Mitochondrial tricarboxylate and dicarboxylate–Tricarboxylate carriers: from animals to plants

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 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 +2 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. 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).

A phylogenetic tree has been calculated from the above-cited alignment (Fig. 1). Figure 1 shows that the represented MCs cluster into three different clades based on their sequence similarity, suggesting a large variety of specialized functions. It should be noted that all CiC proteins are closed in a single clade with the exception of the S. cerevisiae CiC2 that clusters with the ODC proteins. Furthermore, it is also evident that CiC, DTC, DiC, and ODC have been originated from a same clade, suggesting the existence of a common ancestor 56; this ancestor duplicated in some organisms, for example in animals, giving rise to the CiC, OGC, and DiC, whereas in plants it evolved into DTC, which exhibits the combined characteristics of both the OGC and the CiC.

Expression of CiC and DTC in Various Tissues

The human CiC mRNA expression pattern analyzed by Northern blot experiments has revealed an high steady-state levels of CiC mRNA in liver, kidney, and pancreas, lower levels in heart, skeletal muscle, and placenta, and no detectable mRNA in brain and lung 57. High CiC mRNA levels in liver and kidney can be associated with the processes of gluconeogenesis, which mainly occurs in these tissues, and the fatty acid synthesis, which mainly occurs in liver. On the other hand, the low CiC mRNA level in skeletal muscle correlates to the very low activity of gluconeogenesis and fatty acid synthesis in this tissue. The relatively high CiC mRNA level in pancreas could be explained with the role of CiC in regulation of insulin secretion. Because of its role in the acetylation, it would be expected that CiC be expressed ubiquitously, and hence the absence of CiC mRNA in the brain and lung cancer may be owing to the poorly sensitive method used for its determination. More recently, two different isoforms have been detected in prostate epithelial cells 49, the MC isoform with canonical function and a plasma membrane isoform whose function could be the release of citrate in prostatic fluid.

The D. melanogaster CiC mRNA expression pattern analyzed by RT-PCR on mRNAs from wild-type embryos, larvae, pupae, and adults revealed that DmCiC is equally transcribed at each stage, suggesting that its expression is required during the fruit fly development.

The eel CiC mRNA expression pattern analyzed by RT-PCR on RNAs detects the high levels in swim bladder, a weaker but significant signal in brain, gill, intestine, and liver, whereas no band is visible in heart and skeletal muscle. The expression pattern of the CiC is substantially in agreement with the metabolic and environmental necessity of silver eel 35. The huge level of expression observed in swim bladder suggests that this carrier is also necessary to reduce the surface tension and/or to fill this organ 58.

The expression of DTC in A. thaliana and N. tabacum has been examined in different tissues by Northern blot analysis. The presence of DTC transcripts has been found in all plant tissues examined although at different levels. In Arabidopsis, the flower bud shows the highest DTC transcript levels, whereas the root has the lowest. In N. tabacum, DTC expression in flower bud and root appears to be comparable; the highest transcript levels have been detected in sepal, petal, male, and female tissues, whereas somewhat lower amounts have been found in leaf and stem. The observed expression pattern supports the assumption that the DTC fulfills not only a house-keeping role in plant metabolism but also a specialized function during nitrogen assimilation 44.

Expression analysis of the genes encoding V. vinifera DTCs has been studied in grape berry mesocarp (pulp). Three stages of grape berry development have been studied: 1) at the end of the green stage (close to the onset of ripening) 44 days after flowering (DAF); 2) mid-ripening grapes 65 DAF; and 3) mature grapes 98 DAF. At 44 DAF, the complete absence of sugars, berry weight (ca. 50% of mature berries), besides malate concentration, indicates that this sample is close to the onset of ripening. VvDTC2 and VvDTC3 exhibit high expression in this sample and decreasing thereafter, whereas VvDTC1 gene exhibits a relatively low expression in the three analyzed samples. Malate concentration, high at 44 DAF, shows a decrease at 65 DAF, and even more at 98 DAF as expected. These results are consistent with the role in the regulation of malate degradation during grape berry ripening 45.

Functional Characterization of the Human and Rat CiC Gene Promoters

Multiple transcription factor binding sites within the human and rat CiC promoter have been described including sterol regulatory element (SRE), stimulating protein 1 region (Sp1), forkhead box A (FOXA), peroxisome proliferator-activated receptor-responsive element (PPRE) motif, nuclear factor Y (NF-Y) site, and E-box-like site-binding sequences. Furthermore, an inhibitor domain (silencer) and a polyunsaturated fatty acid response region (PUFA-RR) have been identified in human and rat promoter, respectively.

