Publisher Summary This chapter discusses the metabolite carriers in mitochondria. For many metabo... more Publisher Summary This chapter discusses the metabolite carriers in mitochondria. For many metabolic processes in the cell a concerted function of both the cytosolic and the mitochondrial compartment is necessary. Because of the presence of non-specific pore proteins, the outer mitochondrial membrane is permeable to small molecules, such as the metabolites discussed in this chapter. The inner mitochondrial membrane, on the other hand, is impermeable to most solutes. Thus, the presence of specific transporters catalyzing the import into and the export out of the matrix space is essential for mitochondrial function. The metabolic significance of these carriers is reflected in their organ distribution in mammals. The central function of mitochondria is production of ATP, thus the first two carriers involved in energy transfer and oxidative phosphorylation, the ADP/ATP carrier and the phosphate carrier, are present in all mitochondria. Also, the main carriers for import of reducing equivalents or substrates for oxidative phosphorylation into the mitochondrial matrix are widely distributed. This includes the aspartate/glutamate carrier, the oxoglutarate carrier, the pyruvate carrier and the carnitine carrier.
Publisher Summary The tricarboxylate transport protein can be purified approximately six fold by ... more Publisher Summary The tricarboxylate transport protein can be purified approximately six fold by chromatography on hydroxylapatite with a recovery of 50% of the activity present in the submitochondrial particles (SMP) extract. The presence of cardiolipin (DPG) during extraction and purification is required for optimal activity of the reconstituted tricarboxylate carrier. This chapter presents the activity of the reconstituted citrate/citrate exchange of the extracts from liver SMP and of the eluates obtained from HTP, both in the presence and in the absence of DPG. Absorption chromatography on HTP increases the specific activity several fold, with a recovery of the total activity of 50%. Inclusion of DPG in the solubilization medium does not influence the enhancement in purification, but increases the activity of citrate exchange both in the extracts and in the HTP eluates. A similar increase in the activity of the mitochondrial phosphate carrier in the HTP eluate as been observed and it has been suggested that DPG prevents an inactivation of the carrier occurring during its solubilization by Triton. The effect of increasing concentrations of Triton in the solubilization buffer was therefore investigated by measuring the reconstituted citrate exchange activity in both SMP extracts and HTP eluates. In both the cases, the total and the specific activities increase on increasing the concentration of Triton up to 3%, and decrease at higher detergent concentrations although more protein is solubilized. The citrate exchange activity reconstituted from the HTP eluate was characterized by studying the tissue and the substrate specificity.
The oxoglutarate transporter, also known as the oxoglutarate/malate carrier (OGC), is a nuclear-e... more The oxoglutarate transporter, also known as the oxoglutarate/malate carrier (OGC), is a nuclear-encoded protein located in the inner mitochondrial membrane. This enzyme catalyzes the transport of 2-oxoglutarate across the mitochondrial membrane in exchange for malate or other dicarboxylic acids (Palmieri et al., 1972) and plays an important role in several metabolic processes, including the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle, gluconeogenesis from lactate, and nitrogen metabolism (Krämer and Palmieri, 1992). We have purified the OGC from pig heart mitochondria and reconstituted it into liposomes in an active form (Bisaccia et al., 1985). The first amino acid sequence of this transporter was deduced from bovine heart cDNA (Runswick et al., 1990). The protein consists of three tandemly repeated sequences of about 100 amino acids, and these repeats are related to those present in the other functionally characterized members of the mitochondrial carrier family (Palmieri, 1994; Palmieri and van Ommen, 1999). More recently, we have determined the sequences of the human and bovine OGC gene from overlapping genomic clones generated by PCR (Iacobazzi et al., 1992; Accession number X66145). Materials and methods
