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In the structure of bovine F1-ATPase inhibited with residues 1–60 of the bovine inhibitor protein IF1, the α-helical inhibitor interacts with five of the nine subunits of F1-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF1 destabilizes the interaction of the inhibitor with F1-ATPase and may assist in removing the inhibitor from its binding site when F1Fo-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF1 and the C-terminal domains of the βDP-subunit and βTP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the βDP-subunit. Several conserved charged amino acids in the long α-helix of IF1 are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F1-ATPase and occupy aqueous cavities in F1-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme., Graphical Abstract

The catalytic domain of the F-ATPase in mitochondria protrudes into the matrix of the organelle, and is attached to the membrane domain by central and peripheral stalks. Energy for the synthesis of ATP from ADP and phosphate is provided by the transmembrane proton-motive-force across the inner membrane, generated by respiration. The proton-motive force is coupled mechanically to ATP synthesis by the rotation at about 100 times per second of the central stalk and an attached ring of c-subunits in the membrane domain. Each c-subunit carries a glutamate exposed around the midpoint of the membrane on the external surface of the ring. The rotation is generated by protonation and deprotonation successively of each glutamate. Each 360° rotation produces three ATP molecules, and requires the translocation of one proton per glutamate by each c-subunit in the ring. In fungi, eubacteria, and plant chloroplasts, ring sizes of c 10 –c 15 subunits have been observed, implying that these enzymes need 3.3–5 protons to make each ATP, but until now no higher eukaryote has been examined. As shown here in the structure of the bovine F 1 -c-ring complex, the c-ring has eight c-subunits. As the sequences of c-subunits are identical throughout almost all vertebrates and are highly conserved in invertebrates, their F-ATPases probably contain c 8 -rings also. Therefore, in about 50,000 vertebrate species, and probably in many or all of the two million invertebrate species, 2.7 protons are required by the F-ATPase to make each ATP molecule.

A subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been expressed to high levels in a soluble form in Escherichia coli. The subcomplex consists of residues 79-184 of subunit b, residues 1-124 of subunit d and the entire F6 subunit (76 residues). It has been purified and crystallized by vapour diffusion. The morphology and diffraction properties of the crystals of the subcomplex were improved by the presence of thioxane or 4-methylpyridine in the crystallization liquor. With a synchrotron-radiation source, these crystals diffracted to 2.8 A resolution. They belong to the monoclinic space group P2(1).

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.

The ATP synthase enzyme structure includes two stalk assemblies, the central stalk and the peripheral stalk. Catalysis involves rotation of the central stalk assembly together with the membrane-embedded ring of c-subunits driven by the trans-membrane proton-motive force, while the alpha and beta-subunits of F(1) are prevented from co-rotating by their attachment to the peripheral stalk. In the absence of structures of either the intact peripheral stalk or larger complexes containing it, we are studying its individual components and their interactions to build up an overall picture of its structure. Here, we describe an NMR structural characterisation of F(6), which is a 76-residue protein located in the peripheral stalk of the bovine ATP synthase and is essential for coupling between the proton-motive force and catalysis. Isolated F(6) has a highly flexible structure comprising two helices packed together through a loose hydrophobic core and connected by an unstructured linker. Analysis of chemical shifts, (15)N relaxation and RDC measurements confirm that the F(6) structure is flexible on a wide range of timescales ranging from nanoseconds to seconds. The relationship between this structure for isolated F(6) and its role in the intact peripheral stalk is discussed.

Summary The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspar- tate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identi- fied Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomy- ces cerevisiae . On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

In this study, we explore the hypothesis that some member of the mitochondrial carrier family has specific uncoupling activity that is responsible for the basal proton conductance of mitochondria. Twenty-seven of the 35 yeast mitochondrial carrier genes were independently disrupted in Saccharomyces cerevisiae. Six knockout strains did not grow on nonfermentable carbon sources such as lactate. Mitochondria were isolated from the remaining 21 strains, and their proton conductances were measured. None of the 21 carriers contributed significantly to the basal proton leak of yeast mitochondria. A possible exception was the succinate/fumarate carrier encoded by the Xc2 gene, but deletion of this gene also affected yeast growth and respiratory chain activity, suggesting a more general alteration in mitochondrial function. If a specific protein is responsible for the basal proton conductance of yeast mitochondria, its identity remains unknown.

