Download Membrane Vesicles Isolated from Sugar Beet Leaves

Plant Physiol. (1990) 94, 268-277
0032-0889/90/94/0268/1 0/$01 .00/0
Received for publication April 6, 1990
Accepted June 11, 1990
ApH-Dependent Amino Acid Transport into Plasma
Membrane Vesicles Isolated from Sugar Beet Leaves
1. Evidence for Carrier-Mediated, Electrogenic Flux through Multiple Transport Systems
Zhen-Chang Li and Daniel R. Bush*
Photosynthesis Research Unit, U.S. Department of Agriculture, Agricultural Research Service (D.R.B.), and
Department of Plant Biology (D.R.B., Z. -C.L.), University of Illinois, Urbana, Illinois 61801
ABSTRACT
port those amino acids to the heterotrophic tissues that are
dependent upon imported carbon and nitrogen for normal
growth and development. Amino acid transport in plants is
frequently associated with accumulation against a significant
free energy gradient across the plasma membrane. Several
lines of evidence have linked this active transport process to
the proton electrochemical difference generated by the plasma
membrane proton-pumping ATPase (10, 13, 15, 20, 23-25)
and recently, Bush and Langston-Unkefer (6) provided the
first in vitro evidence demonstrating proton-amino acid symport activity in isolated plasma membrane vesicles.
Most amino acid transport experiments have been conducted with intact tissues or suspension culture cells (2, 11,
13, 15, 17, 18, 20, 25). Although these systems have provided
important insight into the nature of amino acid transport,
this experimental approach is limited by a number of problems which can compromise investigations of membrane
bound transport systems. For example, rapid metabolism of
the transported amino acid can lead to an overestimate of
transport stoichiometries and metabolically linked feedback
control systems may significantly influence transport activity
(3, 20, 24). Furthermore, differential rates of accumulation
by the various cell types present in an intact tissue and the
existence of ill-defined diffusion barriers may further complicate interpretation. Nevertheless, results from studies with
intact tissues and cells have lead to the general conclusion
that amino acid transport is mediated by specific carrier
proteins (2, 7, 11, 13, 14, 20, 21, 25), although progress in
characterizing these transport systems has been slow.
Currently, our knowledge of amino acid transport at the
membrane level is quite limited. This is due not only to the
difficulty encountered in the solubilization and reconstitution
of membrane bound transport proteins, but also to the fact
that the amino acid transport systems in plants are not well
understood (20). For example, an amino acid transport protein has yet to be identified and, furthermore, even the
number of independent transport systems is still unknown (2,
7, 14, 18, 20). These observations suggest a simpler experimental system may be more suitable when examining amino
acid transport at the membrane level.
In the work reported here, we used highly purified plasma
membrane vesicles from sugar beet leaves to study amino
acid transport. Isolated membrane vesicles eliminate many of
Amino acid transport into plasma membrane vesicles isolated
from mature sugar beet (Beta vulgaris L. cv Great Westem) leaves
was investigated. The transport of alanine, leucine, glutamine,
glutamate, isoleucine, and arginine was driven by a trans-membrane proton concentration difference. ApH-Dependent alanine,
leucine, glutamine, and glutamate transport exhibited simple Michaelis-Menten kinetics, and double-reciprocal plots of the data
were linear with apparent Km values of 272, 346, 258, and 1981
micromolar, respectively. These results are consistent with carrier mediated transport. ApH-Dependent isoleucine and arginine
transport exhibited biphasic kinetics, suggesting these amino
acids may be transported by at least two transport systems.
Symport mediated alanine transport was electrogenic as demonstrated by the effect of membrane potential (A*) on ApHdependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When A' was
held at 0 millivolt with symmetric potassium concentrations and
valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative A+, alanine transport increased sixfold. These
results are consistent with an electrogenic transport process
which results in a net flux of positive charge into the vesicles.
The effect of changing AI on the kinetics of alanine transport
altered Vmax with no apparent change in Km. Amino acid transport
was inhibited by the protein modifier diethyl pyrocarbonate, but
was insensitive to N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, p-chloromercuribenzenesulfonic acid, phenylglyoxal, and N,N'-dicyclohexylcarbodiimide. Four amino acid
symport systems, two neutral, one acidic, and one basic, were
resolved based on inter-amino acid competition experiments. One
neutral system appears to be active for all neutral amino acids
while the second exhibited a low affinity for isoleucine, threonine,
valine, and proline. Although each symport was relatively specific
for a given group of amino acids, each system exhibited some
crossover specificity for amino acids in other groups.
The concentration of amino acid nitrogen in the sieve tube
sap of castor bean can be as high as 230 mm. In contrast, the
concentration of inorganic nitrogen in the same sap is less
than 2 mM (22). In this system, as in many other higher plants,
amino acids are the primary form of transported nitrogen.
