Functional Properties of the Arabidopsis Peptide Transporters AtPTR1 and AtPTR5
2010; Elsevier BV; Volume: 285; Issue: 51 Linguagem: Inglês
10.1074/jbc.m110.141457
ISSN1083-351X
AutoresUlrich Z. Hammes, Stefan Meier, Daniela Dietrich, John M. Ward, Doris Rentsch,
Tópico(s)Plant Stress Responses and Tolerance
ResumoThe Arabidopsis di- and tripeptide transporters AtPTR1 and AtPTR5 were expressed in Xenopus laevis oocytes, and their selectivity and kinetic properties were determined by voltage clamping and by radioactive uptake. Dipeptide transport by AtPTR1 and AtPTR5 was found to be electrogenic and dependent on protons but not sodium. In the absence of dipeptides, both transporters showed proton-dependent leak currents that were inhibited by Phe-Ala (AtPTR5) and Phe-Ala, Trp-Ala, and Phe-Phe (AtPTR1). Phe-Ala was shown to reduce leak currents by binding to the substrate-binding site with a high apparent affinity. Inhibition of leak currents was only observed when the aromatic amino acids were present at the N-terminal position. AtPTR1 and AtPTR5 transport activity was voltage-dependent, and currents increased supralinearly with more negative membrane potentials and did not saturate. The voltage dependence of the apparent affinities differed between Ala-Ala, Ala-Lys, and Ala-Asp and was not conserved between the two transporters. The apparent affinity of AtPTR1 for these dipeptides was pH-dependent and decreased with decreasing proton concentration. In contrast to most proton-coupled transporters characterized so far, −Imax increased at high pH, indicating that regulation of the transporter by pH overrides the importance of protons as co-substrate. The Arabidopsis di- and tripeptide transporters AtPTR1 and AtPTR5 were expressed in Xenopus laevis oocytes, and their selectivity and kinetic properties were determined by voltage clamping and by radioactive uptake. Dipeptide transport by AtPTR1 and AtPTR5 was found to be electrogenic and dependent on protons but not sodium. In the absence of dipeptides, both transporters showed proton-dependent leak currents that were inhibited by Phe-Ala (AtPTR5) and Phe-Ala, Trp-Ala, and Phe-Phe (AtPTR1). Phe-Ala was shown to reduce leak currents by binding to the substrate-binding site with a high apparent affinity. Inhibition of leak currents was only observed when the aromatic amino acids were present at the N-terminal position. AtPTR1 and AtPTR5 transport activity was voltage-dependent, and currents increased supralinearly with more negative membrane potentials and did not saturate. The voltage dependence of the apparent affinities differed between Ala-Ala, Ala-Lys, and Ala-Asp and was not conserved between the two transporters. The apparent affinity of AtPTR1 for these dipeptides was pH-dependent and decreased with decreasing proton concentration. In contrast to most proton-coupled transporters characterized so far, −Imax increased at high pH, indicating that regulation of the transporter by pH overrides the importance of protons as co-substrate. IntroductionTransporters for di- and tripeptides are found in bacteria, fungi, plants, and animals (1Daniel H. Spanier B. Kottra G. Weitz D. Physiology. 2006; 21: 93-102Crossref PubMed Scopus (138) Google Scholar, 2Rentsch D. Schmidt S. Tegeder M. FEBS Lett. 2007; 581: 2281-2289Crossref PubMed Scopus (268) Google Scholar, 3Tsay Y.F. Chiu C.C. Tsai C.B. Ho C.H. Hsu P.K. FEBS Lett. 2007; 581: 2290-2300Crossref PubMed Scopus (409) Google Scholar, 4Waterworth W.M. Bray C.M. Ann. Bot. 2006; 98: 1-8Crossref PubMed Scopus (29) Google Scholar). The majority of the bacterial peptide transporters characterized so far belong to the ATP-binding cassette transporter family (5Detmers F.J. Lanfermeijer F.C. Poolman B. Res. Microbiol. 