Critical Review Mitochondrial Tricarboxylate and Dicarboxylate–Tricarboxylate Carriers: From Animals to Plants
Vincenza Dolce1* Anna Rita Cappello1 Loredana Capobianco2*
1
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende Cosenza, Italy 2 Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy
Abstract The citrate carrier (CiC), characteristic of animals, and the dicarboxylate–tricarboxylate carrier (DTC), characteristic of plants and protozoa, belong to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors, and nucleotides between the cytoplasm and the mitochondrial matrix. Most of the functional data on these transporters are obtained from the studies performed with the protein purified from rat, eel yeast, and maize mitochondria or recombinant proteins from different sources incorporated into phospholipid vesicles (liposomes). The functional data indicate that CiC is responsible for the efflux of acetyl-CoA from the mitochondria to the cytosol in the form of citrate, the primer for fatty acid, cholesterol synthesis, and histone acetylation. Like the CiC, the citrate exported by DTC
from the mitochondria to the cytosol in exchange for oxaloacetate can be cleaved by citrate lyase to acetyl-CoA and oxaloacetate and used for fatty acid elongation and isoprenoid synthesis. In addition to its role in fatty acid synthesis, CiC is involved in other processes such as gluconeogenesis, insulin secretion, inflammation, and cancer progression, whereas DTC is involved in the production of glycerate, nitrogen assimilation, ripening of fruits, ATP synthesis, and sustaining of respiratory flux in fruit cells. This review provides an assessment of the current understanding of CiC and DTC structural and biochemical characteristics, underlying the structure–function relationship of these carriers. Furthermore, a phylogenetic C 2014 IUBMB relationship between CiC and DTC is proposed. V Life, 66(7):462–471, 2014
Keywords: citrate carrier; dicarboxylate–tricarboxylate carrier; gene expression regulation; mitochondrial carrier; phylogenetic analysis; substrate specificity.
Introduction Abbreviations: BTA, benzenetricarboxylic acid; CiC, citrate (tricarboxylate) carrier; DTC, dicarboxylate/tricarboxylate carrier; FOXA, forkhead box A; MC, mitochondrial carrier (MC); NF-Y, nuclear factor Y; ODC, oxodicarboxylate carrier; OGC, oxoglutarate carrier; PPRE, peroxisome proliferatoractivated receptor-responsive element; PUFA, polyunsaturated fatty acid; Sp1, stimulating protein 1 region; SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein. C 2014 International Union of Biochemistry and Molecular Biology V Volume 66, Number 7, July 2014, Pages 462–471 Address correspondence to: Loredana Capobianco, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce 73100, Italy. Tel.: 139–0832298864. Fax: 139-0-832298626. E-mail: [email protected] Received 28 May 2014; Accepted 22 June 2014 DOI 10.1002/iub.1290 Published online 18 July 2014 in Wiley Online Library (wileyonlinelibrary.com)
462
The mitochondrial carriers (MCs) are nuclear-coded membrane-embedded proteins that, with few exceptions, are localized in the inner membranes of mitochondria. They catalyze the selective transport of specific essential metabolites (di- and tricarboxylates, keto acids, amino acids, nucleotides, and coenzymes/cofactors) across the inner mitochondrial membrane providing a link between mitochondria and cytosol. This link is indispensable as many physiological processes require the participation of both intra- and extramitochondrial enzyme reactions. All MCs of known function exhibit a tripartite sequence structure, consisting of three tandemly repeated homologous domains of about 100 amino acids in length. Each domain contains two hydrophobic stretches, separated by hydrophilic regions, every repeat contains the signature sequence motif PX[DE]XX[RK] (PROSITE