This is especially true for those plants that incorporate reduced
nitrogen into amino acids in the leaf and subsequently trans268
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PROTON-AMINO ACID TRANSPORT
the experimental problems associated with intact tissues and
allow us to manipulate the composition of both the intra- and
extravesicular solutions. The results presented here, based on
this experimental approach, show that the transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine
is mediated by specific carriers and that the driving force for
this process is a proton electrochemical potential difference.
Furthermore, an analysis of inter-amino acid competition
experiments suggests at least four amino acid transport systems can be resolved.
MATERIALS AND METHODS
Plant Material
Sugar beet (Beta vulgaris L. cv Great Western) seedlings
in a 1:1 mixture of vermiculite and sand. When
the seedlings were 1 inch tall, they were transferred to a
hydroponic system, 2 plants per 13 L, containing one-half
strength Hoagland solution (12) and 1 mM NaCl. Plants were
maintained under a 10 h photoperiod (400 ,uE m-2 s-1) at
24°C, 70% RH, and 14 h night period at 18°C, 70% RH.
Only fully expanded leaves were harvested for experimental
analysis.
were grown
Plasma Membrane Vesicle Isolation
Plasma membrane vesicles were prepared at 4°C as described previously (4). Briefly, 35 g of leaves were homogenized with a polytron homogenizer in 350 mL of 240 mM
sorbitol, 50 mm Hepes, 10 mM KC1, 3 mm EGTA, 3 mM
DTT, and 1% of BSA (final pH was adjusted to 8.0 with solid
1,3-bis[tris(hydroxymethyl)methylamino]propane). The homogenate was filtered through four layers of cheesecloth and
the filtrate was centrifuged at 10,000g for 10 min. The supernatant was then centrifuged at 50,000g for 45 min. The
resulting pellets, containing microsomal vesicles, were resuspended in 3 mL 340 mm sorbitol, 5 mM K-phosphate buffer
(pH 7.8) and 0.1 mm DTT. Plasma membrane vesicles were
purified from the resuspended microsomes with the aqueous
phase partitioning method as described elsewhere (4). After
the final phase separation, the plasma membrane vesicles
were washed in 30 mL resuspension buffer containing 330
mM sorbitol, 2 mm Hepes (pH to 8.0 as above), 10 mm KCI,
and 0.1 mM DTT and pelleted with a 45 min centrifugation
at 50,000g. The final vesicle pellet was resuspended in 1 mL
resuspension buffer (z5 mg protein/mL).
Amino Acid Transport Assay
All amino acids
were
L-form in this study. Amino acid
transport was carried out in an acidic transport solution (200400 tL) at 10°C. The transport solution contained resuspension buffer whose pH was adjusted to 6.0 with solid Mes, 5
,AM valinomycin, 0.1 to 0.5 ,Ci of labeled amino acid and
unlabeled amino acid to the desired final concentration. Experiments were initiated by diluting 10 to 20 ,L of the plasma
membrane vesicles into the acidic transport solution. At predetermined time points membrane vesicles were collected and
269
washed with the Millipore' filtration technique as previously
described (4). Accumulated radioactivity was measured with
scintillation spectrometry. ApH-Dependent transport was calculated from the difference between the transport activity in
the absence and that in the presence of 5 ALM CCCP2. Each
experiment was repeated at least three times with multiple
time points sampled for each treatment. All rates were taken
from the best fit lines generated with linear regression analysis.
The results presented are from a single representative experiment.
It has been suggested that, for the analysis of amino acid
transport kinetics and inter-amino acid competition, it is
preferable to avoid extremely high substrate concentrations
(20, 25). This is especially true in competition experiments if
the competing amino acid shifts the total amino acid concentration into a new concentration range (20). Therefore, in the
experiments reported here, substrate and competitor amino
acid concentrations have been maintained near apparent Km
values for transport systems that are active in the 1 mM
concentration range. We have not explored transport systems
that may be active at concentrations exceeding 2 mm.
Inhibition of Amino Acid Transport by Protein Modifiers
To examine the effect of protein modifiers on amino acid
transport activity, 10 to 25 ,L plasma membrane vesicles
were mixed with an equal volume of uptake buffer containing
twice the desired inhibitor concentration. Control samples
were treated in parallel in the absence of inhibitors. After
incubating at 10°C for 15 min, the samples were diluted into
the standard transport solution and transport activity was
measured as described above.
Inter-Amino Acid Competition Experiments
Transport solutions were prepared as above except that, in
addition to the radiolabeled amino acid whose transport was
monitored, a competing amino acid was included at 1 or 10
times the concentration of the transported substrate. Preliminary experiments showed that the relative activity of specific
amino acids as transport antagonists was unaltered when the
results were computed as either a function of total transport
activity or as a function of ApH-dependent transport activity.
Therefore, to conserve radiolabeled stocks, the results reported
here are based on total transport activity.
Protein Determinations
Proteins were determined by the method of Markwell et al.
(16) after solubilization in 0.02% Na-deoxycholate and precipitation in 6.2% TCA. The detergent and TCA step quan'Mention of a trademark, proprietary product, or vendor does not
constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or the University of Illinois and does not
imply its approval to the exclusion of other products or vendors
which may be suitable.