The mitochondrial oxoglutarate carrier exchanges cytosolic malate for 2-oxoglutarate from the mit... more The mitochondrial oxoglutarate carrier exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. Orthologs of the carrier have a high degree of amino acid sequence conservation, meaning that it is impossible to identify residues important for function on the basis of this criterion alone. Therefore, each amino acid residue in the transmembrane alpha-helices H2 and H6 was replaced by a cysteine in a functional mitochondrial oxoglutarate carrier that was otherwise devoid of cysteine residues. The effects of the cysteine replacement and subsequent modification by sulfhydryl reagents on the initial uptake rate of 2-oxoglutarate were determined. The results were evaluated using a structural model of the oxoglutarate carrier. Residues involved in inter-helical and lipid bilayer interactions tolerate cysteine replacements or their modifications with little effect on transport activity. In contrast, the majority of cysteine substitutions in the aqueous cavity had a severe effect on transport activity. Residues important for function of the carrier cluster in three regions of the transporter. The first consists of residues in the [YWLF]- [KR]-G-X-X-P sequence motif, which is highly conserved in all members of the mitochondrial carrier family. The residues may fulfill a structural role as a helix breaker or a dynamic role as a hinge region for conformational changes during translocation. The second cluster of important residues can be found at the carboxy-terminal end of the even-numbered transmembrane alpha-helices at the cytoplasmic side of the carrier. Residues in H6 at the interface with H1 are the most sensitive to mutation and modification, and may be essential for folding of the carrier during biogenesis. The third cluster is at the midpoint of the membrane and consists of residues that are proposed to be involved in substrate binding.
The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shut... more The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle and gluconeogenesis. To establish amino acid residues that are important for function, each residue in the transmembrane alpha-helices H1, H3 and H5 was replaced systematically by a cysteine in a fully functional mutant carrier that was devoid of cysteine residues. The transport activity of the mutant carriers was measured in the presence and absence of sulfhydryl reagents. The observed effects were rationalized by using a comparative structural model of the OGC. Most of the residues that are critical for function are found at the bottom of the cavity and they belong to the signature motifs P-X-[DE]-X-X-[KR] that form a network of three inter-helical salt bridges that close the carrier at the matrix side. The OGC deviates from most other carriers, because it has a conserved leucine (L144) rather than a positively charged residue in the signature motif of the second repeat and thus the salt bridge network is lacking one salt bridge. Incomplete salt-bridge networks due to hydrophobic, aromatic or polar substitutions are observed in other dicarboxylate, phosphate and adenine nucleotide transporters. The interaction between the carrier and the substrate has to provide the activation energy to trigger the re-arrangement of the salt-bridge network and other structural changes required for substrate translocation. For substrates such as malate, which has only two carboxylic and one hydroxyl group, a reduction in the number of salt bridges in the network may be required to lower the energy barrier for translocation. Another group of key residues, consisting of T36, A134, and T233, is close to the putative substrate binding site and substitutions or modifications of these residues may interfere with substrate binding and ion coupling. Residues G32, A35, Q40, G130, G133, A134, G230, and S237 are potentially engaged in inter-helical interactions and they may be involved in the movements of the alpha-helices during translocation.
Publisher Summary This chapter describes the direct methods for studying mitochondrial transport,... more Publisher Summary This chapter describes the direct methods for studying mitochondrial transport, which are necessarily linked to the techniques for separating the mitochondria. Since the rates of transport reactions are higher in mitochondria than in whole cells and bacteria, special emphasis will be devoted to the techniques developed for measuring kinetics. It is possible to study (1) the efflux of endogenous substrates or of substrates from previously loaded mitochondria, (2) the uptake of added metabolites, and (3) the counterexchange by following the in and out movements of the exchanging metabolites. The steady state distribution and the kinetic of transport between the two spaces are also examined. The measurements of metabolite distribution are made after the separation of mitochondria from the incubation medium. It is emphasized that the amount of metabolite within the mitochondria is relatively small, since the volume of the suspending medium is orders of magnitude larger than that of the intramitochondrial space.