IUBMB Life
PS50920 and PFAM PF00153) by which MCF members are recognized (1). One of the well-characterized members of this family in animals is the tricarboxylate (or citrate) carrier (CiC), which catalyzes an electroneutral exchange of a tricarboxylate for another tricarboxylate, a dicarboxylate (L-malate), or phosphoenolpyruvate across the mitochondrial inner membrane (2–7). This carrier protein plays a central role in fatty acid and sterol biosynthesis as it exports citrate from the mitochondria to the cytosol, where citrate is cleaved by ATP-citrate lyase to acetyl-CoA and oxaloacetate. Acetyl-CoA is used for fatty acid and sterol biosynthesis; whereas oxaloacetate is reduced to malate, which in turn is converted to pyruvate via malic enzyme with the production of NADPH plus H1 (1,8). In addition to its role in fatty acid synthesis, CiC is involved in other processes such as gluconeogenesis, insulin secretion, histone acetylation, inflammation, and differentiation of fibroblastes into adipocytes (9–14). Under different metabolic conditions, CiC may play a role in gluconeogenesis from lactate in species (not in man and rat) where phosphoenolpyruvate carboxykinase is located in mitochondria (1). Furthermore, a role of CiC in cancer progression has been postulated (15,16) that was expected as the overexpression of fatty acid synthase, the sole protein in the human genome capable of de novo synthesis of fatty acids from acetyl-CoA (17), has been observed in a wide variety of human tumor types (18) as a consequence of an altered transcriptional regulation (19,20). Very recently, a role of CiC in the alteration of lipid metabolism has been observed in biliary cirrhosis (21). In plants, a related carrier to CiC is the dicarboxylate–tricarboxylate carrier (DTC) that catalyzes an electroneutral transport of a broad spectrum of single protonated tricarboxylate (citrate, isocitrate, and aconitate) in exchange with unprotonated dicarboxylates (oxoglutarate, oxaloacetate, malate, maleate, succinate, and malonate), but not phosphoenolpyruvate. As DTC transports a broad spectrum of dicarboxylates and tricarboxylates, it has been hypothesized that this carrier may play a role in a number of important plant metabolic functions that require organic acid flux to or from the mitochondria. For example, like the CiC, the citrate exported by DTC from the mitochondria to the cytosol in exchange for oxaloacetate can be cleaved by citrate lyase to acetyl-CoA and oxaloacetate and used for fatty acid elongation and isoprenoid synthesis. The malate/oxaloacetate exchange catalyzed by DTC can enable the export of redox equivalents from the mitochondrial matrix that can act as reductants for the production of glycerate during photorespiration in photosynthesizing cells. Furthermore, significances for DTC are its involvement in nitrogen assimilation and in the ripening of fruits, because oxoglutarate is required for the assimilation of ammonium into amino acids by the glutamine synthetase/glutamate synthase pathway (22), whereas malate may be transported to the mitochondria and entered into the tricarboxylic acid cycle to support ATP synthesis and the respiratory flux in fruit cells (23).
Dolce et al.
In this review, we will address biochemical, molecular, and gene phylogenetic relationship of recent investigations on CiC and DTC from various sources, focussing our attention on transport, metabolism, and molecular mechanisms.
