitochondrial permeability transition pore using cyclosporine A (CsA) and bongkrekic acid (BA) in skin epidermal JB6 cells and skin tissues. Our results demonstrated that CsA and BA blocked TPA-induced p53 translocation, leading to protection against the loss of mitochondrial membrane potential and Complex I activity, and eventually suppression of apoptosis. Thus, our results suggest that mitochondrial permeability transition is required for p53 mitochondrial translocation.

The mitochondrial ADP/ATP carriers (AACs) catalyze the exchange of cytosolic ADP for matrix ATP. We have identified and characterized a novel member of the AAC subfamily of mitochondrial metabolite transport proteins, termed AAC4. The AAC4 gene maps to human chromosome 4q28.1, and its product AAC4 is 66–68% identical to human AAC 1–3 and is localized to mitochondria. AAC4 transcripts are exclusively present in liver, testis and brain unlike those of AAC 1–3. Consistent with its belonging to the AAC subfamily, upon heterologous expression and reconstitution into liposomes AAC4 exchanges ADP for ATP by an electrogenic antiport mechanism with high specificity and high sensitivity to carboxyatractyloside and bongkrekic acid.

We have identified a novel protein on the outer membrane of Arabidopsis thaliana mitochondria. This protein displays 67% sequence identity with the 64 kDa translocase of the outer envelope membrane of chloroplasts (Toc). A mitochondrial localisation for this protein was determined by (i) its presence in the proteome of highly purified Arabidopsis mitochondria, (ii) Western blot analysis with antibodies to Toc64 from pea that indicate its presence in Arabidopsis and pea mitochondria, (iii) green fluorescent protein fusion proteins that indicate an exclusive mitochondrial localisation for this protein, and (iv) expression profiles in various tissue types and during development that are more similar to translocase of the outer mitochondrial membrane components than to chloroplastic Toc components. Thus Arabidopsis mitochondria contain a protein with high sequence identity to a plastid protein import receptor.

Protein import into mitochondria is inhibited by protons (IC50 pH 6.5). The channels of the import machinery were examined to further investigate this pH dependence. TOM and TIM23 are the protein translocation channels of the mitochondrial outer and inner membranes, respectively, and their single channel behaviors at various pHs were determined using patch-clamp techniques. While not identical, increasing H+ concentration decreases the open probability of both TIM23 and TOM channels. The pattern of the pH dependences of protein import and channel properties suggests TIM23 open probability can limit import of nuclear-encoded proteins into the matrix of yeast mitochondria.

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP-1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.

Recently the signal transduction function for oxidative phosphorylation was found to be second order in ADP [Jeneson, J.A.L., Wiseman, R.W., Westerhoff, H.V. and Kushmerick, M.J. (1996) J. Biol. Chem. 271, 27995-279981, but the molecular mechanism of signal transduction remained unclear. Previously we described inhibition of cytochrome c oxidase by intramitochondrial ATP, accompanied by a change of hyperbolic into sigmoidal kinetics. The present study describes a sigmoidal relationship also between the ascorbate respiration of reconstituted cytochrome c oxidase and intraliposomal ADP concentration. Its possible role in the control of oxidative phosphorylation and cell respiration is discussed.

The expression of adenine nucleotide translocase isoforms (AAC) during perinatal development of the rat was studied by measuring mRNA transcript levels of AAC1 and AAC2 genes in liver, heart and brain tissue. In contrast to heart and brain, AAC1 mRNA is not present in adult liver tissue, but is transiently expressed around birth. AAC1 expression in liver did not respond to cold stress (examined with adult rats), therefore a possible involvement of AAC1 in the liver thermogenesis of newborns is excluded. Measurement of the [3H]ADP uptake by liver mitochondria revealed that the molecular activity of the AAC protein was significantly higher in mitochondria from 4-day-old neonates compared with adults. We suggest that the transient AAC1 gene expression in the perinatal liver helps to accommodate the mitochondrial ATP supply to the increased cytosolic ATP consumption initiated at birth.

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Delta atm1 cells are deficient in the holoforms, but not the apoforms of heme-carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Delta atm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30-fold higher levels of free iron as compared to wild-type organelles, i.e. three-fold more than mitochondria deficient in frataxin, the protein mutated in Friedreich's ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme-containing proteins in delta atm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.