Publisher Summary Information on the physico-chemical properties of the sulphydryl groups of the ... more Publisher Summary Information on the physico-chemical properties of the sulphydryl groups of the native membrane-bound phosphate carrier can be reached by using paramagnetic analogues of N-ethylmaleimide under conditions in which they react with sulphydryl groups of the phosphate carrier specifically. For this purpose, maleimide spin labels of different chain length are used to characterize the microenvironment of the phosphate carrier sulphydryl groups in situ as well as to sense the average depth at which they are located with respect to the aqueous phase of the membrane. The attempt to use maleimide spin labels for a characterization of the sulphydryl groups of the mitochondrial phosphate carrier are based on the finding that the maleimide spin labels inhibit phosphate transport in mitochondria. The ESR data shows that the sulphydryl groups of the phosphate carrier are at least of two types. One type of the groups (site I) giving rise to a mobile ESR signal is easily accessible from the aqueous phase and its environment does not considerably influence the motion of the bound probe. The second type of sulphydryl groups as sensed by maleimide derivatives (site II) is completely different. The close environment of site II as analyzed by the short chain MSL, strongly restricts the motion of the bound probe, which results in the strongly immobilized ESR signal.
Since several substrates of the mitochondrial enzymes must be transported from the cytosol into t... more Since several substrates of the mitochondrial enzymes must be transported from the cytosol into the matrix and several products have to leave the mitochondria, the inner mitochondrial membrane has to be equipped with transport systems which catalyze metabolic flows between the inner and the outer compartment. The existence of at least 9 transport systems for metabolites has been documented in some detail by studies performed in intact mitochondria (LaNoue & Schoolwerth, 1979; Meijer & Van Dam, 1981; Palmieri et al, 1987). With the exception of the carrier for carnitine and acylcarnitine esters, the other carriers deal with the transport of anions: substrates of oxidative phosphorylation, ADP, ATP and phosphate, fuels of the tricarboxylic acid cycle, pyruvate, β-hydroxybutyrate and acetoacetate, dicarboxylates and tricarboxylates, and substrates of amino acid metabolism. This intense traffic of anions across the mitochondrial membrane is necessary, besides for oxidative phosphorylation, the tricarboxylic acid cycle and the amino acid metabolism, for the transfer of reducing equivalents in both directions and for important metabolic pathways, whose enzymes are partitioned between the cytosol and the mitochondria, such as gluconeogenesis, fatty acid synthesis, ketogenesis, β-oxidation of fatty acids and, in liver, urogenesis. Many of the properties of the proposed mitochondrial anion carriers, i.e. the high specificity, the existence of specific inhibitors, the saturation kinetics, the different distribution in various tissues and species and the inhibition by SH reagents have pointed to the protein nature of these transport systems. Their isolation, however, has been hindered for quite a long time.
Publisher Summary This chapter investigates the partial purification and reconstitution of the tr... more Publisher Summary This chapter investigates the partial purification and reconstitution of the tricarboxylate carrier from rat liver mitochondria. In addition to many other carriers, the inner mitochondrial membrane contains a specific exchange system for the transport of tricarboxylates, malate and phosphoenolpyruvate. This carrier, which is presents in liver but absent in heart and brain, has important functions in the pathways of fatty acid synthesis and gluconeogenesis and in the transfer of reducing equivalents across the inner mitochondrial membrane. The basic properties of the reconstituted citrate carrier, for example, substrate affinity, inhibitor specificity, tissue specificity, and the absolute requirementof an appropriate counteranion, generally resemble those known for this transport system from mitochondria. Externally added cis-aconitate, isocitrate, phosphoenolpyruvate, and malate strongly inhibit the reconstituted citrate or citrate exchange activity. Besides the specific inhibitor benzene 1,2,3-tricarboxylate, also p -iodobenzyl malonate and p-hydroxymercuribenzoate strongly inhibit the exchange of citrate.
