(Received for publication, February 12, 1997, and in revised form, March 13, 1997)
From the Department of Physiology, UCLA School of
Medicine, Los Angeles, California 90095-1751 and the ¶ Institut
für Botanik, Eberhard-Karls-Universität, Auf der
Morgenstelle 1, D-72076 Tübingen, Federal Republic of Germany
The H+-dependent AAP5 amino acid transporter from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used radiotracer flux and electrophysiology methods to investigate its substrate specificity and stoichiometry. Inward currents of up to 9 µA were induced by a broad spectrum of amino acids, including anionic, cationic, and neutral amino acids. The apparent affinity of AAP5 for amino acids was influenced by the position of side chain branches, bulky ring structures, and charged groups. The maximal current was dependent on amino acid charge, but was relatively independent of amino acid structure. A detailed kinetic analysis of AAP5 using lysine, alanine, glutamate, and histidine revealed H+-dependent differences in the apparent affinity constants for each substrate. The differences were correlated to the effect of H+ concentration on the net charge of each amino acid and suggested that AAP5 transports only the neutral species of histidine and glutamate. Stoichiometry experiments, whereby the uptake of 3H-labeled amino acid and net inward charge were simultaneously measured in voltage-clamped oocytes, showed that the charge:amino acid stoichiometry was 2:1 for lysine and 1:1 for alanine, glutamate, and histidine. The results confirm that histidine is transported in its neutral form and show that the positive charge on lysine contributes to the magnitude of its inward current. Thus, the transport stoichiometry of AAP5 is 1 H+:1 amino acid irrespective of the net charge on the transported substrate. Structural features of amino acid molecules that are involved in substrate recognition by AAP5 are discussed.
Transport of amino acids across the plasma membrane of higher plants is mediated by proton-coupled transport proteins that utilize the electrochemical gradient for H+ to drive the uphill transport of amino acids (reviewed in Refs. 1-4). Kinetic analysis of amino acid uptake into plasma membrane vesicles isolated from sugar beet leaves suggests the presence of four H+-coupled amino acid transport systems (5-7), and at least 10 H+/amino acid transporters have been isolated by complementing yeast amino acid transport mutants with plant cDNA libraries (8-12). These transporters have very broad and overlapping specificities. However, each exhibits a preference for amino acids possessing a particular molecular geometry or charge. Analysis of the substrate specificity of amino acid transporters in yeast cells and plasma membrane vesicles is traditionally accomplished by measuring the inhibition of amino acid transport activity by various substrates. Competition experiments yield information on substrates that interact with amino acid transporters, but do not allow a distinction between substrates that are transported and those that act as inhibitors.
We previously analyzed the specificity and kinetic properties of the Arabidopsis AAP1 H+/amino acid transporter by expressing the cloned gene in Xenopus oocytes and measuring substrate-induced currents using electrophysiology methods (13). AAP1 transported anionic and neutral amino acids. However, except for histidine, the transport of cationic amino acids was negligible. In this study, we chose to investigate the specificity of the Arabidopsis AAP5 H+/amino acid transporter, which shares 54% identity and 73% similarity with AAP1. Unlike AAP1, expression of AAP5 in yeast suggests that it efficiently transports anionic, neutral, and cationic amino acids (11), making it an ideal candidate to investigate the effects of a broad spectrum of amino acids on substrate recognition. We expressed AAP5 in Xenopus oocytes and used the two-electrode voltage-clamp method to determine the apparent kinetic parameters (maximal current (imax) and apparent affinity (K0.5)) of various amino acids. For AAP5 and AAP1, a combination of electrophysiology and radiotracer flux methods enabled us to determine the H+:amino acid stoichiometry of neutral, cationic, and anionic amino acids, which revealed the net charge on the transported species. We show that 1) amino acid geometry and charge dramatically affect the substrate specificity of AAP5, and 2) AAP5 transports neutral, anionic, and cationic amino acids with a fixed H+:amino acid stoichiometry. Thus, the kinetic approaches used in this study enabled us to gain insights into the nature of the substrate-binding site and transport mechanism of AAP5.
