AtGAT1, A High Affinity Transporter For -Aminobutyric Acid In ...

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AtGAT1, a High Affinity Transporter for -Aminobutyric Acid
in Arabidopsis thaliana*
Received for publication, October 3, 2005 in revised form, December 26, 2005 Published, JBC Papers in Press, January 10, 2006, DOI 10.1074/jbc.M510766200
Andreas Meyer‡, Sepehr Eskandari§, Silke Grallath‡1, and Doris Rentsch‡2
From the ‡Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern, Switzerland and the §Biological Sciences
Department, California State Polytechnic University, Pomona, California 91768-4032
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 -aminobutyric acid (GABA) transporter. In addition to GABA, other -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.
-Aminobutyric acid (GABA)3 is a four-carbon non-protein amino
acid present in prokaryotes and eukaryotes. Although GABA was discovered in 1949 as a constituent of potato tubers, research on GABA
metabolism and transport advancedmuch faster in the animal system as
GABA turned out to be themost abundant inhibitory neurotransmitter
in the central nervous system (1). Uptake ofGABA into neurons and glia
has been investigated in detail and shown to be mediated by Na-dependent and Cl-facilitated GABA transporters (GATs), thus regulating concentration and duration of the neurotransmitter GABA in the
synapse (2, 3). In addition to its function as a neurotransmitter, GABA
plays a role in the development of the nervous system, influencing proliferation, migration, and differentiation (4).
With the exception of the general amino acid permease BraRI from
Rhizobium leguminosarum, which belongs to the ATP binding cassette
(ABC) transporters, GABA uptake in Gram-negative and Gram-positive bacteria as well as in Saccharomyces cerevisiae is mediated bymembers of the APC (amino acid/polyamine/organocation) superfamily of
transporters (5, 6). In both bacteria and yeast, GABA uptake and biosynthesis aremainly involved in nitrogen and carbonmetabolism (7–9),
although other functions such as GABA synthesis for pH regulation in
Escherichia coli and for normal oxidative stress tolerance in S. cerevisiae
have also been postulated (10, 11).
Much less is known about the role of GABA and its transport across
the plasma membrane in plants. GABA rapidly accumulates under various stress conditions such as low temperature, mechanical stimulation,
and oxygen deficiency (12, 13). As in other organisms, GABA is synthesized in plants primarily by decarboxylation of glutamate and degraded
via succinic semialdehyde to succinate, a pathway that is also called the
GABA shunt (12). Alternatively, succinic semialdehyde can be further
catabolized to -hydroxybutyrate (14). In plants GABA and the GABA
shunt have been discussed as important for regulation of cytosolic pH,
nitrogen storage and metabolism, protection against oxidative stress,
development, and deterrence of insects (12, 13, 15). GABA might also
act as a compatible solute, and more recently its involvement in pollen
tube guidance has been demonstrated, suggesting a role in intercellular
signaling in plants (13, 16, 17). Such functions require both intra- and
intercellular transport of GABA. Indeed, both cellular and vascular
transport of GABA have been documented in physiological experiments (18–20).
So far, only transporters mediating low affinity uptake of GABA (Km
in the millimolar range) have been identified in plants. These GABA
transporters (AtAAP3, ProTs), which belong to the amino acid/auxin
transporter (AAAP) or amino acid transporter (ATF) superfamily (21,
22), might not transport GABA in planta, as their affinity for amino
acids (AtAAP3) (23, 24) or for the compatible solutes proline and glycine betaine (ProTs) (23, 25), respectively, are considerably higher than
for GABA.
In this study we have reported the identification and characterization
of the first high affinityGABA transporter fromArabidopsis, designated
AtGAT1. Characterization in heterologous expression systems showed
that kinetic properties and substrate selectivity of AtGAT1 are similar
but distinct from mammalian, bacterial, and S. cerevisiae GABA transporters described so far.
EXPERIMENTAL PROCEDURES
Plant Material, Growth Conditions, and Stress Treatment—Arabidopsis thaliana L. ecotype Col-0 was grown in soil in a growth chamber
at 22 °C/18 °C, 65% humidity, and 16 h of light. For induction of wounding response, rosette leaves of 4–5-week-old plants were wounded by
scratching them with tweezers. Two 1–1.5-cm-long scratches parallel
to the midrib were made, and wounded leaves were harvested 2, 4, and
24 h after wounding. Low and high temperature treatments were performed by keeping plants in the dark at 4 and 37 °C, respectively; anoxic
stress was applied by submerging whole Arabidopsis plants in water.
