Search

Mitochondrial uncoupling proteins (UCP) transport protons from the intermembrane space to the mitochondrial matrix uncoupling oxidative phosphorylation. In mammals, these proteins have been implicated in several cellular functions ranging from thermoregulation to antioxidant defense. In contrast, their invertebrate homologs have been much less studied despite the great diversity of species. In this study, two transcripts encoding mitochondrial uncoupling proteins were, for the first time, characterized in crustaceans. The white shrimp Litopenaeus vannamei transcript LvUCP4 is expressed in all tested shrimp tissues/organs, and its cDNA includes a coding region of 954 bp long which encodes a deduced protein 318 residues long and a predicted molecular weight of 35.3 kDa. The coding region of LvUCP5 transcript is 906 bp long, encodes a protein of 302 residues with a calculated molecular weight of 33.17 kDa. Both proteins share homology with insect UCPs, their predicted structures show the conserved motifs of the mitochondrial carrier proteins and were confirmed to be located in the mitochondria through a Western blot analysis. The genic expression of LvUCP4 and LvUCP5 was evaluated in shrimp at oxidative stress conditions and results were compared to some antioxidant enzymes to infer about their antioxidant role. LvUCP4 and LvUCP5 genes expression did not change during hypoxia/re-oxygenation, and no coordinated responses were detected with antioxidant enzymes at the transcriptional level. Results confirmed UCPs as the first uncoupling mechanism reported in this species, but their role in the oxidative stress response remains to be confirmed.

The ATP-Mg/phosphate carriers (APCs) modulate the intramitochondrial adenine nucleotide pool size. In this study the concentration-dependent effects of Mg2+ and other divalent cations (Me2+) on the transport of [3H]ATP in liposomes reconstituted with purified human and Arabidopsis APCs (hAPCs and AtAPCs, respectively, including some lacking their N-terminal domains) have been investigated. The transport of Me2+ mediated by these proteins was also measured. In the presence of a low external concentration of [3H]ATP (12 μM) and increasing concentrations of Me2+, Mg2+ stimulated the activity (measured as initial transport rate of [3H]ATP) of hAPCs and decreased that of AtAPCs; Fe2+ and Zn2+ stimulated markedly hAPCs and moderately AtAPCs; Ca2+ and Mn2+ markedly AtAPCs and moderately hAPCs; and Cu2+ decreased the activity of both hAPCs and AtAPCs. All the Me2+-dependent effects correlated well with the amount of ATP-Me complex present. The transport of [14C]AMP, which has a much lower ability of complexation than ATP, was not affected by the presence of the Me2+ tested, except Cu2+. Furthermore, the transport of [3H]ATP catalyzed by the ATP/ADP carrier, which is known to transport only free ATP and ADP, was inhibited by all the Me2+ tested in an inverse relationship with the formation of the ATP-Me complex. Finally, direct measurements of Mg2+, Mn2+, Fe2+, Zn2+ and Cu2+ showed that they are cotransported with ATP by both hAPCs and AtAPCs. It is likely that in vivo APCs transport free ATP and ATP-Mg complex to different degrees, and probably trace amounts of other Me2+ in complex with ATP.

Mitochondria have to import the vast majority of their proteins, which are synthesized as precursors on cytosolic ribosomes. The translocase of the outer membrane (TOM complex) forms the general entry gate for the precursor proteins, which are subsequently sorted by protein machineries into the mitochondrial subcompartments: the outer and inner membrane, the intermembrane space and the mitochondrial matrix. The transport across and into the inner membrane is driven by the membrane potential, which is generated by the respiratory chain. Recent studies revealed that the lipid composition of mitochondrial membranes is important for the biogenesis of mitochondrial proteins. Cardiolipin and phosphatidylethanolamine exhibit unexpectedly specific functions for the activity of distinct protein translocases. Both phospholipids are required for full activity of respiratory chain complexes and thus to maintain the membrane potential for protein import. In addition, cardiolipin is required to maintain structural integrity of mitochondrial protein translocases. Finally, the low sterol content in the mitochondrial outer membrane may contribute to the targeting of some outer membrane proteins with a single α-helical membrane anchor. Altogether, mitochondrial lipids modulate protein import on various levels involving precursor targeting, membrane potential generation, stability and activity of protein translocases.