Identification and Characterization of CiCs and DTCs The existence of a mitochondrial CiC has been postulated for the first time by Chappell (24,25) who at the same time described the structural requirements of the substrates to be transported. The properties of the CiC have been extensively investigated in intact mitochondria (26). Kinetic studies have shown that the activity of the tricarboxylate carrier is high in liver compared to that in heart and brain (27,28) and that the carrier has a single binding site for all its substrates (27). Furthermore, evidence for the presence of a CiC has been given by the studies of the inhibition of citrate transport across the mitochondrial membrane by 1,2,3-benzenetricarboxylic acid (1,2,3-BTA) but not by its 1,2,4- and 1,3,5-isomers (27–29). The protein responsible for citrate transport has been first purified from rat liver mitochondria by solubilization in Triton X-100 and chromatography through hydroxyapatite in the presence of cardiolipin and of the specific inhibitor 1,2,3-BTA (27). To date, different groups have purified this carrier to homogeneity from mitochondria of rat (7,30), eel (6), and yeast (31). The molecular weight of the purified protein has been estimated to be approximately 30–32.5 kDa (6,7,30). The properties of the purified carrier, reconstituted into a liposomal system, are similar to those of CiC from intact mitochondria as far as counter anion requirement, substrate specificity, and inhibitor sensitivity are conserved (26,27,32). Furthermore, the recombinantly expressed and reconstituted CiC of rat, eel, drosophila, and yeast have confirmed that the carrier transports citrate, cis-aconitate, threo-isocitrate, phosphoenolpyruvate, and L-malate. Some activity has also been observed with succinate (31,33–37). The CiC overlaps with oxoglutarate carrier (OGC) by transporting the substrate malate and malonate (38). In yeast, a second isoform of CiC (CiC2, also known as YHM2) (39) has been identified that differs markedly from the yeast CiC (CiC1) previously identified in this organism (31) as this protein efficiently transports citrate, oxoglutarate oxaloacetate, succinate, and fumarate, whereas different from CiC1 the reconstituted CiC2 transports isocitrate, cis-aconitate, and malate with very low efficiency (39). For its substrate, specificity of CiC2 has been identified as a citrate/OGC. In plant, the existence of a tricarboxylate carrier has been suggested by osmotic swelling of isolated mitochondria in ammonium citrate and in the presence of catalytic amount of phosphate and malate (40,41). Later on, a putative CiC from pea and maize mitochondria has been purified (42,43) and its transport specificity has been tested in liposomes. The maize carrier incorporated into liposomes is able to exchange citrate
463
IUBMB LIFE
TABLE 1
Kinetic parameters determined for CiC and DTC citrate homo exchange
Km (mM)
Vmax (mmol/min 3 g protein)
R. norvegicus liver mitochondria
0.12
0.0225
(39)
R. norvegicus native reconstituted
0.13
2.049
(2)
A. anguilla native reconstituted
0.062
9.0
(13)
A. anguilla bacterially expressed
0.068
14.2
(49)
D. melanogaster bacterially expressed
0.132
11.75
(48)
S. cerevisiae (CiC1) bacterially expressed
0.36
2.5
(43)
S. cerevisiae (CiC2) bacterially expressed
0.16
9.8
(50)
Z. mais native reconstituted
0.65
a
A. thaliana bacterially expressedb
13
Reference
(9)
0.95
1.3
(10)
b
1.49
3.9
(10)
b
N. tabacum (NtDTC3) bacterially expressed
1.91
0.31
(10)
A. thaliana bacterially expressedc
N. tabacum (NtDTC1) bacterially expressed
0.15
1.7
(10)
c
N. tabacum (NtDTC1) bacterially expressed
0.24
6.4
(10)
N. tabacum (NtDTC3) bacterially expressedc
0.31
0.38
(10)
0.83
2.03
(11)
b
V. vinifera (VvDTC1) bacterially expressed a
Milligram of total protein at 9 C, pH 7.0. Measured at pH 7.0. c Measured at pH 6.0. b
against citrate, malate, succinate, malonate, and isocitrate as well as differently to CiC animals, oxoglutarate, and oxaloacetate while it is not able to transport phosphoenolpyruvate. In the postgenomic era in Arabidopsis thaliana, Nicotiana tabacum, and Vitis vinifera, a mitochondrial DTC has been identified by overexpression in Escherichia coli (44,45). Like maize CiC, the DTCs are capable of transporting both dicarboxylates (such as oxoglutarate, oxaloacetate, malate, succinate, maleate, malonate, and oxoadipate) and tricarboxylates (such as citrate, isocitrate, cis-aconitate, and trans-aconitate) (44) but not phosphoenolpyruvate. DTCs are also capable of exchanging sulfate. Therefore, maize and pea CiCs (42,43), for their substrate specicity, can’t be considered citrate carriers but they have to be included in plant subfamily DTC. In Plasmodium falciparum, a DTC has been found that is able to transport the dicarboxylates (such as oxoglutarate, oxaloacetate, malate, succinate, and fumarate), the citrate, and the sulfate (46). The kinetic constants (Table 1) of the recombinant purified CiCs have been determined by measuring the initial transport rate at various external [14C]citrate concentrations, in the presence of a constant saturating internal concentration of
464
citrate. The half-saturation constant of CiC for citrate at pH 7 is in the range of 0.062–0.13 mM (2,36,37,47), whereas the citrate Km for yeast CiC1 and CiC2 are 0.36 and 0.16 mM, respectively (33,39). The range of DTC for citrate measured at pH 7 is 0.65–1.91 mM (43–45) and the lower values have been obtained at pH 6 in citrate/citrate exchange in A. thaliana and N. tabacum (0.15–0.31 mM) (44).