Leaves were harvested 2, 4, and 24 h after onset of the treatment. For
dark induction of senescence, green leaves were excised and incubated
on moistened filter paper for 3 or 6 days in the dark. Alternatively,
* This work was supported by Grants 31-64918.01 and 3100A0-107507 from the Swiss
National Foundation (to A. M., S. G., and D. R.) and National Institutes of Health Grant
S06 GM53933 (to S. E.). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Present address: Max-Planck-Institut für Biochemie, Zelluläre Biochemie, 82152
Martinsried, Germany.
2 To whom correspondence should be addressed. Tel.: 41-31-631-4916; Fax: 41-31-6314942; E-mail: doris.rentsch@ips.unibe.ch.
3 The abbreviations used are: GABA, -aminobutyric acid; AAP, amino acid permease;
ATF, amino acid transporter family; GAT, GABA transporter; GabP, GABA permease; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid;
HOMOPIPES, homopiperazine-1,4-bis(2-ethanesulfonic acid); ProT, proline transporter.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 11, pp. 7197–7204, March 17, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
MARCH 17, 2006 • VOLUME 281 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 7197
senescence of attached leaves was assayed by harvesting two batches of
yellowing leaves of different stages of senescence from the same plants.
20% of the leaf area was yellowing in stage I senescent leaves, whereas
50% of the leaf area was yellowing in stage II leaves.
Chlorophyll Extraction—Plant material was extracted three times
with 80% acetone containing 1MKOH, and the chlorophyll content of
the extract was measured spectrophotometrically (26).
Determination of GABAConcentration—150–200mg of plant material was extracted with 500 l of a mixture of methanol:chloroform:
water (12:5:3). After centrifugation, the supernatant was recovered and
188 l of water and 125 l of chloroform were added. The upper phase
of the mixture was dried at room temperature using a speed vacuum
apparatus. The pellet was dissolved in 200 l of water and 300 l of
acetonitrile, and phases were separated by centrifugation. The supernatant was dried, dissolved in 200l of water, and purified using a Sep-Pak
Vac 1cc C18 cartridge (Waters,Milford,MA). The eluate was dried, and
GABA content was measured by high performance liquid chromatography using a modified protocol according to Bidlingmeyer et al. (27).
DNA and RNAWork—TheAtGAT1-cDNAwas isolated by RT-PCR
using primers 5-ACTTATAAAAGTGAGTAGCACC-3, 5-CTCACTTTGCTTTGCATGTTC-3 and RNA extracted from flowers of
A. thaliana L. ecotype Col-0 as template. The AtGAT1-cDNA was
cloned in the EcoRV site of pSK and verified by sequencing. For S.
cerevisiae complementation assays the AtGAT1-cDNA was transferred
into pDR196 using PstI and XhoI (28). The cDNA of At5g41800
was isolated by RT-PCR using primers 5-TATATAAATCATTCACTTGTAG-3, 5-TTTATGTTTTTATCACACTATC-3 and RNA
extracted from flowers of A. thaliana L. ecotype Col-0 as template. The
At5g41800-cDNAwas cloned in the SmaI site of pDR196 and verified by
sequencing.
For translational fusions with GFP, the open reading frame of the
AtGAT1 cDNA was amplified by PCR and cloned in pUC18-spGFP6
and pUC18-GFP5Tsp.4 AtGAT1-GFP fusion: 5-CTAGCTAGCATGGGTGGAGAAGAGAGATC-3, 5-GAAGATCTACATCCGCAAA-
CAACTTATAC-3 (open reading frame cloned into SpeI/BglII site).
GFP-AtGAT1 fusion: 5-CTAGCTAGCATGGGTGGAGAAGAGAGATC-3, 5-CCGCTCGAGTTATACATCCGCAAACAAC-3 (open
reading frame cloned into NheI/SalI site). Sequence identity of all
PCR-amplified fragments was verified by sequencing.
