From the
Three amino acid transporter genes (AAP3-5) were
isolated from Arabidopsis by complementation of a yeast mutant
defective in histidine uptake. Transport is driven against a
concentration gradient and sensitive to protonophores. Analysis of the
substrate specificity demonstrates that the carriers have a broad
substrate specificity covering the major transport forms of reduced
nitrogen, i.e. glutamine and glutamate. The transporters have
similar affinities for glutamate, glutamine, and alanine but differ
with respect to valine, phenylalanine, histidine, arginine, and lysine.
AAP3 and AAP5 efficiently transport arginine and lysine and are
involved in basic amino acid transport. The predicted polypeptides of
53 kDa are highly hydrophobic with 12 putative membrane-spanning
regions and show significant homologies to Arabidopsis amino
acid transporters AAP1 and AAP2. Each of the genes has a different
organ-specific expression in the plant. AAP3 is exclusively
expressed in roots and AAP4 mainly in source leaves, stems,
and flowers, whereas AAP5 is found in all tissues. The
specific distribution in the plant and the different substrate
specificities of AAP transporters may indicate that tissues differ both
qualitatively and quantitatively regarding import or export of amino
acids.
In most plants, the major transport forms for organic nitrogen
are amino acids. A spectrum of different amino acids is found in both
phloem and xylem. Roots or leaves can be sources for amino acids
cycling through the plant, allowing withdrawal in tissues that are
dependent on external supply, e.g. seeds (Pate, 1975).
Composition of phloem and xylem sap and of mesophyll cytosol are highly
similar, indicating that nonspecific loading processes are involved
(Jeschke et al., 1991; Riens et al., 1991). Evidence
for carrier-mediated phloem loading with sucrose has been shown for
several plants including potato, tobacco, and Arabidopsis (von
Schaewen et al., 1990; Riesmeier et al., 1994). It is
therefore probable that similar mechanisms are used for amino acid
transport. Transport measurements have provided evidence for the
presence of low and high affinity amino acid transport systems in a
number of plant species (for recent reviews see Bush(1993); Frommer et al. (1994b)). Analysis of amino acid transport mutants has
indicated that in Nicotiana leaves a single locus is
responsible for uptake of a broad spectrum of amino acids (Marion-Poll et al., 1988). Uptake studies in plasma membrane vesicles from
sugar beet leaves have revealed complex kinetics that were interpreted
to result from the activity of at least four different transporters
specific for acidic, neutral, or basic amino acids (Li and Bush, 1990).
In all cases, transport was active and proton-coupled (Bush, 1993).
Several amino acid transporter genes have been isolated from Arabidopsis by complementation of yeast transport mutants
defective in the uptake of certain amino acids. Up to now, three
families of proteins capable of transporting amino acids have been
found. All members of the first family of permeases (AAPs) transport a
broad spectrum of amino acids and probably function as proton
cotransporters (Frommer et al., 1993; Hsu et al.,
1993; Kwart et al., 1993). A second class of proteins, AAT, is
related to mammalian amino acid transporters and represents a high
affinity, broad specificity system.
Using a sensitive selection system in a yeast
histidine transport mutant, three new amino acid transporters were
identified. To analyze this apparent redundance, sequences were
compared with known amino acid transporters, substrate specificity was
analyzed, and the expression pattern in the plant was determined.
Plants preferentially use certain amino acids or amides such
as glutamic and aspartic acid, glutamine, and aspartate as major
transport forms for long distance translocation (Peoples and Gifford,
1990). Amino acids circulate through the vascular system with multiple
possibilities for carrier-mediated interchange between phloem and xylem
(Feller and Hoelzer, 1991; Pate, 1975). There is a long standing debate
on number and specificity of transport systems involved. The best
evidence derives from an analysis of Nicotiana mutants
resistant to high concentrations of valine due to the lack of high
affinity valine uptake (Marion-Poll et al., 1988). The mutants
are resistant not only to valine but also to a variety of other amino
acids coinciding with a reduction in uptake of acidic and neutral amino
acids, whereas lysine uptake was only slightly affected (Borstlap
1985). These data strongly suggest a mutation in a high affinity, broad
specificity transporter. The complex results of kinetic analyses of
amino acid uptake into plasma membrane vesicles from leaves could be
the result of multiple overlapping broad specificity systems (Li and
Bush, 1990). The data have, however, been interpreted as evidence for
the presence of individual transporters specific for acidic, basic, and
neutral amino acids. Further support for the hypothesis of broad
specific transporters in leaves originates from physiological analyses.
