©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substrate Specificity and Expression Profile of Amino Acid Transporters (AAPs) in Arabidopsis(*)

Wolf-N. Fischer , Marion Kwart , Sabine Hummel , Wolf B. Frommer (§)

From the (1)Institut für Genbiologische Forschung, Ihnestra63, D-14195 Berlin, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.()A third system, NTR1, transports histidine with low efficiency but peptides with high affinity (Frommer et al., 1994a).()

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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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).




DISCUSSION

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.

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).

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.()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.

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).

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

Identity is indicated above the diagonal, and similarity is indicated below.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X77499 (AAP3), X77500 (AAP4), and X77501 (AAP5).

§
To whom correspondence should be addressed. Tel.: 49-30-83000765; Fax: 49-30-83000736; E-mail: Frommer@mpimg-berlin-dahlem.mpg.de.

W. B. Frommer, S. Hummel, M. Unseld, and O. Ninnemann, submitted for publication.

M. Laloi, W. B. Frommer, and D. Rentsch, unpublished observations.

D. Rentsch and W. B. Frommer, unpublished results.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.