Liver mitochondria from rats made hypothyroid by administration of 2-mercapto-1-methylimidazole were less sensitive to the uncoupling effect of myristic acid, as measured by the increase of resting state respiration, than mitochondria from euthyroid animals, whereas subsequent administration to the animals of triiodothyronine (`hyperthyroidism') resulted in an increased uncoupling action of myristate. `Hyperthyroidism' also resulted in doubling of the carboxyatractyloside-sensitive portion of the myristate-stimulated respiration. Parallel to this was a twofold increase of the mitochondrial content of the ADP/ATP carrier protein and an over threefold increase of its activity. The uncoupling effect of phytanic acid was less sensitive to carboxyatractyloside and was increased in the hyperthyroid state to a smaller extent than in the case of myristate. These results provide further support to the thesis [Skulachev, V.P., FEBS Lett. 294 (1991) 158–162] that the ADP/ATP carrier is involved in the mechanism of the uncoupling effect of long-chain fatty acids.

Addition of triiodothyronine at 10 pM in vitro to hypothyroid rat liver mitochondria doubles the rate of the adenine nucleotide transporter at low ADP concentrations. Nicotinamide abolishes this effect in parallel with its inhibition of the ADP-ribosylation of an inner membrane protein identical in size to the transporter. Nicotinamide also renders euthyroid preparations indistinguishable from hypothyroid ones. A mechanism is offered to explain these findings in which it is proposed that the adenine nucleotide transporter is a true allosteric protein and that its covalent modification by ADP-ribosylation increases the stability of the less favoured externally-facing C-conformation and thus increases the proportion of transporters in this orientation: although the C-conformation is significantly more leaky to cations than the tight matrix-facing M-conformation, this enhances ADP import. This model is shown to offer an explanation not only for the transport effects of T3 but also for those of oxidative stress and ADP-ribosylation inhibitors on Ca2+, H+ and K+ transfer across the mitochondrial inner membrane. Ca2+ at 30 nM appears to stabilize the M-conformation of the transporter by a mechanism other than ADP-ribosylation.

Incompletely synthesized polypeptides in the mitochondrial inner membrane are subject to rapid proteolysis. We demonstrate that Yta10p, a mitochondrial homologue of a conserved family of putative ATPases in Saccharomyces cerevisiae, is essential for this proteolytic process. Yta10p-dependent degradation requires divalent metal ions and the hydrolysis of ATP. Yta 10p is an integral protein of the inner mitochondrial membrane exposing the carboxy terminus to the mitochondrial matrix space. Based on the presence of consensus binding sites for ATP, and for divalent metal ions found in a number of metal dependent endopeptidases, a direct role of Yta10p in the proteolytic breakdown of membrane-associated polypeptides in mitochondria is suggested.

The hypothesis on the additional function of the ATP/ADP antiporter (ANT) as uncoupling protein has been tested in proteoliposomes and planar bilayer phospholipid membranes (BLM). It is found that dissipation of the light-induced delta pH in the dark is very much faster in ANT-bacteriorhodopsin proteoliposomes than in proteoliposomes containing bacteriorhodopsin as the only protein. Mersalyl treatment of ANT-bacteriorhodopsin proteoliposomes causes further increase in the delta pH dissipation rate due to formation of a high conductance pore. The properties of this pore are studied on ANT incorporated to BLM. They proved to be similar to those of so-called multiple conductance channel or permeability transition pore of inner mitochondrial membrane. The conductance of the single channel is as high as 2.2 nS. The channel fails to discriminate between K+, Na+, H+ and Cl-. Thus the obtained data are consistent with the assumption that native and modified ANT might function as an H(+)-specific conductor and as a permeability transition pore, respectively.

HSP73, the constitutive form of heat-shock protein 70, has been implicated in the translocation of preproteins across the mitochondrial membranes, being required for maintaining mitochondrial preproteins in an import competent conformation. Here we report that highly purified mitochondrial outer membranes contain a protein indistinguishable from HSP73 as a tightly associated peripheral component of the membrane. This membrane form of HSP73 was photolabelled with [α-32P]ATP and could be released from the outer membrane with sodium carbonate, but not after incubation of the membranes with salt or with ATP. A sensitive immunoassay with an anti-HSP73 monoclonal antibody, revealed the association of HSP73 with mitochondrial outer membrane vesicles at a level similar to that of preprotein import receptors.