CiC and DTC Genes Phylogenetic Relationship of CiC and DTC Proteins with Other Mitochondrial Transporters In 1993, a full-length cDNA encoding the mature rat liver mitochondria CiC has been cloned (48). Subsequently, cDNA sequences encoding the CiC have been identified via overexpression and reconstitution approach from a variety of sources (31,33,35,36). Successively, DTCs have been identified and characterized at a molecular level in plant (44,45), yeast (39), and protozoa (46). Recently, two different isoforms of human CiC, carried out by alternative splicing of the first exon, have been identified in human prostate epithelial cells; the difference between the two
Citrate and Dicarboxylate–Tricarboxylate Carriers
proteins affect the N-terminal region and the subcellular localization being one localized in mitochondria and the other in plasma membrane (49). The human, rat, eel, and fruit fly CiCs possess a presequence of 13, 13, 20, and 26 amino acids, respectively, with a net positive charge of 12 (34,36,49,50). The presequence of rat and eel CiC is dispensable both for targeting to mitochondria and for insertion into the inner membrane (34,50). Interestingly, it has been found that the CiC presequence is important to avoid aggregation of the newly synthesized polypeptide chain, and hence keeping the precursor protein soluble in the cytosol. This presequence is also able to influence the folding state of the precursor protein in the cytosol prior to import into mitochondria. Notably, the chaperoning effect of the presequence is completely retained if the positive charges have been exchanged with negative charges (34). A topologic accepted model based on the sequence features and on the accessibility of CiC to peptide-specific antibodies and proteolytic enzymes is consistent with an arrangement of the carrier into an even number of transmembrane segments with both the N- and the C-termini on the cytosolic side of the mitochondrial inner membrane (51). A multiple sequence alignment (MSA) was obtained using all mature mitochondrial CiCs known (Homo sapiens, Rattus norvegicus, Anguilla anguilla, Drosophila melanogaster, and Saccharomyces cerevisiae); all mitochondrial DTCs known (V. vinifera, N. tabacum, A. thaliana, and P. falciparum); all mitochondrial DiCs known (H. sapiens, R. norvegicus, and D.
Dolce et al.
melanogaster); all mitochondrial OGCs known (R. norvegicus and Bos taurus), all mitochondrial oxodicarboxylate carriers (ODCs) known (H. sapiens and S. cerevisiae), and the S. cerevisiae CiC2 by using ClustalW (52) shows that DTCs despite their capacity to transport citrate and malate present the highest homology with the mitochondrial OGCs sharing of about 40% identical amino acids with this carrier from bovine (53), human (54), and rat (55), whereas only of about 20% is sharing with the CiCs (data not shown). Structural specific data are not disposable for DTC and CiC; nevertheless, the alignment performed using all mature mitochondrial CiCs known and all mitochondrial DTCs known shows that in helices DTC are present in some blocks of conserved residues absent in CiC and vice versa. Furthermore, it could be noted that these regions sometimes present residues with different charges that could be responsible for the different substrate specificities (data not shown).
