AtGAT1, a high affinity transporter for gamma-aminobutyric acid in Arabidopsis thaliana

Abstract

Functional characterization of Arabidopsis thaliana GAT1 in heterologous expression systems, i.e. Saccharomyces cerevisiae and Xenopus laevis oocytes, revealed that AtGAT1 (At1g08230) codes for an H(+)-driven, high affinity gamma-aminobutyric acid (GABA) transporter. In addition to GABA, other omega-aminofatty acids and butylamine are recognized. In contrast to the most closely related proteins of the proline transporter family, proline and glycine betaine are not transported by AtGAT1. AtGAT1 does not share sequence similarity with any of the non-plant GABA transporters described so far, and analyses of substrate selectivity and kinetic properties showed that AtGAT1-mediated transport is similar but distinct from that of mammalian, bacterial, and S. cerevisiae GABA transporters. Consistent with a role in GABA uptake into cells, transient expression of AtGAT1/green fluorescent protein fusion proteins in tobacco protoplasts revealed localization at the plasma membrane. In planta, AtGAT1 expression was highest in flowers and under conditions of elevated GABA concentrations such as wounding or senescence.

FIGURE 1. Phylogenetic relationship between AtGAT1 and related proteins

The analysis was performed using the aligned protein sequences of the Arabidopsis ATF gene family that contains several groups of amino acid permeases (AAPs, LHTs, ANT-like proteins) and proline or compatible solute transporters (ProTs) as well as potential auxin transporters (AUX1-like) (22, 25, 36, 63). Proteins from other plant species were included for the ProT-like (AmT1–3, (64), LeProT1–3, (34), OsProT, (65), HvProT1, (66), AhProT1, AAF76897) and the AtGAT1-like proteins from rice (Oryza sativa OJ1402_H07.15, OJ1007_H05.2, P0407B12.25). The partial AtGAT1-like protein from Cicer arietinum (AJ004959) was not included in the alignment. Maximum parsimony analysis was performed using PAUP 4.0b10 with all characters unweighted and gaps scored as missing characters (67). The complete alignment was based on 800 amino acids; 575 characters were parsimony informative. AUX1 was used as outgroup.

FIGURE 2. Complementation of an S. cerevisiae strain (22574d) deficient in the uptake of proline and GABA by AtGAT1

Growth of 22574d cells expressing AtGAT1, the proline/compatible solute transporter AtProT2 (23, 39), the amino acid permease AtAAP2 (38), and the strain transformed with the vector pDR196 is shown. Minimal medium supplemented with 5 g/liter ammonium sulfate (A), 1 g/liter GABA (B), or 1 g/liter proline (C) as sole nitrogen source.

FIGURE 3. Biochemical properties of AtGAT1 expressed in S. cerevisiae

A, ³H-GABA uptake into S. cerevisiae (22574d) expressing AtGAT1. B, pH dependence of AtGAT1-mediated GABA uptake. C, competition of ³H-GABA uptake in the presence of a 5-fold excess of the respective substrate (GABA-related compounds, black bars; components of the GABA shunt, gray bars; amino acids, white bars; quaternary ammonium compounds, hatched bars). The uncompeted uptake rate was taken as 100% corresponding to 82.7 nmol GABA*min⁻¹*(10⁶ yeast cells)⁻¹. A–C, all values shown are mean ± S.D. from at least three independent experiments. GABA concentrations used were 1–300 μm (A) and 100 μm (B and C).

FIGURE 4. pH-dependent, GABA-evoked currents of oocytes expressing AtGAT1

Current traces were recorded from a control oocyte (A) and an oocyte injected with AtGAT1 cRNA (B) maintained at V_m −50 mV. Oocytes were initially incubated in a solution at pH 7.5, and at the time indicated by the bar the oocyte was perfused with a solution at pH 5.0. In both the control cell and AtGAT1-expressing cell, the increase in the external H⁺ concentration caused an inward current. The magnitude of this current was greater in AtGAT1-expressing cells, suggesting that AtGAT1 may sustain an H⁺ leak in the absence of GABA. The lack of a specific inhibitor of AtGAT1 did not allow us to examine this feature further. At high H⁺ concentration, addition of GABA (1 mm) to the bathing medium of AtGAT1-expressing cell caused an inward current (~180 nA). The GABA-evoked current reached a peak followed by slow decay. No GABA-evoked currents were observed in control oocytes.

FIGURE 5. Kinetics of GABA transport

A, GABA-induced currents are plotted as a function of the external GABA concentration ([H⁺]_out 10 μm and V_m −50 mV). B, voltage dependence of $K_{0.5}^{\mathrm{GABA}}$ at 10 μm [H⁺]_out. C, voltage dependence of $I_{\max }^{\mathrm{GABA}}$ at 10 μm [H⁺]_out. D, GABA-induced currents (1 mm) as a function of the external H⁺ concentration (V_m−50 mV). A–D, values represent the mean ± S.E. of three experiments.

FIGURE 6. Substrate selectivity of AtGAT1

Substrate-induced currents in AtGAT1-expressing oocytes were recorded at substrate concentrations of 1 mm. The holding potential (V_m) was −50 mV, and [H⁺]_out was 10 μm. Substrate-induced currents were normalized with respect to that evoked by GABA (1 mm). No evoked currents were detected when these substrates were tested in control oocytes (data not shown). Values are mean ± S.E. from at least three oocytes. See Table 1 for the structure of compounds.

FIGURE 7. Localization of the GFP-AtGAT1 fusion protein at the plasma membrane of tobacco protoplasts

A and C, confocal laser scanning microscope images. B and D, corresponding bright field images of tobacco protoplasts transiently expressing GFP-AtGAT1 (A, B) or GFP (C, D). Merged images show GFP fluorescence (green) and chlorophyll fluorescence (red). Diameter of protoplasts is ~40 μm.

FIGURE 8. Expression analysis of AtGAT1 in Arabidopsis

Expression was analyzed by relative quantification using real-time PCR with the actin mRNA (At3g18780) as a reference. Expression of AtGAT1 is given relative to actin mRNA levels and is the mean of three replicates ± S.E. Similar results were obtained using RNAs from three independent experiments as template. A, RNA from source leaves, stems, roots, and flowers of soil-grown plants was analyzed. B, AtGAT1 expression in control plants (black bars) and wounded plants (gray bars) 2, 4, and 24 h after treatment. C, RNA from senescing leaves was analyzed for AtGAT1 expression (gray bars) using both detached and naturally senescing leaves at different stages of senescence (see “Experimental Procedures”). Progression of senescence was monitored by measuring the expression of AtSag12 (black bars), a marker gene for senescence (68), and determining the content of chlorophyll. Additionally, GABA concentration in leaves was determined. 100% chlorophyll content corresponds to 0.8 μg of chlorophyll/mg of fresh weight; 100% GABA content corresponds to 34 pmol GABA/mg of fresh weight. Comparable results were obtained in three independent experiments.