2 Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DEPC, diethyl pyrocarbonate; AAH', proton electrochemical
potential difference; A'I, membrane potential
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Plant Physiol. Vol. 94, 1990
Li AND BUSH
270
titatively recover low concentrations of membrane protein
(1).
RESULTS
c
tE
0-
Carrier Mediated and ApH-Dependent Amino Acid
Transport
It has been shown previously that plasma membrane vehicles isolated from mature leaves of sugar beet by the aqueous
two phase partitioning method are highly purified (4). When
these plasma membrane vehicles (equilibrated at pH 8.0) were
diluted into an acidic transport solution at pH 6.0, a stable
trans-membrane proton gradient was established and this
proton electrochemical potential was successfully used to
drive a proton-sucrose symport (4). In this report, the same
experimental approach was used to examine proton-amino
acid symport activity. Amino acid transport was linear for at
least 4 min and exhibited the same ApH-dependence as
previously demonstrated for sucrose flux and for alanine
transport in zucchini plasma membrane vehicles (4, 6). Although transport was linear as a function of time over the
course of each experiment, occasionally a positive, nonzero
intercept of the y axis was observed. We attribute this to a
rapid, initial influx through porters that are poised for substrate binding and translocation.
ApH-dependent alanine, leucine, glutamine, and glutamate
transport exhibited saturable, concentration dependent influx
(Figs. 1, 2, 3, and 4) that is consistent with carrier-mediated
transport. The Lineweaver-Burk plots of these data were
linear, yielding apparent Km values of 272, 346, 258, and
1981 AM, respectively (Figs. 1, 2, 3, and 4, insets). In contrast,
ApH-dependent isoleucine and arginine transport was bi-
_
tCLE
o c
Co
Y-'
Q
a
C-
4 E
S
0
200
400
600
800
1000
Alanine (IM)
Figure 1. Concentration dependence of ApH-dependent alanine
transport into plasma membrane vesicles isolated from sugar beet
leaves. Concentrated plasma membrane vesicles were diluted into
an acidic transport solution and subsequently collected and washed
with the Millipore filtration technique. ApH-Dependent transport was
defined as the difference between transport activity in the absence
and in the presence of of 5 AM CCCP. The inset shows a LineweaverBurk plot of the data with an apparent Km = 276 + 46 AM.
Coe
0.
.-
0
C
E
00
Cj
0
200
400
600
Leucine (pM)
800
1000
Figure 2. Concentration dependence of ApH-dependent leucine
transport into plasma membrane vesicles. Apparent Km = 346 + 25
JIM.
phasic (Figs. 5A and 6A) and the Lineweaver-Burk plots of
these data yielded evidence of high affinity (apparent Km
values 140 Mm) and low affinity (apparent Km values, 1.61.9 mM) transport systems, suggesting more than one carrier
is capable of transporting these two amino acids (Figs. 5B and
6B).
~
Electrogenicity of Alanine Transport
Since ApH-dependent amino acid transport is coupled to
the cotransport of protons into the vesicle, this transport
process should be electrogenic. To explore this possibility, the
affect of membrane potential on ApH-dependent amino acid
transport was examined. The membrane potential of the
isolated plasma membrane vesicles was altered with potassium
gradients and valinomycin. In the presence of 10 mm KCI on
both sides of the vesicle membrane a low rate of alanine
transport was recorded (Fig. 7A). If, however, valinomycin
was included in the transport solution, the resulting increase
in potassium conductance clamped the membrane potential
at zero and a fourfold stimulation in transport activity was
observed (Fig. 7A). When KCl-loaded vesicles were diluted
into a potassium free transport solution in the presence of
valinomycin, a negative membrane potential developed, as
previously demonstrated (5), due to the steep potassium diffusion potential. Under these conditions, alanine transport
was stimulated sixfold (Fig. 7A). We conclude from these
results that alanine transport was electrogenic. Similar changes
in transport activity as a function of membrane potential were
also noted for leucine. A Lineweaver-Burk plot of these data
showed that the apparent Km for alanine was unaffected by
changes in the membrane potential (Fig. 7B).
Inhibition of Amino Acid Transport by Protein Modifiers
Several reagents that modify protein amino acid residues
were tested for their ability to inhibit ApH-dependent amino
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'
PROTON-AMINO ACID TRANSPORT
oE
* 0.
Co
E-
0
0_
=
E
200
400
600
800
Glutamine (pM)
Figure 3. Concentration dependence of ApH-dependent glutamine
transport into plasma membrane vesicles. Apparent Km = 258 + 59
JIM.
acid transport. DEPC was the only compound that inhibited
the transport of Ala, Leu, Gln, Glu, and Ile. In contrast, the
transport of these amino acids was insensitive to 15 min
pretreatments at 10°C with N-ethylmalemide (2.0 mM), 4,4'diisothiocyano-2,2'-stilbene disulfonic acid (2.0 mM), p-chloromercuribenzenesulfonic acid (1.0 mM), N,N'-dicyclohexylcarbodiimide (0.1 mM), or phenylglyoxal (1.0 mM). The inactivation of alanine transport by DEPC was concentration
dependent (Fig. 8) and the I1o ofinactivation was 1 mm DEPC.