FIG 1
Phylogenic tree of amino acid sequences of MCs from various organisms. For comparative purposes, the amino acid sequences of OGC, DTC, DiC, and OGC homologs from various organisms have been used. The dendogram has been constructed with Clustal X using the neighbor-joining method (78) based on the MC sequences retrieved from the GenBank and EMBL data bases. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances have been computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The analysis involved 28 amino acid sequences. All positions with
Vincenza Dolce1* Anna Rita Cappello1 Loredana Capobianco2*
1
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende Cosenza, Italy 2 Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy
Abstract The citrate carrier (CiC), characteristic of animals, and the dicarboxylate–tricarboxylate carrier (DTC), characteristic of plants and protozoa, belong to the mitochondrial carrier protein family whose members are responsible for the exchange of metabolites, cofactors, and nucleotides between the cytoplasm and the mitochondrial matrix. Most of the functional data on these transporters are obtained from the studies performed with the protein purified from rat, eel yeast, and maize mitochondria or recombinant proteins from different sources incorporated into phospholipid vesicles (liposomes). The functional data indicate that CiC is responsible for the efflux of acetyl-CoA from the mitochondria to the cytosol in the form of citrate, the primer for fatty acid, cholesterol synthesis, and histone acetylation. Like the CiC, the citrate exported by DTC
from the mitochondria to the cytosol in exchange for oxaloacetate can be cleaved by citrate lyase to acetyl-CoA and oxaloacetate and used for fatty acid elongation and isoprenoid synthesis. In addition to its role in fatty acid synthesis, CiC is involved in other processes such as gluconeogenesis, insulin secretion, inflammation, and cancer progression, whereas DTC is involved in the production of glycerate, nitrogen assimilation, ripening of fruits, ATP synthesis, and sustaining of respiratory flux in fruit cells. This review provides an assessment of the current understanding of CiC and DTC structural and biochemical characteristics, underlying the structure–function relationship of these carriers. Furthermore, a phylogenetic C 2014 IUBMB relationship between CiC and DTC is proposed. V Life, 66(7):462–471, 2014
Keywords: citrate carrier; dicarboxylate–tricarboxylate carrier; gene expression regulation; mitochondrial carrier; phylogenetic analysis; substrate specificity.
Introduction Abbreviations: BTA, benzenetricarboxylic acid; CiC, citrate (tricarboxylate) carrier; DTC, dicarboxylate/tricarboxylate carrier; FOXA, forkhead box A; MC, mitochondrial carrier (MC); NF-Y, nuclear factor Y; ODC, oxodicarboxylate carrier; OGC, oxoglutarate carrier; PPRE, peroxisome proliferatoractivated receptor-responsive element; PUFA, polyunsaturated fatty acid; Sp1, stimulating protein 1 region; SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein. C 2014 International Union of Biochemistry and Molecular Biology V Volume 66, Number 7, July 2014, Pages 462–471 Address correspondence to: Loredana Capobianco, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce 73100, Italy. Tel.: 139–0832298864. Fax: 139-0-832298626. E-mail: [email protected] Received 28 May 2014; Accepted 22 June 2014 DOI 10.1002/iub.1290 Published online 18 July 2014 in Wiley Online Library (wileyonlinelibrary.com)
462
