Identification and Kinetic Characterization of HtDTC, The Mitochondrial Dicarboxylate–Tricarboxylate Carrier of Jerusalem Artichoke Tubers

Abstract

Jerusalem artichoke (Helianthus tuberosus L.) tubers were reported to be tolerant to cold and freezing. The aim of this study was to perform a kinetic characterization of the mitochondrial dicarboxylate-tricarboxylate carrier (HtDTC) and to assess a possible involvement of this carrier in the cold tolerance of tubers. The HtDTC was purified from isolated mitochondria by sequential chromatography on hydroxylapatite/celite and Matrex Gel Orange A. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31.6 kDa. A polyclonal antibody raised against the tobacco DTC cross-reacted with the purified protein on Western blot analysis. In gel trypsin, digestion of the purified HtDTC yielded peptides that exhibited strong amino acid sequence similarity to previously identified plant DTCs. Furthermore, using degenerate primers, a portion of the Htdtc cDNA was amplified and sequenced; this cDNA encoded for a protein with high sequence similarity to known plant homolog DTCs. When reconstituted in liposomes loaded with dicarboxylate (2-oxoglutarate, malate, malonate, succinate, and maleate) or tricarboxylate anions (citrate, trans-aconitate, and isocitrate), the purified HtDTC transported all these anions in exchange with external [14C]2-oxoglutarate. A kinetic characterization of HtDTC was performed: (a) the half-saturation constant Km and the Vmax at 25 degrees C of the 2-oxoglutarate/2-oxoglutarate exchange by reconstituted HtDTC were found to be 360 microM and 10.9 micromol/(min mg protein), respectively; (b) the activation energy Ea of the succinate/2-oxoglutarate exchange by the reconstituted HtDTC was found to be 50.7 kJ/mol constant between -5 and 35 degrees C. Similarly, the activation energy Ea of succinate respiration of isolated Jerusalem artichoke mitochondria, measured between -2 and 35 degrees C, was shown to be constant (65.3 kJ/mol). The physiological relevance of kinetic properties and temperature dependence of transport activities of HtDTC is discussed with respect to the cold tolerance ability of Jerusalem artichoke tubers.

Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (Helianthus tuberosus L.) heterotrophic tissues

“…The purity of the mitochondrial preparations was checked by assaying marker enzymes as reported in ''Materials and methods''. In accordance with the data of Spagnoletta et al (2006) this analysis showed that extramitochondrial contamination was negligible (data not shown). To monitor DW, we used safranin O as a fluorescence probe as reported in ''Materials and methods'' (Table 1).…”

“…The purity of the mitochondrial preparations was checked by assaying marker enzymes for endoplasmic reticulum, peroxisomes, plasma membranes, and vacuoles, as described in Spagnoletta et al (2006).…”

Plant Mitochondrial Carriers: Molecular Gatekeepers That Help to Regulate Plant Central Carbon Metabolism

“…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].…”

Drought-Responsive Mechanisms in Plant Leaves Revealed by Proteomics

“…Two mitochondrial carrier proteins (i.e., dicarboxylate/tricarboxylate carrier (DTC) and 2-oxoglutarate/malate carrier protein (OMC)) were decreased in T. aestivum [35] and G. max [17], respectively. DTC and OMC can catalyze the transport of various metabolites (e.g., dicarboxylates, tricarboxylates, amino acids, and keto acids) across the inner mitochondrial membrane, and play an important role in several metabolic processes, such as the gluconeogenesis, nitrogen metabolism, as well as biotic stress [178]. However, three voltage-dependent anion channel proteins (VDAC) in T. aestivum [35] and two mitochondrial outer membrane porin 1-like proteins in B. napus [47] were significantly increased under drought stress.…”