These results suggest that the imidazole ring of a histidine
residue is important in proton-coupled amino acid transport.
Amino Acid Transport Competition
Alanine, leucine, glutamate, arginine, isoleucine, and glutamine were chosen as the focus of a broad study of interamino acid transport competition. These amino acids were
selected as representatives of the neutral, acidic, and basic
amino acids. The inhibition of alanine and leucine transport
by the other 20 common amino acids is shown in Table I.
From these results, the 20 amino acids can be divided into
two groups. The first group includes the acidic amino acids
(Glu, Asp), basic amino acids (Arg, Lys, and His), and five
neutral amino acids (Thr, Ile, Val, Pro, and OH-Pro). In this
group, little or no inhibition was observed at equimolar
concentrations, although some inhibition (from 10 to 20%)
was observed when the concentration of the competing amino
acid was an order of magnitude higher than the transported
substrate. The second group identified in this experiment
(Table I) included the remaining neutral amino acids (Ala,
Leu, Ser, Gly, Met, Cys, Gln, Asn, Tyr, Phe, and Trp) that
exhibited intermediate to strong inhibition of alanine and
leucine transport.
When glutamate and arginine transport were measured in
the presence of other amino acids, the acidic and basic amino
acids can be separated into two distinct groups (Table II). The
basic amino acids had no effect on glutamate transport,
271
whereas aspartate showed strong inhibition. Generally, those
amino acids that significantly decreased alanine and leucine
transport showed little inhibition of glutamate transport, although methionine, alanine, leucine, glutamine, and asparagine exhibited some activity. In contrast, the acidic and aromatic amino acids showed no inhibition of arginine transport
and a moderate inhibition of arginine transport was observed
for the remaining neutral amino acids, with the exception of
asparagine. These results (Table II) suggest that the acidic and
basic amino acids are transported through independent transport systems and that each system exhibits some sensitivity
to different groups of neutral amino acids.
To examine whether the neutral amino acids are transported by more than one transport system, the effect of the
other common amino acids on isoleucine and glutamine
transport was also examined (Table III). All neutral amino
acids were effective inhibitors of isoleucine transport while
the basic and acidic amino acids had little impact. Although
the pattern of sensitivity was similar for glutamine transport,
it is noteworthy that several neutral amino acids (Thr, Val,
Ile, and Pro) exhibited little transport inhibition. These neutral amino acids also displayed little activity against alanine
and leucine transport (Table I). Based on these observations,
we conclude two neutral amino acid transport systems are
active in the isolated plasma membrane vesicles.
DISCUSSION
Isolated plasma membrane vesicles and imposed proton
electrochemical potential differences (AAH+) were used in the
present investigation to study the transport of alanine, leucine,
glutamate, arginine, glutamine, and isoleucine. This group of
amino acids includes representatives of the various structural
and charge configurations displayed by this biologically important class of compounds. The transport of each of these
amino acids was driven by an imposed proton concentration
difference and each exhibited concentration dependent influx
that was consistent with one or two saturable transport sys800
c 600
0E
c
0
- C. 400
E E
a
C. 200
=
0
0
400
800
Glutamate (pM)
1200
1600
Figure 4. Concentration dependence of ApH-dependent glutamate
transport into plasma membrane vesicles. Apparent Km = 1981 +
272 AM.
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272
Plant Physiol. Vol. 94, 1990
Li AND BUSH
800
,
s
E 600
a. c
(go
"L 400
._
m
0)
<,o E0. 2000
_
_
0
0
400
800
1200
1600
Isoleucine (gM)
,-
0.01
-0.00
0.02
0.01
0.03
0.04
11S
Figure 5. Concentration dependence of ApH-dependent isoleucine
transport into plasma membrane vesicles. (A) Concentration-dependent uptake. (B) Lineweaver-Burk plot. Transport was biphasic with a
high affinity component (K, = 156 ± 27 gM) and a low affinity
component (Km = 1925 ± 579 ltM).
potential would contribute to the total proton electrochemical
potential difference. These results are consistent with electrogenic transport and support previous electrophysiological
studies using intact cells (10, 13, 15).
Interestingly, the impact of membrane potential on alanine
transport altered Vmax with no apparent effect on Km (Fig.
7B). This suggests that the binding affinity of the porter was
not sensitive to AI. We conclude from these results that AI
exerts its influence over a critical charge translocation step,
which may be rate limiting, in the translocation process.
Similar results were also observed for the proton-sucrose
transport system in plasma membrane vesicles isolated from
sugar beet leaves (5).