The mitochondrial carriers (MCs) are nuclear-coded membrane-embedded proteins that, with few exceptions, are localized in the inner membranes of mitochondria. They catalyze the selective transport of specific essential metabolites (di- and tricarboxylates, keto acids, amino acids, nucleotides, and coenzymes/cofactors) across the inner mitochondrial membrane providing a link between mitochondria and cytosol. This link is indispensable as many physiological processes require the participation of both intra- and extramitochondrial enzyme reactions. All MCs of known function exhibit a tripartite sequence structure, consisting of three tandemly repeated homologous domains of about 100 amino acids in length. Each domain contains two hydrophobic stretches, separated by hydrophilic regions, every repeat contains the signature sequence motif PX[DE]XX[RK] (PROSITE
IUBMB Life
PS50920 and PFAM PF00153) by which MCF members are recognized (1). One of the well-characterized members of this family in animals is the tricarboxylate (or citrate) carrier (CiC), which catalyzes an electroneutral exchange of a tricarboxylate for another tricarboxylate, a dicarboxylate (L-malate), or phosphoenolpyruvate across the mitochondrial inner membrane (2–7). This carrier protein plays a central role in fatty acid and sterol biosynthesis as it exports citrate from the mitochondria to the cytosol, where citrate is cleaved by ATP-citrate lyase to acetyl-CoA and oxaloacetate. Acetyl-CoA is used for fatty acid and sterol biosynthesis; whereas oxaloacetate is reduced to malate, which in turn is converted to pyruvate via malic enzyme with the production of NADPH plus H1 (1,8). In addition to its role in fatty acid synthesis, CiC is involved in other processes such as gluconeogenesis, insulin secretion, histone acetylation, inflammation, and differentiation of fibroblastes into adipocytes (9–14). Under different metabolic conditions, CiC may play a role in gluconeogenesis from lactate in species (not in man and rat) where phosphoenolpyruvate carboxykinase is located in mitochondria (1). Furthermore, a role of CiC in cancer progression has been postulated (15,16) that was expected as the overexpression of fatty acid synthase, the sole protein in the human genome capable of de novo synthesis of fatty acids from acetyl-CoA (17), has been observed in a wide variety of human tumor types (18) as a consequence of an altered transcriptional regulation (19,20). Very recently, a role of CiC in the alteration of lipid metabolism has been observed in biliary cirrhosis (21). In plants, a related carrier to CiC is the dicarboxylate–tricarboxylate carrier (DTC) that catalyzes an electroneutral transport of a broad spectrum of single protonated tricarboxylate (citrate, isocitrate, and aconitate) in exchange with unprotonated dicarboxylates (oxoglutarate, oxaloacetate, malate, maleate, succinate, and malonate), but not phosphoenolpyruvate. As DTC transports a broad spectrum of dicarboxylates and tricarboxylates, it has been hypothesized that this carrier may play a role in a number of important plant metabolic functions that require organic acid flux to or from the mitochondria. For example, like the CiC, the citrate exported by DTC from the mitochondria to the cytosol in exchange for oxaloacetate can be cleaved by citrate lyase to acetyl-CoA and oxaloacetate and used for fatty acid elongation and isoprenoid synthesis. The malate/oxaloacetate exchange catalyzed by DTC can enable the export of redox equivalents from the mitochondrial matrix that can act as reductants for the production of glycerate during photorespiration in photosynthesizing cells. Furthermore, significances for DTC are its involvement in nitrogen assimilation and in the ripening of fruits, because oxoglutarate is required for the assimilation of ammonium into amino acids by the glutamine synthetase/glutamate synthase pathway (22), whereas malate may be transported to the mitochondria and entered into the tricarboxylic acid cycle to support ATP synthesis and the respiratory flux in fruit cells (23).
Dolce et al.
In this review, we will address biochemical, molecular, and gene phylogenetic relationship of recent investigations on CiC and DTC from various sources, focussing our attention on transport, metabolism, and molecular mechanisms.
