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Originally published In Press as doi:10.1074/jbc.M405192200 on May 11, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30150-30157, July 16, 2004
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Mechanisms and Functional Properties of Two Peptide Transporters, AtPTR2 and fPTR2*

Chien-Sung Chiang{ddagger}, Gary Stacey§, and Yi-Fang Tsay{ddagger}

From the {ddagger}Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan and the §Department of Plant Microbiology and Pathology, University of Missouri, Columbia, Missouri 65211

Received for publication, May 10, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Arabidopsis AtPTR2 and fungal fPTR2 genes, which encode H+/dipeptide cotransporters, belong to two different subgroups of the peptide transporter (PTR) (NRT1) family. In this study, the kinetics, substrate specificity, stoichiometry, and voltage dependence of these two transporters expressed in Xenopus oocytes were investigated using the two-microelectrode voltage-clamp method. The results showed that: 1) although AtPTR2 belongs to the same PTR family subgroup as certain H+/nitrate cotransporters, neither AtPTR2 nor fPTR2 exhibited any nitrate transporting activity; 2) AtPTR2 and fPTR2 transported a wide spectrum of dipeptides with apparent affinity constants in the range of 30 µM to 3 mM, the affinity being dependent on the side chain structure of both the N- and C-terminal amino acids; 3) larger maximal currents (Imax) were evoked by positively charged dipeptides in AtPTR2- or fPTR2-injected oocytes; 4) a major difference between AtPTR2 and fPTR2 was that, whereas fPTR2 exhibited low Ala-Asp– transporting activity, AtPTR2 transported Ala-Asp– as efficiently as some of the positively charged dipeptides; 5) kinetic analysis suggested that both fPTR2 and AtPTR2 transported by a random binding, simultaneous transport mechanism. The results also showed that AtPTR2 and fPTR2 were quite distinct from PepT1 and PepT2, two well characterized animal PTR transporters in terms of order of binding of substrate and proton(s), pH sensitivity, and voltage dependence.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although nitrate and peptides are quite different in chemical structure, nitrate transporters and peptide transporters show significant sequence homology and belong to the same family of peptide transporters (PTR)1 (alternative name of this family is POT standing for proton-coupled oligopeptide transporters (1, 2)). The first member of the PTR family identified was the Arbidopsis nitrate transporter gene, CHL1 (AtNRT1) (3). In 1994, 1 year after the identification of CHL1, several peptide transporter genes were independently isolated by functional cloning from rabbit (PepT1) (4), Arabidopsis (AtPTR2) (5, 6), Saccharomyces cerevisiae (PTR2) (7), and the Gram-positive bacterium, Lactococcus lactis (DtpT) (8). These peptide transporters were found to share sequence similarity with CHL1, and were classified together with CHL1 to form the new transporter family, PTR, this name being preferred because the majority of members are peptide transporters.

CHL1 and these peptide transporters then served as prototypes to search for homologs in other organisms or tissues. Today, more than 20 PTR family members with transport activities are known (9). Phylogenetic analysis of the nitrate and peptide transporters of the PTR family indicates that they can be classified into four groups (Fig. 1). Group I contains transporters from bacteria, group II transporters from animals, group III transporters from fungi, and group IV transporters from higher plants and histidine/peptide transporters from mammals. The four groups also have different topologies (Fig. 1). All are predicted to have 12 transmembrane (TM) domains. Members of group I are compact, with only short hydrophilic loops between the TM domains, whereas the members of the other groups contain a long hydrophilic loop (~100 amino acid long), which lies between TM domains 9 and 10 in group II, between TM domains 7 and 8 in group III, and between TM domains 6 and 7 in group IV.