The inhibition of amino acid transport by various compounds that chemically modify specific amino acid residues
within proteins was investigated. Only DEPC, which forms
covalent bonds with the imidazole ring of histidine, inhibited
the transport of each amino acid examined. Inhibition of
amino acid transport by DEPC suggests that a histidine residue may play an important role in the reaction sequence
which couples proton and amino acid transport. Similar
sensitivity to DEPC has been observed for the leaf protonsucrose symport and for the proton-lactose symport of Escherichia coli (4, 19). Although N,N'-dicyclohexylcarbodiimide
was an effective inhibitor of alanine transport in zucchini
hypocotyl plasma membrane vesicles (6) and p-chloromer0.40
0.
tems (Figs. 1-6). Since we limited our study to substrate
concentrations less than 2 mm, linear rates of flux in a higher
concentration range (e.g. 5-50 mM) cannot be excluded. The
data presented here are similar to those previously reported
for alanine transport into membrane vesicles isolated from
zucchini hypocotyls (6) and, taken together, we conclude that
amino acid transport across the plant plasma membrane is
mediated by proton-coupled cotransport systems.
ApH-dependent alanine transport was electrogenic. This
conclusion was based on experiments wherein the membrane
potential of the isolated vesicles was altered with potassium
concentration differences and valinomycin. Under conditions
of minimum charge compensation, low rates of amino acid
transport were recorded (Fig. 7A). When the membrane potential was clamped at zero or set at a negative potential,
alanine transport was stimulated four- and sixfold, respectively. These data are consistent with an electrogenic transport
process which results in the net flux of positive charge into
the vesicle. In the absence of significant charge compensation,
lower relative rates of flux would be expected due to the build
up of positive charge as protons enter the vesicle with alanine.
Charge accumulation results in the generation of a positive
membrane potential that thermodynamically brakes the symport. In vesicles clamped at zero potential, where potassium
ions exit the vesicle as protons enter, this would not occur.
Similarly, in vesicles clamped with a negative potential, increased rates of flux would occur since a negative membrane
co
.=E
ZsE
4 308
1
1
400
1200
1600
0.200.10
0.000
800
Arginine (gM)
B
12.0-
8.0
4.0
0.0-
.
-0.00
l/S
.
0.01
0.02
Figure 6. Concentration dependence of ApH-dependent arginine
transport into plasma membrane vesicles. (A) Concentration-dependent uptake. (B) Lineweaver-Burk plot. Transport was biphasic with a
high affinity component (Km = 136 ± 26 AM) and a low affinity
component (Km = 1657 ± 1458 jAM).
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PROTON-AMINO ACID TRANSPORT
U
A
c
E
-Q
a -C
6.0
00
-0.
s0
'c0 cm
/
4.0
'E
E 2.0
/
c
w/
0.0
0
200
400
600
8t 10
200
400
600
800
Alanine (pM)
2.0
response to changes in At, was not examined in these intact
cell systems. In contrast, such indirect interactions were easily
controlled for in the experiments reported here and, based on
our observations with isolated plasma membrane vesicles, we
conclude that four independent amino acid transport systems
can be resolved.
Two neutral amino acid symport systems were identified
in this study. One system appears to transport all neutral
amino acids while the second system, which also exhibits
broad specificity, does not transport Thr, Val, Ile, and Pro.
These four neutral amino acids displayed a unique pattern of
transport inhibition. Although they were effective transport
inhibitors within their own group (i.e. Val, Thr, and Pro
inhibition of Ile: Table III), they were not competitive against
alanine, leucine, and glutamine transport (Tables I and III).
In contrast, isoleucine transport was sensitive to all neutral
amino acids. Therefore, we conclude two neutral amino acid
transport systems are active in isolated plasma membrane
vesicles. It is noteworthy that threonine, isoleucine, and valine
also exhibited a unique pattern of transport characteristics in
B
Chlorella vulgaris (21).
1.0
00o
273
~
-
,
-0.000
0.004
0.008
Vs
Figure 7 Concentration dependence of ApH-dependent alanine
transport as a function of membrane potential. Plasma membrane
vesicles Mvere diluted into an acidic transport solution in the presence
(0, U) or absence (0) of 10 mm KCI and in the presence (, 0) or
absence (0) of 5 mM valinomycin. These treatments resulted in
rh_r
rmraneainrI_i
1 Arejlm"rwi
meMDrane poILeniLai
m1nimum cnarge
ciampea
compensaxlontm), ae r%mnmkr,%no
(U), and a negative membrane potential (0). (A) Concentration-dependence of Aph-dependent alanine transport. (B) Lineweaver-Burk plots of the data from A.
at 0 mV
curibenzenesulfonic acid was a potent inhibitor of the protonsucrose symport of sugar beet (4), these compounds exhibited
little activity against the amino acid transport systems found
in plasma membrane vesicles isolated from sugar beet leaves.