The sequences of 42 subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria have been described previously. Seven are encoded by mitochondrial DNA, whereas the remaining 35 are nuclear gene products imported into the organelle from the cytoplasm. An additional protein, which does not correspond to any previously known subunit of the complex I assembly, has now been detected. Denaturing gels of subcomplex Ilambda, the hydrophilic arm of complex I, clearly show a hitherto unidentified band, which was digested with trypsin and subjected to mass-spectrometric analysis to provide several peptide sequences, used in cDNA cloning and sequencing. Measurement of the intact protein mass indicated that the N terminus is acetylated. The new complex I subunit (B16.6) is the bovine homolog of GRIM-19, the product of a cell death regulatory gene induced by interferon-beta and retinoic acid, thus providing a new link between the mitochondrion and its electron-transport chain and apoptotic cell death.

Bovine IF1 is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F1F0 ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF1 (44–84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.

In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysine.

The nuclear genome of Saccharomyces cerevisiae encodes 35 members of a family of membrane proteins. Known members transport substrates and products across the inner membranes of mitochondria. We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria. They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism. Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported [14C]oxoglutarate uptake. The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism.

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F1Fo ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.

Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1).

The genome of Saccharomyces cerevisiae encodes 35 members of a family proteins thattransport metabolites and substrates across the inner membranes of mitochondria. They includethree isoforms of the ADP/ATP translocase and the phosphate and citrate carriers. At the startof our work, the functions of the remaining 30 members of the family were unknown. We areattempting to identify these 30 proteins by overexpression of the proteins in specially selectedhost strains of Escherichia coli that allow the carriers to accumulate at high levels in the formof inclusion bodies. The purified proteins are then reconstituted into proteoliposomes wheretheir transport properties are studied. Thus far, we have identified the dicarboxylate,succinate-fumarate and ornithine carriers. Bacterial overexpression and functional identification, togetherwith characterization of yeast knockout strains, has brought insight into the physiologicalsignificance of these transporters. The yeast dicarboxylate carrier sequence has been used toidentify the orthologous protein in Caenorhabditis elegans and, in turn, this latter sequencehas been used to establish the sequence of the human ortholog.

The dicarboxylate carrier (DIC) is a nuclear-encoded protein located in the mitochondrial inner membrane. It catalyses the transport of dicarboxylates such as malate and succinate across the mitochondrial membrane in exchange for phosphate, sulphate and thiosulphate. We have determined the sequences of the human cDNA and gene for the DIC. The gene sequence was established from overlapping genomic clones generated by PCRs by use of primers and probes based upon the human cDNA sequence. It is spread over 8.6 kb of human DNA and is divided into 11 exons. Five short interspersed repetitive Alu sequences are found in intron I. The protein encoded by the gene is 287 amino acids long. In common with the rat protein, it does not have a processed presequence to help to target it into mitochondria. It has been demonstrated by Northern- and Western-blot analyses that the DIC is present in high amounts in liver and kidney, and at lower levels in all the other tissues analysed. The positions of introns contribute towards an understanding of the processes involved in the evolution of human genes for carrier proteins.