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  		FIG. 1.
Phylogenetic tree for members of the PTR family. The phylogram was created from a multiple protein sequence comparison using the Pile-Up, Distances, and GrowTree programs of the GCG package (the Genetic Computer Group Inc., Madison, WI) with a gap penalty of 1 and a gap length penalty of 1. LlDtpT and LhDtpT are peptide transporters from L. lactis and Lactococcus helveticus, respectively (8, 27). YhiP and CePTR2 are putative gene products from Escherichia coli and C. elegans, respectively (28, 29). HsPepT2, RnPepT2, and OcPepT2 are renal peptide transporters from man (30), rat (31), and rabbit (32); HsPepT1, RnPepT1, and OcPepT1 are intestinal peptide transporters from man (33), rat (34, 35), and rabbit (4). RnPHT1 and RnPHT2 are rat peptide/histidine transporters expressed in brain and the lymphatic system, respectively (19, 36). DmOPT1 is a peptide transporter expressed in nurse cells of Drosophila ovaries (37, 38). Fungal fPTR2 (13), AtPTR2 (NTR1) (6, 12) from Arabidopsis, ScPTR2 from yeast (7), and CaPTR2 from Candida albicans (39) are peptide transporters cloned by complementing a yeast peptide uptake mutant. AtPTR2 is the same as AtNTR1, originally cloned by weak complementation of a yeast histidine uptake mutant (5). HvPTR1 is a barley peptide transporter isolated using degenerated primers (40). AtCHL1 is an Arabidopsis nitrate transporter cloned using a T-DNA-tagged chlorate-resistant mutant (3). BnNRT1;2 from Brassica (41) and LeNRT1-1 and LeNRT1-2 from tomato (42) were cloned by homologous hybridization using AtCHL1 cDNA as probe. AtNRT1:2 from Arabidopsis and OsNRT1 from rice were isolated by a sequence homology search of the EST data base using the CHL1 protein sequence (10, 11). LJNOD65 is a Lotus japonicus nodule-specific gene of unknown function (43). c, cytosolic side.

 
All members of groups I, II, and III are peptide transporters, whereas group IV consists of a mixture of peptide and nitrate transporters (Table I), which cannot be easily classified into two subgroups on the basis of sequence similarity. This raises the question whether nitrate transporters in this family can also transport peptides and whether the peptide transporters in this family can also transport nitrate. As addressed in our previous studies, the nitrate transporters AtNRT1:2 and Os-NRT1 do not transport peptides (10, 11). However, none of the peptide transporters in the PTR family have been tested for their ability to transport nitrate. In this study, we chose two peptide transporters, AtPTR2 (group IV) and fPTR2 (group III), to address this question.


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  		TABLE I
Substrate specificities of members of groups III and IV of the PTR family

 
AtPTR2 is an Arabidopsis peptide transporter gene isolated by complementing the yeast peptide transport mutant ptr2 with an Arabidopsis cDNA library (6). In situ hybridization analysis showed that AtPTR2 is expressed in the embryo (12). The peptide transporter gene fPTR2 (originally named AtPTR2A) was also isolated by functional complementation of a yeast peptide transport mutant using an Arabidopsis cDNA library (13), but was later found not to be an Arabidopsis gene, but probably derived from a fungal contaminant (14).

In this report, the substrate specificity and kinetic properties of AtPTR2 and fPTR2 were extensively examined by electrophysiological analysis of cRNA-injected Xenopus oocytes. The results revealed functional similarities and differences between peptide transporters in different groups of the PTR family. A mode of action of the two transporters is also proposed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Subcloning of AtPTR2 and fPTR2 cDNA into pGEMHE—To enhance the expression of AtPTR2 and fPTR2 in Xenopus oocytes, cDNA fragments were cloned into plasmid pGEMHE (15). The ~2.0-kb NotI cDNA fragment of AtPTR2 or fPTR2 was excised from plasmids pDTF1 (6) or pDTF4 (13), respectively, and cloned into pBluescript SK+ (Stratagene, La Jolla, CA) to produce the restriction sites for the subsequent construction. For AtPTR2, the cDNA fragment containing 25 bp of the 5' untranslated region, 1758 bp of the coding region, and 141 bp of the 3' untranslated region were generated by digestion with EcoRI and XhaI (both in the multiple cloning site) and cloned into the EcoRI and XhaI sites of pGEMHE to give plasmid pGEMHE-AtPTR2. For fPTR2, the cDNA fragment containing 293 bp of the 5' untranslated region, 1830 bp of the coding region, and 315 bp of the 3' untranslated region was generated by digestion with EcoRI (in the cDNA) and XhoI (in the multiple cloning site), the XhoI end was converted into a blunt end by Klenow filling, and the insert cloned into XbaI-digested (followed by blunting with Klenow) and EcoRI-digested pGEMHE to give plasmid pGEMHE-fPTR2.