A screening for yeast mutants impaired in mitochondrial protein import led to the identification of two genes (MPI1 and MPI2) encoding the essential components MIM44 and MIM17 of the inner membrane import machinery. We analyzed twelve additional mutants obtained in the screening and found two further complementation groups. One group represents mutants of SSC1, the gene encoding mitochondrial hsp70, an essential matrix protein required for protein import across the inner membrane. The second complementation group represents mutants of a new gene (MPI3) encoding a 23 kDa integral inner membrane protein (MIM23). MIM23 is synthesized without a presequence, and its import to the inner membrane requires a membrane potential. MIM23 contains a domain homologous to half of MIM17. We speculate that MIM23 is a new member of the protein import machinery of the mitochondrial inner membrane.

Saccharomyces cerevisiae strains expressing a single type of ADP/ATP carrier (AAC) protein were prepared from a mutant in which all AAC genes were disrupted, by transformation with plasmids containing a chosen AAC gene. As demonstrated by measurements of [14C]ADP specific binding and transport, all three translocator proteins, AAC1, AAC2 and AAC3 when present in the mitochondrial membrane, exhibited similar translocation properties. The disruption of some AAC genes, however, resulted in phenotypes indicating that the function of these proteins in whole cells can be quite different. Specifically, we found that the disruption of AAC1 gene, but not AAC2 and AAC3, resulted in a change in colony phenotype.

The study of mitochondrial ion channels changed our perception of these double-wrapped organelles from being just the power house of a cell to the guardian of a cell’s fate. Mitochondria communicate with the cell through these special channels. Most of the time, the message is encoded by ion flow across the mitochondrial outer and inner membranes. Potassium, sodium, calcium, protons, nucleotides, and proteins traverse the mitochondrial membranes in an exquisitely regulated manner to control a myriad of processes, from respiration and mitochondrial morphology to cell proliferation and cell death. This review is an update on both well established and putative mitochondrial channels regarding their composition, function, regulation, and therapeutic potential.

2-h-old neonatal liver mitochondria, when depleted of adenine nucleotides, showed an ‘ohmic’ current-voltage relationship and a higher passive proton permeability of the membrane, resembling fetal mitochondrial behaviors for the proton conductance. Incubation of fetal mitochondria with ATP, GDP or carboxyatractyloside promoted a significant reduction in the passive proton permeability of the membrane and the appearance of the characteristic biphasic behavior for the proton conductance. It is concluded that the postnatal increase in intramitochondrial adenine nucleotide concentration promotes, by the interaction of the nucleotides with the adenine nucleotide translocase, the reduction in the passive proton permeability of the mitochondrial membrane, allowing efficient energy conservation in the neonatal liver.

The influence of the conformational state of ADP/ATP antiporter on the efficiency of the inhibitory effect of cyclosporine A on the Ca2+-induced nonspecific permeability of the inner mitochondrial membrane has been studied. Carboxyatractiloside, the inhibitor of ADP/ATP-antiporter, was shown to prevent the cyclosporine A-induced suppression of the nonspecific permeability. The Carboxyatractiloside enect was displayed only in mitochondria depleted of adenine nucleotides. Bifunctional SH reagent, phenylarsine oxide, was also able to reverse the effect of cyclosporine A. The data are consistent with the suggestion that cyclosporine A causes suppression of the nonspecific permeability due to its effect on the ADP/ATP antiporter conformation.

The influenza A virus PB1-F2 protein predominantly localizes in the mitochondria of virus-infected cells. A series of cDNAs encoding N- and C-terminal deletion mutants and site-directed mutagenesis of the basic residues of PB1-F2 appended to 3xFLAG revealed the domain from residues 46 to 75 to be both necessary and sufficient for mitochondrial targeting. In addition, the subdomain residues 63–75 and both Lys73 and Arg75 are minimally required for mitochondrial localization. Transfection of untagged- and 3xFLAG tagged-PB1-F2 into Vero, HeLa and MDCK cells changed the mitochondrial morphology from a filamentous to a dotted structure and suppressed the inner-membrane potential.

