*2.2. Dicarboxylate*/*Tricarboxylate Carrier (DTC)*

DTCs facilitate the transport of dicarboxylates such as malate and 2-OG and tricarboxylates such as citrate [48]. Our phylogenetic analysis revealed that DTC homologs are present in all included plant species and in some fungal species (Figure 1). DTCs and OGCs share a common ancestor. The relative position of the two subgroups indicates that the ancestral gene is closely related to DICs. We hypothesize that the common ancestral gene was duplicated and underwent neofunctionalization upon speciation. The animal copy became OGC while in plants it became DTC. Alternatively, neofunctionalization happened in the last common ancestor and the plant and animal lineages received DTC and OGC, respectively, upon speciation. Hints to support the second hypothesis are provided by the proteomes of some protists. There are three species that contain both OGC-like and DTC-like proteins: *Aureococcus anophage*ff*erens* (Class Pelagophyceae), *Emiliania huxleyi* (Class Prymnesiophyceae), and *Tetrahymena thermophila* (Class Oligohymenophorea). However, other protists possess only OGC-like transporters as in *Leishmania major* and *Trypanosoma brucei* and yet other protists, such as *Plasmodium falciparum*, only have DTC-like transporters.

DTCs are among the most abundant MC proteins in the Arabidopsis IMM comprising 0.8% of the total IMM area, i.e., 6836 protein copies per mitochondria [43]. Unlike the other three highly abundant MC proteins: ADP/ATP carriers (AtAAC1-3; 6.2% of IMM area; 53,065 protein copies/mitochondria); mitochondrial phosphate carriers (AtMPT2-3; 2.5% of IMM area; 21,325 protein copies/mitochondria); and, uncoupling proteins (AtUCP1-3, 1.0% of IMM area; 8595 protein copies/mitochondria), there is only one DTC homolog in Arabidopsis. DTCs have been studied in a few plants including Arabidopsis and *Nicotiana tabacum* [48], *Vitis vinifera* (grapes) [49], *Helianthus tuberosus* (Jerusalem artichoke) [50], and *Citrus junos* (yuzo) [51]. The purification and characterization of a citrate transporter in maize have been described [52]. The reported activity may represent the MzDTC homolog since the transport substrates of the maize citrate transporter closely resemble that of AtDTC. In the plant kingdom, the numbers of DTC homologs vary without any clear pattern. A single homolog was found in *Chlamydomonas reinhardtii*, while there are two and three homologs in the mosses *S. moellendor*ffi*i* and *P. patens*, respectively. In the *Brassica* genus, the number of DTC homologs varies from one in *A. thaliana*, Arabidopsis *lyrata*, and *Capsella rubella*, two in *Brassica oleracea*, and three in *Brassica rapa*. *N. tabacum* has four homologs (NtDTC1-4) consistent with that reported in literature [48]. Regalado et al. (2013) [49] reported that there were three DTC homologs in *V. vinifera* (VvDTC1-3) and that VvDTC2 and VvDTC3 reached high transcript levels in the berry mesocarp at the onset of ripening. However, the phylogenetic analysis here showed that only two of these homologs clustered with plant DTCs. Our analysis revealed that VvDTC1 (vitvi\_GSVIVT01025463001) is similar to AtDTC. An investigation of VvDTC2 and VvDTC3 revealed that these two proteins have the same locus tag indicating they are likely the same gene, and this would correspond to the second VvDTC observed in our analysis. Finally, a single DTC homolog was cloned from *C. junos* [51] and, similarly, we were able to detect only one DTC homolog in the close relative *Citrus sinensis*.

AtDTC and NtDTCs have been assigned a transport function that involves an obligatory electroneutral exchange of dicarboxylates such as malate and 2-OG and tricarboxylates such as citrate [48]. It was demonstrated that DTCs were able to catalyze homoexchange transport, i.e., dicarboxylate/dicarboxylate and tricarboxylate/tricarboxylate on top of the dicarboxylate/tricarboxylate transport modality. It remains to be seen, however, which of these modalities are relevant *in planta*. It is clear from the in vitro transport data [48] that DTCs are promiscuous in terms of transport substrate. DTCs can transport almost all the intermediates of the TCA cycle except fumarate and succinyl-CoA for which there is no available data. For AtDTC, the homoexchange kinetic constants measured for different substrates in two different pH values showed that regardless of the substrate, the *K*m and Vmax changed as a function of pH. The *K*m values generally increased at pH 7, indicating that substrate affinities were decreased; Vmax values were also decreased at pH 7. These changes in the transport kinetics as a function of pH is critical because it has been shown that in Arabidopsis, the pH of the MM is slightly basic (pH 8.1) and that cytosolic pH is close to neutral, pH 7.3 [53]. It is, however, important to note that these in vitro data were not obtained under conditions representative of physiological conditions. To our knowledge, there are no reports where the external and the internal pH mimic physiological values in DTC transport measurements.