Functional Expression of AtPTR2 or fPTR2 in Xenopus Oocytes—For in vitro transcription, pGEMHE-AtPTR2 were linearized with NheI, and plasmid pGEMHE-fPTR2 was linearized with SphI and blunted with Klenow. Capped cRNA was transcribed in vitro using mMESSAGE mMACHINE kits (Ambion, Austin, TX). Oocytes were isolated as described (16) and defoliculated by incubation for 1 h in Ca2+-free ND-96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5) (17) containing 1 mg/ml collagenase A (Type II, Sigma). Defoliculated mature oocytes (stage IV–V) were selected, injected with 50 ng of cRNA, then incubated for 2 days at 18 °C for translation.

Electrophysiological Measurements—Measurements were made in a solution containing 230 mM mannitol, 0.3 mM CaCl2, and 10 mM MES/Tris, pH 7.4, then perfused with 220 mM mannitol, 0.3 mM CaCl2, 10 mM MES/Tris at the pH indicated (pH 4.5–7.4 by mixing different ratios of MES and Tris), plus dipeptides, histidine, or HNO3. The oocytes were voltage-clamped at —60 mV with a TEV-200 two-electrode amplifier (Dagan, Minneapolis, MN). In the I/V relationship studies, the oocyte plasma membrane was held at —60 mV, then 300-ms pulses of step changes (0 mV (or —40 mV) to —160 mV in 20 mV decrements) in membrane potential were applied. The current evoked at the end of the 300-ms pulses was plotted against the membrane potential. Measurements were recorded using the AXOTAPE and pCLAMP6 programs (Axon Instruments, Inc., Foster City, CA).

Kinetic Analysis—The currents elicited by different concentrations of peptides at each membrane potential (Vm) were fitted to the equation, by a nonlinear least squares method using Program Original 5.0 (Microcal Software, Northampton). I is the evoked current (i.e. the current difference in the presence and absence of substrate), [S] the peptide concentration, the maximal current at a saturating concentration of peptide, and is the peptide concentration at which the current is half-maximal. The currents elicited by dipeptide at different pH values were fitted to the equation, by a nonlinear least squares method using Program Original 5.0. [H+] is the proton concentration, the maximal current elicited by a saturating proton concentration, the proton concentration at which the current is half-maximal, and nH the Hill coefficient.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
AtPTR2 and fPTR2 Lack Nitrate Transport Activity—Oocytes injected with AtPTR2 or fPTR2 were voltage clamped at —60 mV, then perfused with 10 mM nitrate or dipeptide Gly-Gly at pH 5.5. As shown in Table II, AtPTR2-or fPTR2-injected oocytes responded to Gly-Gly with an inward current, but showed little or no current changes in response to nitrate, showing that AtPTR2 and fPTR2 transport peptide, but not nitrate. The Arabidopsis nitrate transporter, CHL1, was included for comparison and, consistent with our results for the Arabidopsis nitrate transporter, AtNRT1:2 (10), and the rice nitrate transporter, OsNRT1 (11), was found to transport nitrate, but not Gly-Gly (Table II).


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  		TABLE II
Whole cell current measurements of oocytes voltage-clamped at -60 mV

10 mM nitrate, Gly-Gly, or histidine was applied at pH 5.5. n, number of oocytes tested. The values are the mean ± S.D.

 
In addition to nitrate and peptide, histidine can be transported by two group IV members. For example, using the Xenopus oocyte expression system, the Brassica transporter, Bn-NRT1;2, was shown to transport both nitrate and histidine (18), and the rat transporter PHT1 to transport both peptide and histidine (19). As shown in Table II and Fig. 2, little or no current change was elicited by histidine in AtPTR2- or fPTR2-injected oocytes (Table II and Fig. 2).