Publisher Summary This chapter discusses the metabolite carriers in mitochondria. For many metabo... more Publisher Summary This chapter discusses the metabolite carriers in mitochondria. For many metabolic processes in the cell a concerted function of both the cytosolic and the mitochondrial compartment is necessary. Because of the presence of non-specific pore proteins, the outer mitochondrial membrane is permeable to small molecules, such as the metabolites discussed in this chapter. The inner mitochondrial membrane, on the other hand, is impermeable to most solutes. Thus, the presence of specific transporters catalyzing the import into and the export out of the matrix space is essential for mitochondrial function. The metabolic significance of these carriers is reflected in their organ distribution in mammals. The central function of mitochondria is production of ATP, thus the first two carriers involved in energy transfer and oxidative phosphorylation, the ADP/ATP carrier and the phosphate carrier, are present in all mitochondria. Also, the main carriers for import of reducing equivalents or substrates for oxidative phosphorylation into the mitochondrial matrix are widely distributed. This includes the aspartate/glutamate carrier, the oxoglutarate carrier, the pyruvate carrier and the carnitine carrier.
Publisher Summary The tricarboxylate transport protein can be purified approximately six fold by ... more Publisher Summary The tricarboxylate transport protein can be purified approximately six fold by chromatography on hydroxylapatite with a recovery of 50% of the activity present in the submitochondrial particles (SMP) extract. The presence of cardiolipin (DPG) during extraction and purification is required for optimal activity of the reconstituted tricarboxylate carrier. This chapter presents the activity of the reconstituted citrate/citrate exchange of the extracts from liver SMP and of the eluates obtained from HTP, both in the presence and in the absence of DPG. Absorption chromatography on HTP increases the specific activity several fold, with a recovery of the total activity of 50%. Inclusion of DPG in the solubilization medium does not influence the enhancement in purification, but increases the activity of citrate exchange both in the extracts and in the HTP eluates. A similar increase in the activity of the mitochondrial phosphate carrier in the HTP eluate as been observed and it has been suggested that DPG prevents an inactivation of the carrier occurring during its solubilization by Triton. The effect of increasing concentrations of Triton in the solubilization buffer was therefore investigated by measuring the reconstituted citrate exchange activity in both SMP extracts and HTP eluates. In both the cases, the total and the specific activities increase on increasing the concentration of Triton up to 3%, and decrease at higher detergent concentrations although more protein is solubilized. The citrate exchange activity reconstituted from the HTP eluate was characterized by studying the tissue and the substrate specificity.
The oxoglutarate transporter, also known as the oxoglutarate/malate carrier (OGC), is a nuclear-e... more The oxoglutarate transporter, also known as the oxoglutarate/malate carrier (OGC), is a nuclear-encoded protein located in the inner mitochondrial membrane. This enzyme catalyzes the transport of 2-oxoglutarate across the mitochondrial membrane in exchange for malate or other dicarboxylic acids (Palmieri et al., 1972) and plays an important role in several metabolic processes, including the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle, gluconeogenesis from lactate, and nitrogen metabolism (Krämer and Palmieri, 1992). We have purified the OGC from pig heart mitochondria and reconstituted it into liposomes in an active form (Bisaccia et al., 1985). The first amino acid sequence of this transporter was deduced from bovine heart cDNA (Runswick et al., 1990). The protein consists of three tandemly repeated sequences of about 100 amino acids, and these repeats are related to those present in the other functionally characterized members of the mitochondrial carrier family (Palmieri, 1994; Palmieri and van Ommen, 1999). More recently, we have determined the sequences of the human and bovine OGC gene from overlapping genomic clones generated by PCR (Iacobazzi et al., 1992; Accession number X66145). Materials and methods