The number of carriers which mediate amino acid transport
into the higher plant cell has yet to be determined and, in
fact, this number is rather controversial. Some workers suggest
a single carrier is responsible for the influx of all amino acids
while others have proposed two or three separate systems (2,
8, 11, 14, 18, 20, 21, 25). This situation is not surprising
because in many of the experiments these conclusions are
based on utilized intact tissues or cultured cell systems. Intact
tissues and cells can contribute to experimental ambiguity
because it is difficult sometimes to differentiate between specific mechanisms of interaction (7, 20). For example, the
observation that the transport of any single amino acid is
inhibited in the presence of any one of the other 20 amino
acids has been interpreted as evidence for a single carrier with
broad specificity ( 1 8). However, the possibility that decreased
transport in the presence of another amino acid may be the
result of indirect effects, such as competition for ApH or in
The specific activity of the neutral amino acid transport
systems varies with tissue and species (Z-C Li, DR Bush,
unpublished data). In sugar beet leaf plasma membrane vesicles,
d , .alanine transport is >10 times higher than isoleucine
transport. In cucumber leaf plasma membrane vesicles, alanine transport is only twice as high. This variation in the
relative abundance of alanine and isoleucine transport provides further evidence in support of the notion that multiple
neutral amino acid transport systems are active in the plant
plasma membrane. Whether these systems are differentially
expressed within single cells or between specific tissue types
cannot be deduced from the heterogeneous plasma membrane
vesicles used in these experiments.
Acidic amino acids were transported by a third system.
This is supported by the fact that glutamate transport was not
inhibited by the basic amino acids (Table II) and little or no
inhibition was exerted by most of the neutral amino acids. In
contrast, aspartate was an effective inhibitor of glutamate
100
-
0-
a..
(U
0
C
-
-o
o-
0.0
1.0
3.0
DEPC (mM)
2.0
4.0
5.0
Figure 8. Concentration dependence of DEPC inactivation of ApHdependent alanine transport. Plasma membrane vesicles were pretreated with DEPC for 15 min and then diluted into the acidic transport
solution containing 100 yM alanine, 0.2 ACi [14C]L-alanine, 5 4M
valinomycin, and plus or minus 5 Mm CCCP. Transport activity was
expressed as a percentage of the control.
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274
LI AND BUSH
Plant Physiol. Vol. 94, 1990
Table I. Inhibition of Alanine and Leucine Transport by Different Amino Acids
Plasma membrane vesicles were diluted into the transport solution containing 100 AM of [14C]alanine
or [14C]leucine and the antagonist amino acid at 1 or 10 times the concentration of the radiolabeled
substrate. Transport activity was determined after 1 min as described in the "Materials and Methods."
Antagonist Amino
Acid
Controlb
Glutamate
Aspartate
Lysine
Histidine
Arginine
Threonine
Proline
OH-Proline
Valine
Isoleucine
Asparagine
Glutamine
Serine
Cysteine
Methionine
Glycine
Alanine
Antagonist Concentration
1x
lOx
% alanine transport'
100
99 ± 4
65 ± 10
94 ± 3
71 ± 3
100 ± 6
87 ± 9
94 ± 6
85 ± 12
93 ±13
65 ± 12
96 ± 1
88 ± 8
98±2
91±2
102 ± 5
95 ± 4
101±3
89±4
107 ± 2
91 ± 4
85 ± 1
48 ± 7
75 ± 16
33 ± 5
84± 11
41±7
80 ± 6
36 ± 7
55 ± 5
20 ± 5
97 ± 5
56 ± 5
1x
lOx
% leucine transport'
100
93 ± 3
83 ± 11
106 ± 4
95 ± 16
97 ± 2
105 ± 7
106 ± 2
92 ± 5
99 ± 2
81 ± 6
96 ± 4
83 ± 16
104±6
90±8
108 ± 8
89 ± 4
99±4
94± 14
103 ± 3
87 ± 16
90 ± 5
47 ± 8
77 ± 5
38 ± 6
85+7
53±9
80 ± 6
40 ± 7
52 ± 4
23 ± 9
92 ± 2
54 ± 5
74 ± 5
36 ± 4
Leucine
83 ± 10
36 ± 6
Tyrosine
91 ± 2
67 ± 2
86 ± 4
53 ± 2
Tryptophan
89 ± 7
52 ± 9
90 ± 2
47 ± 7
Phenylalanine
93 ± 7
56 ± 7
88 ± 2
52 ± 6
Sucrose (1 mM)
91 + 3
96 + 4
a
b
The results are presented as mean % ± SD (n = 3) of alanine or leucine transport.
Control rates:
Ala = 2.2 nmol/mg protein/min; Leu = 1.1 nmol/mg protein min.
transport, suggesting this pathway is specific for acidic amino
acids. When the competing amino acid concentration was
increased 10-fold higher than that of glutamate, however, a
few neutral amino acids partially inhibited glutamate uptake,
suggesting that these amino acids have a measurable affinity
for the acidic transport system. Asparagine and glutamine, at
10 times the concentration of glutamate, were especially
competitive against glutamate transport (Table II). These two
neutral amino acids exhibit some structural similarity to
glutamate and, thus, they may share the glutamate transport
system.