Identification and Characterization of CiCs and DTCs The existence of a mitochondrial CiC has been postulated for the first time by Chappell (24,25) who at the same time described the structural requirements of the substrates to be transported. The properties of the CiC have been extensively investigated in intact mitochondria (26). Kinetic studies have shown that the activity of the tricarboxylate carrier is high in liver compared to that in heart and brain (27,28) and that the carrier has a single binding site for all its substrates (27). Furthermore, evidence for the presence of a CiC has been given by the studies of the inhibition of citrate transport across the mitochondrial membrane by 1,2,3-benzenetricarboxylic acid (1,2,3-BTA) but not by its 1,2,4- and 1,3,5-isomers (27–29). The protein responsible for citrate transport has been first purified from rat liver mitochondria by solubilization in Triton X-100 and chromatography through hydroxyapatite in the presence of cardiolipin and of the specific inhibitor 1,2,3-BTA (27). To date, different groups have purified this carrier to homogeneity from mitochondria of rat (7,30), eel (6), and yeast (31). The molecular weight of the purified protein has been estimated to be approximately 30–32.5 kDa (6,7,30). The properties of the purified carrier, reconstituted into a liposomal system, are similar to those of CiC from intact mitochondria as far as counter anion requirement, substrate specificity, and inhibitor sensitivity are conserved (26,27,32). Furthermore, the recombinantly expressed and reconstituted CiC of rat, eel, drosophila, and yeast have confirmed that the carrier transports citrate, cis-aconitate, threo-isocitrate, phosphoenolpyruvate, and L-malate. Some activity has also been observed with succinate (31,33–37). The CiC overlaps with oxoglutarate carrier (OGC) by transporting the substrate malate and malonate (38). In yeast, a second isoform of CiC (CiC2, also known as YHM2) (39) has been identified that differs markedly from the yeast CiC (CiC1) previously identified in this organism (31) as this protein efficiently transports citrate, oxoglutarate oxaloacetate, succinate, and fumarate, whereas different from CiC1 the reconstituted CiC2 transports isocitrate, cis-aconitate, and malate with very low efficiency (39). For its substrate, specificity of CiC2 has been identified as a citrate/OGC. In plant, the existence of a tricarboxylate carrier has been suggested by osmotic swelling of isolated mitochondria in ammonium citrate and in the presence of catalytic amount of phosphate and malate (40,41). Later on, a putative CiC from pea and maize mitochondria has been purified (42,43) and its transport specificity has been tested in liposomes. The maize carrier incorporated into liposomes is able to exchange citrate
463
IUBMB LIFE
TABLE 1
Kinetic parameters determined for CiC and DTC citrate homo exchange
Km (mM)
Vmax (mmol/min 3 g protein)
R. norvegicus liver mitochondria
0.12
0.0225
(39)
R. norvegicus native reconstituted
0.13
2.049
(2)
A. anguilla native reconstituted
0.062
9.0
(13)
A. anguilla bacterially expressed
0.068
14.2
(49)
D. melanogaster bacterially expressed
0.132
11.75
(48)
S. cerevisiae (CiC1) bacterially expressed
0.36
2.5
(43)
S. cerevisiae (CiC2) bacterially expressed
0.16
9.8
(50)
Z. mais native reconstituted
0.65
a
A. thaliana bacterially expressedb
13
Reference
(9)
0.95
1.3
(10)
b
1.49
3.9
(10)
b
N. tabacum (NtDTC3) bacterially expressed
1.91
0.31
(10)
A. thaliana bacterially expressedc
N. tabacum (NtDTC1) bacterially expressed
0.15
1.7
(10)
c
N. tabacum (NtDTC1) bacterially expressed
0.24
6.4
(10)
N. tabacum (NtDTC3) bacterially expressedc
0.31
0.38
(10)
0.83
2.03
(11)
b
V. vinifera (VvDTC1) bacterially expressed a
Milligram of total protein at 9 C, pH 7.0. Measured at pH 7.0. c Measured at pH 6.0. b
against citrate, malate, succinate, malonate, and isocitrate as well as differently to CiC animals, oxoglutarate, and oxaloacetate while it is not able to transport phosphoenolpyruvate. In the postgenomic era in Arabidopsis thaliana, Nicotiana tabacum, and Vitis vinifera, a mitochondrial DTC has been identified by overexpression in Escherichia coli (44,45). Like maize CiC, the DTCs are capable of transporting both dicarboxylates (such as oxoglutarate, oxaloacetate, malate, succinate, maleate, malonate, and oxoadipate) and tricarboxylates (such as citrate, isocitrate, cis-aconitate, and trans-aconitate) (44) but not phosphoenolpyruvate. DTCs are also capable of exchanging sulfate. Therefore, maize and pea CiCs (42,43), for their substrate specicity, can’t be considered citrate carriers but they have to be included in plant subfamily DTC. In Plasmodium falciparum, a DTC has been found that is able to transport the dicarboxylates (such as oxoglutarate, oxaloacetate, malate, succinate, and fumarate), the citrate, and the sulfate (46). The kinetic constants (Table 1) of the recombinant purified CiCs have been determined by measuring the initial transport rate at various external [14C]citrate concentrations, in the presence of a constant saturating internal concentration of
464
citrate. The half-saturation constant of CiC for citrate at pH 7 is in the range of 0.062–0.13 mM (2,36,37,47), whereas the citrate Km for yeast CiC1 and CiC2 are 0.36 and 0.16 mM, respectively (33,39). The range of DTC for citrate measured at pH 7 is 0.65–1.91 mM (43–45) and the lower values have been obtained at pH 6 in citrate/citrate exchange in A. thaliana and N. tabacum (0.15–0.31 mM) (44).