2001; 152: 245-258Crossref PubMed Scopus (85) Google Scholar). Some prokaryotic as well as most of the di- and tripeptide transporters of eukaryotes are members of the PTR 5The abbreviations used are: PTRpeptide transporterTEVCtwo-electrode voltage clampingCHES2-(cyclohexylamino)ethanesulfonic acid. /NRT1 (peptide transporter/nitrate transporter 1) family (1Daniel H. Spanier B. Kottra G. Weitz D. Physiology. 2006; 21: 93-102Crossref PubMed Scopus (138) Google Scholar, 3Tsay Y.F. Chiu C.C. Tsai C.B. Ho C.H. Hsu P.K. FEBS Lett. 2007; 581: 2290-2300Crossref PubMed Scopus (409) Google Scholar), which belongs to the major facilitator superfamily (6Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 2000; 64: 354-411Crossref PubMed Scopus (683) Google Scholar). In plants, the PTR/NRT1 gene family is much larger than in other kingdoms and consists of 53 genes in Arabidopsis (3Tsay Y.F. Chiu C.C. Tsai C.B. Ho C.H. Hsu P.K. FEBS Lett. 2007; 581: 2290-2300Crossref PubMed Scopus (409) Google Scholar). Functional di-/tripeptide transport has been shown for members from Arabidopsis (AtPTR1, AtPTR2, AtPTR3, and AtPTR5 (2Rentsch D. Schmidt S. Tegeder M. FEBS Lett. 2007; 581: 2281-2289Crossref PubMed Scopus (268) Google Scholar, 3Tsay Y.F. Chiu C.C. Tsai C.B. Ho C.H. Hsu P.K. FEBS Lett. 2007; 581: 2290-2300Crossref PubMed Scopus (409) Google Scholar, 7Komarova N.Y. Thor K. Gubler A. Meier S. Dietrich D. Weichert A. Suter Grotemeyer M. Tegeder M. Rentsch D. Plant. Physiol. 2008; 148: 856-869Crossref PubMed Scopus (133) Google Scholar)), faba bean (VfPTR1 (8Miranda M. Borisjuk L. Tewes A. Dietrich D. Rentsch D. Weber H. Wobus U. Plant Physiol. 2003; 132: 1950-1960Crossref PubMed Scopus (53) Google Scholar)), barley (HvPTR1 (9West C.E. Waterworth W.M. Stephens S.M. Smith C.P. Bray C.M. Plant J. 1998; 15: 221-229Crossref PubMed Scopus (56) Google Scholar)), and Hakea actites (HaPTR4 (10Paungfoo-Lonhienne C. Schenk P.M. Lonhienne T.G. Brackin R. Meier S. Rentsch D. Schmidt S. J. Exp. Bot. 2009; 60: 2665-2676Crossref PubMed Scopus (40) Google Scholar)). For most plant PTR/NRT1 proteins, the substrate selectivity has not been determined yet, but it is clear that some transport substrates other than peptides. For example, various Arabidopsis PTR/NRT1 members mediate low affinity uptake or export of nitrate (3Tsay Y.F. Chiu C.C. Tsai C.B. Ho C.H. Hsu P.K. FEBS Lett. 2007; 581: 2290-2300Crossref PubMed Scopus (409) Google Scholar, 11Segonzac C. Boyer J.C. Ipotesi E. Szponarski W. Tillard P. Touraine B. Sommerer N. Rossignol M. Gibrat R. Plant Cell. 2007; 19: 3760-3777Crossref PubMed Scopus (143) Google Scholar). Furthermore, a nitrate/histidine transporter from Brassica napus (BnNRT1;2 (12Zhou J.J. Theodoulou F.L. Muldin I. Ingemarsson B. Miller A.J. J. Biol. Chem. 1998; 273: 12017-12023Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar)) and a carboxylate transporter from alder were functionally characterized (13Jeong J. Suh S. Guan C. Tsay Y.F. Moran N. Oh C.J. An C.S. Demchenko K.N. Pawlowski K. Lee Y. Plant Physiol. 2004; 134: 969-978Crossref PubMed Scopus (83) Google Scholar), and a chloroplast nitrite transporter was described (14Sugiura M. Georgescu M.N. Takahashi M. Plant Cell Physiol. 