Cloning, Expression and Functional Characterization of V. vinifera CAT2 Arginine Transporter

Abstract

The amino acid membrane transporters of grape species take part in metabolic pathways that play crucial roles in nitrogen trafficking and in the synthesis of secondary metabolites. Therefore, identifying these amino acid transporters and defining their functional properties might have further applications in crop improvement and, hence, relevance to human nutrition. The VvCAT2 (Cation Amino acid Transporter) transporter cDNA has been isolated and cloned into a specific plasmid for over-expression in Escherichia coli. The expressed protein, after purification by Ni²⁺-chelating chromatography, has been functionally characterized in an experimental model of proteoliposomes by measuring the uptake of radiolabeled compounds. Arginine was revealed to be the best substrate, confirming the role of CAT2 in nitrogen trafficking in plant cells and within sub-cellular spaces, given its plausible localization in vacuoles. The transporter activity is modulated by pH, osmotic imbalance and ATP. The transport kinetics have been measured. Overall, the obtained data indicate the capacity of VvCAT2 in transporting arginine, making it a possible target for crop improvement with a relevance to human health.

Keywords: amino acid; plant nitrogen; secondary metabolites; transporter.

Figure 1

Purification of VvCAT2 transporter. (a) Proteins were separated by SDS-PAGE: lane 1, page ruler prestained plus marker; lane 2: insoluble fraction of induced cell lysate solubilized before IMAC loading; lane 3: passthrough fraction containing unbound proteins; lane 4: fraction of proteins eluted with washing buffer with 10 mM imidazole added; and lane 5: fraction of protein eluted with 50 mM imidazole. (b) Western blotting of sample loaded as in (a).

Figure 2

Effect of Cholesteryl HemiSuccinate (CHS) on transport activity of VvCAT2. VvCAT2 was purified and reconstituted in proteoliposomes, as described in Section 4. Transport was measured by adding 100 µM [³H]arginine to proteoliposomes containing 15 mM ATP. Proteoliposomes were prepared in the absence (○) or in the presence of 0.5 mg (●) or 1 mg (□) CHS, which is a more soluble analogue of cholesterol. Data were plotted by first-order rate equation. Results are means ± SD from three different experiments (n = 3).

Figure 3

Effect of osmotic pressure on transport activity of VvCAT2. VvCAT2 was purified and reconstituted in proteoliposomes, as described in Section 4. Transport was measured in 20 min by adding 100 µM [³H]arginine to proteoliposomes containing 15 mM ATP and the indicated concentrations of sucrose. Results are means ± SD from three different experiments (n = 3).

Figure 4

Effect of pH on transport activity of VvCAT2. VvCAT2 was purified and reconstituted in proteoliposomes, as described in Section 4. Transport was measured in 20 min by adding 100 µM [³H]arginine to proteoliposomes containing 15 mM ATP. The pH conditions were varied and kept equal in both internal and external proteoliposome compartments. Results are means ± SD from three different experiments (n = 3).

Figure 5

Effect of ATP on transport activity of VvCAT2. VvCAT2 was purified and reconstituted in proteoliposomes, as described in Section 4. Transport was started by adding 100 μM [³H]arginine to proteoliposomes reconstituted in absence (○) or presence (●) of intraliposomal 15 mM ATP. As control, according to preparations of commercially available ATP, ATP was replaced by 30 mM NaCl (□). Data were plotted by first-order rate equation. Results are means ± SD from three different experiments (n = 3).

Figure 6

Transport of other substrates mediated by reconstituted VvCAT2. VvCAT2 was purified and reconstituted in proteoliposomes as described in Section 4. Transport was started by adding 100 μM of [³H]arginine (●) or [³H]ornithine (○), or [³H]glutamine (□) or [³H]histidine (■) to proteoliposomes containing 15 mM ATP. Data were plotted by first-order rate equation. Results are means ± SD from three different experiments (n = 3).

Figure 7

Effect of counter substrates on transport activity of VvCAT2. VvCAT2 was purified and reconstituted in proteoliposomes as described in Section 4. Transport was started by adding 100 μM of [³H]arginine to proteoliposomes containing 15 mM ATP in absence of internal substrate (○), or in presence of 10 mM internal arginine (●) or ornithine (□). As control, 10 mM internal sucrose was introduced in proteoliposomes in place of substrates (■). Data were plotted by first-order rate equation. Results are means ± SD from three different experiments (n = 3).

Figure 8

Effect of inhibitors on transport activity of VvCAT2. VvCAT2 was purified and reconstituted in proteoliposomes as described in Section 4. Transport was measured in 20 min by adding 100 μM [³H]arginine along with 10 mM of indicated molecules to proteoliposomes containing 10 mM arginine and 15 mM ATP. Percentage of residual transport activity (nmoles/20 min/mg proteins) with respect to control (absence of inhibitor) is reported. Results are means ± SD from three different experiments (n = 3). Significantly different at p < 0.01, as calculated from Student’s t test analyses. Exact p values are reported. BTA (benzyltriethylammonium); TMA (tetramethylammonium); and TEA (tetraethylammonium).

Figure 9

Dose–response analysis of inhibition of VvCAT2 by ornithine. VvCAT2 was purified and reconstituted in proteoliposomes as described in Section 4. Transport was measured in 20 min by adding 100 µM [³H]arginine together with indicated concentrations of ornithine to proteoliposomes containing 10 mM internal arginine and 15 mM ATP. Percentage of residual transport activity (nmoles/20 min/mg proteins) with respect to control (absence of inhibitor) is reported. Results are means ± SD from three different experiments (n = 3).

Figure 10

Kinetic analysis of ornithine inhibition. VvCAT2 was purified and reconstituted in proteoliposomes as described in Section 4. Transport rate was started by adding [³H]arginine at indicated concentrations to proteoliposomes containing 10 mM internal arginine and 15 mM ATP. Transport was measured in 15 min in absence (○) or presence of (●) 5 mM ornithine. Data are analyzed, according to Lineweaver–Burk, as reciprocal transport rate versus reciprocal arginine concentration. Results are means ± SD from three different experiments (n = 3).

Figure 11

Effect of oxidant compounds on transport activity of VvCAT2. VvCAT2 was purified and reconstituted in proteoliposomes as described in Section 4. Transport was measured in 20 min by adding 100 µM of [³H]arginine, in absence (none) or presence of oxidants copper phenanthroline (Cu-Phe) or hydrogen peroxide (H₂O₂), to proteoliposomes containing 10 mM internal arginine and 15 mM ATP. Percentage of residual transport activity (nmoles/20 min/mg proteins) with respect to control (absence of inhibitor) is reported. Results are means ± SD from three different experiments (n = 3). Significantly different a p < 0.01, as calculated from Student’s t test analyses. Exact p values are reported.