Mitochondrial adenine nucleotide translocase 1 (ANT1), but not ANT2, can dominantly induce apoptosis [Bauer et al. (1999) J. Cell Biol. 439, 258–262]. Nothing is known, however, about the apoptotic activity of ANT3. We have transfected HeLa cells with the three human ANT isoforms to compare their potential as inducers of apoptosis. Transient overexpression of ANT3 resulted, like ANT1, in apoptosis as shown by an increase in the sub-G1 fraction, annexin V staining, low ΔΨm, and activation of caspases 9 and 3. Moreover, the apoptosis produced by ANT3 was inhibited by bongkrekic acid and by cyclosporin A. The pro-apoptotic activities of the ANT1 and ANT3 isoforms contrast with the lack of apoptotic activity of ANT2. This finding may help to identify the specific factors associated with the pro-apoptotic activities of ANT isoforms.

The effect of aging and treatment with acetyl-l-carnitine on the activity of cytochrome oxidase and adenine nucleotide translocase in rat heart mitochondria was studied. It was found that the activity of both these mitochondrial protein systems was reduced (by around 30%) in aged animals. Treatment of aged rats with acetyl-l-carnitine almost completely reversed this effect. Changes in the mitochondrial cardiolipin content appear to be responsible for these effects of acetyl-l-carnitine.

Using a cytofluorimetric approach, we studied intramitochondrial cardiolipin (CL) distribution in HCW-2 cells, an apoptosis-resistant clone of human HL-60 cells. In HL-60, about 50% of total CL is distributed in the outer leaflet of mitochondrial inner membrane, while in HCW-2 a significantly higher amount of CL (about 65%) is in that site. In basal conditions, HSW-2 cells also show a reduced mitochondrial membrane potential even if they are able to proliferate as the parental line. Taking into account the complex functions that CL plays in the regulation of mitochondrial activity, it is likely that HCW-2 could produce ATP utilizing more glycolytic pathways rather than mitochondrial respiratory chain.

The effect of the expression of murine Bax protein on growth and vitality was examined in Saccharomyces cerevisiae and compared with the effect of Bax in mutant cells lacking functional mitochondria. The cytotoxic effect of Bax on yeast does not require functional oxidative phosphorylation, respiration, or mitochondrial proteins (ADP/ATP carriers) implicated in the formation of the permeability transition pore in mammalian mitochondria. In the wild type S. cerevisiae the expression of Bax does not result in a severe effect on mitochondrial membrane potential and respiration. On the basis of Bax induced differences in the fluorescence of green fluorescent protein fused to mitochondrial proteins, it is proposed that Bax may interfere with one essential cellular process in yeast: the mitochondrial protein import pathway that is specific for the proteins of the mitochondrial carrier family.

In the living cellular environment, DNAs exist in a compact state in the presence of a polyamine, such as spermidine. We found that the hydrolysis of ATP into ADP induces the folding of elongated DNAs, by the single-chain observation of individual T4 DNA molecules. This result is discussed in relation to the possible role of ATP as a regulatory factor in genetic activity, in addition to its well-established role as an energy source.

The action of ATP/ADP-antiporter inhibitors upon the uncoupling effect of palmitate, detergents and ‘classical’ uncouplers has been studied. The uncoupling effect was estimated by stimulation of succinate oxidation and of H+ permeability of rat liver mitochondria in the presence of oligomycin. It is shown that carboxyatractylate (CAtr) and pyridoxal 5-phosphate (PLP) suppress the uncoupling induced by palmitate and the anionic detergents SDS and cholate, but do not affect that induced by the cationic detergents CTAB, by the non-ionic detergent Triton X-100, as well as by the ‘classical’ uncouplers FCCP and DNP. The results are discussed in terms of a concept assuming that the ATP/ADP-antiporter facilitates the electrophoretic export of hydrophobic anions from mitochondria.