For electrophysiological studies AtGAT1 was transferred from
pDR196 to pBF1 (29) using BamHI and ClaI. AtGAT1-GFP and GFPAtGAT1 were transferred from pUC18-vectors to pBF1 using XmaI
and PstI. cRNA was synthesized using the AMBION SP6 mMessage
mMachine kit (Ambion, Austin, TX) following the manufacturer’s
instructions.
For quantification of expression, RNA was extracted using a method
based on phenol extraction (30) including an additional DNase I treatment. Reverse transcription was performed using the RETROscript kit
(Ambion) according to the manufacturer’s instructions with oligo(dT)
primers and 2g of total RNA as template. Relative quantification using
real-time PCR was performed on a LightCycler instrument (Roche
Diagnostics). The FastStart DNAMaster SYBRGreen I kit (RocheDiagnostics) was used according to the manufacturer’s instructions with
MgCl2 at a final concentration of 4 mM and 10 pmol of each primer
(AtGAT1: 5-ATACCTGAAATTCAGGCAACAA-3, 5-TAGGTT-
GTAGGTATACCACAG-3). Actin (AtAct2) was used as a reference
gene (5-ATTCAGATGCCCAGAAGTCTTGTT-3, 5-GAAACAT-
TTTCTGTGAACGATTCCT-3). AtSag12 was used as a marker for
senescence (5-CGAAGGCGGTTTAATGGATA-3, 5-CACCTCCTTCAATTCCAACG-3).
Yeast Growth, Transformation, and Selection—S. cerevisiae strain
22574d (MAT ura3–1, gap1–1, put4–1, uga4–1) (31) was transformed according to Dohmen et al. (32), and transformants were
selected on synthetic dextrose minimal medium (33). To test for substrate specificity, transformants were selected onminimalmedium supplemented with 20 g/liter of glucose and 1 g/liter of proline, GABA, or
citrulline as sole nitrogen source.
Transport Assays—Transport assays using S. cerevisiae were done
essentially as described previously (34) using a final concentration of
2  108 cells/ml, 1.85–55.5 kBq 3H-GABA (Amersham Biosciences)
and appropriate amounts of the respective unlabeled GABA.
Expression in Xenopus Oocytes—Stage V-VI Xenopus laevis oocytes
were injected with 50 ng (50 nl) ofAtGAT1 cRNA and were maintained
in Barth’s medium (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41
mMCaCl2, 0.82mMMgSO4, 2.4mMNaHCO3, 10mMHEPES, pH7.4, 50
g/ml of gentamicin, 100 g/ml of streptomycin, and 100 units/ml of
penicillin) at 18 °C for 1–3 days until used in experiments. All of the
experimentswere performed at 21 1 °C. Experimentswere performed
in a NaCl buffer containing (in mM): 100 NaCl, 2 KCl, 1 CaCl2, 1MgCl2,
10 PIPES, and 10 HOMOPIPES, pH 7.4. Substrates were added to the
buffer solutions as indicated, and the necessary pH adjustments were
made. All reagents were purchased from Sigma.
Oocytes were voltage clamped by using the Warner Oocyte Clamp
(OC-725C; Warner Instrument Corp., Hamden, CT). In the recording
experimental chamber, oocytes were initially stabilized in the NaCl
buffer, and the composition of the bath was changed as indicated. In all
of the experiments, the reference electrodes were connected to the
experimental oocyte chamber via agar bridges (3% agar in 3 M KCl). For
continuous holding current measurements, the oocyte membrane
potential (Vm) was clamped at 50 mV unless otherwise indicated.
Currents were low pass filtered at 100 Hz (LPF 8; Warner Instrument
Corp.), sampled at 10Hz (pCLAMP 8.1; Axon Instruments, Union City,
CA). Substrate-induced currents were determined by subtracting the
base-line current present in NaCl buffer from the evoked current
observed after addition of the substrate.
The effects of substrate concentration on the steady-state kinetics
were determined by non-linear curve fitting of the induced currents (I)
to the Michaelis-Menten equation as shown in Equation 1,
I 
Imax
S  S
K0.5
S  S
(Eq. 1)
where S is the substrate, ImaxS is the maximal substrate-induced current,
and K0.5S is the substrate concentration at half ImaxS (half-maximal concentration). Curve fittings were performed by using SigmaPlot (SPSS
Science, Chicago, IL).