The 2-oxoglutarate carrier (OGC) belongs to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors and nucleotides between the cytoplasm and mitochondrial matrix. Initially, OGC was characterized by determining substrate specificity, kinetic parameters of transport, inhibitors and molecular probes that form covalent bonds with specific residues. It was shown that OGC specifically transports oxoglutarate and certain carboxylic acids. The substrate specificity combination of OGC is unique, although many of its substrates are also transported by other mitochondrial carriers. The abundant recombinant expression of bovine OGC in Escherichia coli and its ability to functionally reconstitute into proteoliposomes made it possible to deduce the individual contribution of each and every residue of OGC to the transport activity by a complete set of cys-scanning mutants. These studies give experimental support for a substrate binding site constituted by three major contact points on the even-numbered α-helices and identifies other residues as important for transport function through their crucial positions in the structure for conserved interactions and the conformational changes of the carrier during the transport cycle. The results of these investigations have led to utilize OGC as a model protein for understanding the transport mechanism of mitochondrial carriers.

Mitochondria are essential components of eukaryotic cells and are involved in a diverse set of cellular processes that include ATP production, cellular signalling, apoptosis and cell growth. These organelles are thought to have originated from a symbiotic relationship between prokaryotic cells in an effort to provide a bioenergetic jump and thus, the greater complexity observed in eukaryotes (Lane and Martin 2010). Mitochondrial processes are required not only for the maintenance of cellular homeostasis, but also allow cell to cell and tissue to tissue communication (Nunnari and Suomalainen 2012). Mitochondrial phospholipids are important components of this system. Phospholipids make up the characteristic outer and inner membranes that give mitochondria their shape. In addition, these membranes house sterols, sphingolipids and a wide variety of proteins. It is the phospholipids that also give rise to other characteristic mitochondrial structures such as cristae (formed from the invaginations of the inner mitochondrial membrane), the matrix (area within cristae) and the intermembrane space (IMS) which separates the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). Phospholipids are the building blocks that make up these structures. However, the phospholipid composition of the OMM and IMM is unique in each membrane. Mitochondria are able to synthesize some of the phospholipids it requires, but the majority of cellular lipid biosynthesis takes place in the endoplasmic reticulum (ER) in conjunction with the Golgi apparatus (Fagone and Jackowski 2009). In this review, we will focus on the role that mitochondrial phospholipids play in specific cellular functions and discuss their biosynthesis, metabolism and transport as well as the differences between the OMM and IMM phospholipid composition. Finally, we will focus on the human diseases that result from disturbances to mitochondrial phospholipids and the current research being performed to help us gain a better understanding of their function.

BCL-2 family members are pivotal regulators of the apoptotic process. Mitochondria are a major site-of-action for these proteins. Several prominent alterations occur to mitochondria during apoptosis that seem to be part of the "mitochondrial apoptotic program." The BCL-2 family members are believed to be the major regulators of this program, however their exact mechanism of action still remains a mystery. BID, a pro-apoptotic BCL-2 family member plays an essential role in initiating this program. Recently, we have revealed that in apoptotic cells the activated/truncated form of BID, tBID, interacts with a novel, uncharacterized protein named mitochondrial carrier homolog 2 (Mtch2). Mtch2 is a conserved protein that is similar to members of the mitochondrial carrier protein (MCP) family. This review summarizes the current knowledge regarding BCL-2 family members and the mitochondrial apoptotic program and examines the possible involvement of Mtch2 in this program.

Sequence alignment of conserved signature motifs predicts the existence of the uncoupling protein 5 (UCP5)/brain mitochondrial carrier protein (BMCP1) homologue in Drosophila melanogaster (Hanak P. and Jezek P. (2001). FEBS Lett.495, pp. 137–141.). Here we demonstrate the functional characterization of the Drosophila melanogaster UCP5 protein (DmUCP5) in the heterologous yeast system, the first insect UCP reported to date. We show that physiological levels of DmUCP5 expression are responsible for an increase in state 4 respiration rates and a decrease in mitochondrial membrane potential. Furthermore, similar to UCP1, UCP2, and UCP3, the uncoupling activity of DmUCP5 is augmented by fatty acids and inhibited by the purine nucleotide GDP. Thus, DmUCP5 shares the mechanisms known to regulate the UCPs characterized to date. A lack of growth inhibition observed in DmUCP5 expressing yeast is consistent with the notion that physiological uncoupling has a minimal effect on cell growth. Finally, semiquantitative RT-PCR analysis shows a distinctive pattern of DmUCP5 expression predominantly localized in the adult head, similar to the expression pattern of its mammalian homologues. The conserved regulation of the expression of this gene from mammals to fruit flies suggests a role for UCP5 in the brain.

