Purification and functional characterization of the vacuolar malate transporter tDT from Arabidopsis

Abstract

The exact transport characteristics of the vacuolar dicarboxylate transporter tDT from Arabidopsis are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of tDT overexpressors, ¹³CO₂ feeding and quantification of ¹³C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. tdt knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes. Interestingly, under the latter two conditions, tDT overexpressors exhibited malate and citrate levels opposite to tdt knockout plants. Highly purified tDT protein transports malate and citrate in a 1:1 antiport mode. The apparent affinity for malate decreased with decreasing pH, whereas citrate affinity increased. This observation indicates that tDT exhibits a preference for dianion substrates, which is supported by electrophysiological analysis on intact vacuoles. tDT also accepts fumarate and succinate as substrates, but not α-ketoglutarate, gluconate, sulfate, or phosphate. Taking tDT as an example, we demonstrated that it is possible to reconstitute a vacuolar metabolite transporter functionally in proteoliposomes. The displayed, so far unknown counterexchange properties of tDT now explain the frequently observed reciprocal concentration changes of malate and citrate in leaves from various plant species. tDT from Arabidopsis is the first member of the well-known and widely present SLC13 group of carrier proteins, exhibiting an antiport mode of transport.

Keywords: Arabidopsis; Arabidopsis thaliana; citrate; dicarboxylate carrier; malate; organelle; transporter; tricarboxylic acid cycle (TCA cycle) (Krebs cycle); vacuolar transporter; vacuole.

Figure 1.

Malate and citrate contents in Arabidopsis leaves. Shown are malate (A) and citrate (B) contents in plants from wildtype (black circles), tDt1 (white squares), and tDT overexpressor plants oex15 (white triangles) and oex24 (black diamonds). Plants were grown under standard conditions for 5 weeks. Samples were harvested in a diurnal rhythm at the given time points. Black bars, periods of darkness. Each data point represents the mean value of four biological replicates ± S.E. (error bars).

Figure 2.

Changes in citrate and malate contents in Arabidopsis leaves after 14 days of drought. WT, knockout (tdt1), and tDT overexpressor plants (Oex15 and Oex24) were grown under standard conditions for 3 weeks. Subsequent to this, drought was applied by withholding water for 14 days (white). A, malate levels; B, citrate levels. Control plants were still watered (dark gray). Samples were harvested at the end of the dark period. Each data point represents the mean value of 14 biological replicates ± S.E. (error bars). Asterisks indicate statistically significant differences analyzed with Student's t test (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Figure 3.

Time dependence of tDT-mediated malate and citrate uptake into proteoliposomes. Proteoliposomes were incubated with either 50 μm ¹⁴C-labeled citrate or malate, respectively, for the indicated time. Concentration of preloaded carboxylates was 5 mm. Uptake was stopped by separation of external carboxylates from proteoliposomes by anion-exchange chromatography. A, time dependence of malate uptake into malate-loaded (circles), citrate-loaded (squares), or unloaded proteoliposomes (triangles). B, time dependence of citrate uptake into malate-loaded (circles), citrate-loaded (squares), or unloaded proteoliposomes (triangles). Luminal pH was always set to 7. Each data point represents the mean value of at least five independent experiments ± S.E. (error bars).

Figure 4.

Concentration dependence of ¹⁴C-labeled malate or citrate uptake into tDT-containing proteoliposomes. A, uptake of ¹⁴C-labeled malate into proteoliposomes preloaded with 5 mm malate. B, uptake of ¹⁴C-labeled citrate into proteoliposomes preloaded with 5 mm citrate. pH levels on the luminal site of the proteoliposomes were adjusted to pH 7.0. Substrates from the external site were supplied at pH ranging from alkaline (pH_ex 7.8) to neutral (pH_ex 7.0) to acidic (pH_ex 6.2). Each data point represents the mean value of at least five individual replicates ± S.E. (error bars).

Figure 5.

Influence of different internal substrates on ¹⁴C-labeled malate or citrate import into tDT proteoliposomes and counterexchange stoichiometry. Proteoliposomes were incubated with 50 μm ¹⁴C-labeled citrate or malate, respectively, and uptake was stopped after 5 min by separation of external carboxylates from proteoliposomes by anion-exchange chromatography. A, uptake of ¹⁴C-labeled malate into preloaded proteoliposomes at external pH values of 7.8 (dark gray) or 6.2 (light gray). Inset, comparison of [¹⁴C]citrate import versus [¹⁴C]malate export. [¹⁴C]Citrate (50 μm) import (dark gray bar) was measured on proteoliposomes preloaded with 50 μm unlabeled malate, and [¹⁴C]malate export (50 μm luminal concentration; light gray bar) was measured at an external citrate concentration of 50 μm. Transport was allowed for 2 min and stopped by anion-exchange chromatography. Rates are given in pmol/mg protein. B, uptake of ¹⁴C-labeled citrate into preloaded proteoliposomes at external pH values of 7.8 (dark gray) or 6.2 (light gray). Concentration of loaded substrates were 5 mm. Luminal pH was always set to 7. Each data point represents a mean value of at least five independent experiments ± S.E. (error bars). Asterisks indicate statistically significant differences analyzed with Student's t test (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Figure 6.

Uptake of ¹⁴C-radioactively labeled citrate into proteoliposomes under different pH conditions and occurrence of different citrate ions dependent on pH. Citrate uptake into proteoliposomes preloaded with 50 μm malate (black diamonds) was compared with the distribution of citrate ions at different pH conditions. pH on the luminal site of the proteoliposomes was adjusted to pH 7.0. Uptake of ¹⁴C-labeled citrate was quantified at different pH values, ranging from 4.0 to 7.0. Each data point represents the mean value of three individual replicates ± S.E. Calculation of citrate ion distribution was done using CurTiPot–pH and acid–base titration curves: Analysis and Simulation software, version 4.2.0: citrate H₃ (red circles), citrate¹⁻ H₂ (yellow squares), citrate²⁻ H (blue triangles), citrate³⁻ (purple triangles).

Figure 7.

¹³C enrichment (percentage) in malate. Rosette leaves of WT (white bars), knockout (gray bars), and tDT overexpressor plants (black bars) were labeled with ¹³CO₂ for 0, 3, 10, 20, and 40 min. Each data point represents the mean value of three biological replicates ± S.D. (error bars). Due to technical difficulties, here are only two replicates for WT labeled for 20 min and one sample for the tdt1 line labeled for 20 min. Significant differences from values obtained in WT according to Student's t test are indicated by asterisks (*, p < 0.05; **, p < 0.01).