Evidence has been presented that the cytosolic amino acid composition
of mesophyll cells determines the composition of phloem sap (Riens et al., 1991). The simplest explanation for such a
nonselective process would be the presence of broad specificity
transporters.
AAP4 and AAP5 are expressed in mature leaves, indicating a role in phloem
loading of amino acids. This is supported by the finding that, similar
to the sucrose transporter, the expression of AAP4 and AAP5 is low in young leaves not yet capable of export
(Riesmeier et al., 1993). None of the transporters identified
so far is expressed in sink leaves. However, a further member of this
family (AAP6) that was isolated by a different approach is
expressed at higher levels in sink as compared with source
leaves.
AAP3 transcripts were exclusively
found in roots where the transporter might function in uptake and
retrieval of amino acids from the soil. Based on the finding that
aminoethylcysteine-resistant mutants of barley were deficient only in
the uptake of basic amino acids into roots, a high affinity transport
system for basic amino acids was postulated (Bright et al.,
1983). Provided that roots contain both general amino acid transporters
such as AAP3 and transporters that transport only acidic and neutral
amino acids, a deficiency in the barley counterpart of AAP3 could also
explain the lack of basic amino acid uptake since the second system
would still be present and able to transport acidic and neutral amino
acids. AAP5, which is expressed only at low levels in roots, on the
other hand could serve as a transporter for basic amino acids in other
tissues.
It has been shown previously that AAP1 and AAP2 were preferentially expressed in developing siliques and
in the vascular system of cotyledons, indicating that both transporters
on one hand are involved in the supply of developing seeds with organic
nitrogen and on the other hand in remobilization of stored nitrogen
from developing seedlings (Kwart et al., 1993).
Identity is indicated above the diagonal,
and similarity is indicated below.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
We are very grateful to Jean Claude Jauniaux
(Université Libre de Bruxelles, Belgium), Per Ljungdahl, and
Gerald Fink (MIT, Cambridge, MA) for providing the yeast mutants and
Michèle Minet and Francois Lacroute (Centre National de la
Recherche Scientifique, Gif sur Yvette, France) for the excellent Arabidopsis cDNA library.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)A third
system, NTR1, transports histidine with low efficiency but peptides
with high affinity (Frommer et al., 1994a).
(
)
Materials
Saccharomyces cerevisiae strain 22574d (MAT-, gap 1-1, put 4-1, uga
4-1, ura 3-1) (Jauniaux et al., 1987) and strain
JT16 (Mat-a, hip1-614, his4-401, can1, ino1, ura3-52)
(Tanaka and Fink, 1985) were used. Arabidopsis thaliana L.
Heynh. ecotype C24 was used.
Yeast Growth, Transformation, and Selection
The
yeast strain JT16 was transformed with an expression library derived
from Arabidopsis seedlings (Dohmen et al., 1991;
Minet et al., 1992). Transformants were selected on SC medium
supplemented with 6 mM histidine. For nonselective conditions,
the medium was supplemented with 30 mM histidine. Colonies
that were able to grow were reselected in liquid medium, and plasmid
DNA was isolated and reintroduced into the mutant JT16. AAP3-5 were able to restore the growth of the mutant on
6 mM histidine. The proline uptake-deficient yeast strain
22574d was transformed with the cDNAs AAP1-5 (this
paper; Kwart et al., 1993). Transformants were selected on
nitrogen-free medium (Difco) containing 0.5 mg/ml L-proline as
nitrogen source.