2007; 48: 1022-1035Crossref PubMed Scopus (92) Google Scholar).In other kingdoms, only a few members of the PTR/NRT1 family are present, which primarily mediate proton-coupled transport of di- and tripeptides, as well as structurally related compounds (15Biegel A. Knütter I. Hartrodt B. Gebauer S. Theis S. Luckner P. Kottra G. Rastetter M. Zebisch K. Thondorf I. Daniel H. Neubert K. Brandsch M. Amino Acids. 2006; 31: 137-156Crossref PubMed Scopus (78) Google Scholar). In mammals, four peptide transporters (PepT1, PepT2, PHT1, and PHT2) show 21–28% amino acid identity to AtPTR1, AtPTR2, and AtPTR5, whereas sequence identity among these three plant transporters is 59–74% (supplemental Table 1). Two of the four mammalian peptide transporters mediate transport of free histidine, in addition to di- and tripeptides (16Daniel H. Kottra G. Pflugers Arch. 2004; 447: 610-618Crossref PubMed Scopus (385) Google Scholar). The transporters from rat and human have been investigated in detail; one of the mammalian transporters mediates uptake of peptides in the small intestine functioning as a main pathway for the absorption of dietary nitrogen (17Daniel H. Annu. Rev. Physiol. 2004; 66: 361-384Crossref PubMed Scopus (450) Google Scholar). Moreover, mammalian PEPT1 and PEPT2 also transport modified peptides, including β-lactam antibiotics, enzyme inhibitors, and other peptide-like drugs (15Biegel A. Knütter I. Hartrodt B. Gebauer S. Theis S. Luckner P. Kottra G. Rastetter M. Zebisch K. Thondorf I. Daniel H. Neubert K. Brandsch M. Amino Acids. 2006; 31: 137-156Crossref PubMed Scopus (78) Google Scholar).Studies on the substrate selectivity of PEPT1 and PEPT2, using peptides with proteinogenic amino acids and natural peptides, as well as pharmacologically interesting compounds with a similar structure (>350 different compounds (15Biegel A. Knütter I. Hartrodt B. Gebauer S. Theis S. Luckner P. Kottra G. Rastetter M. Zebisch K. Thondorf I. Daniel H. Neubert K. Brandsch M. Amino Acids. 2006; 31: 137-156Crossref PubMed Scopus (78) Google Scholar)), confirmed the predicted low selectivity. That work also revealed important features of compounds to be recognized as substrates by PEPT1 and/or PEPT2 (15Biegel A. Knütter I. Hartrodt B. Gebauer S. Theis S. Luckner P. Kottra G. Rastetter M. Zebisch K. Thondorf I. Daniel H. Neubert K. Brandsch M. Amino Acids. 2006; 31: 137-156Crossref PubMed Scopus (78) Google Scholar, 16Daniel H. Kottra G. Pflugers Arch. 2004; 447: 610-618Crossref PubMed Scopus (385) Google Scholar). The substrate selectivity of plant peptide transporters has been investigated only for Arabidopsis AtPTR1 and AtPTR2, which, like the mammalian transporters, recognize a large number of di- and tripeptides with moderate to high apparent affinity (18Chiang C.S. Stacey G. Tsay Y.F. J. Biol. Chem. 2004; 279: 30150-30157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 19Dietrich D. Hammes U. Thor K. Suter-Grotemeyer M. Flückiger R. Slusarenko A.J. Ward J.M. Rentsch D. Plant J. 2004; 40: 488-499Crossref PubMed Scopus (75) Google Scholar, 20Rentsch D. Laloi M. Rouhara I. Schmelzer E. Delrot S. Frommer W.B. FEBS Lett. 1995; 370: 264-268Crossref PubMed Scopus (273) Google Scholar). Affinity of AtPTR2 is dependent on both the N- and C-terminal amino acids and is largely independent of the membrane potential (18Chiang C.S. Stacey G. Tsay Y.F. J. Biol. Chem. 2004; 279: 30150-30157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Chiang et al. (18Chiang C.S. Stacey G. Tsay Y.F. J. Biol. Chem. 2004; 279: 30150-30157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) also established that AtPTR2 transports peptides and protons simultaneously by a random binding mechanism.Transporters for di/tripeptides have important functions in plants. Experiments using antisense repression of AtPTR2 showed the importance of peptide transport for flowering and seed development (21Song W. Koh S. Czako M. Marton L. Drenkard E. Becker J.M. Stacey G. Plant Physiol. 