Possible involvement of the ATP/ADP antiporter and uncoupling protein (UCP) in thermoregulatory uncoupling of oxidative phosphorylation in heart muscle has been studied. To this end, effects of carboxyatractylate (cAtr) and GDP, specific inhibitors of the antiporter and UCP, on the membrane potential of the oligomycin-treated mitochondria from cold-exposed (6 degrees C, 48 h) and control rats have been measured. It is found that cAtr increases the membrane potential level in both cold-exposed and non-exposed groups, the effect being strongly enhanced by cooling. As for GDP, it is effective only in mitochondria from the cold-exposed rats. In these mitochondria, the coupling effect of GDP is smaller than that of cAtr. CDP, which does not interact with UCP, is without any influence on membrane potential. The cold exposure is found to increase the uncoupling efficiency of added natural (palmitate) or artificial (SF6847) uncouplers, the increase being cAtr- and GDP-sensitive in the case of palmitate. The fatty acid-free bovine serum albumin enhances delta psi in both cold-exposed and control groups, the effect being much larger in the former case. It is concluded that in heart muscle mitochondria the ATP/ADP antiporter is responsible for the 'mild uncoupling' under normal conditions and for major portion of the thermoregulatory uncoupling in the cold whereas the rest of thermoregulatory uncoupling is served by UCP (presumably by UCP2 since the UCP2 mRNA level is shown to strongly increase in rat heart muscle under the cold exposure conditions used).

We show here that TPP→TMP conversion can take place in rat liver mitochondria. This occurs via the novel, putative TPP pyrophosphatase localised in the mitochondrial matrix, as shown both by digitonin titration and by an HPLC enzyme assay carried out on the mitochondrial matrix fraction. Certain features of the reaction, including the substrate and pH dependence, are reported. Additional evidence is given that externally added TMP can cross the mitochondrial membrane in a manner consistent with the occurrence of a carrier-mediated process. This can occur both via the TPP translocator and via a novel translocator, inhibited by CAT but different from the ADP/ATP carrier.

Patch-clamp techniques were used to characterize the channel activity of mitochondrial inner membranes of two human osteosarcoma cell lines: a mitochondrial genome-deficient (ρ0) line and its corresponding parental (ρ+) line. Previously, two high conductance channels, mitochondrial Centum picoSiemen (mCS) and multiple conductance channels (MCC), were detected in murine mitochondria. While MCC was assigned to the protein import in yeast mitochondria, the role of mCS is unknown. This study demonstrates that mCs and MCC activities from mouse mitochondria are indistinguishable from those of human mitochondria. The channel activities and their functional expression levels are not altered in cells lacking mtDNA. Hence, ρ0 cells may provide a model system for elucidating the role of mitochondrial channels in disease processes and apoptosis.

The mechanism by which non-esterified long-chain fatty acids (FFA) promote mitochondrial permeability transition (MPT) is not clear. We examined with energized rat liver mitochondria the role of two possible actions of FFA in MPT, (i) the reduction of the transmembrane potential (delta psi) and (ii) the increase of the negative surface charge of the inner mitochondrial membrane [Broekemeier, K.M. and Pfeiffer, D.G., Biochemistry 43, (1995) 16440-16449]. It was found that the ability of FFA to stimulate large amplitude swelling is clearly related to their uncoupling activity. Moreover, compared with classical protonophores (FCCP) FFA increase the sensitivity of the pore opening process to delta psi changes. In addition, FFA interact like their thioester derivatives in a structure-dependent manner with the ADP/ATP carrier (measured as inhibition of [3H]atractyloside binding to the AAC protein). It is suggested that not only the protonophoric action of FFA, but also a presumable stabilization of the 'cytosolic' conformation of AAC contribute to the FFA-promoted MPT.

Fatty acids stimulate the oxidation rate of mitochondria isolated from the wild-type Saccharomyces cerevisiae, but do not affect significantly the respiration of mitochondria isolated from mutants, in which the ADP/ATP carrier (AAC) was either modified (R96H) or deleted (delta aac2). Similarly as in mammalian mitochondria, the transmembrane electrical potential difference (delta psi) in the wild-type yeast mitochondria was dissipated by low concentrations of free fatty acids, and this was partially inhibited by bongkrecate. In contrast to the wild-type mitochondria, the addition of increasing concentrations of fatty acids to the op1 (R96H) mutant mitochondria abolished only a small portion of delta psi, as compared to the change induced by classical uncouplers. The different effects of fatty acids on both, the respiration and the delta psi of mitochondria isolated from the wild-type and the aac mutants, respectively, demonstrates that the intact AAC is essential for the fatty acids induced H+ permeability of mitochondrial membrane.

Genes that encode mitochondrial homologues to the bacterial enzymes of fatty acid synthesis were found in various eukaryotic species. Inactivation of these genes leads to a disturbed mitochondrial respiration and an increase in mitochondrial lysophospholipids. We postulate that there is a mitochondrial biosynthetic system providing fatty acids for phospholipid repair. The mitochondrial acyl carrier protein may also play another role, supporting the formation of the respiratory NADH:ubiquinone oxidoreductase.