Human CiC Promoter. Structural and functional analyses of human CiC promoter have revealed the presence of the following elements: the FOXA, the SRE at −1,969/−1,689 bp; the inhibitor domain (silencer) and the Sp1 (Fig. 2).

The role of FOXA site at −1,098/−1,088 bp in the regulation of CiC gene expression has been investigated in HepG2 and INS-1 cells 59. In HepG2 cells, FOXA site acts as a strong enhancer of the CiC gene transcription. Thus, as demonstrated by the observation that the wild-type (but not the mutated) FOXA site markedly enhances gene reporter expression activity, and that the wild-type FOXA-driven gene reporter activity is further enhanced in cells expressing the transcription factor FOXA1. The role of FOXA1 in the regulation of CiC gene transcription has also been shown by the increase in the levels of both CiC transcript and protein produced by overexpression of FOXA1. Vice versa, silencing not only of FOXA1 but also of the other FOXA subfamily transcription factors (FOXA2 and FOXA3) strongly decreases CiC gene expression as measured by transcript and protein levels. With regard to the role that CiC plays in the fatty acid synthesis, it could be possible that the CiC transcriptional activation by FOXA may be another mechanism by which FOXA regulates lipogenesis. The experiments reported in this study 59 also demonstrate that FOXA1 controls glucose-stimulated insulin secretion in INS-1 cells. This finding is consistent with the report showing that FOXA1-deficient mouse islets exhibit a severe defect of glucose-stimulated insulin secretion 60. On the basis of these results, the effect of FOXA1 on glucose-stimulated insulin secretion can be explained by its transcriptional regulation of CiC gene expression. This interpretation is also supported by the previous observation that CiC silencing in INS-1 cells inhibits glucose-stimulated insulin secretion without affecting the cytosolic ATP/ADP ratio 9. Furthermore, the role of some antipsychotic drugs on the expression of FOXA1 has been investigated in HepG2 cells. It has been demonstrated that clozapine, the antipsychotic drug, enhances FOXA1 DNA binding and its transcriptional activity, increasing mitochondrial CiC gene expression, whereas haloperidol, another antipsychotic drug, does not determine any increase of FOXA1 gene expression. It has also been demonstrated that clozapine upregulates FOXA1 and CiC gene expression in INS-1 cells only at basal glucose concentration. In addition, it has been found that abnormal insulin secretion in basal glucose conditions could be completely abolished by FOXA1 silencing in INS-1 cells treated with clozapine 61.

The promoter activity of the CiC SRE site at −1,696/−1,686 bp has been tested by transfecting HepG2 cells with pGL3 basic-LUC vector containing the −1,785 to −20 bp region of the CiC gene, whereas the binding activity of the CiC promoter SRE has been investigated by electrophoretic mobility shift assay (EMSA) experiments using nuclear extracts of HepG2 cells and a labeled probe from −1,700 to −1,679 bp encompassing the SRE region 62. In this report, it has been found that insulin upregulates and PUFA downregulates CiC gene transcription via the SREBP-1. The CiC transcriptional activation by insulin and suppression by PUFA are clearly mediated by the SRE/SREBP-1 regulatory system as their effects on CiC promoter-driven LUC activity are abolished by mutations in the SRE site of the CiC gene. These findings are in agreement with the observations, made in the context of the transcription of other enzymes, that insulin upregulates and PUFA downregulates SREBP-1 63. Furthermore, it is known that the level of SREBP-1 is decreased by starvation 64. Therefore, these results provide a molecular basis and explain, at least in part, the changes in CiC activity or/and CiC level previously observed in diabetic rats before and after insulin administration 65, 66, in rats fed with a PUFA-enriched diet 67, 68, and in starved rats 69, 70.

By transfection experiments and EMSA, an inhibitory domain (silencer) (at −595/−569 bp) has also been identified within the CiC gene promoter. The transcription factor that binds to the silencer region has been purified from HepG2 cell nuclear extracts by DNA affinity and identified as ZNF224 71.