The mitochondrial oxoglutarate carrier exchanges cytosolic malate for 2-oxoglutarate from the mit... more The mitochondrial oxoglutarate carrier exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. Orthologs of the carrier have a high degree of amino acid sequence conservation, meaning that it is impossible to identify residues important for function on the basis of this criterion alone. Therefore, each amino acid residue in the transmembrane alpha-helices H2 and H6 was replaced by a cysteine in a functional mitochondrial oxoglutarate carrier that was otherwise devoid of cysteine residues. The effects of the cysteine replacement and subsequent modification by sulfhydryl reagents on the initial uptake rate of 2-oxoglutarate were determined. The results were evaluated using a structural model of the oxoglutarate carrier. Residues involved in inter-helical and lipid bilayer interactions tolerate cysteine replacements or their modifications with little effect on transport activity. In contrast, the majority of cysteine substitutions in the aqueous cavity had a severe effect on transport activity. Residues important for function of the carrier cluster in three regions of the transporter. The first consists of residues in the [YWLF]- [KR]-G-X-X-P sequence motif, which is highly conserved in all members of the mitochondrial carrier family. The residues may fulfill a structural role as a helix breaker or a dynamic role as a hinge region for conformational changes during translocation. The second cluster of important residues can be found at the carboxy-terminal end of the even-numbered transmembrane alpha-helices at the cytoplasmic side of the carrier. Residues in H6 at the interface with H1 are the most sensitive to mutation and modification, and may be essential for folding of the carrier during biogenesis. The third cluster is at the midpoint of the membrane and consists of residues that are proposed to be involved in substrate binding.
The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shut... more The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle and gluconeogenesis. To establish amino acid residues that are important for function, each residue in the transmembrane alpha-helices H1, H3 and H5 was replaced systematically by a cysteine in a fully functional mutant carrier that was devoid of cysteine residues. The transport activity of the mutant carriers was measured in the presence and absence of sulfhydryl reagents. The observed effects were rationalized by using a comparative structural model of the OGC. Most of the residues that are critical for function are found at the bottom of the cavity and they belong to the signature motifs P-X-[DE]-X-X-[KR] that form a network of three inter-helical salt bridges that close the carrier at the matrix side. The OGC deviates from most other carriers, because it has a conserved leucine (L144) rather than a positively charged residue in the signature motif of the second repeat and thus the salt bridge network is lacking one salt bridge. Incomplete salt-bridge networks due to hydrophobic, aromatic or polar substitutions are observed in other dicarboxylate, phosphate and adenine nucleotide transporters. The interaction between the carrier and the substrate has to provide the activation energy to trigger the re-arrangement of the salt-bridge network and other structural changes required for substrate translocation. For substrates such as malate, which has only two carboxylic and one hydroxyl group, a reduction in the number of salt bridges in the network may be required to lower the energy barrier for translocation. Another group of key residues, consisting of T36, A134, and T233, is close to the putative substrate binding site and substitutions or modifications of these residues may interfere with substrate binding and ion coupling. Residues G32, A35, Q40, G130, G133, A134, G230, and S237 are potentially engaged in inter-helical interactions and they may be involved in the movements of the alpha-helices during translocation.
Publisher Summary This chapter describes the direct methods for studying mitochondrial transport,... more Publisher Summary This chapter describes the direct methods for studying mitochondrial transport, which are necessarily linked to the techniques for separating the mitochondria. Since the rates of transport reactions are higher in mitochondria than in whole cells and bacteria, special emphasis will be devoted to the techniques developed for measuring kinetics. It is possible to study (1) the efflux of endogenous substrates or of substrates from previously loaded mitochondria, (2) the uptake of added metabolites, and (3) the counterexchange by following the in and out movements of the exchanging metabolites. The steady state distribution and the kinetic of transport between the two spaces are also examined. The measurements of metabolite distribution are made after the separation of mitochondria from the incubation medium. It is emphasized that the amount of metabolite within the mitochondria is relatively small, since the volume of the suspending medium is orders of magnitude larger than that of the intramitochondrial space.