Transient Expression in Protoplasts—Transient expression of GFP
fusion proteins in tobacco protoplasts was done as described previously
(35), and the samples were examined by using a SP2 AOBS confocal
microscope (Leica Microsystems, Wetzlar, Germany). Filter settings
were 500–520 nm for GFP and 628–768 nm for chlorophyll epifluorescence detection.
RESULTS
The Arabidopsis genome contains two members of the ATF amino
acid transporter gene family, At1g08230 (AtGAT1) and At5g41800,
which exhibit a higher degree of homology to the proline/compatible
solute transporters (ProTs) than to members of other subfamilies (Fig.4 M. Suter Grotemeyer and D. Rentsch, unpublished information.
AtGAT1, a High Affinity GABA Transporter of Arabidopsis
7198 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 11 • MARCH 17, 2006
1) (22, 36). Interestingly, AtGAT1 showed higher homology to a rice
protein (OJ1402_H07.15; 62.3% amino acid identity) than to its closest
Arabidopsis homolog, At5g41800 (45.8% amino acid identity). Similarly, At5g41800 showed highest homology to a partial mRNA from
chickpea (Cicer arietinum AJ004959) and to two rice proteins
(OJ1007_H05.2 and P0407B12.25). This is in contrast tomembers of the
ProT subfamily, where proteins from tomato, mangrove, rice, and Arabidopsis display a higher degree of homology within a species than
between species. Similar to predictions for other ATF family members,
AtGAT1 andAt5g41800 encode proteinswith amolecularmass of 49.69
and 49.86 kDa, respectively, and 9–12 predicted transmembrane
domains (37). Based on the relatively low sequence identity of AtGAT1
andAt5g41800 to ProTs (26–27.2% identity), wewonderedwhether the
newly identified genes code for compatible solute transporters or represent a separate group of amino acid transporters.
AtGAT1 Transports GABA with High Affinity—To determine
whetherAtGAT1differs in its substrate specificity frommembers of the
ProT gene family, the cDNAs of AtGAT1 (At1g08230) and At5g41800
were isolated by RT-PCR. Subsequently, AtGAT1 and At5g41800 were
expressed under the control of the strong PMA1 promoter (vector
pDR196) in the S. cerevisiae strain 22574d (28, 31). The S. cerevisiae
mutant 22574d carries mutations in the general amino acid (gap1), proline (put4), andGABA (uga4) permeases and therefore is unable to grow
on citrulline, proline, or GABA as the sole nitrogen source. As control,
strain 22574d was transformed with the expression vector pDR196 and
pDR195 harboring the cDNAs of the proline/compatible solute transporterAtProT2 or of the amino acid permeaseAtAAP2 (38, 39). Growth
under selective conditions showed that, likeAtProT2, AtGAT1was able
to mediate growth on GABA. However, AtGAT1 could not mediate
growth on proline or citrulline (Fig. 2 and data not shown) and histidine
was not a substrate for AtGAT1 (data not shown; strain JT16) (40). This
behavior distinguishedAtGAT1 from all transporters of the ATF family
characterized so far. In contrast, At5g41800 could not mediate growth
on any of the tested substrates. Therefore, At5g41800 was not included
in further functional studies.
Previous studies described the ProTs as low affinity GABA transporters (Km, 1.7–5mM) (23, 25). To examinewhetherAtGAT1 differed in its
kinetic properties, 3H-GABA uptake experiments were performed in
22574d cells expressing AtGAT1. Transport assays showed that
AtGAT1 has a much higher affinity for GABA (Km 10 3 M) than any
of the plant transporters characterized before (Fig. 3A). As shown for
other transporters of the ATF family, transport rates increased with
decreasing pH (Fig. 3B) (39, 41). Competition experiments for
3H-GABA uptake in the presence of a 5-fold excess of competitors
showed that the GABA-related compounds -aminobutyric acid and
-alanine reduced GABA transport rates by 30%, whereas -aminobutyric acid did not compete for GABA transport (Fig. 3C). In addition, compounds involved in GABA metabolism (i.e. glutamate, succinic semialdehyde, and succinate) were not competitors for AtGAT1mediated GABA uptake. Alanine slightly reduced GABA transport
rates, whereas histidine as well as compounds that were good competitors for ProT-mediated GABA transport (i.e. D- and L-proline, glycine
betaine, and choline) did not reduce GABA uptake activity (23). That
none of the substrates tested efficiently competed for GABA uptake
suggested that AtGAT1 is a highly selective, high affinity GABA
transporter.