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  		FIG. 2.
Current-voltage relationship recorded in AtPTR2- or fPTR2-injected oocytes. Average currents evoked using 10 mM nitrate, histidine, or Gly-Gly at pH 5.5 in three different oocytes expressing AtPTR2 (A) or fPTR2 (B). The currents presented are the difference between the currents measured in the presence or absence of the substrate and are presented as the mean ± S.D.

 
In AtPTR2- or fPTR2-injected oocytes, Gly-Gly evoked a current increase at hyperpolarizing membrane potentials, whereas nitrate or histidine evoked little or no current change at any membrane potential tested up to —160 mV (Fig. 2). Nitrate transporters in the PTR family are electrogenic (3, 10, 18, 20), the absence of current elicited by nitrate in AtPTR2- or fPTR2-injected oocytes is therefore indicative of no nitrate transport activity in AtPTR2 and fPTR2. To rule out the possibility that nitrate might be transported through AtPTR2 and fPTR2 in an electroneutral manner, effect of nitrate on peptide transport activity was determined. As shown in the Supplemental Materials (Fig. S1), the current elicited by Gly-Gly in the fPTR2-injected oocyte was not inhibited by the presence of nitrate. Taken together, these data indicate that, despite their sequence similarity, the nitrate transporter, CHL1, transports nitrate, but not peptide, whereas the peptide transporters, AtPTR2 and fPTR2, transport peptide, but not nitrate.

Optimal Peptide Length for AtPTR2 and fPTR2—In AtPTR2- or fPTR2-injected oocytes, the current elicited by Gly-Gly-Gly was about 60% of that elicited by Gly-Gly, whereas that elicited by Gly or Gly-Gly-Gly-Gly was ≤10% of that elicited by Gly-Gly (all at 10 mM) (Fig. 3). This is consistent with the inhibitory effect of di- and tripeptides containing toxic amino acids on the growth of yeast transformed with AtPTR2 or fPTR2 (6, 13). Thus, like PepT1 and PepT2 (4), AtPTR2 and fPTR2 respond optimally to di- and tripeptides.



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  		FIG. 3.
Optimal peptide length for AtPTR2 and fPTR2. Current responses to bath application of 10 mM glycine peptides measured at pH 5.5 and a holding potential of —60 mV. The substrate-induced currents are expressed as a percentage of the Gly-Gly-induced current. n, number of oocytes tested. The values are the mean ± S.D.

 
Amino Acid Side Chain Specificity of AtPTR2 and fPTR2—To elucidate the amino acid side chain specificity of AtPTR2 and fPTR2, injected oocytes were voltage-clamped at —60 mV, then exposed to various dipeptides at different concentrations. The normalized Imax values calculated for the dipeptides tested are shown in Fig. 4 and the calculated affinities and permeabilities are shown in Table III. For both AtPTR2 and fPTR2, the Imax for positively charged peptides (Ala-His, His-Leu, Lys-Leu, and Ala-Lys) were larger than that for the other peptides tested. The major difference between AtPTR2 and fPTR2 was their response to the negatively charged dipeptide, Ala-Asp, which evoked relatively large currents in AtPTR2-injected oocytes, but smaller currents in fPTR2-injected oocytes (normalized Imax for Ala-Asp ~500 and ~150%, respectively, in AtPTR2- and fPTR2-injected oocytes, see Fig. 4).



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  		FIG. 4.
Maximal currents induced by neutral and charged dipeptides. Oocytes injected with AtPTR2 (left) or fPTR2 (right) were voltage-clamped at —60 mV and the currents were elicited by various concentrations of dipeptides measured at pH 5.5. The current/concentration curves were fitted to the Michaelis-Menten equation to obtain the maximal current (Imax) and apparent affinity (Km, shown in Table III). The Imax for each dipeptide was expressed as a percentage of that elicited by 1 mM Gly-Gly in the same oocyte. With the exception of the data for the His-Leu- and Lys-Leu-induced currents in AtPTR2-injected oocytes, which were obtained from a single oocyte, the data are the mean ± S.D. for the results from two to four different oocytes.