1997; 114: 927-935Crossref PubMed Scopus (53) Google Scholar). Furthermore, work with knock-out mutants (atptr1 and atptr5) and overexpression lines (35S-AtPTR5) demonstrated that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds and provided evidence that peptide transporters facilitate uptake of nitrogen from the rhizosphere (7Komarova N.Y. Thor K. Gubler A. Meier S. Dietrich D. Weichert A. Suter Grotemeyer M. Tegeder M. Rentsch D. Plant. Physiol. 2008; 148: 856-869Crossref PubMed Scopus (133) Google Scholar). These physiological functions indicated a role in transport of protein degradation products, which is consistent with the low selectivity of these transporters for different di- and tripeptides.Detailed knowledge of transport characteristics and kinetics is essential to understand the physiological role in planta. Here, we report the comprehensive characterization of the biochemical properties of AtPTR1 and AtPTR5 using heterologous expression in Xenopus oocytes and two-electrode voltage clamping (TEVC). The results show functional similarities and differences between these two peptide transporters as well as profound differences compared with the properties of AtPTR2.RESULTSDipeptide-induced currents were analyzed in X. laevis oocytes injected with AtPTR1 or AtPTR5 cRNA using TEVC. At pH 7.5 and pH 5.5, the addition of 1 mm Ala-Lys to AtPTR5- or AtPTR1-expressing oocytes induced inward currents (Fig. 1, B and C). Current amplitude was dependent on the batch of oocytes and time after RNA injection. No dipeptide-induced currents were detected in water-injected oocytes (Fig. 1A). The addition of 1 mm Ala-Ala also induced inward currents (data not shown). As Ala-Ala is present in its zwitterionic form at pH 5.5, the induced inward current indicates a co-transport of cations. Ala-Ala transport was confirmed using radio-tracer flux experiments in AtPTR1- and AtPTR5-expressing oocytes, which accumulated [3H]Ala-Ala (Fig. 2). Furthermore, under TEVC inward (leak) currents were observed when the pH was shifted from pH 7.5 to pH 5.5 in AtPTR5- and AtPTR1-injected oocytes in the absence of substrate (Fig. 1, B and C). These results are consistent with a co-transport of protons and peptides as described for AtPTR2 and the mammalian peptide transporters (16Daniel H. Kottra G. Pflugers Arch. 2004; 447: 610-618Crossref PubMed Scopus (385) Google Scholar, 18Chiang C.S. Stacey G. Tsay Y.F. J. Biol. Chem. 2004; 279: 30150-30157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar).FIGURE 2Uptake of [3H]Ala-Ala in AtPTR1- and AtPTR5-expressing oocytes at high and low pH. Uptake of [3H]Ala-Ala was determined in AtPTR1- (black) and AtPTR5 (white)-injected oocytes for 1 h at pH 5.5 and pH 7.5 (± S.E.; n = 6). Student's t test was performed, and asterisks indicate significant differences, p < 0.05, between transport rates at pH 5.5 and pH 7.5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Dependence of Apparent Affinity and Maximal Transport Rate on Membrane PotentialThe dependence of K0.5 and Imax on the membrane potential was determined in the range of Vm −140 to +20 mV for both AtPTR1 and AtPTR5. Ala-Ala, Ala-Lys, and Ala-Asp were chosen as representatives for neutral, cationic, and anionic dipeptides. At a membrane potential of −140 mV and pH 5.5, the apparent affinity of AtPTR5 was determined as K0.5Ala-Ala 23 ± 4 μm, K0.5Ala-Lys 167 ± 12 μm, and K0.5Ala-Asp 129 ± 4 μm, respectively (Table 1), the latter two affinities were thus comparable with the apparent affinity of AtPTR5 for Ala-Lys (K0.5Ala-Lys 163 μm) and Ala-Asp (K0.5Ala-Asp 131 μm) determined previously (n = 4 oocytes (7Komarova N.Y. Thor K. Gubler A. Meier S. Dietrich D. Weichert A. Suter Grotemeyer M. Tegeder M. Rentsch D. Plant. Physiol. 