AAP5 and AAP1 were polyadenylated as described previously (13). The resulting plasmids, pKAAP5 and pKAAP1, were linearized with KpnI, and capped cRNA was transcribed in vitro using T7 RNA polymerase and an RNA transcription kit (Ambion Inc., Austin, TX).
Oocyte PreparationXenopus oocytes were isolated and injected with 25-50 ng (1 µg/µl) of cRNA encoding AAP5 or AAP1 or with 50 nl of water (control oocytes) and were maintained in Barth's medium for up to 5 days post-injection as described previously (13).
Uptake ExperimentsThe amount of amino acid transported into oocytes under non-voltage-clamp conditions was determined using a radiotracer method. Groups of 8-10 cRNA- or water-injected oocytes were incubated in transport buffer (100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM PIPES,1 and 10 mM HOMOPIPES) containing 0.032 or 10 µM H+ and 100 µM 3H-labeled alanine, lysine, histidine, or glutamate (Amersham International, Buckinghamshire, United Kingdom). After 30 min at 22 °C, the oocytes were washed three times in 5 ml of ice-cold buffer and lysed in 10% sodium dodecyl sulfate, and the amount of radioactivity was determined by liquid scintillation counting.
Electrophysiology ExperimentsAll experiments were done
using the two-electrode voltage-clamp method (13-15). The membrane
potential was clamped at 50 mV, and steady-state currents were
recorded 50 ms after the onset of voltage pulses ranging from
150 to
50 mV (20-mV increments). Steady-state amino acid-induced currents were
obtained by taking the difference between steady-state currents in the
presence and absence of amino acid. The apparent affinities for amino
acids and protons (K0.5aa
and K0.5H, respectively)
and their maximal currents
(imaxaa and
imaxH, respectively) were
obtained by fitting the steady-state amino acid-induced currents at
each test potential (Vm) to Equation 1,
![]() |
(Eq. 1) |
To
determine the H+:amino acid transport ratio, oocytes were
voltage-clamped, and inward fluxes of 3H-labeled amino
acids and net inward amino acid-induced currents were measured
simultaneously (15).2 Throughout the
experiment, substrate-induced currents were recorded using Fetchex
software (Axon Instruments, Inc., Foster City, CA). Oocytes were
clamped at potentials ranging from 10 to
90 mV and superfused with
transport buffer at a rate of 160 µl/min. Current traces were
monitored until they reached a steady base line, after which 0.5 mM 3H-labeled amino acid was superfused for
30 s to 10 min while recording the amino acid-induced current. The
oocyte was washed in the absence of amino acid until the current
returned to base-line levels. The oocyte was quickly removed from the
chamber, washed three times in 5 ml of ice-cold buffer, and lysed in
10% sodium dodecyl sulfate, and the amount of radioactivity was
determined by liquid scintillation counting. The total inward charge
was calculated by subtracting the base-line current and integrating the
area under the current versus time curve. The
H+:amino acid transport ratio is presented as pmol of net
inward charge/pmol of amino acid transport.
The results are representative of experiments that were repeated at least three times with oocytes from different donor frogs. All experiments were carried out at 22 °C, and all amino acids used in this study were L-isomers. Chemicals were purchased from Sigma.
Crystal Structure ComparisonsThe Cambridge
Crystallographic Data Base was searched to obtain the x-ray crystal
structures of the -amino acids lysine, ornithine, histidine,
cysteine, arginine, methionine, serine, threonine, glycine, leucine,
glutamine, glutamate, alanine, citrulline, isoleucine, valine, proline,
phenylalanine, aspartate, tryptophan, and asparagine. The crystal
structure of homoarginine is not available, and this molecule was drawn
using the molecular modeling program Hyperchem (Version 4.5, Hypercube,
Waterloo, Ontario, Canada).