The fourth transport system appears to be mainly responsible for the basic amino acids. Since glutamate and aspartate
do not inhibit arginine transport (Table II), this porter is not
shared by the acidic amino acids. However, this system is
shared differentially by the neutral amino acids. Most neutral
amino acids exhibited little evidence of transport competition,
yet alanine and methionine were very effective. The two
groups of neutral amino acids that were evident in Table I
can not be distinguished based on the inhibition of arginine
transport (Table II). Such differential sensitivity is consistent
with multiple transporters. The inhibition of arginine transport by lysine and histidine was low at the concentrations
tested. One explanation for this is that there may be more
than one system for the basic amino acids. Further study is
needed to resolve this observation.
Sucrose was used as an internal control in each competition
experiment and it showed little or no inhibition of amino
acid transport (Tables I-III). Testing the effect of sucrose on
amino acid transport was important because it demonstrates
that competition for the proton motive force is not a reasonable explanation for the amino acid-dependent transport inhibition observed in the competition studies reported here.
Since sucrose is actively transported by a proton-symport in
the plasma membrane vesicles used in these experiments, any
significant sucrose-dependent decrease in AILH+ would have
resulted in a change in amino acid flux. The absence of such
a decrease suggests symport activity is too slow to significantly
alter AAH' over the time course of the competition experiments. Since these experiments were also performed with the
membrane potential clamped at zero, we conclude that amino
acid-dependent decreases in transport activity is due to direct
interactions at the transport proteins and cannot be assigned
to indirect effects on ApH or membrane potential.
The resolution of four transport systems was based on the
results of inter-amino acid competition (Tables I-III). It
should be noted, however, that considerable variation exists
within these broad classifications. For example, some amino
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PROTON-AMINO ACID TRANSPORT
275
Table II. Inhibition of Arginine and Glutamate Transport by Different Amino Acids
Plasma membrane vesicles were diluted into the transport solution containing 50 lM [14C]arginine or
25 /M [14C]glutamate and the antagonist amino acid at 1 or 10 times the concentration of the radiolabeled
substrate. Transport activity was determined after 2 min of uptake.
Antagonist Concentration
Antagonist
lx
lOx
% arginine transporta
1x
lox
% glutamate transporta
100
Controlb
100
Glutamate
96 ± 2
90 ± 3
Aspartate
95 ± 1
104 ± 11
76 ± 2
63 ± 3
77±4
104±16
Lysine
93±1
95±2
87 ± 2
Histidine
88 ± 3
83 ± 1
96 ± 3
94 ± 1
111 ± 12
Arginine
84 ± 1
Threonine
70 ± 8
75 ± 14
83 ± 2
97 ± 4
88 ± 7
93 ± 4
105 ± 11
Proline
97 ± 3
105 ± 8
104 ± 7
OH-proline
111 ± 10
91±2
84±3
100±16
Valine
98±2
94 ± 8
85 ± 4
96 ± 2
Isoleucine
111 + 11
91 ± 4
101 ± 10
83 ± 7
63 ± 12
Asparagine
62 ± 9
77 ± 10
66 ± 2
Glutamine
90 ± 1
91 ± 1
58 ± 7
87 ± 7
Serine
106 ± 15
94 ± 2
56 ± 8
92 ± 9
82 ± 12
Cysteine
79 ± 7
48 ± 7
79 ± 5
72 ± 15
Methionine
89±3
61±7
89±7
77±14
Glycine
82 ± 1
77 ± 4
77 ± 8
Alanine
46 ± 2
84± 16
Leucine
56±6
91±3
91±3
Tyrosine
95±2
70±3
103±1
88±5
87 ± 7
93 ± 3
92 ± 6
124 ± 17
Tryptophan
95 ± 1
89 ± 8
98 ± 2
122 ± 9
Phenylalanine
91 2
Sucrose ( 1 mM)
100 4
b
±
Control
Results are presented as mean % SD (n = 3) of glutamine or isoleucine transport.
rates: Arg = 0.06 nmol/mg protein min; Glu = 0.02 nmol/mg protein/min
acids inhibited the transport of a given amino acid to a lesser
extent than other amino acids within the same group. Methionine, alanine, and cysteine were strong inhibitors of leucine
and glutamine transport (Tables I and III) while glycine and
serine were not as effective. Similarly, glutamine and asparagine were stronger inhibitors ofglutamate transport than were
the other neutral amino acids. The broad specificity of the
four transport systems identified here has also been observed
in the major amino acid transport systems described in animal
cells (9). Ultimately, differential inhibition of specific symports or purification and characterization of individual systems will be necessary to unequivocally determine the number
of amino acid transport systems in the plasma membrane of
sugar beet leaves.