 


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  		TABLE III
Comparison of kinetic parameters of AtPTR2 and fPTR2 for neutral and charged dipeptides

All measurements were made at –60 mV. The Km and Imax were calculated by fitting the Michaelis-Menten equation. The relative Imax/Km ratio is given as an indicator of permeability.

 
As shown in Table III, the affinities of the dipeptides tested in AtPTR2- and fPTR2-injected oocyte were in the range of ~30 µM to ~2 mM. With the exception of His-Leu and Tyr-Leu, the affinities of the different dipeptides were quite similar for AtPTR2 and fPTR2, with Leu-Leu showing the highest affinity for both. The studies of L. lactis peptide transporter DtpT (21) and the rat kidney oligopeptide/H+ symporter (22) showed that increasing hydrophobicity in both the N- and C-terminal side chains increases the affinity of a dipeptide substrate. However, in the cases of AtPTR2 and fPTR2, no such clear relationship was found. For example, when dipeptides with a fixed C terminus (Leu), the preferred residues in the N-terminal are in the order of LeuH > TyrP > Lys+ > AlaH > His+ > GlyP (H: hydrophobic, P: polar, +: positively charged) for AtPTR2 and LeuH > His+ > Lys+ > GlyP > AlaH > TyrP for fPTR2 (Table III). Similarly, when dipepdies with a fixed N-terminal (Ala), the preferred residues in the C-terminal are in the order of PheH > LeuH > GlyP > His+ > Asp— for AtPTR2 and LeuH > Lys+ > GlyP >PheH > Asp— > His+. Nevertheless, a common feature among AtPTR2, fPTR2, DtpT, and the rat kidney oligopeptide/H+ symporter is that dipeptides with Phe and Leu residues exhibited the highest affinity.

Influence of the Membrane Potential on Substrate Affinity— The neutral, anionic, and cationic dipeptides, Ala-Gly, Ala-Asp, and Ala-His, were chosen to investigate the effect of voltage on the substrate affinity of fPTR2 and AtPTR2 at pH 5.5. Xenopus oocytes injected with either fPTR2 or AtPTR2 were voltage-clamped at —60 mV, then subjected to voltage pulses ranging from —40 to —160 mV for 300 ms in 20-mV decrements. As shown in Fig. 5, the apparent affinity constant (Km) of AtPTR2 and fPTR2 for the three dipeptides tested was relatively voltage-insensitive. At a hyperpolarizing Vm (—80 to —160 mV), the Km values of AtPTR2 and fPTR2 for all three dipeptides were voltage-independent, whereas at membrane potentials more positive than —80 mV, the Km values either remained voltage-insensitive (e.g. AtPTR2 for all three dipeptides and fPTR2 for Ala-Asp), or increased slightly (e.g. fPTR2 for Ala-Gly and Ala-His+).



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  		FIG. 5.
Voltage dependence of the Km for neutral and charged dipeptides determined at pH 5.5. The apparent affinities for AtPTR2 (A–C) and fPTR2 (D–F) were determined by fitting the currents elicited by 0.05, 0.1, 0.25, 0.5, 1, 2.5, and 10 mM dipeptides to the Michaelis-Menten equation at each membrane potential. The data are the mean ± S.D. for the results from three different oocytes.

 
Stoichiometry—To estimate how many protons were transported with peptide in each transport cycle, injected oocytes were exposed to 2 mM peptide at different pH values. As shown in Fig. 6, for fPTR2, the apparent coupling coefficient, n, for proton(s) calculated at various membrane potentials, was close to 1 for both Ala-Asp and Ala-Gly. In the case of AtPTR2, n was close to 1 for the neutral dipeptide Ala-Gly, but close to 2 for the negatively charged dipeptide Ala-Asp.