The mitochondrial F 1 -ATPase inhibitor protein, IF 1 , inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF 1 occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF 1 with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1–60 of bovine IF 1 with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione- S -transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

The delta-subunit of bovine F1-ATPase was expressed from a bacterial vector at fairly high level in Escherichia coli, but the yield of bovine epsilon-subunit was rather low under similar conditions. However, co-expression of the proteins from a dicistronic operon delta-epsilon in the same expression vector, produced both of them in good yield in a soluble form in the bacterial cytoplasm, and by chromatography it was found that the delta- and epsilon-subunits were associated in a stable complex. The amino groups in the complex were labelled exhaustively by chemical reaction under denaturing conditions with ethyl-[1-14C]acetimidate. The alpha-amino groups of the proteins were unmodified, but complete reaction of all epsilon-amino groups in both proteins was demonstrated by determination of the molecular masses of the modified proteins by electrospray MS. The modified subunits were separated by denaturing gel electrophoresis, and from measurements of the ratio of incorporated radioactivities and the lysine contents of the proteins, it was calculated that the subcomplex contains equimolar amounts of the two proteins. As the apparent molecular mass of the complex determined by gel filtration was 29 kDa, it appears that the complex contains one copy of each protein. It is likely that the delta- and epsilon subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the delta-subunit, they interact with the gamma- and beta-subunits.

The F1 globular catalytic domain and the Fo intrinsic membrane domain of the F1Fo-ATPases in bacteria, chloroplasts, and mitochondria are connected by a slender stalk. In the F1Fo complex from bovine heart mitochondria, the stalk is thought to contain subunits OSCP, d, and F6, and the globular part of the membrane bound subunit b, referred to as b‘. It has been shown previously that the OSCP, b‘, d, and F6 proteins can be assembled in vitro into a water soluble complex named the “stalk”. The stalk and F1-ATPase together form another complex named F1·stalk. In this paper, the molar ratios of the OSCP, b (or b‘), d, and F6 in the stalk, F1·stalk, and F1Fo-ATPase complexes have been investigated by three independent methods. By quantitation of radioactivity incorporated by S-carboxymethylation with iodo-2-[14C]acetic acid into a stalk complex containing a form of F6 with the mutation Glu3-Cys, it was shown that the stalk consists of equimolar quantities of its four constituent proteins. In the stalk complex ...

Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c 8 -ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c 8 -ring. The e- and g-subunits extend from the a-subunit density distal to the c 8 -ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers.

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F0. Otherwise the F0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F1 and F0 domains of the F1F0-ATPase complex.

Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli . They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d. Likewise, fragments b′, bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment ′ (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression. These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase. Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase. When fragments b′, bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1·OSCP·b′, F1-OSCP-bI or F1·OSCP·bC were formed, but b′, bI and bC appeared to be present in sub-stoichiometric amounts. When F6 was added also, then the stoichiometric quaternary complexes F1·OSCP·b′·F6 and F1·OSCP·bI·F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6. Finally, three pentameric complexes F1·OSCP·b′·F6·d, F1·OSCP·b′·F6·′ and F1·OSCP·b·F6·d were isolated. In a further series of reconstitution experiments, the binary complexes b′·OSCP and b′·d, the ternary complex b′·′·F6, and the quaternary complex OSCP·b′·F6·d were obtained. The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase. It was shown by S-carboxymethylation of cysteine residues with iodo-[2-14C]acetic acid that bovine F1F0-ATPase and the reconstituted F1·stalk complex, F1·OSCP·b′·d·F6, each contained one copy per complex of subunits b (or b′), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1 : 1 : 1. The ratio of b to d in purified F0 was 1 : 1. Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold. These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.