This paper describes the effect of intramitochondrial ATP/ADP ratios on the H+/e− stoichiometry of reconstituted cytochrome c oxidase (COX) from bovine heart. At 100% intraliposomal ATP the H+/e− stoichiometry of the reconstituted enzyme is decreased to half of the value measured below 98% intraliposomal ATP (above 2% ADP), while it remains constant up to 100% ADP. The decrease is obtained with different COX preparations, independent of the absolute value of the H+/e− stoichiometry. Decrease of H+/e− stoichiometry is prevented by preincubation of the enzyme with a tissue-specific monoclonal antibody to subunit VIa-H (heart-type). Tissue-specific regulation of the efficiency of energy transduction in COX of muscle mitochondria could have a physilogical function in maintaining the body temperature at rest or sleep, i.e. at low ATP expenditure.

Characterization of two mitochondrial proteins of M1 42 and 18 kDa, respectively, phosphorylated by the cAMP-dependent protein kinase of bovine heart mitochondria (mtPKA), is presented. A 42 kDa protein is found to be loosely associated to complexes I, III and IV of the respiratory chain and complex V (ATP synthase) in the inner mitochondrial membrane. An 18 kDa protein is associated to complex I in the inner membrane and in a purified preparation of this complex where it can be phosphorylated by the isolated catalytic subunit of PKA.

Mitochondrial precursor proteins are known to be imported at sites of close contact between mitochondrial outer and inner membranes. We have identified translocation intermediates exposed to the intermembrane space, including the precursor of the ADP/ATP carrier accumulated at the general insertion site GIP, and the precursor of F1-ATPase subunitβ accumulated on its import pathway at low levels of ATP. These results suggest that mitochondrial contact sites are not sealed structures, but that polypeptides pass (at least partly) through the intermembrane space on their route from the outer membrane to the inner membrane.

We previously reported that hsp70 in the mitochondrial matrix (mt-hsp70 = Ssc1p) is required for import of precursor proteins destined for the matrix or intermembrane space. Here we show that mt-hsp70 is also needed for the import of mitochondrial inner membrane proteins. In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt-hsp70 for import, suggesting a general role of mt-hsp70 in membrane translocation of precursors. Moreover, a precursor arrested in contact sites was specifically co-precipitated with antibodies directed against mt-hsp70. We conclude that mt-hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane.

20-30 ng atom magnesium [l-3], about half of which is located in the matrix compartment 123. They also contain about 10 nmol adenine nucleotides per mg protein [4], most of it in the matrix. Although the form of magnesium and adenine nucleotides present in the mitochondrial matrix is not known precisely, it seems likely that most of the nucleotides are complexed by Mg. On the other hand, it has been suggested [5-71 that free ADP and ATP, and not their magnesium complexes, are transported across the mitochondrial membrane by the specific adenine nucleotide translocase [8]. Experimental evidence is a decrease of the translocation rate by increasing external Mg2+ concentration [7]. Thus, a very low intramitochondrial concentration of free, non- complexed, ADP and ATP might be one of the factors controlling the rate of the translocation. One of the intriguing features of the translocase is its preference for external ADP over external ATP [5,9]. An explanation for this can be provided by assuming that the exchange of ATP against ADP is mostly electrogenic [5,10,1 l] and is therefore driven in one direction only by the mitochondrial trans- membrane potential which is negative inside and positive outside. An alternative explanation assumes

Introduction Differences in the content and kinetic properties of the ADP, ATP translocator in mitochondria from various sources have been demonstrated (reviewed [ 1,2] ). Recently the translocator protein was isolated from several organs [3-51 and data were reported suggesting differences between immunological properties of this protein isolated from heart, liver and kidney mitochondria [5]. In this study a comparison of immunological properties of translocator protein of rat heart, liver and hepatoma by a radioimmunological technique revealed that the same antigenic determinant(s) related to ADP, ATP translocator protein are present in the mitochondria of all the above tissues. In addi- tion, the data obtained suggest that antigenic proper- ties of translocator protein isolated from different sources are unequally modified during the isolation. 2. Materials and