Electrophysiological Assay of AtGAT1GABATransport Kinetics—To
determine whether weakly competing compounds were substrates of
AtGAT1 and to examine the kinetic properties of AtGAT1 in more
detail, substrate-induced currents were analyzed in X. laevis oocytes
injected with AtGAT1 cRNA. At a membrane potential of 50 mV and
a pHof 5.0, addition of 1mMGABA to the bathingmediumofAtGAT1expressing oocytes induced inward currents ranging from 96 to 191 nA
(Fig. 4B). Current amplitude was dependent on the batch of oocytes and
incubation time after cRNA injection. As is commonly observed with
many other electrogenic transporters expressed inX. laevis oocytes, the
AtGAT1 substrate-evoked current reached a peak followed by slow
decay in the presence of the substrate (Fig. 4B) (e.g. Refs. 42 and 43). No
GABA-evoked currents were observed in control oocytes (Fig. 4A).
Fig. 5A shows the GABA activation curve at a holding potential of
50 mV and an external proton concentration ([H]out) of 10 M (pH
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.
AtGAT1, a High Affinity GABA Transporter of Arabidopsis
MARCH 17, 2006 • VOLUME 281 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 7199
5). The apparent affinity of AtGAT1 for GABA was 43  7 M (n 3).
Likewise, the H activation curve at 50 mV and 1 mM GABA was
hyperbolic with aK0.5 of 440 40 nM (Fig. 5D). The apparent affinity for
GABA (K0.5GABA) appeared not to be voltage dependent in the range
from 90 to 10 mV (Fig. 5B), whereas the maximum transport rate
(ImaxGABA) increased slightly at depolarizedmembrane potentials (Fig. 5C).
Substrate Selectivity of AtGAT1—Uptake studies such as those
shown in Fig. 3C demonstrate whether a compound can alter AtGAT1mediated GABA transport, presumably by competing for the GABA
FIGURE 3. Biochemical properties of AtGAT1 expressed in S. cerevisiae. A, 3H-GABA
uptake into S. cerevisiae (22574d) expressing AtGAT1. B, pH dependence of AtGAT1mediated 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 GABAmin1(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 M (A) and
100 M (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 Vm 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 GABAevoked 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 M and Vm 50 mV). B, voltage
dependence of K0.5
GABA at 10 M [H]out. C, voltage dependence of Imax
GABA at 10 M [H]out.
D, GABA-induced currents (1 mM) as a function of the external H concentration (Vm 50
mV). A–D, values represent the mean  S.E. of three experiments.
AtGAT1, a High Affinity GABA Transporter of Arabidopsis
7200 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 11 • MARCH 17, 2006
binding site of AtGAT1. However, these competition studies do not
reveal whether a tested compound is in fact a transported substrate of
AtGAT1. Thus, to further examine the substrate selectivity of AtGAT1,
electrophysiological assays were performed (Fig. 6). GABA, GABA analogs, and other substrates were applied at a concentration of 1 mM,
whereas [H]out was 10 M and Vm was 50 mV (Fig. 6). An inward
current evoked by a substrate was taken as H-driven, AtGAT1-mediated substrate translocation into the cell. L-Alanine, -aminobutyric
acid, and -alanine, which only weakly competed for GABA uptake in
S. cerevisiae (see Fig. 3C), induced inward currents comparable in magnitude with that induced by GABA. Other GABA-related compounds
with longer carbon chains, such as 5-aminovaleric acid, 6-aminocaproic
acid, and 8-aminocaprylic acid, were also good substrates. Interestingly,
the current induced by butylamine, which lacks the carboxyl group, was
similar to that induced by GABA. None of these substrates induced
currents in control oocytes.
Similar to competition experiments using S. cerevisiae, D- and L-proline as well as glycine betaine and choline were not recognized as substrates (see Figs. 3C and 6). In addition, substrates of the GABA shunt
(succinate and glutamate) did not induce currents in AtGAT1-expressing oocytes. Moreover, trigonelline, a betaine present at high concentrations in many legume seeds (44), and the amino acids histidine, glutamine, and norvaline were not transported. The rigid GABA analog
nipecotic acid and the amino sulfonate taurine, both substrates of neuronal GABA transporters in mouse (mGAT3 and mGAT4) (2, 45, 46),
did not induce currents in AtGAT1-expressing oocytes.