The role of Sp1 sites (at −285/−81 bp) in the regulation of CiC gene expression has been investigated in HepG2 and SK-N-SH cells. It has been demonstrated that CiC gene is derepressed in HepG2 cells and repressed in SK-N-SH cells as revealed by an approximately fourfold higher level of both CiC transcript and protein in the former with respect to the latter 72. This is likely owing to the fact that the −335/−20 bp region of the CiC promoter is largely demethylated and associated with Sp1 and acetylated histones in HepG2 cells but not in SK-N-SH cells. The following evidence strongly supports this interpretation. SP1 and acetylated histones have been found to be associated with the CiC proximal promoter of HepG2 cells even when untreated with the demethylating agent 5-azacytidine (AzaC) and the acetylating agent trichostatin (TSA); in contrast, Sp1 is associated with the CiC proximal promoter of SK-N-SH cells after treatment with AzaC, and acetylated histone H3 after treatment with TSA. AzaC and TSA added alone or in combination do not affect CiC gene expression in HepG2 cells contrary to the stimulating effect in SK-N-SH cells. In addition, CiC mRNA and protein levels have been markedly decreased by silencing Sp1 in untreated HepG2 cells and in SK-N-SH cells when treated with AzaC + TSA. These results show that different post-transcriptional modifications can influence the transcriptional control activity and stability of Sp1. In addition to these modifications, an alternative splice isoform of Sp1 named Sp1c has been identified 73. This variant, generated through alternative splice acceptor site usage by exclusion of a short domain, has been designated α. Although at very low levels, Sp1c is ubiquitously expressed. A preliminary characterization of Sp1c shows that Sp1c works as stronger activator of transcription than full-length Sp1 and the percentage of HEK293 Sp1c-overexpressing cells is higher in G1 phase and lower in S phase than the percentage of HEK293 Sp1-overexpressing cells.

Rat CiC Promoter. Structural and functional analyses of rat CiC promoter (Fig. 3) have revealed the presence of two start sites at positions −59 and −61 bp with respect to the AUG translation initiator codon, the absence of the canonical polymerase II transcription element TATA box, and the presence of GC-rich stretches with the consensus sequence for six Sp1 transcription factors (at positions −187, −160, −129, −118, −92, and −62 bp), this latter characteristic has been reported for the promoter of several TATA-less genes.

Furthermore, in the promoter region, PPRE motif is present at position −625 bp and a PUFA response region (PUFA-RR) is constituted by the putative binding sites for several transcription factors such as NF-Y site (−43 bp), E-box-like site (−72 bp), SRE1-like site (−67 bp), and Sp1 site (−62 bp) (Fig. 3).

The role of PPRE site (at −625/−613 bp) in the regulation of CiC gene expression has been investigated in rat BRL-3A hepatocytes and murine 3T3-L1 adipocytes 14, 74. It is worth noting that this region of rat CiC promoter shows a very high degree of sequence similarity with the corresponding portion of the murine CiC genes (approximately, 94% identity). Evidences demonstrate that CiC expression is regulated by PPARα and PPARγ in hepatocytes and adipocytes, respectively. CiC expression increased in rat BRL-3A hepatocytes treated with WY-14,643, agonist of PPARα, and in murine 3T3-L1 adipocytes treated with rosiglitazone (BRL), agonist of PPARγ. The overexpression of PPARα/RXRα and PPARγ/RXRα heterodimer enhanced CiC promoter activity in BRL-3A and 3T3-L1, respectively. Luciferase reporter gene and EMSA have indicated that a functional PPRE, identified in the CiC promoter, confers responsiveness to activation by PPARs. The binding of PPRE of CiC promoter by PPARα and PPARγ in vivo has been confirmed by chromatin immunoprecipitation (ChIP) assay in BRL-3A and 3T3-L1 cells, respectively 74. Furthermore, the role of PPARγ, on the regulation of CiC, has been studied in 3T3-L1 fibroblasts and in adipocytes 14. These data contradict the previous findings, indicating that PPARγ ligands increased CiC expression in adipocytes 74 although the latter measurements have been performed at 7 days after differentiation induction. Functional experiments using a reporter gene containing rat CiC promoter show that BRL enhances, whereas the PPARγ antagonist GW9662 reverses CiC promoter activity, addressing how this effect is mediated by PPARγ. Mutagenesis studies, EMSA, and ChIP analysis reveal that upon BRL treatment, PPARγ and Sp1 are recruited on the Sp1 site positioned at −92 bp within the CiC promoter, leading to an increase in CiC expression. In addition, mithramycin, a specific inhibitor for Sp1–DNA binding activity, abolished the PPARγ-mediated upregulation of CiC in fibroblasts. Furthermore, Re-ChIP assays showed that in fibroblast cells, after BRL stimulation, an enhanced recruitment of PGC-1α and ARA-70 on the Sp1 site of the CiC promoter has been evidenced. In contrast, it has been observed that mature adipocytes treated with BRL show an increased recruitment of SMRT corepressor to the Sp1 site within the CiC promoter along with no changes in the occupancy of RNA POLII 14. Finally, it has been demonstrated about a direct involvement of SMRT in the loss of CiC promoter responsiveness to the BRL in mature adipocytes using a specific SMRT siRNA. In conclusion, this study proposes a novel molecular mechanism through which PPARγ modulates CiC expression in which fibroblasts treated with BRL, PPARγ/Sp1 complex along with PGC1α, and ARA-70 coactivators are recruited on the Sp1-containing region of the CiC promoter, and hence increasing CiC expression, whereas in adipocytes treated with BRL, PPARγ/Sp1 complex is associated with an enhanced recruitment of SMRT corepressor on the Sp1 site of CiC promoter, resulting in an inhibition of CiC transcription 14.