2008; 148: 856-869Crossref PubMed Scopus (133) Google Scholar)) and similar to the apparent affinity of AtPTR1 for these dipeptides determined under comparable experimental conditions (19Dietrich D. Hammes U. Thor K. Suter-Grotemeyer M. Flückiger R. Slusarenko A.J. Ward J.M. Rentsch D. Plant J. 2004; 40: 488-499Crossref PubMed Scopus (75) Google Scholar). At pH 5.5, K0.5 of AtPTR1 for Ala-Lys decreased with more negative membrane potentials, whereas the apparent affinity for Ala-Ala was not affected by the membrane potential, and K0.5Ala-Asp was lower at less negative membrane potentials (Fig. 3A). In contrast, K0.5 of AtPTR5 was constant for Ala-Ala and Ala-Lys and increased with more negative membrane potential for Ala-Asp (Fig. 3C). On the other hand −Imax increased at more negative membrane potentials for all three dipeptides in both AtPTR- and AtPTR5-injected oocytes (Fig. 3, B and D).TABLE 1Apparent affinities of AtPTR1 and AtPTR5 for different dipeptides at pH 5.5, measured at Vm −140 mV and Vm −60 mVK0.5 (μm) at Vm = −140 mVK0.5 (μm) at Vm = −60 mVAla-AlaAla-LysAla-AspAla-AlaAla-LysAla-AspAtPTR157 ± 2142 ± 12409 ± 1758 ± 3247 ± 38632 ± 112AtPTR523 ± 4167 ± 12129 ± 419 ± 3134 ± 971 ± 6 Open table in a new tab FIGURE 3Voltage dependence of K0.5 and −Imax in AtPTR1- and AtPTR5-injected oocytes at pH 5. 5. Voltage dependence of K0.5 (A and C) and −Imax (B and D) was determined at 3.2 μm [H+]out for AtPTR1- (A and B) and AtPTR5 (C and D)-injected oocytes. For each oocyte, currents were normalized to the current induced by 500 μm Ala-Ala at pH 5.5 and a membrane potential of −140 mV. Values represent mean ± S.E. of at least three oocytes. ○, Ala-Ala; ▾, Ala-Lys; ●, Ala-Asp.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Apparent Affinity and Maximal Transport Rates Are Dependent on the Proton ConcentrationApparent affinity and Imax were dependent on the proton concentration. Measurements for AtPTR1 revealed that at a membrane potential of −140 mV K0.5 decreased with increasing proton concentration for all three dipeptides (Fig. 4, A–C). Similarly, −Imax for Ala-Ala and Ala-Lys decreased with increasing proton concentration (Fig. 4, D and E). Only −Imax of Ala-Asp was not changed by pH over a broad range and sharply decreased at very low proton concentrations, indicating that the aspartate residue in Ala-Asp might be transported in its protonated form (Fig. 4F). AtPTR5 also showed higher currents at saturating concentrations of Ala-Ala at high pH (pH 7.5) (Fig. 5), demonstrating that higher transport rates at low proton concentrations is a common feature of both transporters and possibly other transporters of this clade. This trend was robust until pH 8.5 was reached, whereas at higher pH values, 10 mm Ala-Ala-induced currents decreased considerably (Fig. 6). This suggests that within the physiological range, the intrinsic pH dependence of the transporter itself overrules the dependence on protons as co-substrate.FIGURE 4Dependence of K0.5 and −Imax of AtPTR1 on pH. K0.5 (A–C) and −Imax (d–F) values of AtPTR1 for Ala-Ala (A and