Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. (1) Phosphate-transport protein and phosphate-transport protein β (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. (2) Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. (3) Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. (4) Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. (5) Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate: roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport

The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424-432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys(185). In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.     

Sequence of the bovine mitochondrial phosphate carrier protein: structural relationship to ADP/ATP translocase and the brown fat mitochondria uncoupling protein.

A cDNA encoding the precursor of the bovine mitochondrial phosphate carrier protein has been cloned from a bovine cDNA library using a mixture of 128 different 17-mer oligonucleotides as hybridisation probe. The protein has an N-terminal extension of 49 amino acids not present in the mature protein. This extension has a net positive charge and is presumed to direct the import of the protein from the cytoplasm to the mitochondrion. Comparison of the protein sequence of the mature phosphate carrier with itself, with ADP/ATP translocase and with the uncoupling protein from brown fat mitochondria shows that all three proteins contain a 3-fold repeated sequence approximately 100 amino acids in length, and that the repeats in the three proteins are related to each other. This implies that the three proteins have related three-dimensional structures and mechanisms and that they share a common evolutionary origin. The distribution of hydrophobic residues in the phosphate carrier protein suggests that each repeated 100 amino acid element is composed of two membrane-spanning alpha-helices linked by an extensive hydrophilic domain. This model is similar to that first proposed for the ADP/ATP translocase and later for the brown fat mitochondria uncoupling protein.     

How does the mitochondrial ADP/ATP carrier distinguish transportable ATP and ADP from untransportable AMP and GTP?Dynamic modeling of the recognition/translocation process in the major substrate binding region

To understand the transport mechanism of the bovine heart mitochondrial ADP/ATP carrier at the atomic level, we studied the four-dimensional features of the interaction of various purine nucleotides with the adenine nucleotide binding region (ABR) consisting of Arg(151)-Asp(167) in the second loop facing the matrix side. After three-dimensional modeling of ABR based on the experimental results, its structural changes on interaction with purine nucleotides were examined by molecular dynamics computation at 300 K. ATP/ADP were translocated to a considerable degree from the matrix side to the inner membrane region accompanied by significant backbone conformational changes, whereas neither appreciable translocation nor a significant conformational change was observed with the untransportable nucleotides AMP/GTP. The results suggested that binding of the terminal phosphate group and the adenine ring of ATP/ADP with Arg(151) and Lys(162), respectively, and subsequent interaction of a phosphate group(s) other than the terminal phosphate with Lys(162) triggered the expansion and subsequent contraction of the backbone conformation of ABR, leading to the translocation of ATP/ADP. Based on a simplified molecular dynamic simulation, we propose a dynamic model for the initial recognition process of ATP/ADP with the carrier.     

Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant

Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.     

Sequence of the N-terminal formic acid fragment and location of the N-ethylmaleimide-binding site of the phosphate transport protein from beef heart mitochondria

The N-terminal formic acid fragment (FA1) of the N-[3H]ethylmaleimide-labeled and carboxymethylated bovine mitochondrial phosphate transport protein (PTPN*CM) has been purified and completely sequenced: NH2-Ala-Val-Glu-Glu-Gln-Tyr-Ser-Cys-Asp-Tyr10-Gly-Ser-Gly-Arg-Phe- Phe-Ile-Leu-Cys- Gly20-Leu-Gly-Gly-Ile-Ile-Ser-Cys-Gly-Thr-Thr30-His-Thr -Ala-Leu-Val-Pro-Leu-Asp- -Leu-Val40-Lys-Cys(N-[3H]ethylmaleimide)-Arg-Met-Gln-Val-Asp- COOH. By thermolysin digestion of FA1 and high-performance liquid chromatography isolation of the radioactive subfragment Leu39-Arg43, the sole N-ethylmaleimide-binding residue has been identified as Cys42. FA1 contains a high mole percentage of cysteine (8.5%) and shows silver staining anomaly. Its sequence reveals significant homology in the triplicated gene regions (Pro27,132,229) of the mitochondrial ADP/ATP carrier from beef heart and Neurospora crassa. The hydropathic profile suggests that FA1 contains a transmembrane segment (Phe15-Val40) with only one basic (His31) and one acidic (Asp38) residue. The presence of the phosphate transport protein gene among nuclear genes is suggested from a lack of significant homology between the reverse-translated FA1 (mitochondrial codons) and the bovine mitochondrial genome. The inhibitory action of N-ethylmaleimide on the phosphate transport mechanism is discussed.     