Although the inter-amino acid transport competition studies reported here suggest four amino acid transport systems
are present in isolated plasma membrane vesicles, this conclusion is not always consistent with the concentration-dependent transport kinetics reported in Figures 1 to 6. For example,
concentration-dependent alanine transport exhibited simple
saturation kinetics and yielded linear Lineweaver-Burk plots
of the data that are consistent with a single transport system
within the concentration range tested (Fig. 1). Yet, alanine
was a potent transport inhibitor of amino acids representing
three of the four symports reported here. If alanine is transported through all three systems, why did not this show up as
complex, multiphasic transport kinetics in Figure 1 (note that
the amino acid concentrations used in all inter-amino acid
competition experiments were kept within the same range as
those used in the kinetic experiments to avoid complications
from low affinity porters). One possible explanation for these
data is that the apparent Km for alanine for all three symports
is approximately the same and/or that a high Vma for the
dominant alanine symport overshadows the other transport
systems. An alternative explanation is essentially a special
case of the above wherein alanine binds to the active site of
each symport but this binding is not translated into efficient
translocation into the vesicle. As a small neutral amino acid
it is possible that alanine can enter the active site of a symport
system without binding in the necessary configuration that
results in translocation across the membrane. Yet, the presence of alanine in the active site would competitively limit
access to the natural substrate and result in a significant
decrease in transport. This kind of binding would not show
up in alanine transport kinetics and this explanation is consistent with reports that alanine is a potent, across the board
inhibitor of amino acid transport in a variety of experimental
system (14, 25). In contrast to the alanine data, the biphasic
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Li AND BUSH
276
Plant Physiol. Vol. 94, 1990
Table ll. Inhibition of Glutamine and Isoleucine Transport by Different Amino Acids
Plasma membrane vesicles were diluted into the transport solution containing 100 AM [14C]glutamine
or 25 AM [14C]isoleucine and the antagonist amino acid at 1 or 10 times the concentration of the
radiolabeled substrate. Transport activity was determined after 2 min of uptake.
Antagonist Concentration
Antagonist
Controlb
Glutamate
Aspartate
Lysine
Histidine
Arginine
Threonine
Proline
OH-proline
Valine
Isoleucine
Asparagine
Glutamine
Serine
Cysteine
Methionine
Glycine
Alanine
Leucine
Tyrosine
Tryptophan
lox
1x
% glutamine transport'
100
100 ± 1
73±2
87±12
83±7
101 ±3
104±4
99 ±1
93±5
100±3
109±6
110±8
109 ± 1
98±3
101±8
92±5
77±1
81±1
86±2
97±3
89±4
89 ± 2
51±1
92±3
90 ±5
60±4
99 ± 6
81±3
90±1
93±1
57±4
57±4
24±2
61±1
97±4
35±0
47±2
58±0
57±3
61 ± 5
lox
1x
% isoleucine transporta
100
91±2
98±2
100±5
103±13
102±7
88± 15
74±7
85±4
104±4
78±5
103±12
101±2
87±5
81±9
43±3
54±4
104±2
45±1
89±10
80±13
72±3
62±15
43±2
84±6
55±4
63±10
95±2
97±10
64 ± 4
70±1
46±2
47±7
34±6
22±2
44±4
27±2
31±3
60±2
80±3
40 ± 1
97 ± 4
Phenylalanine
104 1
Sucrose (1 mM)
98±6
±
=
a Results are presented as mean %
SD (n 3) of glutamine or isoleucine transport.
rates: Gln = 1.1 nmol/mg protein/min; lie = 0.05 nmol /mg protein/min.
transport kinetics of isoleucine and arginine
are
consistent
with the inter-amino acid competition experiments that suggest more than one transport system is present in the isolated
membrane vesicles.
CONCLUSION
Isolated plasma membrane vesicles and imposed proton
electrochemical potential differences were used to examine
proton-coupled amino acid transport. ApH-dependent alanine, leucine, glutamine, glutamate, isoleucine, and arginine
transport exhibited saturable transport kinetics and sensitivity
to protein modification. Alanine transport was electrogenic
and both components of AAH', ApH and At, contributed to
b
Control
transport activity. From these results, we conclude that amino
acid transport is mediated by specific proton-amino acid
symports.
The results of inter-amino acid competition experiments
suggest at least four transport systems can be resolved in
plasma membrane vesicles isolated from sugar beet leaves
(Table IV). Although a given transport system may be the
primary porter for a specific group of amino acids, the data
reported here also suggest that structural similarity between
the various amino acids results in an unusual lack of specificity. Since many soluble enzymes that interact with amino
acids do not exhibit such broad specificity, structural similarity is not an impenetrable biophysical barrier. We conclude,
therefore, that evolutionary pressures have selected for gen-
Table IV. Summary of Amino Acid Transport Systems in the plasma membrane of sugar beet leaves.
Four amino acid transport systems were resolved here based on transport kinetics and inter-amino
acid competition.
Transport System
Amino Acid Transported
Neutral system (low abundance)
All neutral amino acids
Neutral system 11 (high abundance)
All neutral amino acids, but low affinity for
lie, Thr, Val, and Pro
Acidic system
Glu, Asp
Basic system
Arg, Lys, His
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PROTON-AMINO ACID TRANSPORT
eralized amino acid transport systems rather than highly
specific, single substrate porters.
12.
ACKNOWLEDGMENT
13.
We thank Cara Ripperda for her expert technical assistance.
14.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
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