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  		FIG. 6.
Hill analysis of the proton activation of dipeptide-evoked currents. The currents evoked by 2 mM Ala-Asp (A and C) or Ala-Gly (B and D) were measured at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5 in oocytes expressing AtPTR2 (A and B) or fPTR2 (C and D). The Hill coefficient for protons (nH) was obtained by fitting the current elicited at each voltage to the equation, . Representative data from a single oocyte are shown; the error bars represent the error in the estimate. Similar results were obtained for two to five different oocytes.

 
Kinetics of Ala-His Transport—Of the dipeptides tested, Ala-His elicited the largest current in both fPTR2- and AtPTR2-injected oocytes (Fig. 4) and was therefore used to investigate the kinetics of AtPTR2 and fPTR2. As shown in Figs. 7, A and C, the maximal current induced by protons exhibited a supralinear dependence on voltage and increased as the [Ala-His]0 increased. Similarly, as shown in Fig. 7, B and D, at a hyperpolarizing Vm (less than —60 mV for AtPTR2-injected oocytes and less than —20 mV for fPTR2-injected oocytes), the maximal current induced by Ala-His increased as the proton concentration increased, but, at depolarizing potentials (—60 to 0 mV for AtPTR2-injected oocytes and —20 to 0 mV for fPTR2-injected oocytes), the remained the same when the proton concentration was increased from 1 µM, pH 6, to 10 µM, pH 5.



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  		FIG. 7.
Voltage dependence of the as a function of the external [Ala-His] and of the as a function of the external [H+rsqb]. A and C, the I/V relationships for AtPTR2-(A) or fPTR2-(C) injected oocytes, were obtained at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5 using 2 and 0.5 mM Ala-His. The currents elicited were plotted as a function of the external proton concentration at each voltage and the curves fitted to the equation, to obtain the values for the and (see Fig. 8). B and D, the I/V relationships for AtPTR2-(B) or fPTR2-(D) injected oocytes were obtained at pH 6.0 and 5.0 using 0.05, 0.1, 0.25, 0.5, 1, 2.5, and 10 mM Ala-His. The currents elicited were plotted as a function of the external Ala-His concentration at each voltage and the curves fitted to the equation, to obtain the values for the and (see Fig. 8). Representative data from a single oocyte are shown; the error bars represent the error in the fit. Similar results were obtained in two other oocytes.

 
The affinity for protons was relatively voltage-independent at 2 and 0.5 mM Ala-His (Fig. 8, A and C). The affinity for Ala-His was also voltage-independent at 10 µM H+ (Fig. 8, B and D), but voltage-dependent at 1 µM H+. The KHm increased when the Ala-His concentration was decreased from 2 to 0.5 mM, and the also increased when the proton concentration was decreased from 10 to 1 µM.



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  		FIG. 8.
Voltage dependence of the as a function of the external [Ala-His] and of the as a function of the external [H+]. The and were obtained for AtPTR2-(A and B) and fPTR2-(C and D) injected oocytes, as described in the legend to Fig. 7.

 
and shown in Fig. 8 were calculated according to the total amount of Ala-His added. Because pK of the histidine side chain is about 6.0, the ratio of [Ala-His0]/[Ala-His+] will be about 0.1 at pH 5, and 1 at pH 6. Taking this into consideration, the calculated or will still increase when pH increases from pH 5 to 6 (Supplemental Materials Table S1). Similarly, the , recalculated if AtPTR2 transports the neutral form of Ala-His, will still increase when the peptide concentration increases from 0.5 to 2 mM. And, when the peptide concentration increases from 0.5 to 2 mM, , recalculated if AtPTR2 transports positively charged Ala-His+, will remain the same at —160 to —120 mV and increase at —100 to —60 mV. Therefore, overall, regardless of which form of Ala-His was transported by these two transporters, the affinity for proton and Ala-His depended on the concentration of their respective co-substrates.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
fPTR2 and AtPTR2 Are Peptide Transporters with No Nitrate Uptake Activity
We previously showed that nitrate transporters in the PTR family lacked peptide transport activity. Several peptide transporters in the PTR family have been identified from a wide range of organisms, including bacteria, fungi, animals, and plants. However, none of these peptide transporters have been tested for possible nitrate transport activity. In this study, two peptide transporters were shown to have no nitrate uptake activity. Together with our previous studies on nitrate transporters (10, 11), it appears that that PTR family peptide transporters and nitrate transporters are two distinct transporter subtypes.