One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (Kd) of 0.3–0.5 and 23–92 μM. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a Kd of greater than 100 μM, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-14C] ATP and [8-14C] ADP with Kds of 4–5 and 16–18 μM, respectively. Mg2+ (2 mM) reduced the apparent ATP affinity to 53 μM, an effect entirely explained by chelation of ATP; with Mg2+, Kd using calculated free ATP was 3 μM. A combination of gel filtration, Cu2+-phenanthroline cross-linking, and ultracentrifugation indicated that 75–80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg2+-free nucleotides: the Kd for ATP is about 3–5 μM and for Mant-ATP it is about 10 times lower; and (c) in C12E9 detergent, the monodisperse protein may be in dimeric form.

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.

An energetically significant leak of protons occurs across the mitochondrial inner membranesof eukaryotic cells. This seemingly wasteful proton leak accounts for at least 20% of thestandard metabolic rate of a rat. There is evidence that it makes a similar contribution tostandard metabolic rate in a lizard. Proton conductance of the mitochondrial inner membranecan be considered as having two components: a basal component present in all mitochondria,and an augmentative component, which may occur in tissues of mammals and perhaps ofsome other animals. The uncoupling protein of brown adipose tissue, UCP1, is a clear exampleof such an augmentative component. The newly discovered UCP1 homologs, UCP2, UCP3,and brain mitochondrial carrier protein 1 (BMCP1) may participate in the augmentativecomponent of proton leak. However, they do not appear to catalyze the basal leak, as this isobserved in mitochondria from cells which apparently lack these proteins. Whereas UCP1plays an important role in thermogenesis, the evidence that UCP2 and UCP3 do likewiseremains equivocal.

Nonesterified long-chain fatty acids have long been known as uncouplers of oxidative phosphorylation. They are efficient protonophores in the inner mitochondrial membrane but not so in artificial phospholipid membranes. In the un-ionized form, they undergo a rapid spontaneous transbilayer movement (flip-flop). However, the transbilayer passage of the dissociated (anionic) form is hindered by the negatively charged hydrophilic carboxylic group. In the inner mitochondrial membrane, the transfer of fatty acid anions is mediated by the adenine nucleotide translocase, the dicarboxylate carrier, and the glutamate/aspartate carrier. As a result, the passage of protons and electric charges is a concerted effect of the spontaneous flip-flop of the undissociated (protonated) form in one direction and carrier-facilitated transfer of the ionized (deprotonated) form in the other direction. In addition, fatty acids also promote opening of the mitochondrial permeability transition pore, presumably due to their interaction with one of its constituents, the adenine nucleotide translocase, thus forming an additional route for dissipation of the proton gradient. Structural prerequisites for these proton-conducting mechanisms are (1) a weakly ionized carboxylic group and (2) a hydrocarbon chain of appropriate length without substituents limiting its mobility and hydrophobicity.

This review is primarily focused on the contribution of our laboratory to study of the mitochondrial uncoupling UCPs. The initial stage was the description of a 32-kDa membranous protein specifically induced in brown adipose tissue mitochondria of cold-adapted rats. This protein was then shown by others to be responsible for brown fat thermogenesis and was referred to as the uncoupling protein-UCP (recently renamed UCP1). cDNA and genomic clones of UCP1 were isolated and used to investigate the topology and functional organization of the protein in the membrane and the mechanisms of control of UCP1 gene transcription. Orientation of the transmembrane fragments was proposed and specific amino acid residues involved in the inhibition of UCP1 by purine nucleotides were identified in recombinant yeast. A potent enhancer mediating the response of the UCP1 gene to retinoids and controlling the specific transcription in brown adipocytes was identified using transgenic mice. More recently, we identified UCP2, an UCP homolog widely expressed in human and rodent tissues we also collaborated to characterize the plant UCP. Although the biochemical activities and physiological roles of the novel UCPs are not well understood, these recent data stimulate research on mitochondrial carriers, mitochondrial bioenergetics, and energy expenditure.

The effect of pyridoxal 5'-phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by pyridoxal 5'-phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal 5'-phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]pyridoxal 5'-phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by pyridoxal 5'-phosphate. These results indicate that pyridoxal 5'-phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site.