We reasoned that the compounds that did not compete for GABA
uptake in S. cerevisiae but induced currents in AtGAT1-expressing
oocytes were low affinity substrates of AtGAT1. Thus, we determined
the affinity of AtGAT1 for various substrates. Substrate-induced currentsweremeasured during applications of substrates at concentrations
between 5 M and 100 mM. Currents were plotted against the substrate
concentration, and curves were fitted to Equation 1. The apparent affinity of AtGAT1 for individual substrates varied by a factor of 1000 (Table
1). AtGAT1 showed the highest affinity for butylaminewith aK0.5 2-fold
lower than that for GABA. Moving the amino group closer to the carboxyl group (such as in -aminobutyric acid and -aminobutyric acid)
progressively reduced the apparent affinity (see Table 1). Increasing the
carbon chain backbone from GABA to 8-aminocaprylic acid only marginally affected the K0.5 values (30–80 M). In addition, 5-aminolevulinic acid was transported with an affinity comparable with that of
GABA. Reducing the carbon chain length (e.g. -alanine) resulted in a
much lower substrate affinity (K0.5 200M). A similar affinity was determined for 4-aminophenylacetic acid, a peptide mimic lacking a peptide
bond (47). For compounds with even shorter carbon chains (i.e. L-alanine), the affinity dropped even further. The apparent affinity for L-glycine and L--aminobutyric acid, which had been shown to induce
currents in AtGAT1-expressing oocytes (Fig. 6), as well as for L-2,4diaminobutyric acid and the dipeptide glycyl-glycine, was
10 mM.
Although the K0.5 values of the different substrates varied considerably,
FIGURE 6. Substrate selectivity of AtGAT1. Substrate-induced currents in AtGAT1-expressing oocytes were recorded at substrate concentrations of 1 mM. The holding potential (Vm) was 50 mV, and [H
]out was 10 M. 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.
TABLE 1
Kinetics of AtGAT1-mediated substrate transport
Substrate-induced currents were recorded atVm 50mVand 10M [H]out, and
the apparent affinities (K0.5) were determined according to Equation 1. For each
substrate, the maximum transport rate was normalized with respect to that
observed for GABA in the same cell. Values are mean  S.E. from at least three
independent experiments.
AtGAT1, a High Affinity GABA Transporter of Arabidopsis
MARCH 17, 2006 • VOLUME 281 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 7201
the maximum transport rate (Imaxsubstrate) for all substrates remained relatively constant (20%) (see Table 1).
AtGAT1 Is Localized at the PlasmaMembrane—Functional complementation of the S. cerevisiaeGABA transport mutant by AtGAT1 and
functional expression in X. laevis oocytes showed that at least a fraction
of the protein is targeted to and localized at the plasma membrane in
both heterologous expression systems. To assess its cellular localization
in planta, fusion proteins of AtGAT1 and GFP were transiently
expressed in tobacco protoplasts under the control of the cauliflower
mosaic virus 35S promoter (Fig. 7). Fluorescent images obtained by
confocal laser scanning microscopy showed that the signal from the
GFP-AtGAT1 fusion protein was present as a single fluorescent ring at
the periphery of the protoplast, suggestive of its localization at the
plasma membrane (Fig. 7A). In addition, the protoplasts showed some
GFP fluorescence on internalmembranes. Similar results were obtained
for the AtGAT1-GFP fusion (data not shown). FreeGFP localized to the
cytosol (Fig. 7C). When expressed in Xenopus oocytes, both fusion proteins, AtGAT1-GFP and GFP-AtGAT1, were able to mediate GABA
transport with affinities comparable with that of AtGAT1 (K0.5GABA was
58  6 M for AtGAT1-GFP and 36  5 M for GFP-AtGAT1; n 3).