The effect of wedeloside, an atractyloside analogue occurring in the Australian weed Wedelia asperrima, on the ADP/ATP carrier is investigated on 3 levels; the ADP stimulated respiration, the ADP/ATP exchange, and the binding in competition with atractylate (ATR) and carboxyatractylate (CAT). The inhibition of respiration and of ADP/ATP exchange by wedeloside is nearly uncompetitive with ADP. The competitive binding with [3H]cat and [3H]ATR reveals a high binding affinity of wedeloside similar to that of CAT. The titration of the ADP/ATP exchange and of the binding with wedeloside gives a titer of 0.35 μmolg protein for rat liver protein and the titer of the binding gives 2.7 μmolg protein for beef heart mitochondria. The 1.8-times higher titers than with CAT may indicate that two molecules of wedeloside bind to one ADP/ATP carrier dimer in contrast to the half site reactivity known for the binding of the other ligands.

Methods for isolation of the ADP/ATP carrier (AAC) from yeast (Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast. The procedure varies according to whether one wishes to obtain a stable CAT-AAC complex, the free and active AAC for reconstitution, or the SDS-denatured pure AAC peptide. CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC-1 and AAC-2 recently found in yeast, due to the exclusive occurrence of a methionine (M-115) residue at the end of the first domain in AAC-2. Thus the AAC isolated from wild-type yeast is primarily or exclusively AAC-2. The isolated AAC is active in ADP/ATP exchange in reconstituted liposomes with a Vmax of 1100 mumol/min per g protein and Km = 15 microM for ADP, and a Vmax of 900 mumol/min per g protein and Km = 9 microM for ATP.

Bongkrekic acid causes fatal food poisoning which is associated with hyperglycaemia. Here we demonstrate that bongkrekic acid, a potent inhibitor of the mitochondrial ATP/ADP translocase, inhibits glucose-induced electrical activity in the pancreatic β-cell through the simulation of ATP-sensitive potassium channel (K-ATP-channel) activity. By comparison of its effects with those of oligomycin, we suggest that bongkrekic acid acts by the inhibition of glucose metabolism and may induce hyperglycaemia by impairing β-cel function.

Members of the mitochondrial carrier family transport compounds over the inner mitochondrial membrane to link the biochemical pathways in the cytosol with those in the mitochondrial matrix. X-ray crystallography has recently provided us with the first atomic model of the bovine ADP/ATP carrier, which is a member of this family. The structure explains the typical three-fold sequence repeats and signature motif of mitochondrial carriers. However, the carrier was crystallised as a monomer in detergent, which is inconsistent with the consensus that mitochondrial carriers exist as homo-dimers. The projection structure of the yeast ADP/ATP carrier by electron crystallography shows that carriers could form homo-dimers in the membrane.

The pH effect on carboxyatractylate-, glutamate- and aspartate-induced recoupling of palmitate-uncoupled rat liver mitochondria has been studied. Stimulation of respiration by low palmitate concentrations (5–20 μM) in the presence of 3 mM MgCl2 is shown to be pH-independent within the 7.0–7.8 range. The recoupling effect of glutamate (or aspartate) decreases and that of carboxyatractylate increases with increase in pH. The recoupling effect of a combination of carboxyatractylate and glutamate (aspartate) appears to be constant at these pH values, being as high as about 80%. It is concluded that uncoupling by low palmitate in liver mitochondria is mediated mainly by ATP/ADP and aspartate/glutamate antiporter.

Human uncoupling protein 3 (UCP3) has two RNA transcripts that arise from the differential processing of the same gene product. One encodes the full length protein (UCP3L) while the other encodes a truncated version (UCP3S) lacking the sixth membrane spanning domain. The roles of the two isoforms are not known, but a mutation that decreases the proportion of UCP3L decreases fat oxidation and increases susceptibility to obesity. In the ADP/ATP carrier, a protein closely related to UCP3, the sixth membrane spanning domain is required for insertion into the inner membrane. Therefore, defective membrane insertion of UCP3S may account for the different effects of the two isoforms in vivo. We investigated mitochondrial import of the two UCP3 isoforms. When epitope-tagged versions of UCP3S and UCP3L were expressed in COS7 cells, both were inserted into the mitochondrial inner membrane. Translation in vitro followed by incubation with isolated mitochondria showed that both isoforms were inserted into the inner membrane, however, the insertion of UCP3S was significantly slower.