Mitochondrial transmembrane carrier deficiencies are a recently discovered group of disorders, belonging to the so-called mitochondriocytopathies. We examined the human tissue distribution of carriers which are involved in the process of oxidative phosphorylation (adenine nucleotide translocator, phosphate carrier, and voltage-dependent anion channel) and some mitochondrial substrate carriers (2-oxoglutarate carrier, carnitine-acylcarnitine carrier, and citrate carrier). The tissue distribution on mRNA level of mitochondrial transport proteins appears to be roughly in correlation with the dependence of these tissues on mitochondrial energy production capacity. In general the main mRNA expression of carriers involved in mitochondrial energy metabolism occurs in skeletal muscle and heart. Expression in liver and pancreas differs between carriers. Expression in brain, placenta, lung, and kidney is lower than in the other tissues. Western and Northern blotting experiments show a comparable HVDAC1 protein and mRNA distribution for the tested tissues. Patient's studies showed that cultured skin fibroblasts may not be a reliable alternative for skeletal muscle in screening for human mitochondrial carrier defects.

Mitochondrial biogenesis requires the import of hundreds of different proteins from the cytosol. Protein import into mitochondria is a multistep pathway that includes recognition of precursor proteins by machinery both in the cytoplasm and on the mitochondrial surface, translocation of the precursor across one or both mitochondrial membranes, and folding of the protein after its import into the organelle. Over the past several years, many components of the import machinery have been identified using both biochemical and genetic methods. Recently, significant progress has been made determining the function of some of these import proteins. One purpose of this minireview is to summarize our current understanding of the import pathway, and to introduce the topics of the minireviews that will follow. The other goal of this minireview is to discuss recent findings suggesting that proteins are translocated across both the mitochondrial inner and outer membranes through aqueous channels.

A dynamic complex between the mitochondrial cognate of hsp70 (mthsp70) and the inner membrane protein tim44 couples energy derived from ATP hydrolysis to drive multiple steps in the mitochondrial protein import pathway: (1) The ΔΨ dependent import step and the mthsp70/tim44 complex cooperate to facilitate the unidirectional transfer of the mitochondrial targeting signal across the inner membrane. (2) The mthsp70/tim44 complex helps to unfold domains on precursors proteins that arrive at the import apparatus in a folded conformation on the cis side of the outer membrane. (3) Completion of import is then driven by the mthsp70/tim44 complex in a manner that is independent of ΔΨ. Mechanisms proposed to explain how the mthsp70/tim44 complex harvests chemical energy to drive these aspects of the import process are discussed.

Nuclear-encoded mitochondrial proteins are imported into mitochondria due to the presence of a targeting sequence, the presequence, on their amino termini. Presequences, which are typically proteolyzed after a protein has been imported into a mitochondrion, lack any strictly conserved primary structure but are positively charged and are predicted to form amphiphilic α-helices. Studies with synthetic peptides corresponding to various presequences argue that presequences can partition nonspecifically into the mitochondrial outer membrane and that the specificity of translocation of precursors into mitochondria may depend on interactions of the presequence with the electrical potential of the inner membrane. Although proteins of the outer membrane that are necessary for the translocation of precursor proteins have been proposed to function as receptors for presequences, the binding of presequences to these proteins has not been demonstrated directly. Proteins of the mitochondrial outer membrane may not be responsible for the specificity of translocation of precursors but may instead function, together with cytosolic molecular chaperones, to maintain precursor proteins in conformations that are competent for translocation as the precursors associate with the mitochondrial surface.

The peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 has very recently been found to transport NAD+, NADH, AMP and ADP. In this work we have reinvestigated the substrate specificity and the transport properties of PXN by using a wide range of potential substrates. Heterologous expression in bacteria followed by purification, reconstitution in liposomes, and uptake and efflux experiments revealed that PNX transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3′, 5′-phosphate (PAP), besides NAD+, NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport and was strongly inhibited by pyridoxal 5′-phosphate, bathophenanthroline and tannic acid. Transport was saturable with a submillimolar affinity for NAD+, CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD+ and CoA.