Even though peptide transporters and nitrate transporters in the PTR family are functionally distinct, they cannot be easily classified into two groups on the basis of sequence similarity. In the Arabidopsis genome, there are 53 transporter genes predicted to belong to the PTR family (23). Thus, because only 6 PTR members are found in the human genome, 4 in the Caenorhabditis elegans genome, 3 in the Drosophila genome, and 2 in the yeast genome, plants seem to have evolved high copy numbers of PTR transporters. This has led to the speculation that: 1) plants might use peptide hormones (23), or 2) PTR transporters might be able to transport substrates other than peptides or nitrate. One interesting potential substrate is indole acetic acid (IAA)-amino acid conjugates. IAA is an important plant growth hormone and 95% of the IAA pool is sequestered in an inactivated conjugated form, e.g. the amino acid-conjugated form. However, in AtPTR2- and fPTR2-injected oocytes, IAA-Ala, IAA-Leu, or IAA-Phe did not induce any current changes (data not shown). It remains to be determined whether other Arabidopsis PTR members have IAA-amino acid conjugate transporting activity.

A Random Binding, Simultaneous Transport Model
Fig. 8 shows that the decreased at all membrane potentials when the Ala-His concentration was increased and that the decreased when the proton concentration was increased. This increase in the apparent affinity of the transporter for proton and Ala-His as the concentration of their respective co-substrate increased suggests positive cooperativity between the two ligands for binding to their respective sites on the transporter. This suggests that both ligands bind to the transporter before being transported across the membrane, i.e. that the transporter operates via a simultaneous transport mechanism instead of a sequential transport mechanism (Fig. 9).



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  		FIG. 9.
A kinetic model for the operation of fPTR2 and AtPTR2. [T] is unloaded transporter; [TS], transporter loaded with substrate; [TH], transporter loaded with protons; and [THS], transporter loaded with both substrate and protons. The kinetic characteristics of fPTR2 and AtPTR2 suggest that they operate through a random binding, simultaneous transport mechanism when the membrane potential is more negative than —20 mV for fPTR2 and more negative than —60 mV for AtPTR2. As indicated by the heavier arrows, at more positive membrane potentials, fPTR2 and AtPTR2, prefer to bind proton and dipeptides in an orderly fashion (proton first, then dipeptide). Because leaking currents were not observed in the absence of dipeptide for either fPTR2- or AtPTR2-injected oocytes, translocation of [TH]o and [TH]i is unlikely.

 
The increased as the [Ala-His]o increased at all membrane potentials (Fig. 7, A and C) and the increased as the [H+]o increased at hyperpolarizing potentials (more negative than —20 mV for fPTR2 and —60 mV for AtPTR2) (Fig. 7, B and D). This means that, at hyperpolarizing potentials, the Imax values for both H+ and Ala-His were dependent on the concentration of their respective co-substrate, suggesting that AtPTR2 and fPTR2 operate by a random-binding transport model (Fig. 9). However, at depolarizing potentials (greater than —20 mV for fPTR2 and greater than —60 mV for AtPTR2), AtPTR2 and fPTR2 function through an ordered binding mode in which H+ binds first (heavier arrows in Fig. 9). Because the membrane potentials of plant cells are usually more negative than —60 mV, our data suggest that, in the physiological range of membrane potentials, AtPTR2 and fPTR2 transport proton and dipeptide via a random binding, simultaneous transport model.