DNA Work
cDNAs were excised with NotI and
subcloned into pBluescript SK (Stratagene,
Heidelberg), and both strands were sequenced. Sequences were analyzed
using UWGCG programs (Devereux et al., 1984). Southern and
Northern blot analysis were performed as described (Frommer et
al., 1994a).
Transport Measurements
For uptake studies, yeast
cells were harvested at A = 0.5, washed,
and resuspended in 50 mM potassium phosphate (pH 4) to a final A
= 12. To start the reaction, 100 µl
of the cell suspension were added to 100 µl of buffer containing
18.5 kBq of
C-labeled L-proline, L-valine, L-arginine, or L-histidine
(Amersham Corp.) and 0.5 mM of the respective unlabeled amino
acids. Since yeast cells contain endogenous uptake activities for some
of the amino acids tested, the transport activity of 22574d transformed
with the empty vector pFL61 was subtracted as background. Endogenous
uptake rates of the mutant corresponded to 2.3 (proline), 669 (valine),
640 (histidine), 100 (lysine), 110 (arginine), and 2.9 (citrulline)
pmol min
mg cells
. Samples were
removed after 20, 60, 120, and 180 s, transferred to 4 ml of ice-cold
water, filtered on glass fiber filters, and washed with 8 ml of water.
Competition for proline uptake was performed in the presence of a
10-fold molar excess of the respective competitors. To determine the
capacity of accumulating proline against concentration gradients,
uptakes were measured over a period of 80 min. Aliquots were removed
every 10 min, and cells were sedimented, resuspended in 200 µl of
50 mM potassium phosphate (pH 4), filtered on glass fiber
filters, and washed with 8 ml of water. Radioactivity was determined in
sediment and supernatant by liquid scintillation spectrometry.
Transport measurements were repeated independently and represent the
mean of at least three experiments.
Complementation of a Histidine Uptake
Deficiency
The Saccharomyces cerevisiae strain JT16,
carrying mutations in histidine and arginine permease genes and the His4 gene, requires high concentrations of histidine (30
mM) for efficient growth (Tanaka and Fink, 1985). To identify
new plant amino acid transporter genes, JT16 was transformed with a
yeast expression cDNA library from Arabidopsis seedlings that
allows constitutive expression from the phosphoglycerate kinase
promoter in pFL61 (Minet et al., 1992). Transformants were
selected directly on SC medium supplemented with 6 mM histidine. Despite the high concentration of histidine, this
condition does not allow background growth of JT16 transformed with
pFL61 alone. Plasmid DNA was isolated, and several classes of clones
could be identified. Regarding the restriction map, several clones that
were different from the previously isolated AAP, AAT,
and NTR genes were identified and analyzed further (Kwart et al., 1993; Frommer et al., 1994a, 1995). To
confirm that no reversion, e.g. of the HIS4 gene or
suppressor mutation had occurred, the recombinant plasmids were
reintroduced into the mutant. Among the candidates that were able to
grow, three new clones enabling efficient growth on histidine were
identified (AAP3, AAP4, and AAP5).
Biochemical Characterization of the AAPs
To
determine whether the new transporters represent broad specific
systems, the proline uptake-deficient yeast mutant 22574d was
transformed with the three cDNAs, and transformants were selected on
media containing proline as the sole nitrogen source (Jauniaux et
al., 1987). All three cDNAs mediated growth on proline when
expressed in the yeast mutant 22574d (Fig. 1). The capability to
transport proline was confirmed by uptake studies using L-[C]proline. Transport activities were
linear for at least 30 min and had Michaelis-Menten constants of
100-250 µM (data not shown). To be able to compare
the properties with those of AAP1 and AAP2 directly, further uptake
experiments were performed using L-[
C]proline. Parallel competition
experiments for proline uptake at a 10-fold excess of different amino
acids as competitors were performed with yeast strains expressing any
of the five transporters AAP1-5 (Fig. 2A).