The apparent kinetic
parameters for various amino acids were obtained by expressing AAP5 in
Xenopus oocytes and measuring the steady-state amino
acid-induced currents as a function of membrane voltage and external
[amino acid]o at 10 µM H+o.
The amino acid-induced currents obtained at 150 mV were plotted
against [amino acid]o, and the concentration/current curves
were fitted to Equation 1. Table I shows the apparent affinity (K0.5) and three-dimensional x-ray
crystal structures of the amino acids. The K0.5
values for glutamate and histidine were adjusted to account for the net
charge on the transported species: the neutral species of histidine and
glutamate are transported by AAP5 (see below and "Discussion").
AAP5 had the highest apparent affinity for arginine, histidine,
homoarginine, and methionine (~0.1-0.3 mM), followed by
lysine, ornithine, alanine, and glycine (~0.4-0.5 mM).
Serine, glutamine, glutamate, citrulline, and cysteine all had
K0.5 values <1 mM. The
K0.5 values for threonine, homoserine (1.6 ± 0.1 mM), and leucine were higher, between ~2 and 4 mM. For proline, hydroxyproline (32 ± 10 mM), tryptophan, and valine, the
K0.5 values increased by at least 2 orders of
magnitude over that of arginine to between 20 and 33 mM.
The current/concentration curves for isoleucine, phenylalanine, and
asparagine were far from saturation even at 50 mM amino
acido, with estimated K0.5 values of
~40, 80, and >100 mM, respectively. Neither aspartate nor the
-amino acids
-aminobutyric acid and
-alanine were
transported by AAP5. Aspartate does not interact with AAP5: the
magnitude of the lysine-induced currents was the same in the absence
and presence of 50 mM aspartate.
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Fig. 1 shows a comparison of the maximal currents
induced by amino acids as a percentage of the lysine-induced current.
The highest imax values were obtained for lysine
and ornithine, followed by histidine, arginine, and homoarginine.
Except for tryptophan, which had an imax of
~8% of the lysine-induced current, all other amino acids had
imax values between 30 and 50% of that for
lysine. The activation curves for phenylalanine, isoleucine, and
asparagine did not saturate. Thus, the imax
values for these amino acids are not included in Fig. 1.
Fig. 2 shows representative, normalized current/voltage
relationships obtained with 20 mM alanine, glutamate,
histidine, and lysine at 10 µM H+o.
For each amino acid, the voltage dependence of the inward currents was
identical: they increased supralinearly with membrane
hyperpolarization, appeared to asymptote toward zero at positive
potentials (more than +50 mV), and did not reverse. The qualitative and
quantitative characteristics of the current/voltage curves were not
altered when Na+ or Li+ was substituted for
choline in the transport buffer at 0.032 and 10 µM
H+o. Negligible currents (<5 nA) were induced by
amino acid application in oocytes injected with water.
Kinetics of Alanine, Lysine, Glutamate, and Histidine Transport
Alanine, lysine, glutamate, and histidine carry
different net charges in solution over the range of
[H+]o (0.032-10 µM) used in this
study (see Fig. 7 and "Discussion"). Therefore, as a first step to
investigate the effect of substrate charge on the specificity of AAP5,
the apparent kinetic parameters for each amino acid were obtained by
varying [amino acid]o at fixed [H+]o,
varying [H+]o at fixed [amino acid]o,
and fitting the concentration/current data to Equation 1.
Fig. 3A shows the normalized amino acid
activation curves obtained at 150 mV and 10 µM
H+o. At test potentials between
150 and
30 mV
and when [H+]o was decreased, the curves were
hyperbolic with n = 1. Likewise, the H+
activation curves obtained with alanine, lysine, and histidine were
hyperbolic with n = 1 irrespective of the applied
potential and [amino acid]o. However, the H+
activation curves obtained with glutamate were sigmoidal. Fig. 3B shows representative H+ activation curves
obtained with 20 mM amino acids at
150 mV and with 5 mM glutamate at
30 mV.