D), Ala-Lys (B and E), and Ala-Asp (C and F) were determined at Vm of −140 mV and increasing proton concentrations. Values are mean ± S.E. from at least three oocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Dependence of Ala-Ala transport on pH and sodium chloride. Ala-Ala (1 mm)-induced currents were determined at pH 7.5 and 5.5 in the presence of increasing concentrations (0, 50, and 100 mm) of NaCl in AtPTR1- (black bars) and AtPTR5 (white bars)-injected oocytes (±S.E.; n = 3; Vm −140 mV). Substrate-induced currents were normalized to 500 μm Ala-Ala-induced currents at pH 5.5 in sodium-free Ringer's solution and Vm −140 mV set at 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6AtPTR1-mediated Ala-Ala transport at high pH. The currents induced by 10 mm Ala-Ala were determined at pH 5.5 and between pH 7.5 and pH 10 in oocytes expressing AtPTR1 (Vm −140 mV). Substrate-induced currents were normalized on 500 μm Ala-Ala-induced currents at pH 5.5 set at 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Voltage dependence of peptide transport was also influenced by the proton concentration. Maximum transport rates of AtPTR1-mediated dipeptide transport decreased supralinearly with hyperpolarizing voltages at pH 5.5 (Fig. 3B), 6.5, and 7.5 (supplemental Fig. 1). At all membrane potentials, Imax was most negative at high pH for Ala-Ala and Ala-Lys and similar at all pH values for Ala-Asp. Although at pH 5.5 a decrease in apparent affinity at depolarized voltages was only shown for Ala-Lys (Fig. 3A), at pH 7.5 this effect was observed for all three dipeptides (supplemental Fig. 1).To test whether higher −Imax values at high pH were mediated by co-transport of Na+ instead of H+, currents induced by 1 mm Ala-Ala were measured at pH 5.5 and 7.5 in the presence of different concentrations of NaCl (Fig. 5). Increasing the Na+ concentration did not change currents of AtPTR1-injected oocytes at both high and low pH and slightly increased Ala-Al- induced currents for AtPTR5 at pH 7.5, indicating that elevated substrate-induced current at high pH was due to pH regulation of the transporter rather than Na+ replacing H+ as the coupling ion. This was confirmed by experiments showing that at pH 7.5, both AtPTR1- and AtPTR5-expressing oocytes accumulated significantly more [3H]Ala-Ala than at low pH (pH 5.5) (Fig. 2).To further examine the nature of the coupling ion and the functional groups involved in substrate binding or transport, AtPTR1 and AtPTR5 were expressed in S. cerevisiae and Ala-Ala transport rates determined in the presence of various well established inhibitors (Table 2). Carbonyl cyanide m-chlorophenylhydrazone, a protonophore, reduced uptake rates of [3H]Ala-Ala by AtPTR1 and AtPTR5 to 26 and 20%, respectively, consistent with protons as coupling ions. [3H]Ala-Ala uptake by both transporters was sensitive to the sulfhydryl group-modifying agent N-ethylmaleimide, similar to the effect of this inhibitor on AtPTR2 (20Rentsch D. Laloi M. Rouhara I. Schmelzer E. Delrot S. Frommer W.B. FEBS Lett. 1995; 370: 264-268Crossref PubMed Scopus (273) Google Scholar), and also diethyl pyrocarbonate, a histidyl-modifying agent, reduced uptake rates of [3H]Ala-Ala of both transporters to ∼1%.TABLE 2Effects of inhibitors on AtPTR1- and AtPTR5-mediated Ala-Ala uptake rates in S. cerevisiaeInhibitorAtPTR1 relative uptakeAtPTR5 relative uptake%%None10010010 mm CCCP26.16 ± 2.320.48 ± 1.71 mm NEM12.46 ± 1.014.00 ± 2.01 mm DEPC0.42 ± 0.20.55 ± 0.2 Open table in a new tab AtPTR1 and AtPTR5 Have Different Substrate