The Fo membrane domain of the F1Fo-ATP synthase complex has been purified from bovine heart mitochondria. The purification procedure involves the removal of peripheral membrane proteins, including F1-ATPase, from submitochondrial particles with guanidine hydrochloride, followed by extraction of Fo and other membrane proteins from the stripped membranes in the presence of the detergent n-dodecyl beta-D-maltoside. Fo was then purified by ion-exchange and dye ligand chromatography in the presence of the same detergent. Approximately 15 mg of pure Fo was recovered from 1.8 g of mitochondrial membrane protein. The purified Fo is a complex of nine different polypeptides. They are subunits a, b, c, d, e, F6, and A6L characterized before in F1Fo-ATPase preparations, and two new hitherto undetected subunits, named f and g. The sequences of subunits f and g have been determined. They are not related significantly to any known protein, but subunit f appears to contain a membrane-spanning alpha-helix. Proteins f and g are also present in approximately stoichiometric amounts in a highly purified preparation of intact F1Fo-ATPase, and so it is concluded that they are authentic subunits of the bovine enzyme with unknown functions. Dibutyltin 3-hydroxyflavone, an inhibitor of F1Fo-ATPase, also binds to the purified Fo in detergent and competes for binding with venturicidin. In the presence of F1 and OSCP, the purified Fo was reassembled into the intact F1Fo-ATPase complex. Therefore, this procedure provides a relatively abundant source of pure and functional Fo that is suitable for structural analysis.

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

The ADP/ATP, phosphate, and oxoglutarate/malate carrier proteins found in the inner membranes of mitochondria, and the uncoupling protein from mitochondria in mammalian brown adipose tissue, belong to the same protein superfamily. Established members of this superfamily have polypeptide chains approximately 300 amino acids long that consist of three tandem related sequences of about 100 amino acids. The tandem repeats from the different proteins are interrelated, and probably have similar secondary structures. The common features of this superfamily are also present in nine proteins of unknown functions characterized by DNA sequencing in various species, most notably in Caenorhabditis elegans and Saccharomyces cerevisiae. The high level expression in Escherichia coli of the bovine oxoglutarate/malate carrier, and the reconstitution of active carrier from the expressed protein, offers encouragement that the identity of superfamily members of known sequence but unknown function may be uncovered by a similar route.

NADH: ubiquinone oxidoreductase, the first enzyme in the respiratory electron transport chain of mitochondria, is a membrane-bound multi-subunit assembly, and the bovine heart enzyme is now known to contain about 40 different polypeptides. Seven of them are encoded in the mitochondrial DNA; the remainder are the products of nuclear genes and are imported into the organelle. The primary structures of 12 of the nuclear coded subunits have been described and those of a further 20 are described here. The subunits have been sequenced by following a strategy based on the polymerase chain reaction. This strategy has been tailored from existing methods with the twofold aim of avoiding the use of cDNA libraries, and of obtaining a cDNA sequence rapidly with minimal knowledge of protein sequence, such as can be determined in a single N-terminal sequence experiment on a polypeptide spot on a two-dimensional gel. The utility and speed of this strategy have been demonstrated by sequencing cDNAs encoding 32 nuclear-coded-membrane associated proteins found in bovine heart mitochondria, and the procedures employed are illustrated with reference to the cDNA sequences of the 20 subunits of NADH: ubiquinone oxidoreductase that are presented. Extensive use has also been made of electrospray mass spectrometry to measure molecular masses of the purified subunits. This has corroborated the protein sequences of subunits with unmodified N terminals, and their measured molecular masses agree closely with those calculated from the protein sequences. Nine of the subunits, B8, B9, B12, B13, B14, B15, B17, B18 and B22 have modified α-amino groups. The measured molecular masses of subunits B8, B13, B14 and B17 are consistent with the post-translational removal of the initiator methionine and N-acetylation of the adjacent amino acid. The initiator methionine of subunit B18 has been removed and the N-terminal glycine modified by myristoylation. Subunits B9 and B12 appear to have N-terminal and other modifications of a hitherto unknown nature. The sequences of the subunits of bovine complex I provide important clues about the location of iron-sulphur clusters and substrate and cofactor binding sites, and give valuable information about the topology of the complex. No function has been ascribed to many of the subunits, but some of the sequences indicate the presence of hitherto unsuspected biochemical functions. Most notably the identification of an acyl carrier protein in both the bovine and Neurospora crassa complexes provides evidence that part of the complex may play a role in fatty acid biosynthesis in the organelle, possibly in the formation of cardiolipin. The complexity of the mitochondrial enzyme is emphasized by the calculation that the total length of protein sequence in the 39 characterized subunits of bovine NADH: ubiquinone oxidoreductase is 7724 amino acid residues, and exceeds the total protein sequences present in the Escherichia coli ribosome. The calculated molecular mass of bovine complex I is greater than 880,061, assuming that one copy of each subunit is present in each assembly.