Table II shows that
K0.5H values determined in
20 mM alanine and lysine were lower than in glutamate and
histidine, and when [amino acid]o decreased,
K0.5H increased. Thus, AAP5
had a very high apparent affinity for H+ (0.2 µM, pH 6.7) when [lysine]o and
[alanine]o were saturating such that both amino acids induced
inward currents in the absence of a downhill H+ gradient
across the oocyte plasma membrane (cytoplasmic [H+] = ~0.04 µM) (16). Due to substrate-dependent
differences in the apparent affinity of AAP5 for H+,
apparent kinetic parameters were obtained at 0.032 and 10 µM H+o for lysine and alanine and at
1 and 10 µM H+o for glutamate and
histidine. Mean kinetic values obtained from three experiments at 150
mV are shown in Fig. 4. Fig. 4A shows that
increasing [H+]o increased the maximal current
for amino acids. A 300-fold increase in [H+]o
increased imaxLys 4-fold
and imaxAla 5-fold; a
10-fold increase in [H+]o increased
imaxGlu 2-fold and
imaxHis 1.5-fold. For each
amino acid, the imaxaa
versus voltage curves were supralinear (data not shown).
Fig. 4B shows that the apparent affinity constants decreased
as [H+]o increased. A 300-fold increase in
[H+]o decreased
K0.5Lys from 1.5 to 0.4 mM and K0.5Ala
from 6 to 0.5 mM. A 10-fold increase in
[H+]o decreased
K0.5Glu from 50 to 5 mM, whereas the decrease in
K0.5His was much less (3 to
2 mM). The K0.5 values for histidine
and glutamate shown in Table I were adjusted to account for the net charge on the transported species (see "Discussion").
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The steady-state kinetic analysis of AAP5 can be summarized as follows. 1) The transport of neutral, anionic, and cationic amino acids is electrogenic. 2) The voltage dependence of the saturating substrate-induced currents is the same, suggesting that a voltage-dependent step in the reaction cycle is independent of the net charge on the substrate. According to the models proposed for the H+/dipeptide transporter and the Na+/glucose cotransporters, this step is probably the reorientation of the empty carrier from the cytoplasmic to the extracellular surface (14, 17). 3) H+ and amino acids increased the imax and K0.5 values for their respective cosubstrates. Positive cooperativity between the two ligands suggests that AAP5 operates via a simultaneous mechanism (18). Also, H+ acts as an essential activator probably by orientating the amino acid-binding site to the external membrane surface (13, 15, 19).
Uptake ExperimentsTo show that the inward currents induced
by amino acids were due to their uptake into oocytes, radiotracer flux
experiments were performed on unclamped oocytes with 100 µM 3H-labeled alanine, lysine, histidine, and
glutamate at 0.032 and 10 µM H+o.
After 30 min at 0.032 µM H+o, uptake
was as follows: alanine lysine > glutamate
histidine (108 ± 5, 94 ± 14, 46 ± 5, and 1.4 ± 0.3 pmol/oocyte, respectively). Thus, although glutamate-induced
currents could not be measured at 0.032 µM
H+o, tracer experiments showed that glutamate is
transported by AAP5 at low [H+]o. At 10 µM H+o, uptake was as follows:
alanine > lysine > glutamate > histidine (520 ± 47, 316 ± 29, 127 ± 5, and 76 ± 6 pmol/oocyte, respectively). These results were surprising since the currents induced
by lysine were significantly higher than the currents induced by
alanine, and both substrates had similar K0.5
values and voltage dependences at 10 µM
H+o. This suggests that an extra charge accompanies
lysine transport and contributes to the magnitude of the lysine-induced current, which has important implications for the transport mechanism of AAP5.