AtGAT1 Expression in Arabidopsis Is Induced byWounding and during Senescence—AtGAT1 expression in Arabidopsis was extremely
weak and hardly detectable by RNA gel blot analysis. Relative quantification using real-time PCR, with the actin mRNA (AtACT2) as a reference, demonstrated the highest levels of AtGAT1 expression in flowers
and low expression levels in roots, leaves, and stems (Fig. 8A). Microarray analyses (48) showed that in flowers AtGAT1 expression is highest
in sepals, lower in petals and carpels, and very low in stamen. In agreement with our own data, only low AtGAT1 transcript levels were found
in pollen (data not shown). It was shown previously that GABA accumulates under various stress conditions (12). However, cold, heat, and
anoxia did not significantly alter AtGAT1 mRNA levels (not shown).
Mechanical wounding caused a transient increase ofAtGAT1 transcript
levels that dropped below control values after 24 h (Fig. 8B). A similar
time course ofAtGAT1 expression after wounding has been observed in
microarray analyses (TAIR Accession: ExpressionSet 1007966439).
Interestingly,AtGAT1 expression also increased during leaf senescence
of both detached andnaturally senescing leaves (Fig. 8C), corresponding
to results obtained inmicroarray analyses (48). Differences in induction
during dark-induced and natural senescence might result from
impaired export of compounds in detached leaves or may reflect differences in carbon:nitrogen ratio as described for other genes (49). In both
experiments, the increase in AtGAT1 expression correlates with an
increase in GABA content in the corresponding tissue (Fig. 8C).
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 GFPAtGAT1 (A, B) or GFP (C, D). Merged images show GFP fluorescence (green) and chlorophyll fluorescence (red). Diameter of protoplasts is 40 m.
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 soilgrown 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 g 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.
AtGAT1, a High Affinity GABA Transporter of Arabidopsis
7202 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 11 • MARCH 17, 2006
DISCUSSION
Expression of AtGAT1 in S. cerevisiae and Xenopus oocytes enabled
us to demonstrate that AtGAT1 mediates H-dependent, high affinity
transport of GABA and GABA-related compounds. The characteristics
of AtGAT1 differ significantly from those of the most closely related
proteins of the ProT-family, as glycine betaine and proline are not recognized and as GABA is transported with very high affinity. Amino
acids, which are substrates for othermembers of theATF family, are not
recognized byAtGAT1 (24). The comparison of the substrate selectivity
of AtGAT1 determined in S. cerevisiae and Xenopus oocytes showed
that some compounds that only weakly (-Ala, -ABA) or marginally
(L-Ala) competed for GABA uptake in S. cerevisiae induced currents in
AtGAT1-expressing oocytes. It was determined that these compounds
were transported in Xenopus oocytes with medium or low apparent
affinity, thus corroborating the substrate selectivity determined using S.
cerevisiae.
GABA transporters that evolved in bacteria, yeast, and mammals
belong to different gene families and do not show sequence homology to
AtGAT1. Although both the primary structure and driving force for
transport (ATP, Na, or H gradient) are diverse, their high affinity for
GABA is a common feature of GABA transporters from the different
kingdoms, whereas their affinity for other substrates is less conserved.
GABA transporters frommammals5 and E. coli (50) have been shown
to transport 5-aminovaleric acid in addition to GABA. Similarly,
AtGAT1 was able to recognize GABA-related compounds with longer
carbon backbone. Interestingly, not only 5-aminovaleric acid but also
6-aminocaproic acid, 7-aminoheptanoic acid, and 8-aminocaprylic acid
induced currents with similar apparent affinities as GABA. In mammals, 7-aminoheptanoic acid and 8-aminocaprylic acid are not transported by members of the GAT family.5 The proton-coupled animal
peptide transporter PepT1 does not recognize GABA but transports
both 5-aminovaleric acid and 6-aminocaproic acid, and even 8-aminocaprylic acid and 11-amino-undecanoic acid were recognized (51).
However, these long chain -aminofatty acids are not substrates for
other peptide transporters (i.e. for animal PepT2, Lactococcus lactis
DtpT, or Arabidopsis AtPTR1) and thus their transport is not a general
feature of peptide transporters (35, 52, 53). In addition, -aminolevulinic acid, a precursor of porphyrin biosynthesis, is transported not only
by AtGAT1 and GABA transporters from mammals (GATs) (54) and
S. cerevisiae (Uga4) (55) but also by proton-coupled intestinal and renal
peptide transporters (PEPT1 and PEPT2) (56).