Immunological characterization of the mitochondrial 2-oxoglutarate carrier from liver and heart. Organ specificity

Antibodies have been prepared against the 2-oxoglutarate transport proteins purified from bovine heart and rat liver mitochondria. The anti-heart antiserum cross-reacts with the 2-oxoglutarate carrier (OGC) from beef, pig, rat and rabbit heart, but not with the OGC from liver of the same animals. Conversely, the anti-liver antiserum recognizes the carrier protein from liver of all species tested but not from heart. Immunoinactivation of oxoglutarate transport activity by the antibodies is also tissue specific. Peptide maps of purified OGC show structural differences between the carrier from heart and liver of the same animal species. These results indicate the existence of isoforms of the OGC in heart and liver.     

Isolation and functional reconstitution of the aspartate/glutamate carrier from mitochondria

The aspartate/glutamate carrier from beef heart mitochondria was solubilized by the detergent dodecyloctaoxyethylene ether (C12E8) in the presence of high concentrations of ammonium acetate. After separating the bulk amount of contaminating proteins by differential solubilization and by hydroxyapatite centrifugation chromatography, the aspartate/glutamate carrier was purified by high-performance liquid chromatography on hydroxyapatite. During the purification process, the aspartate/glutamate carrier as well as other transport proteins was identified by functional reconstitution. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis the purified aspartate/glutamate carrier protein appears as a protein band with an apparent molecular mass of 68 kDa. Small amounts of some contaminating proteins mainly at 31 kDa were also found. Since the ADP/ATP carrier has an apparent molecular mass of 31 kDa in SDS-gel electrophoresis, possible contamination by the nucleotide carrier was analyzed by immunological methods. The enrichment of the aspartate/glutamate carrier--based on functional reconstitution--was about 570-fold, the protein yield was 0.1%.     

Specific elution from hydroxylapatite of the mitochondrial phosphate carrier by cardiolipin

The role of cardiolipin in the purification of the mitochondrial phosphate carrier by hydroxylapatite has been investigated. Without added cardiolipin, the reconstituted phosphate-transport activity in the hydroxylapatite eluate is small and only confined to the first fraction. With cardiolipin added to the extract, the eluted activity is much higher and present until fraction 6. The activity retained by hydroxylapatite in the absence of cardiolipin is eluted after addition of this phospholipid to the column. The requirement of added cardiolipin diminishes on increasing the concentration of solubilized mitochondria. The hydroxylapatite eluate contains five protein bands in the Mr-region of 30 000-35 000, which are differently distributed in the various fractions. Among these, only the presence and the relative amount of band 3 of Mr 33 000 corresponds to the phosphate transport activity. Cardiolipin is the only phospholipid tested which causes elution of band 3 from hydroxylapatite; on the other hand, it prevents the elution of band 2 and retards that of band 5 (the ADP/ATP carrier). Band 1 starts to appear in the second fraction even without cardiolipin. On increasing the concentration of cardiolipin, in the first fraction of the hydroxylapatite eluate band 3 increases and the contamination of band 4 decreases. Under optimal conditions a preparation of band 3 about 90% pure and with high reconstituted phosphate transport activity is obtained. It is concluded that the elution of the phosphate carrier from hydroxylapatite requires cardiolipin and that the phosphate carrier is identical with (or with part of) band 3 of the hydroxylapatite eluate.     

Solute carriers involved in energy transfer of mitochondria form a homologous protein family

The sequences of three mitochondrial carriers involved in energy transfer, the ADP/ATP carrier, phosphate carrier and uncoupling carrier, are analyzed. Similarly to what has been previously reported for the ADP/ATP carrier and the uncoupling protein, now also the phosphate carrier is found to have a tripartite structure comprising three similar repeats of approx. 100 residues each. The three sequences show a fair overall homology with each other. More significant homologies are found by comparing the repeats within and between the carriers in a scheme where the sequences are spliced into repeats, which are arranged for maximum homology by allowing possible insertions or deletions. A striking conservation of critical residues, glycine, proline, of charged and of aromatic residues is found throughout all nine repeats. This is indicative of a similar structural principle in the repeats. Hydropathy profiles of the three proteins and a search for amphipathic alpha-spans reveal six membrane-spanning segments for each carrier, providing further support for the basic structural identity of the repeats. The proposed folding pattern of the carriers in the membrane is exemplified with the phosphate carrier. A possible tertiary arrangement of the repeats and the membrane-spanning helices is shown. The emergence of a mitochondrial carrier family by triplication and by divergent evolution from a common gene of about 100 residues is discussed.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Mitochondrial phosphate transport. N-ethylmaleimide insensitivity correlates with absence of beef heart-like Cys42 from the Saccharomyces cerevisiae phosphate transport protein

The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.     