The mitochondrial ATP-Mg/P(i) carrier functions to modulate the matrix adenine nucleotide pool size (ATP + ADP + AMP). Micromolar Ca2+ is required to activate the carrier. Net adenine nucleotide transport occurs as an electroneutral divalent exchange of ATP-Mg2- for HPO4(2-). A steady-state adenine nucleotide pool size is attained when the HPO4(2-) and ATP-Mg2- matrix/cytoplasm concentration ratios are the same. This means that ATP-Mg2- can be accumulated against a concentration gradient in proportion to the [HPO4(2-)] gradient that is normally maintained by the P(i)/OH- carrier. In liver, changes in matrix adenine nucleotide concentrations that are brought about by the ATP-Mg/P(i) carrier can affect the activity of adenine nucleotide-dependent enzymes that are in the mitochondrial compartment. These enzymes in turn contribute to the overall regulation of bioenergetic function, flux through the gluconeogenesis and urea synthesis pathways, and organelle biogenesis. The ATP-Mg/P(i) carrier is distinct from other mitochondrial transport systems with respect to kinetics and to substrate and inhibitor sensitivity. It is the only carrier regulated by Ca2+. This carrier is present in kidney and liver mitochondria, but not in heart.

Eight mitochondrial carrier proteins were solubilized and purified in the authors' laboratories using variations of a general procedure based on hydroxyapatite and Celite chromatography. The molecular mass of all the carriers ranges between 28 and 34 kDa on SDS-PAGE. The purified carrier proteins were reconstituted into liposomes mainly by using a method of detergent removal by hydrophobic chromatography on polystyrene beads. The various carriers were identified in the reconstituted state by their kinetic properties . A complete set of basic kinetic data including substrate specificity, affinity, interaction with inhibitors, and activation energy was obtained. These data closely resemble those of intact mitochondria, as far as they are available from the intact organelle. Mainly on the basis of kinetic data, the asymmetric orientation of most of the reconstituted carrier proteins were established. Several of their functional properties are significantly affected by the type of phospholipids used for reconstitution. All carriers which have been investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport; i.e., a ternary complex, made up of two substrates and the carrier protein, is involved in the catalytic cycle. The only exception was the carnitine carrier, where a ping-pong mechanism of transport was found. By reaction of particular cysteine residues with mercurial reagents, several carriers could be reversibly converted to a functional state different from the various physiological transport modes. This "unphysiological" transport mode is characterized by a combination of channel-type and carrier-type properties.

Phosphate and oxoglutarate carriers transport phosphate and oxoglutarate across the inner membranes of mitochondria in exchange for OH- and malate, respectively. Both carriers belong to the mitochondrial carrier protein family, characterized by a tripartite structure made up of related sequences about 100 amino acids in length. The results obtained on the topology of the phosphate and oxoglutarate carriers are consistent with the six α-helix model proposed by Saraste and Walker. In both carriers the N- and C-terminal regions are exposed toward the cytosol. In addition, the oxoglutarate carrier has been shown to be a dimer by means of cross-linking studies. The bovine and human genes coding for the oxoglutarate carrier are split into eight and six exons, respectively, and five introns are found in the same position in both genes. The bovine and human phosphate carrier genes have the same organization with nine exons separated by eight introns at exactly the same positions. The phosphate carrier of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to the oxoglutarate carrier and the other members of the mitochondrial carrier family. The precursor of the phosphate carrier is efficiently imported, proteolytically processed, and correctly assembled in isolated mitochondria. The presequence-deficient phosphate carrier is imported with an efficiency of about 50% as compared with the precursor of the phosphate carrier and is correctly assembled, demonstrating that the mature portion of the phosphate carrier contains sufficient information for import and assembly into mitochondria. © 1993 Plenum Publishing Corporation.

In the process of oxidative phosphorylation, the exchange of cytosolic ADP3- against mitochondrial ATP4- across the inner mitochondrial membrane is mediated by a specific carrier protein. Two different conformations for this carrier have been demonstrated on the basis of interactions with specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA). The two conformations, referred to as CATR and BA conformations, are interconvertible, provided that ADP or ATP are present. The functional ADP/ATP carrier is probably organized as a tetramer. In the presence of CATR or BA the tetramer is split into two dimers combined with either of the two inhibitors. The amino acid sequence of the beef heart carrier monomer (297 residues) contains three repeats of about 100 residues each. Experimental results obtained through different approaches, including photolabeling, immunochemistry, and limited proteolysis, can be interpreted on the basis of a model with five or six transmembrane alpha helices per carrier monomer. Two mobile regions involved in the binding of nucleotides and accessible to proteolytic enzymes have been identified. Each of them may be visualized as consisting of two pairs of short amphipathic alpha helices, which can be juxtaposed to form hydrophilic channels facilitating the nucleotide transport. Mutagenesis in yeast is currently being used to detect strategic amino acids in ADP/ATP transport.