The amino-acid sequence of a subunit of NADH:ubiquinone oxidoreductase from bovine heart mitochondria has been determined and is closely related to those of acyl carrier proteins that are involved in fatty acid biosynthesis in Escherichia coli and plants. Evidence for the presence of covalently attached pantetheine-4′-phosphate in the bovine protein has been obtained by determination of the molecular mass of the isolated subunit by electrospray mass spectrometry, before and after incubation of the protein at alkaline pH under reducing conditions. This decreased the molecular mass from 10751.6 to 10449.4, a difference of 302.2 mass units; the value calculated from the protein sequence with one covalently attached pantetheine-4′-phosphate is 10449.8. The acyl group which is removed by alkaline reduction, appears to be attached via a thioester linkage, By analogy with the bacterial protein it is likely that the attachment site of the pantetheine-4-phosphate is serine-44, which is found in a highly conserved region of the sequence. At present the function of the acyl carrier protein in mitochondrial complex I is not understood.

The amino acid sequence of the 10 kDa subunit of the flavoprotein (FP) fragment of complex I from bovine heart mitochondria has been determined by protein sequence analysis, thereby completing the sequence of the FP fragment. The calculated molecular weight of the 10 kDa subunit agrees exactly with the value of 8438 determined by electrospray mass spectrometry, and further confirmation of the sequence has been obtained by sequencing cDNA amplified from total bovine heart cDNA by the polymerase chain reaction, using mixed oligonucleotides based upon the protein sequence as primers and hybridization probes. The sequence of the 10 kDa subunit is not related to that of any known protein. Being devoid of cysteine residues, it has none of the characteristic features of known iron-sulfur proteins and it is improbable that it is involved in liganding Fe-S centers in the FP fragment.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

The amino acid sequence of the 2-oxoglutarate/malate carrier protein, a component of the inner membranes of mitochondria, has been deduced from the sequences of overlapping cDNA clones. These clones were generated in polymerase chain reactions using, in the first instance, complex mixtures of oligonucleotides as primers and probes, with sequences based upon partial protein sequences of cyanogen bromide fragments of the purified protein. The protein sequence of the carrier, including the initiator methionine, is 314 amino acids long. The mature protein has a modified alpha-amino group, but the nature of this modification and the precise position of the mature N-terminal amino acid have not been ascertained, although it must lie in amino acids 1-4 of the deduced protein sequence. Comparison of the protein sequence with itself and with those of 3 other mitochondrial carrier proteins, ADP/ATP translocase, the phosphate carrier, and the uncoupling protein from brown fat, shows that all 4 proteins contain a 3-fold repeated sequence about 100 amino acids in length, and all the repeats are interrelated. This suggests that the members of this family of proteins have similar structures and mechanisms and that they have evolved from a common origin. The distribution of hydrophobic amino acids in the oxoglutarate/malate carrier supports the view that the domains are folded into similar structural motifs, possibly consisting of two transmembrane alpha-helices joined by an extensive extramembranous hydrophilic region. Clones of cDNA arising from a longer related transcript of the oxoglutarate/malate carrier gene have also been analyzed. They contain 271 additional nucleotides in the 3' noncoding region.(ABSTRACT TRUNCATED AT 250 WORDS)