Regarding the competition of proline uptake by glutamate, glutamine,
alanine, proline, and citrulline, the five AAPs were comparable since
all five amino acids compete for proline uptake. Differences were,
however, detected for competition by valine, phenylalanine, and
arginine. Phenylalanine strongly competed for proline uptake mediated
by AAP2 and AAP4, whereas AAP5 was much less efficient in recognizing
phenylalanine. Proline transport by AAP2, AAP3, and AAP4 was more
sensitive to valine, and AAP3 and AAP5 had higher affinities for basic
amino acids arginine and lysine. Since competition experiments do not
indicate whether an inhibitor is actually transported, results were
confirmed by direct uptake measurements with radiolabeled amino acids (Fig. 2B). In contrast to the other transporters, AAP2
and AAP4 were able to mediate significant valine transport. In
accordance with the competition experiments, arginine and lysine were
transported only by AAP3 and AAP5.
Figure 1:
Relative growth rates of the
yeast amino acid uptake mutant 22574d transformed with the vector pFL61
or the transporter cDNAs AAP3, AAP4, and AAP5.
Figure 2:
Specificity of the amino acid transporters
AAP1-5 from Arabidopsis.A, competition of L-[C]proline uptake into yeast cells
(22574d-AAP1-5) in the presence of a 10-fold excess (5 mM final competitor concentration) of respective amino acids.
Uncompeted uptake rates were taken as 100% and correspond to 0.64
(AAP1), 0.63 (AAP2), 0.40 (AAP3), 1.18 (AAP4), and 1.98 nmol of proline
min
mg cells
(AAP5),
respectively. B, direct uptake of radiolabeled L-amino acids (proline, valine, histidine, arginine, and
lysine) into yeast cells (22574d-AAP1-5) after subtraction of
background uptake into the mutant strain transformed with
pFL61.
Active Transport into Yeast Cells
The transport
activity of AAP3-5 increased when the pH of the medium was
decreased (Fig. 3A). Furthermore, proline and citrulline
uptake activity was highly sensitive to 2,4-dinitrophenol, confirming
the finding that AAP transport activities are sensitive toward
protonophores (Fig. 3, B-D, ; Kwart et al., 1993). To determine whether the transport occurs in an
active manner, the accumulation of citrulline in AAP3-5-expressing yeast cells was analyzed. Within 120
min, the cells accumulated L-[C]citrulline up to 450-fold inside
the cells (AAP3, 85-fold; AAP4, 450-fold, and AAP5, 80-fold; Fig. 3, B-D). Taken together, these data argue for
an active proton-coupled transport mechanism.
Figure 3:
pH
dependence of proline uptake and accumulation of L-[C]citrulline against a concentration
gradient in yeast cells (22574d) expressing AAP3-5. A, pH dependence of AAP3-5. The 100% values correspond
to 770 (AAP3), 1400 (AAP4), and 250 (AAP5) pmol of proline
min
mg cells
. B-D,
accumulation of citrulline against a concentration gradient in yeast
cells 22574d transformed with pFL61 (B) or AAP3 (B), AAP4 (C), and AAP5 (D). +DNP shows the accumulation of
citrulline when 100 µM dinitrophenol (DNP) was
added 15 min after the start of the reaction.
Sequence Analysis of AAP Genes
The cDNAs have open
reading frames encoding proteins of 476 (AAP3), 467 (AAP4), and 482
amino acids (AAP5) and a molecular mass of 53 kDa. Sequence comparisons
show that all three genes are related to previously identified members
of the AAP family (; Fig. 4). The high homology
between AAP2 and AAP4 reflects the similarity in substrate specificity.
The predicted proteins are highly hydrophobic and contain 10-12
putative membrane-spanning regions (Fig. 5; Kyte and Doolittle,
1982).
Figure 4:
Comparison of the deduced amino acid
sequences of AAP1-5 (Frommer et al., 1993; Kwart et
al., 1993; this work). The first amino acid residue of the
translation start site was designated number 1 (PILEUP,
PRETTYBOX).
Figure 5:
Prediction of putative membrane-spanning
regions in AAP1-5 using PEPALLWINDOW with the window set to
15.