The steady-state kinetic data
suggested that the H+:amino acid stoichiometry was 1:1 for
alanine, lysine, and histidine, but >1:1 for glutamate. To determine
the H+:amino acid stoichiometry directly rather than
relying on Hill coefficients and to investigate the discrepancy between
the magnitude of the steady-state currents and amount of substrate
transported by AAP5, we simultaneously measured the amino
acid-induced current and uptake of 3H-labeled amino acid in
voltage-clamped oocytes. This method was chosen because it gives a more
direct measurement of stoichiometry, whereas a thermodynamic approach
requires a knowledge of the internal ligand concentrations. Fig.
5A is a typical current trace obtained from
an oocyte voltage-clamped at 50 mV and superfused with 0.5 mM [3H]lysine at 10 µM
H+o. Addition of lysine to the oocyte transport
buffer induced a large inward current (~600 nA) that declined with
time. When lysine was removed, the currents returned to the base-line levels. The current traces were qualitatively similar irrespective of
the amino acid under investigation. Under voltage-clamp conditions, amino acids accumulated above [amino acid]o. For example, oocytes clamped at
50 mV and superfused with 0.15 mM
lysineo for 10 min accumulated lysine up to ~15-fold (2.2 mM) above [lysine]o. This was calculated assuming
that the volume of the stage V oocytes used in this study was ~900
nl. The uptake of amino acids into voltage-clamped water-injected
oocytes was negligible (<5 pmol, 5.0 µM) over the same
time scale.
Fig. 5B shows plots of amino acid transported (pmol/oocyte)
versus amount of charge transported (pmol/oocyte) for
oocytes superfused with 0.5 mM lysine or alanine and 10 µM H+o at 50 mV. Regression
analysis yielded straight lines with slopes corresponding to the
charge:amino acid stoichiometry: 2.1 ± 0.04:1
(r2 = 0.99) for lysine and 1.1 ± 0.07:1
(r2 = 0.97) for alanine. Thus, the net single
positive charges on lysine and, presumably, ornithine, homoarginine,
and arginine contribute to the magnitude of their induced currents.
At 10 µM H+o and 0.5 mM
lysine, the charge:lysine stoichiometry was
voltage-dependent and increased from 1.9 ± 0.1:1 at
10 mV to 2.5 ± 0.05:1 at
90 mV. At 0.032 µM
H+o and 0.5 mM lysine, the charge:amino
acid stoichiometry was 1.6 ± 0.03:1 at
90 mV. These results
suggest that there is an uncoupled transport of protons through AAP5
and is supported by the increase in the base-line current when
[H+]o was increased from 0.032 to 10 µM (see Fig. 5A).
Fig. 6 shows a comparison of the charge:amino acid
stoichiometries of AAP5 and AAP1 obtained with 0.5 mM
3H-labeled amino acids and 10 µM
H+o at 50 mV. For both transporters, the
transport stoichiometry of histidine, glutamate, and alanine was ~1
charge:1 amino acid compared with ~2 charges:1 lysine for AAP5 (AAP1
does not transport lysine) and are conclusive evidence that histidine
is transported in its neutral form (see "Discussion"). Assuming
that the single inward charge accompanying the transport of each amino
acid is due to the transport of H+, we conclude that the
H+:amino acid stoichiometry (1:1) is the same irrespective
of the net charge of the amino acid in solution.
Expression of the H+/amino acid transporter AAP5 in Xenopus oocytes enabled us to use electrophysiology and radiotracer flux measurements to determine 1) the preferred molecular geometry of amino acids for transport, 2) the net charge on the transported amino acid species, 3) the H+:amino acid coupling stoichiometry.