SelectivityPrevious analyses of substrate selectivity showed that AtPTR1 recognized di- and tripeptides with different composition of the side chains (19Dietrich D. Hammes U. Thor K. Suter-Grotemeyer M. Flückiger R. Slusarenko A.J. Ward J.M. Rentsch D. Plant J. 2004; 40: 488-499Crossref PubMed Scopus (75) Google Scholar). Here, we analyzed the ability of AtPTR1 and AtPTR5 to recognize dipeptides with different hydrophobic or aromatic amino acids at the N- or C-terminal position. At a concentration of 1 mm dipeptide, Vm −140 mV, and pH 5.5, the currents induced by various substrates differed considerably (Fig. 7). Whereas Ala-Ile and Ile-Ala induced comparable currents in AtPTR5-injected oocytes, Ile-Ala was transported more efficiently than Ala-Ile by AtPTR1. Ala-containing dipeptides with the aromatic amino acids Trp or Phe induced inward currents only when the hydrophobic residue was at the C-terminal position. Tyr-Ala also induced less current than Ala-Tyr in both AtPTR1- and AtPTR5-injected oocytes. The exception was Phe-Phe, which induced currents comparable with Ala-Ala or Ala-Phe in AtPTR5 but not in AtPTR1-expressing oocytes. Transport of Ala-Phe, Phe-Ala, and Phe-Phe was dependent on pH and was considerably higher at low proton concentrations (Fig. 8).FIGURE 7Substrate selectivity of AtPTR1 and AtPTR5 for dipeptides with hydrophobic or aromatic residues. 1 mm dipeptide-induced currents were determined at Vm −140 mV and pH 5.5 in AtPTR1- (black bars) and AtPTR5 (white bars)-injected oocytes. No current was detected when these substrates were tested in control oocytes (data not shown). Currents were normalized on 500 μm Ala-Ala-induced current at pH 5.5 set at 1. Values are mean ± S.E. (AtPTR1, n = 3–5; AtPTR5, n = 3–6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8pH dependence of Ala-Phe, Phe-Ala, and Phe-Phe transport. 1 mm Ala-Phe-, Phe-Ala-, and Phe-Phe-induced current was determined at Vm −140 mV and pH 5.5 and 7.5 in AtPTR1- (black bars) and AtPTR5 (white bars)-injected oocytes. Values are mean ± S.E. (n ≥3 oocytes). All currents were normalized to currents recorded with 500 μm Ala-Ala at pH 5.5, Vm −140 mV, for each oocyte. Data for pH 5.5 are extracted from Fig. 6.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Surprisingly, and in contrast to Ala-Phe, at pH 5.5 the dipeptide Phe-Ala but also Trp-Ala and Phe-Phe reduced the background leak current in AtPTR1-expressing oocytes (Fig. 7). Interestingly, the Phe-Ala- and Phe-Phe-induced reduction of the leak current was pH-dependent and could not be observed at pH 7.5 (Fig. 8). The Phe-Ala-mediated reduction in current was concentration-dependent and could be described by Michaelis-Menten kinetics. The apparent affinity of the Phe-Ala-induced reduction of the leak current was 9.2 ± 1.8 μm (n = 3 oocytes; Fig. 9A). To test if the reduction is due to occupation of the substrate-binding site, the inhibition constant, Ki (i.e. 50% reduction of Ala-Lys induced current at its K0.5 [100 μm] (19Dietrich D. Hammes U. Thor K. Suter-Grotemeyer M. Flückiger R. Slusarenko A.J. Ward J.M. Rentsch D. Plant J. 2004; 40: 488-499Crossref PubMed Scopus (75) Google Scholar)), was determined by increasing concentrations of Phe-Ala to be 24.2 ± 3.3 μm (Fig. 9B). These results indicate that Phe-Ala is indeed recognized by AtPTR1 with high apparent affinity, even though it does not seem to be transported at pH 5.5. In oocytes expressing AtPTR5, only Phe-Ala reduced the leak current, whereas Trp-Ala induced small inward currents, and Phe-Phe currents were comparable