Publisher Summary Information on the physico-chemical properties of the sulphydryl groups of the ... more Publisher Summary Information on the physico-chemical properties of the sulphydryl groups of the native membrane-bound phosphate carrier can be reached by using paramagnetic analogues of N-ethylmaleimide under conditions in which they react with sulphydryl groups of the phosphate carrier specifically. For this purpose, maleimide spin labels of different chain length are used to characterize the microenvironment of the phosphate carrier sulphydryl groups in situ as well as to sense the average depth at which they are located with respect to the aqueous phase of the membrane. The attempt to use maleimide spin labels for a characterization of the sulphydryl groups of the mitochondrial phosphate carrier are based on the finding that the maleimide spin labels inhibit phosphate transport in mitochondria. The ESR data shows that the sulphydryl groups of the phosphate carrier are at least of two types. One type of the groups (site I) giving rise to a mobile ESR signal is easily accessible from the aqueous phase and its environment does not considerably influence the motion of the bound probe. The second type of sulphydryl groups as sensed by maleimide derivatives (site II) is completely different. The close environment of site II as analyzed by the short chain MSL, strongly restricts the motion of the bound probe, which results in the strongly immobilized ESR signal.
Since several substrates of the mitochondrial enzymes must be transported from the cytosol into t... more Since several substrates of the mitochondrial enzymes must be transported from the cytosol into the matrix and several products have to leave the mitochondria, the inner mitochondrial membrane has to be equipped with transport systems which catalyze metabolic flows between the inner and the outer compartment. The existence of at least 9 transport systems for metabolites has been documented in some detail by studies performed in intact mitochondria (LaNoue & Schoolwerth, 1979; Meijer & Van Dam, 1981; Palmieri et al, 1987). With the exception of the carrier for carnitine and acylcarnitine esters, the other carriers deal with the transport of anions: substrates of oxidative phosphorylation, ADP, ATP and phosphate, fuels of the tricarboxylic acid cycle, pyruvate, β-hydroxybutyrate and acetoacetate, dicarboxylates and tricarboxylates, and substrates of amino acid metabolism. This intense traffic of anions across the mitochondrial membrane is necessary, besides for oxidative phosphorylation, the tricarboxylic acid cycle and the amino acid metabolism, for the transfer of reducing equivalents in both directions and for important metabolic pathways, whose enzymes are partitioned between the cytosol and the mitochondria, such as gluconeogenesis, fatty acid synthesis, ketogenesis, β-oxidation of fatty acids and, in liver, urogenesis. Many of the properties of the proposed mitochondrial anion carriers, i.e. the high specificity, the existence of specific inhibitors, the saturation kinetics, the different distribution in various tissues and species and the inhibition by SH reagents have pointed to the protein nature of these transport systems. Their isolation, however, has been hindered for quite a long time.
Publisher Summary This chapter investigates the partial purification and reconstitution of the tr... more Publisher Summary This chapter investigates the partial purification and reconstitution of the tricarboxylate carrier from rat liver mitochondria. In addition to many other carriers, the inner mitochondrial membrane contains a specific exchange system for the transport of tricarboxylates, malate and phosphoenolpyruvate. This carrier, which is presents in liver but absent in heart and brain, has important functions in the pathways of fatty acid synthesis and gluconeogenesis and in the transfer of reducing equivalents across the inner mitochondrial membrane. The basic properties of the reconstituted citrate carrier, for example, substrate affinity, inhibitor specificity, tissue specificity, and the absolute requirementof an appropriate counteranion, generally resemble those known for this transport system from mitochondria. Externally added cis-aconitate, isocitrate, phosphoenolpyruvate, and malate strongly inhibit the reconstituted citrate or citrate exchange activity. Besides the specific inhibitor benzene 1,2,3-tricarboxylate, also p -iodobenzyl malonate and p-hydroxymercuribenzoate strongly inhibit the exchange of citrate.
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Papers by Ferdinando Palmieri