Complexes made up of the kinases, hexokinase and glycerol kinase, together with the outer mitochondrial membrane voltage-dependent anion channel (VDAC) protein, porin, and the inner mitochondrial membrane protein, the adenine nucleotide translocator, are involved in tumorigenesis, diabetes mellitus, and central nervous system function. Identification of these two mitochondrial membrane proteins, along with an 18 kD protein, as components of the peripheral benzodiazepine receptor, provides independent confirmation of the interaction of porin and the adenine nucleotide translocator to form functional contact sites between the inner and outer mitochondrial membranes. We suggest that these are dynamic structures, with channel conductances altered by the presence of ATP, and that ligand-mediated conformational changes in the porin-adenine nucleotide translocator complexes may be a general mechanism in signal transduction.

Recent progress is summarized on the structure, function, and regulation of the tricarboxylate (i.e., citrate) transport protein (CTP) from the rat liver mitochondrial inner membrane. The transporter has been purified and its reconstituted function characterized. A cDNA clone encoding the CTP has been isolated and sequenced, thus enabling a deduction of the complete amino acid sequence of this 32.6 kDa transport protein. Dot matrix analysis and sequence alignment indicate that based on structural considerations the CTP can be assigned to the mitochondrial carrier family. Hydropathy analysis of the transporter sequence indicates six putative membrane-spanning alpha-helices and has permitted the development of an initial model for the topography of the CTP within the inner membrane. The questions as to whether more than one gene encodes the CTP and whether more than one isoform is expressed remain unanswered at this time. Studies documenting a diabetes-induced alteration in the function of several mitochondrial anion transporters, which can be reversed by treatment with insulin, provide a physiologically/pathologically relevant experimental system for studying the molecular mechanism(s) by which mitochondrial transporters are regulated. Potential future research directions are discussed.

The tricarboxylate carrier has recently been purified from rat liver mitochondria by three distinct scientific groups using different methods. A 37-38-kDa protein has been prepared by silca gel 60 chromatography by our group (Claeys and Azzi, 1989; Glerum et al., 1990). The specific citrate transport activity of this preparation is not significantly different from that measured in mitochondria and it is inhibitable by 1,2,3-benzenetricarboxylic acid. Bisaccia et al. (1990) have reported the isolation of a 30-kDa protein by Celite 535 chromatography, and Kaplan's group (Kaplan et al., 1990) have isolated a 32.5-kDa protein by Matrex Orange, Matrex Blue, and Affi-Gel chromatography. Peptide mapping has failed to support any structural homologies between the 37-38-kDa and the 30-32.5-kD proteins. The 38-kD protein is N-terminally blocked. The peptides obtained by several cleavage procedures have been partially sequenced. Their sequence information has been used to obtain different cDNA clones by a dual approach, the polymerase chain reaction and screening of a lambda ZAP cDNA library. The largest cDNA which could be isolated is 2,986 bp in length and contains a 1071-bp-long open reading frame and an unusually long 3' untranslated region, both of which have been completely sequenced. The protein sequence of the carrier from the first in-frame methionine is 322 amino acids in length and exhibits a molecular mass of 35,546. Comparison of the protein sequence to the sequences of the four members of the mitochondrial carrier protein family (ADP/ATP carrier, phosphate carrier, 2-oxoglutarate/malate carrier, and uncoupling protein) does not reveal significant similarity (cf. Walker et al., 1987). A tripartite internal homology, which is a characteristic of these proteins, is not present in the sequence of the tricarboxylate carrier protein. The mRNA for the tricarboxylate carrier is expressed in rat liver and brain, but not in rat heart.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than "normal" mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.

There is a renewed interest in the structure and functioning of the mitochondrial respiratory chain with the realization that a number of genetic disorders result from defects in mitochondrial electron transfer. These so-called mitochondrial myopathies include diseases of muscle, heart, and brain. The respiratory chain can be fractionated into four large multipeptide complexes, an NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol oxidoreductase (complex III), and cytochromec oxidase (complex IV). Mitochondrial myopathies involving each of these complexes have been described. This review summarizes compositional and structural data on the respiratory chain proteins and describes the arrangement of these complexes in the mitochondrial inner membrane. This biochemical information is provided as a framework for the diagnosis and molecular characterization of mitochondrial diseases.

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H+-ATPase can proceed in a mtDNA-less (rho degree) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H+-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized beta-subunit of the H+-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H+-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.