Unlike the E. coli GabP, animal GATs, or AtGAT1, the GabP-mediated GABA transport in B. subtilis is not inhibited by the larger GABA
analog 5-aminovaleric acid and shows equal preference for the 3-carbon
-alanine and the 4-carbon GABA. In addition, transport is not inhibited by conformationally constrained compounds such as nipecotic
acid. Thus it was suggested that B. subtilis has a more stringent and less
spacious domain for substrate recognition than the other GABA transporters (50). The functional characteristics of AtGAT1 with a preference for longer chain GABA-related compounds and a reduced affinity
for 3- and 2-carbon amino acids place AtGAT1 closer to E. coli GabP
and the animal GATs than to B. subtilis GabP. Nevertheless, AtGAT1
differs substantially from these transporters in its inability to transport
nipecotic acid and its high affinity for several long chain -aminofatty
acids as discussed above. Additionally, in contrast to the neuronal
GABA transporters mGAT3 and mGAT4 from mouse,5 the aminosulfonic acid taurine was not able to induce currents in AtGAT1-expressing oocytes (Fig. 6). Thus, a sulfate group cannot substitute for a carboxyl group. However, the carboxyl group is not required for substrate
recognition byAtGAT1, as butylamine was able to induce currents with
high apparent affinity. Concentrations of butylamine and long chain
-aminofatty acids in plants are unknown, but it is assumed that concentrations are rather low. Therefore, in planta, AtGAT1 might function primarily as a GABA transporter.
Potential Function of AtGAT1 in Arabidopsis—GABA is present in
the cytosol, where it is synthesized, but can also be found in chloroplasts,
the vacuole, and in the apoplast (57). In addition, GABA has to be
translocated to the mitochondria for degradation. Thus, GABA transporters are required at several cellular membranes. Our results demonstrate that the AtGAT1/GFP fusion proteins are targeted to the plasma
membrane when transiently expressed in protoplasts, indicating that in
Arabidopsis AtGAT1 mediates GABA uptake from the apoplast and
might be important in the reallocation or retrieval of GABA.
High GABA concentrations have been detected under a variety of
stress conditions such as low temperature, mechanical stimulation, and
oxygen deficiency (12, 13). In soybean plants, GABA concentrations
increase under water stress in all plant tissues. Elevated GABA concentrations can also be found in the xylem of nodulated soybean subjected
to drought or hypoxic stress (58). In anoxically grownRicinus communis
seedlings, GABA is one of the main amino acids in the phloem (20),
indicating that translocation ofGABA in the phloemmight be increased
under stress conditions. Although in our experiments no induction of
AtGAT1 expression was detectable under low oxygen availability, elevated AtGAT1 mRNA levels were found upon wounding and during
senescence, two conditions under which GABA concentrations are elevated (59, 60).
Under non-stress conditions,AtGAT1 expressionwas higher in flowers when comparedwith other organs. Palanivelu et al. (17) showed that
a GABA gradient is important for pollen tube growth and guidance
through female tissue to the micropyle. AtGAT1 expression was very
low in pollen and only slightly elevated in carpels, thus not strongly
supporting a role of AtGAT1 in this process. However, AtGAT1might
be expressed in germinating pollen, and AtGAT1 activity in carpels
might contribute in establishing such a GABA gradient important for
pollen tube guidance. Thus, a possible role of AtGAT1 in this process
remains to be further investigated. The higherAtGAT1 transcript levels
in sepals and petals correlate with increased expression of other senescence-associated genes, e.g. pheophorpide a oxygenase, which again
points to a role of AtGAT1 during senescence (61, 62). The role of
GABA during senescence is not clear, but Bouché and Fromm (13)
speculated that GABA might act as a signaling molecule to coordinate
carbon:nitrogen balance in changing nutrient environments, as it
occurs during senescence (49). In addition, GABA may play an anaplerotic role during senescence (60).
In summary, our data show that AtGAT1 is an H-driven, plasma
membrane-localized transporter that recognizes GABA and several
GABA-related compoundswith high affinity. The expression implicates
a role of AtGAT1 in conditions with elevated GABA concentrations as
observed during senescence and wounding.
Acknowledgments—We thank Michael J. Errico, William Lee, Erik B. Malarkey, and Stefan Meier for preparation of oocytes and Christopher Ball and
Rebecca Alder for taking care of the plants.
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AtGAT1, a High Affinity GABA Transporter of Arabidopsis
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