The epsilon-subunit of ATP synthase from bovine heart mitochondria is assembled into the extrinsic membrane sector, F1-ATPase. The mature protein is 50 amino acid residues in length and its function is unknown. It is a nuclear gene product that is imported into the organelle. A mixture of 64 oligonucleotides 17 bases long, designed on the basis of the known protein sequence, was synthesized and used as a hybridization probe to isolate a cognate cDNA clone from a bovine library. The DNA sequence of this clone was determined, and the protein sequence of the epsilon-subunit deduced from it agrees exactly with that determined by direct sequence analysis of the protein isolated from bovine hearts. The bovine cDNA was used as a hybridization probe to examine the expression of the epsilon-subunit in various bovine tissues. mRNAs related to the cDNA are found in all of these tissues, and no evidence was obtained of the presence of mRNAs for the epsilon-subunit with similar coding sequences and dissimilar 3′ non-coding regions. By hybridization experiments with digests of DNA from cow, man and rat it has been shown that sequences related to the bovine cDNA are present in the genomes of all three species. More than one related sequence was detected in all cases, indicating the presence in all three genomes of more than one gene and/or pseudogenes.

ATP synthase is found in bacteria, chloroplasts and mitochondria. The simplest known example of such an enzyme is that in the eubacterium Escherichia coli ; it is a membrane-bound assembly of eight different polypeptides assembled with a stoichiometry of x 3 β 3 γ 1 δ 1 ε 1 a 1 b 2 c 10-12 . The first five of these constitute a globular structure, Fj-ATPase, which is bound to an instrinsic membrane domain, F0, an assembly of the three remaining subunits. ATP synthases driven by photosynthesis are slightly more complex. In chloroplasts, and probably in photosynthetic bacteria, they have nine subunits, all homologues of the components of the E. coli enzyme; the additional subunit is a duplicated and diverged relation of subunit b. The mammalian mitochondrial enzyme is more complex. It contains 14 different polypetides, of which 13 have been characterized. Two membrane components, a (or ATPase-6) and A6L, are encoded in the mitochondrial genome in overlapping genes and the remaining subunits are nuclear gene products that are translated on cytoplasmic ribosomes and then imported into the organelle. The sequences of the proteins of ATP-synthase have provided information about amino acids that are important for its function. For example, amino acids contributing to nucleotide binding sites have been identified. Also, they provide the basis of models of secondary structure of membrane components that constitute the transmembrane proton channel. An understanding of the coupling of the transmembrane potential gradient for protons, A/%+, to ATP synthesis will probably require the determination of the structure of the entire membrane bound complex. Crystals have been obtained of the globular domain, Fj-ATPase. They diffract to a resolution of 3^4 A f and data collection is in progress. As a preliminary step towards crystallization of the entire complex, we have purified it from bovine mitochondria and reconstituted it into phospholipid vesicles.

The peripheral stalk of ATP synthase holds the alpha3beta3 catalytic subcomplex stationary against the torque of the rotating central stalk. In bovine mitochondria, the N-terminal domain of the oligomycin sensitivity conferral protein (OSCP-NT; residues 1-120) anchors one end of the peripheral stalk to the N-terminal tails of one or more alpha-subunits of the F1 subcomplex. Here we present the solution structure of OSCP-NT and an NMR titration study of its interaction with peptides representing N-terminal tails of F1 alpha-subunits. The structure comprises a bundle of six alpha-helices, and its interaction site contains adjoining hydrophobic surfaces of helices 1 and 5; residues in the region 1-8 of the alpha-subunit are essential for the interaction. The OSCP-NT is similar to the N-terminal domain of the delta-subunit from Escherichia coli ATP synthase (delta-NT), except that their surface charges differ (basic and acidic, respectively). As the charges of the adjacent crown regions in their alpha3beta3 complexes are similar, the OSCP-NT and delta-NT probably do not contact the crowns extensively. The N-terminal tails of alpha-subunit tails are probably alpha-helical, and so this interface, which is essential for the rotary mechanism of the enzyme, appears to consist of helix-helix interactions.