Role of Charged Groups in Substrate Specificity and Stoichiometry of AAP5To undertake a thorough investigation of the substrate
specificity of AAP5, it was necessary to determine the net charge on the transported amino acid species. For each amino acid investigated under the experimental conditions used in this study (0.032-10 µM H+o), the -carboxyl and
amino groups were ionized, and their net charge was dependent on
the nature of the side chain. Fig. 7 shows the effect of
[H+]o on the distribution of charged species of
alanine, lysine, glutamate, and histidine. About 100% of lysine
carries a single net positive charge (cationic), and ~100% of
alanine carries no net charge (neutral). Glutamate is predominantly
negatively charged (anionic). However, there is a significant amount of
neutral glutamate (~0.06% at 0.032 µM
H+o increasing to 15% at 10 µM
H+o). Between 0.032 and 10 µM
H+o, the amount of cationic histidine increases
from 9 to 91%, and the amount of neutral histidine decreases from 91 to 9%. Of the other amino acids tested, arginine, ornithine, and homoarginine carry a net positive charge; the charge on aspartate is
similar to that on glutamate; and the other amino acids are neutral.
The stoichiometry data showed that, for both AAP5 and AAP1, a single net inward positive charge accompanied the uptake of alanine, histidine, and glutamate, whereas two inward charges were cotransported with lysine by AAP5. At 10 µM H+o, the net charge on alanine is zero, and the net charge on lysine is +1. Assuming the extra inward charge was carried by H+, the H+:amino acid stoichiometry for alanine and lysine was 1:1 with the charge on lysine contributing to the magnitude of its induced current. Thus, AAP5 recognizes the cationic species of lysine and probably the cationic species of arginine, homoarginine, and ornithine.
Since the neutral species of histidine is transported by AAP5, the values of K0.5His obtained at 1 and 10 µM H+o (3 and 2 mM, respectively) were recalculated. The amount of neutral histidine at 1 µM H+o is 50% compared with 9% at 10 µM H+o, yielding "real" K0.5His values of 1.5 and 0.18 mM, respectively. Also, the H+ activation data for histidine shown in Table II must be re-examined. These data were obtained by varying [H+]o at fixed [histidine]o. However, to obtain H+ activation data using the same concentration of neutral histidine (e.g. 20 mM), we would need to vary [H+]o and [histidine]o.
The stoichiometry data suggest either that neutral glutamate is transported with one proton or that anionic glutamate is transported with two protons. The glutamate activation curves obtained at fixed [H+]o were hyperbolic, whereas the H+ activation curves obtained at fixed [glutamate]o were sigmoidal, suggesting that the H+:amino acid stoichiometry was >1:1. However, when [H+]o was decreased from 10 to 1 µM, K0.5Glu increased 10-fold, which is consistent with a decrease in the amount of neutral glutamate; K0.5His increased only 1.5-fold over the same range of [H+]o. Thus, the shape of the H+ activation curves can also be explained if glutamate is transported in its neutral form. For example, a 20 mM glutamate solution would contain 12 µM neutral glutamate at 0.032 µM H+o and 3 mM at 10 µM H+o (see Fig. 7), which would explain the observed lag in the H+ activation curves. If glutamate is transported as the neutral species, the real K0.5Glu at 10 µM H+o is 0.75 mM, which is similar to the K0.5 for glutamine (0.78 mM), a neutral amino acid that has a similar three-dimensional structure to glutamate (see Table I). Like histidine, the H+ activation data for glutamate shown in Table II must be re-examined using the same concentrations of neutral glutamate at each [H+]o.