with Ala-Phe-induced currents. This indicates both differences but also common characteristics in substrate recognition of peptide transporters of this clade.FIGURE 9Recognition of Phe-Ala by AtPTR1. A, reduction of leak current in the absence of substrate at Vm −140 mV and pH 5.5 by increasing concentrations of Phe-Ala follows a Michaelis-Menten kinetic (K0.5 9.2 ± 1.8 μm). B, competition of Ala-Lys (100 μm)-induced current in the presence of increasing concentrations of Phe-Ala, Ki 24.2 ± 3.3 μm. Values are mean ± S.E. of at least three oocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Results from S. cerevisiae strain LR2 expressing AtPTR1 or AtPTR5 showed that both Ala-Phe and Phe-Ala inhibited growth on His-Ala as a sole source of histidine (data not shown). This independently supports the finding that Phe-Ala interacts with the substrate-binding site and not only blocks leak currents.DISCUSSIONPeptide transporters are generally considered to play a role in translocation of peptides generated by protein degradation. Because di- and tripeptides produced by protein hydrolysis vary in size, charge, and hydrophobicity, it is expected that these transporters have a low selectivity regarding the composition of the side chains. Chiang et al. (18Chiang C.S. Stacey G. Tsay Y.F. J. Biol. Chem. 2004; 279: 30150-30157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) showed that the K0.5 of AtPTR2 for most neutral peptides is in the range of 50–400 μm and is 1830 μm for Ala-Asp (pH 5.5 and Vm 60 mV). The affinities of AtPTR1 and AtPTR5 were in a similar range for Ala-Ala and Ala-Lys, although the apparent affinity for Ala-Asp was higher than shown for AtPTR2 (i.e. at pH 5.5, Vm −60 mV K0.5Ala-Asp: AtPTR5 71 μm, AtPTR1 632 μm; pH 5.5, Vm −140 K0.5Ala-Asp: AtPTR5 129 μm, AtPTR1 409 μm ((7Komarova N.Y. Thor K. Gubler A. Meier S. Dietrich D. Weichert A. Suter Grotemeyer M. Tegeder M. Rentsch D. Plant. Physiol. 2008; 148: 856-869Crossref PubMed Scopus (133) Google Scholar, 18Chiang C.S. Stacey G. Tsay Y.F. J. Biol. Chem. 2004; 279: 30150-30157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 19Dietrich D. Hammes U. Thor K. Suter-Grotemeyer M. Flückiger R. Slusarenko A.J. Ward J.M. Rentsch D. Plant J. 2004; 40: 488-499Crossref PubMed Scopus (75) Google Scholar), see Table 1 for comprehensive overview of the kinetic parameters at different membrane potentials).For AtPTR1 and AtPTR5, higher dipeptide-induced currents were observed when the aromatic amino acids Trp, Tyr, or Phe were present at the C-terminal position. Ile-Ala was transported as efficiently as Ala-Ile in AtPTR5 but not in AtPTR1-injected oocytes, indicating slight differences in substrate recognition. Unexpectedly, some of the dipeptides with an aromatic amino acid at the N-terminal position did not induce inward currents but rather reduced the leak current observed at pH 5.5 in a concentration-dependent manner and thereby provided evidence that the leak current originated from the flow of the co-transported ion (FIGURE 7, FIGURE 8, FIGURE 9). Whether the block of the leak current is due to the inhibition of the conformational changes required to facilitate transport into the cell, to masking of the proton-binding site, or whether the (high affinity) binding of Phe-Ala prevents substrate translocation or hinders a coordinated release of both substrates remains elusive. A model illustrating the reduction of the leak current is presented in supplemental Fig. 2.The Phe-Ala-induced reduction of the leak current could be competed for b
Referência(s)