ADENINE NUCLEOTIDE-INDUCED CONTRACTION OF THE INNER MITOCHONDRIAL MEMBRANE : I. General Characterization

The inner membranes of isolated bovine heart mitochondria undergo pronounced contraction upon being exposed to exogenous adenosine diphosphate (ADP), adenosine triphosphate (ATP), and certain other high-energy phosphate compounds. Contraction results in decrease of inner membrane expanse which in turn results in decrease of intracristal space and increase of mitochondrial optical density (OD). The magnitude of the OD change appears to be proportional to the degree of contraction Half-maximal contraction can be achieved with ADP or ATP at concentrations as low as about 0 3 µM. Atractyloside at concentrations as low as about 1.2 nmol/mg mitochondrial protein completely inhibits the contraction. It is concluded from these and other observations that inner membrane contraction occurs as a result of adenine nucleotide binding to the carrier involved in the exchange of adenine nucleotides across the inner mitochondrial membrane.     

Two bovine genes for mitochondrial ADP/ATP translocase expressed differences in various tissues

Two different bovine cDNAs have been characterized that encode closely related homologues of the mitochondrial membrane carrier protein ADP/ATP translocase. One of them codes for the protein that has been characterized previously from bovine heart mitochondria, and the other codes for a protein that differs from it in 33 amino acids out of 297. Including the base substitutions required to bring about these changes in amino acid sequence, the coding regions of the cDNAs differ at 184 positions. In addition, they are extensively diverged in their 3' noncoding sequences, which differ greatly in both length and sequence, and these segments of the cDNAs have been used as hybridization probes to demonstrate that the expression of the two genes giving rise to the two proteins is very different in various bovine tissues. Expression of one gene predominates in heart muscle and that of the other in intestine. Hybridization experiments with digests of genomic DNA have shown the presence of numerous sequences related to the two cDNAs in both the bovine and human genomes. Some of these probably arise from pseudogenes, but three expressed genes have been detected in the human genome. The study of the regulation of the expression of these genes may help to illuminate the basis of tissue-specific human mitochondrial diseases which arise because of defects in mitochondrial enzymes only in the affected tissue and not in other tissues of the same individual.     

Brown adipose tissue mitochondria: recoupling caused by substrate level phosphorylation and extramitochondrial adenosine phosphates.

1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 muM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicatdd by a dramatic increase in the phosphorylation state of adenine nucleotides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix. 2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 muM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20-50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP. 3. GDP (100 muM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier. 4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.     

Mitochondrial phosphate transport. Import of the H+/Pi symporter and role of the presequence

The rat liver mitochondrial phosphate transporter contains a 44-amino acid presequence. The role of this presequence is not clear since the ADP/ATP carrier and the brown fat uncoupling protein, related members of a family of inner membrane anion transporters, lack a presequence and contain targeting information within the mature protein. Here, we present evidence that the rat liver mitochondrial phosphate transporter can be synthesized in vitro, imported into mitochondria, and processed to a protein of Mr 33,000. Import requires the membrane potential and external nucleotide triphosphate. The presequence inserts into the outer mitochondrial membrane, and import proceeds via a process similar to other proteins destined for the inner membrane or matrix. A mutant phosphate transporter lacking 35 amino acids at the NH2 terminus of the presequence has little capacity for mitochondrial import. The rat liver phosphate transporter is also imported and processed by rat kidney mitochondria and by mitochondria from the yeast Saccharomyces cerevisiae. A site-directed mutation of the N-ethyl-maleimide reactive cysteine 41 does not affect import or processing. The results presented show that optimal import of the mitochondrial phosphate transporter, unlike the ADP/ATP carrier and the brown fat uncoupling protein, is dependent on a presequence. As these carriers are believed to have evolved from a single gene, it seems likely that the H+/Pi carrier, known to be present in prokaryotes, appeared first and that subsequent evolutionary events leading to the other anion carriers eliminated the presequence.     

The carboxyl-terminal two-thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria

The precursor of the mitochondrial inner membrane protein ADP/ATP carrier is cytoplasmically synthesized without an amino-terminal peptide extension. We constructed a truncated precursor lacking the 103 amino acids from the amino terminus (about a third of the protein). Import of the truncated precursor into mitochondria showed the import characteristics of the authentic precursor, including nucleoside triphosphate dependence, requirement for a protease-sensitive component on the mitochondrial surface, two-step specific binding to the outer membrane, and membrane potential-dependent translocation into the inner membrane. We conclude that, in contrast to all other mitochondrial precursor proteins studied so far, domains of the ADP/ATP carrier distant from the amino terminus can carry specific targeting information for transport into mitochondria.     

Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana

Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner-membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant Deltamir1 Deltapic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic-like proteins are also able to complement the double mutant Deltamir1 Deltapic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions.     

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[alpha-32P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[alpha-32P]ADP in the dark with a Kd value of congruent to 8 microM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[alpha-32P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[alpha-32P]ADP, both the ADP/ATP carrier and the beta subunit of the membrane-bound F1-ATPase are covalently labeled. The binding specificity of 2-azido-[alpha-32P]ADP for the beta subunit of F1-ATPase is ascertained by prevention of photolabeling of isolated F1 by preincubation with an excess of ADP.