The PUFA–RR of rat CiC gene promoter resides within (−147/+35 bp) and its alignment with the corresponding portion of the human CiC gene showed a low similarity 75. In particular, the E-box-like site is responsible for both sterol regulatory element-binding protein-1c (SREBP-1c) transactivation and PUFA inhibition of the CiC promoter. The observation that the PUFA–RR of rat CiC promoter contains a functional E-box-like and NF-Y and Sp1 sites suggests that SREBP-1c activates the promoter in synergy with NF-Y and Sp1 75. Overexpression of nuclear SREBP-1c overrides arachidonic acid (AA) suppression but does not prevent the repression by docosahexaenoic acid (DHA). ChIP assays show that DHA affects the NF-Y, Sp1, and SREBP-1c binding to the PUFA–RR of CiC gene promoter, whereas AA alters only the binding of SREBP-1c. Overall, these data show that PUFA inhibition of hepatic CiC transcription is mediated not only by the nuclear level of SREBP-1c but also might involve a reduction of Sp1 and NF-Y DNA binding, suggesting differential mechanisms in the CiC gene regulation by different PUFAs 75.

The role of SREBP-1c on the reduction of CiC gene expression observed in diabetes, the effects of insulin withdrawal on the precursor (pSREBP-1c), and mature (nSREBP-1c) forms of SREBP-1c protein content and on the SREBP-1c mRNA abundance has been investigated. In this study, it has been shown that SREBP-1c expression decreases in rat hepatocytes cultured in the absence of insulin when compared to control cells. Transfection experiments demonstrate that insulin withdrawal causes a reduction of CiC gene expression that can be ascribed to a decrease of transactivation of its promoter by SREBP-1c. In addition, the results of ChIP assay demonstrate that the binding of SREBP-1c to CiC promoter is reduced in streptozotocin (STZ)-diabetic with respect to control rats, whereas no significant change in the binding of SREBP-1c to the CiC promoter is observed in insulin-treated STZ-diabetic rats when compared to control rats. Furthermore, in this study, the molecular mechanism(s) underlying the CiC activity reduction in liver mitochondria from diabetic rats has been investigated by Western blot analysis and ribonuclease protection assay, showing that STZ-induced diabetes lead to a reduction in the CiC mRNA abundance, and in turn to a proportionate decrease of CiC protein content. It has been shown that the transcriptional rate of CiC mRNA is reduced in nuclei from diabetic as compared to control rats and that CiC RNA splicing is a regulative step in nuclei of diabetic rat liver. Indeed, the reduced ratio of spliced to unspliced RNA, observed in diabetic when compared to control rats, indicates that the splicing reaction itself is inhibited. The accumulation of uncleaved versus polyadenylated RNA measured by RNase protection assay and the length of the poly(A) tail, determined by RNase H analysis in control and diabetic rats, show that the rate of formation of the 3′-end of CiC RNA is not affected by diabetes. Thus, once fully processed, CiC mRNA is stable and no change in its rate of degradation is detectable in diabetic rats.

Altogether, these findings allow concluding that the regulation of CiC expression observed in the liver of diabetic rats occurs at both the transcriptional and the post-transcriptional level.