CiC and DTC Genes Phylogenetic Relationship of CiC and DTC Proteins with Other Mitochondrial Transporters In 1993, a full-length cDNA encoding the mature rat liver mitochondria CiC has been cloned (48). Subsequently, cDNA sequences encoding the CiC have been identified via overexpression and reconstitution approach from a variety of sources (31,33,35,36). Successively, DTCs have been identified and characterized at a molecular level in plant (44,45), yeast (39), and protozoa (46). Recently, two different isoforms of human CiC, carried out by alternative splicing of the first exon, have been identified in human prostate epithelial cells; the difference between the two
Citrate and Dicarboxylate–Tricarboxylate Carriers
proteins affect the N-terminal region and the subcellular localization being one localized in mitochondria and the other in plasma membrane (49). The human, rat, eel, and fruit fly CiCs possess a presequence of 13, 13, 20, and 26 amino acids, respectively, with a net positive charge of 12 (34,36,49,50). The presequence of rat and eel CiC is dispensable both for targeting to mitochondria and for insertion into the inner membrane (34,50). Interestingly, it has been found that the CiC presequence is important to avoid aggregation of the newly synthesized polypeptide chain, and hence keeping the precursor protein soluble in the cytosol. This presequence is also able to influence the folding state of the precursor protein in the cytosol prior to import into mitochondria. Notably, the chaperoning effect of the presequence is completely retained if the positive charges have been exchanged with negative charges (34). A topologic accepted model based on the sequence features and on the accessibility of CiC to peptide-specific antibodies and proteolytic enzymes is consistent with an arrangement of the carrier into an even number of transmembrane segments with both the N- and the C-termini on the cytosolic side of the mitochondrial inner membrane (51). A multiple sequence alignment (MSA) was obtained using all mature mitochondrial CiCs known (Homo sapiens, Rattus norvegicus, Anguilla anguilla, Drosophila melanogaster, and Saccharomyces cerevisiae); all mitochondrial DTCs known (V. vinifera, N. tabacum, A. thaliana, and P. falciparum); all mitochondrial DiCs known (H. sapiens, R. norvegicus, and D.
Dolce et al.
melanogaster); all mitochondrial OGCs known (R. norvegicus and Bos taurus), all mitochondrial oxodicarboxylate carriers (ODCs) known (H. sapiens and S. cerevisiae), and the S. cerevisiae CiC2 by using ClustalW (52) shows that DTCs despite their capacity to transport citrate and malate present the highest homology with the mitochondrial OGCs sharing of about 40% identical amino acids with this carrier from bovine (53), human (54), and rat (55), whereas only of about 20% is sharing with the CiCs (data not shown). Structural specific data are not disposable for DTC and CiC; nevertheless, the alignment performed using all mature mitochondrial CiCs known and all mitochondrial DTCs known shows that in helices DTC are present in some blocks of conserved residues absent in CiC and vice versa. Furthermore, it could be noted that these regions sometimes present residues with different charges that could be responsible for the different substrate specificities (data not shown).
FIG 1
Phylogenic tree of amino acid sequences of MCs from various organisms. For comparative purposes, the amino acid sequences of OGC, DTC, DiC, and OGC homologs from various organisms have been used. The dendogram has been constructed with Clustal X using the neighbor-joining method (78) based on the MC sequences retrieved from the GenBank and EMBL data bases. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances have been computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The analysis involved 28 amino acid sequences. All positions with