Functional Differences between AtPTR2 and fPTR2
In terms of structure, the Arabidopsis peptide transporter, AtPTR2, and the "fungal" peptide transporter, fPTR2, differ in having a long hydrophilic loop between TM domains 7–8 and 6–7, respectively (Fig. 1). In this study, most of the functional properties involved in the transport of neutral and positively charged dipeptides were quite similar between these two transporters. However, the two transporters differed in terms of the transport of the negatively charged peptide, Ala-Asp. First, Ala-Asp elicited a large current in AtPTR2-injected oocytes, but only a small current in fPTR1-injected oocytes (Fig. 4); this was unique to the negatively charged Ala-Asp, because positively charged and neutral dipeptides had a similar relative Imax in AtPTR2 or fPTR2-injected oocytes. Second, the apparent coupling coefficient, n, for H+ is ~2 for AtPTR2 and ~1 for fPTR2 (Fig. 5), but further experiments are needed to clearly define the stoichiometry.

Functional Differences between the Arabidopsis Peptide Transporter, AtPTR2, and the Animal Peptide Transporters, PepT1 and PepT2
The two best characterized peptide transporter members of the PTR family, PepT1 and PepT2, are animal transporters and belong to group II. AtPTR2 and fPTR2 belong to a different group (groups IV and III, respectively, Fig. 1). Our study showed that, in terms of their functional properties, AtPTR2 and fPTR2 were quite similar to each other, but distinct from the animal peptide transporters, PepT1 and PepT2, in several aspects, as described below.

Random Binding Versus Ordered Binding—All four transporters transport dipeptide and proton by a simultaneous transport mode. However, PepT1 and PepT2 use an ordered binding mechanism in which the proton binds first (24, 25), whereas AtPTR2 and fPTR2 mainly use a random binding mechanism (Fig. 9).

Voltage Sensitivity of the Dipeptide Affinity—Several studies of PepT1 and PepT2 have shown that their apparent affinities for dipeptides, particularly charged dipeptides, are strongly voltage-dependent, decreasing about 10–30-fold when the membrane is hyperpolarized from —50 to —160 mV (24–26). However, our present study of AtPTR2 and fPTR2 showed that their affinities were more or less voltage-insensitive or slightly increased (not decreased) on hyperpolarization from —50 to —160 mV (Figs. 5 and 8).

Lack of Inhibition at a Low pH—Several studies (24–26) have shown that, for both PepT1 and PepT2, the current evoked declines with increasing proton concentration, particularly at a hyperpolarized Vm and a low substrate concentration. In contrast, our data showed that, for both AtPTR2 and fPTR2, the current evoked, even at —160 mV, did not decline with increasing proton concentration (see Supplemental Materials Fig. S2).

Lack of a Hyperpolarization Inactivation Effect—Hyperpolarization inhibits the transport of PepT1 and PepT2, and this effect is enhanced by a low pH and a low substrate concentration (24, 25). However, our data showed that, under similar conditions, no hyperpolarization-mediated inhibition was seen with AtPTR2- or fPTR2-injected oocytes (Fig. 2 and Supplemental Materials Fig. S3).

In contrast to animal cells, in which the Vm is about —50 mV, the membrane potential of a plant cell is about —120 mV and sometimes as low as —200 mV. In addition, because of the presence of H+-ATPase on the plasma membrane, the external pH of a plant cell is maintained at around pH 5.5. This suggests that the lack of low pH and hyperpolarization inactivation in plant peptide transporters might have evolved to ensure that they can function under physiological conditions. As shown in Fig. 1, the major structural differences between groups II (animal group), III (fungal group), and IV (plant group) are their long hydrophilic loops, which may be responsible for the observed functional differences.


   FOOTNOTES
 
* This work was supported by National Science Council Grants NSC-89-2311-B-001-033 and NSC-89-2311-B-001-124 (to Y.-F. T.) and National Science Foundation Grant MCB-0235286 (to G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Figs. S1–S3 and Tables S1 and S2. Back

To whom correspondence should be addressed. E-mail: yftsay{at}gate.sinica.edu.tw.

1 The abbreviations used are: PTR, peptide transporters; TM, transmembrane; MES, 4-morpholineethanesulfonic acid; IAA, indole acetic acid. Back



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 DISCUSSION
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