Bovine complex I is an assembly of 46 different proteins. Seven of them are encoded in mitochondrial DNA, and the rest are nuclear gene products that are imported into the organelle. Fourteen of the nuclear encoded subunits have modified N termini. Many of these post-translational modifications have been deduced previously from intact protein masses. These assignments have been verified by mass spectrometric analysis of peptides. Thirteen of them are N-alpha-acetylated, and a 14th, subunit B18, is N-alpha-myristoylated. Subunit B18 forms part of the membrane arm of the complex, and the myristoyl group may attach subunit B18 to the membrane. One subunit, B12, has a particularly complex pattern of post-translational modification that has not been analyzed before. It is a mixture of the N-alpha-acetylated form and the form with a free N terminus. In addition, it has one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations. The predominant form is methylated on residues 4 and 6. There is no evidence for the methylation of histidine 2. Subunit B12 is also part of the membrane arm of complex I, and it probably spans the membrane once, but as its orientation is not known, the methylation sites could be in either the matrix or the intermembrane space. These experiments represent another significant step toward establishing the precise chemical composition of mammalian complex I.

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.

Subunit h is a component of the peripheral stalk region of ATP synthase from Saccharomyces cerevisiae. It is weakly homologous to subunit F6 in the bovine enzyme, and F6 can replace the function of subunit h in a yeast strain from which the gene for subunit h has been deleted. The removal of subunit h (or F6) uncouples ATP synthesis from the proton motive force. A biotinylation signal has been introduced following the C terminus of subunit h. It becomes biotinylated in vivo, and allows avidin to be bound quantitatively to the purified enzyme complex in vitro. By electron microscopy of the ATP synthase–avidin complex in negative stain and by subsequent image analysis, the C terminus of subunit h has been located in a region of the peripheral stalk that is close to the Fo membrane domain of ATP synthase. Models of the peripheral stalk are proposed that are consistent with this location and with reconstitution experiments conducted with isolated peripheral stalk subunits.

In Saccharomyces cerevisiae, at least three proteins (IF(1), STF(1), and STF(2)) appear to be involved in the regulation of ATP synthase. Both IF(1) and STF(1) inhibit F(1), whereas the proposed function for STF(2) is to facilitate the binding of IF(1) and STF(1) to F(1). The oligomerization properties of yeast IF(1) and STF(1) have been investigated by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that IF(1) and STF(1) oligomerize in opposite directions in relation to pH, suggesting that both proteins might regulate yeast F(1)F(0)-ATPase under different conditions. Their effects on bovine F-ATPases are also described. Whereas bovine IF(1) inhibits yeast F(1)-ATPase even better than yeast IF(1) or STF(1), the capability of yeast IF(1) to inhibit the bovine enzyme is very low and decreases with time. Such an effect is also observed in the study of the homologous inhibition of yeast F(1)-ATPase. Yeast inhibitors are not as effective as their bovine counterpart, and the complex seems to dissociate gradually.

In this study, we explore the hypothesis that some member of the mitochondrial carrier family has specific uncoupling activity that is responsible for the basal proton conductance of mitochondria. Twenty-seven of the 35 yeast mitochondrial carrier genes were independently disrupted in Saccharomyces cerevisiae. Six knockout strains did not grow on nonfermentable carbon sources such as lactate. Mitochondria were isolated from the remaining 21 strains, and their proton conductances were measured. None of the 21 carriers contributed significantly to the basal proton leak of yeast mitochondria. A possible exception was the succinate/fumarate carrier encoded by the Xc2 gene, but deletion of this gene also affected yeast growth and respiratory chain activity, suggesting a more general alteration in mitochondrial function. If a specific protein is responsible for the basal proton conductance of yeast mitochondria, its identity remains unknown.