The stoichiometry experiments showed that, irrespective of the net charge on the transported substrate, amino acid transport by AAP5 occurs with a H+:amino acid coupling stoichiometry of 1:1. Our results for histidine concur with those of Wyse and Komor (20), who concluded that neutral histidine is cotransported with 1 H+ across the plant plasma membrane. However, the results for lysine contradict previous studies that suggested that the transport of lysine was facilitative (20, 21). Also, our steady-state kinetic data showed that lysine transport was H+-coupled: increasing [H+]o decreased K0.5Lys and increased imaxLys; lysine transport was concentrative; and a 1 H+:1 lysine coupling ratio was predicted from the hyperbolic lysine and H+ activation curves (n = 1). Similarly, Sanders et al. (22) showed that H+ accompanied the transport of cationic amino acids in Neurospora and that the H+:amino acid stoichiometry was the same for neutral and cationic amino acids. Based on activation curves, Mackenzie et al. (23) showed that the human hPEPT1 H+/dipeptide transporter cotransports anionic, cationic, and neutral dipeptides with 1 H+. That glutamate is transported in its neutral form with 1 H+ also contradicts the results of Kinraide and Etherton (21) and Wyse and Komor (20), who suggested that glutamate was cotransported with two cations. However, others have suggested that the neutral forms of anionic substrates are transported by the mammalian ASCT2 neutral amino acid transporter (24) and the mammalian hPEPT1 and rPEPT1 dipeptide transporters (25, 26).
Structural Determinants on Amino Acids Affecting Specificity of AAP5As in amino acid transport into sugar beet leaves (7), the
-amino and
-carboxyl groups are essential for transport of amino
acids by AAP5:
-aminobutyric acid and
-alanine were
non-interacting substrates. At 10 µM
H+o, the apparent affinity of AAP5 for amino acids
ranged over 4 orders of magnitude (~0.1 to >100 mM), and
these differences were dependent upon amino acid structure and charge,
but were independent of their hydrophobicity. AAP5 had a high apparent affinity for arginine, homoarginine, histidine, lysine, and ornithine, which are highly polar and, except for histidine, possess long linear
side chains. Arginine and homoarginine have highly reactive, terminal
guanidinium groups, which probably accounts for their high apparent
affinity. Neutral, nonpolar methionine is also transported with high
apparent affinity. Therefore, the substrate-binding site of AAP5 is
relatively long and can accommodate amino acids >8 Å in length.
Although citrulline, glutamine, and glutamate are polar molecules with
long side chains, their apparent affinities were ~4-fold lower
compared with arginine, probably due to a slight destabilizing effect
of the distal amide or carboxylate groups: distal amide or carboxylate
groups are not discriminated by AAP5. That positively charged amino
acids are transported with high apparent affinity is probably a
consequence of their structure rather than the presence of the positive
charge: methionine and citrulline are neutral amino acids. Reducing the
length of the glutamine side chain by one carbon to yield asparagine
dramatically decreased the apparent affinity by at least 3 orders of
magnitude. Thus, an amide group on the
-carbon yields favorable
interactions with the substrate-binding site, whereas an amide group on
the
-carbon is in an unfavorable position for binding.
Groups attached to the -carbon were very important in determining
substrate specificity. Two methyl groups on the
-carbon of valine
decreased the apparent affinity by an order of magnitude over leucine,
which has two methyl groups on the
-carbon. Similarly, the apparent
affinity for isoleucine, phenylalanine, tryptophan, and the imino acids
(proline and hydroxyproline) was reduced by methyl groups, aromatic
residues, or branching at the
-carbon. Adding a methyl group to the
-carbon of serine to give threonine decreased the apparent affinity
by an order of magnitude, whereas a sulfhydryl on the
-carbon
(cysteine) maintained a high apparent affinity. A hydroxyl residue on
the
-carbon was not as restrictive as a methyl group at this
position: the apparent affinities for homoserine and hydroxyproline
were not significantly different from those for serine and proline.
AAP5 had a high apparent affinity for alanine and glycine, which are
not branched at the
-carbon. Aspartate did not interact with AAP5,
suggesting that substrate binding is prevented by a
-carboxylate.
This restriction was relaxed when the side chain was extended by
one carbon to yield glutamate. Asparagine has a similar structure to
aspartate, with a high electron density near the
-carbon.
Thus, the apparent affinity for asparagine was very low (>100
mM).