Organ-specific Expression of AAP Genes
To exclude
artifacts in the Northern analysis by cross-hybridization of different
genes, Southern blot hybridizations of Arabidopsis DNA were
performed. Under stringent conditions, all three genes showed patterns
with low complexity, indicating that only a single gene was hybridizing (Fig. 6). To analyze the expression profile in mature plants, RNA
was isolated from different organs (Fig. 7). All three genes
showed differential expression patterns that were different from that
of AAP1 and AAP2 (Kwart et al., 1993). AAP3 is selectively expressed in roots, whereas AAP4 was expressed in leaves, stems, and flowers. AAP5 mRNA
was found in all organs although at different levels, i.e. at
the highest levels in leaves, stems, and flowers. Both AAP4 and AAP5 are under developmental control in leaves as
only low levels of expression could be detected in sink leaves.
Figure 6:
Southern
blot analysis of AAP3-5. Genomic DNA (50 µg) from
mature greenhouse-grown plants was digested with restriction enzymes
and analyzed by Southern blot hybridization under stringent conditions
using P-labeled cDNAs of AAP3 (A), AAP4 (B), and AAP5 (C) as
probes.
Figure 7:
Organ-specific expression of AAP1-5 in Arabidopsis. Total RNA from
developing, mature, and cauline leaves, flowers, siliques, and roots
(20 µg) was analyzed by Northern blot hybridization using P-labeled cDNAs as probes (data for AAP1 and AAP2 from Kwart et al.,
1993).
Molecular Analysis of Amino Acid Transport in Plants
To
dissect the apparent complexity described at the physiological level,
respective transporter genes were isolated by complementation of yeast
amino acid transport mutants. The AAPs represent broad specificity
transporters encoded by a gene family consisting of at least five
members (Frommer et al., 1993; Hsu et al., 1993,
Kwart et al., 1993; this paper). The transporters contain
10-12 putative transmembrane helices and share a minimum of 54%
identical amino acids. Amino acid transport mediated by the AAPs is
sensitive to protonophores, increases with decreasing pH, and occurs
against a concentration gradient, suggesting active transport via a
H-symport mechanism. All systems including AAT1, which
constitutes a high affinity uptake system for basic amino acids, share
a low specificity toward amino acid side chains (Frommer et
al., 1995). All systems are capable of transporting the major
transport forms of reduced nitrogen, i.e. glutamine,
glutamate, asparagine, and aspartate. Based on their differential
activity toward basic amino acids, the AAPs can be grouped into two
subfamilies: (i) broad specific transporters that recognize acidic and
neutral amino acids and ureides such as AAP1, AAP2, and AAP4 and (ii)
general amino acid transporters that besides acidic and neutral amino
acids also recognize basic amino acids like AAP3 and AAP5.
Different Functions for Individual Amino Acid
Transporters
All transporters identified so far show a different
expression pattern covering all tissues of the plant. The cell
specificity of the individual members still remains to be shown, but at
least in cotyledons, the expression was associated with the vascular
system (Kwart et al., 1993).
(
)Since AAP4 efficiently transports
valine, a mutation in the AAP4 homologue of tobacco might
explain valine resistance in the mutants (Marion-Poll et al.,
1988). In addition to their expression in leaves, AAP4 and AAP5 are expressed at different levels in other organs. AAP2, AAP4, and AAP5 transcripts were found in stems,
where they might be involved in storage, retrieval along the
translocation pathway, or interchange between xylem and phloem. It
remains to be shown whether this represents an actual redundance or
whether the three transporters serve different functions in different
cell types of the stem.
Multiplicity of Amino Acid Transporters in
Plants
The presence of several high affinity transporters with
overlapping specificities and differential expression patterns could
explain the complex physiological, biochemical, and genetic data
obtained for amino acid transport. It is interesting to note that
despite the broad specificity, the individual transporters differ with
respect to their affinities toward individual amino acids. This might
point to different requirements of different cell types and preferences
for certain amino acids in different cell types.
Table: Sensitivity of proline transport activity of the
AAPs in 22574d to 100 µM 2,4-dinitrophenol
Table: Sequence similarities between the amino acid
transporters AAP1-5
/EMBL Data Bank with accession
number(s) X77499 (AAP3), X77500 (AAP4), and X77501 (AAP5).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.