In mitochondria, the hydrolytic activity of ATP synthase is regulated by an inhibitor protein, IF(1). Its binding to ATP synthase depends on pH, and below neutrality, IF(1) is dimeric and forms a stable complex with the enzyme. At higher pH values, IF(1) forms tetramers and is inactive. In the 2.2 A structure of the bovine IF(1) described here, the four monomers in the asymmetric unit are arranged as a dimer of dimers. Monomers form dimers via an antiparallel alpha-helical coiled coil in the C-terminal region. Dimers are associated into oligomers and form long fibres in the crystal lattice, via coiled-coil interactions in the N-terminal and inhibitory regions (residues 14-47). Therefore, tetramer formation masks the inhibitory region, preventing IF(1) binding to ATP synthase.

The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle.

The synthesis of DNA in mitochondria requires the uptake of deoxynucleotides into the matrix of the organelle. We have characterized a human cDNA encoding a member of the family of mitochondrial carriers. The protein has been overexpressed in bacteria and reconstituted into phospholipid vesicles where it catalyzed the transport of all four deoxy (d) NDPs, and, less efficiently, the corresponding dNTPs, in exchange for dNDPs, ADP, or ATP. It did not transport dNMPs, NMPs, deoxynucleosides, nucleosides, purines, or pyrimidines. The physiological role of this deoxynucleotide carrier is probably to supply deoxynucleotides to the mitochondrial matrix for conversion to triphosphates and incorporation into mitochondrial DNA. The protein is expressed in all human tissues that were examined except for placenta, in accord with such a central role. The deoxynucleotide carrier also transports dideoxynucleotides efficiently. It is likely to be medically important by providing the means of uptake into mitochondria of nucleoside analogs, leading to the mitochondrial impairment that underlies the toxic side effects of such drugs in the treatment of viral illnesses, including AIDS, and in cancer therapy.

The genome of Saccharomyces cerevisiae encodes 35 putative members of the mitochondrial carrier family. Known members of this family transport substrates and products across the inner membranes of mitochondria. We are attempting to identify the functions of the yeast mitochondrial transporters via high-yield expression in Escherichia coli and/or S. cerevisiae, purification and reconstitution of their protein products into liposomes, where their transport properties are investigated. With this strategy, we have already identified the functions of seven S. cerevisiae gene products, whose structural and functional properties assigned them to the mitochondrial carrier family. The functional information obtained in the reconstituted system and the use of knock-out yeast strains can be usefully exploited for the investigation of the physiological role of individual transporters. Furthermore, the yeast carrier sequences can be used to identify the orthologous proteins in other organisms, including man.

The mitochondrial carrier protein for carnitine has been identified in Saccharomyces cerevisiae. It is encoded by the gene CRC1 and is a member of the family of mitochondrial transport proteins. The protein has been over-expressed with a C-terminal His-tag in S. cerevisiae and isolated from mitochondria by nickel affinity chromatography. The purified protein has been reconstituted into proteoliposomes and its transport characteristics established. It transports carnitine, acetylcarnitine, propionylcarnitine and to a much lower extent medium- and long-chain acylcarnitines.

Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [(35)S]sulfate/sulfate and [(35)S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase.

The protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown. The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid. The protein was over-produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl. The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate. Its physiological role in S. cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate. This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S. cerevisiae on ethanol or acetate as the sole carbon source.

The inner membranes of mitochondria contain a family of transport proteins of related sequence and structure. The DNA sequence of the genome of Saccharomyces cerevisiae encodes at least 35 members of this family. Three of them can be recognised as known isoforms of the ADP-ATP translocase and two others as the phosphate and citrate carriers. The transport functions of the remainder cannot be identified with certainty. One of them, encoded on yeast chromosome xii, shows a fairly close sequence relationship to the known sequence of the bovine mitochondrial oxoglutarate-malate carrier. The yeast protein has been obtained by over-expression in Escherichia coli, reconstituted into phospholipid vesicles and shown to have transport properties characteristic of the mitochondrial carrier for dicarboxylate ions, such as malate, and also phosphate, previously biochemically characterised, but not sequenced, from both mammalian and yeast mitochondria. This is the first example of the biochemical identification of an unknown membrane protein encoded in the yeast genome since the completion of the genomic sequence.