The imax values for lysine and ornithine were high compared with those for most other amino acids, with the single net positive charge carried by these amino acids contributing to the magnitude of their induced currents. The imaxHis was ~75% of the lysine-induced current. If we remove the contribution of the net single positive charge on lysine and ornithine to the magnitude of their inward currents, then AAP5 transports neutral histidine with the highest maximal transport rate. Positively charged homoarginine and arginine have considerably lower imax values than lysine and ornithine, probably due to the large, terminal guanidinium group, which may restrict their movement through the transporter. Except for tryptophan, the imax values for the other amino acids were between 28 and 51% of imaxLys. The bulky, aromatic side chain on tryptophan probably accounts for the low maximal rate of transport for this molecule.
Why are the charged species of glutamate and histidine excluded by
AAP5? Unpaired oxygens on the deprotonated form of glutamate and
protonation of the imidazole ring of histidine may produce unfavorable
steric interactions that block access to the substrate-binding site.
The positive charge on the protonated imidazole ring lies ~3.6 Å from the -carbon, whereas for arginine, homoarginine, ornithine, and
lysine, the positive charge lies at least 5 Å from the
-carbon.
Although the three-dimensional amino acid structures may be altered in
solution due to hydration, [H+]o at the
substrate-binding site, and interactions with amino acid residues, the
data suggest that charged groups close to the
-carboxyl and
-amino groups prevent substrate binding. Why is the neutral form of
histidine transported with such high apparent affinity? Histidine,
phenylalanine, and tryptophan all have bulky, aromatic rings with high
electron densities close to the
-carbon, yet the
K0.5 values for phenylalanine and tryptophan are
between 150- and 450-fold higher than that for histidine. Unlike
phenylalanine and tryptophan, histidine possesses two nitrogen atoms in
the imidazole ring, which must confer a high apparent affinity for
AAP5. AAP5 has similar apparent affinities for histidine, arginine, and
homoarginine, all of which possess two nitrogen atoms at their distal
end.
How do the results of this study relate to the transport of amino acids in Arabidopsis? Unfortunately, the composition of free amino acids in Arabidopsis is unknown. Aspartate, glutamate, and glutamine are found at high concentrations in many plants: up to 30, 90, and 20 mM, respectively, depending on the plant species (27-29). In contrast, the concentration of lysine is typically low: <1 mM in sugar beet leaves (30) and ~2 mM in barley leaves (28). With the exception of sink leaves, AAP5 is expressed throughout the plant, where it may play a central role in the high affinity transport of lysine. The low affinity amino acids will only be transported by AAP5 if they occur at high concentrations. Since AAP5 does not transport aspartate and since many of the low affinity amino acids such as valine, asparagine, and phenylalanine occur at low concentrations in plants, other members of the AAP family of amino acid transporters are probably responsible for the transport of these amino acids. For example, AAP1 transports valine, asparagine, and aspartate with K0.5 values of ~0.7, 25, and 80 mM.3 It will be interesting to correlate the apparent affinity of each AAP transporter and their expression pattern in Arabidopsis to the abundance of particular amino acids within the plant.
ConclusionsWe have shown that AAP5 recognizes and transports a broad spectrum of amino acids differing in geometry and charge, albeit with different apparent affinities and maximal velocities. Stoichiometry experiments enabled us to determine the charge on the transported amino acid species and showed that AAP5 transports anionic, cationic, and neutral amino acids via the same mechanism, i.e. with a fixed H+:amino acid coupling stoichiometry. Thus, in planta, the energy consumption for H+/amino acid transport will be independent of the net charge on the amino acid. Future experiments will include a detailed investigation of the substrate specificity of other members of the AAP family of transporters and mutant transporters to identify amino acid residues involved in substrate recognition.
We thank Manoli Contreras for the preparation and injection of oocytes and Bruce Hirayama, Don Loo, Eric Turk, Wolf Frommer, and Ernest Wright for useful discussions during the preparation of this manuscript.