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Peptide uptake in the ectomycorrhizal fungus Hebeloma cylindrosporum : characterization of two di‐ and tripeptide transporters (HcPTR2A and B)

2006; Wiley; Volume: 170; Issue: 2 Linguagem: Inglês

10.1111/j.1469-8137.2006.01672.x

1469-8137

Mariam Benjdia, Enno Rikirsch, Tobias Müller, Mélanie Morel‐Rouhier, Claire Corratgé‐Faillie, Sabine Zimmermann, Michel Chalot, Wolf B. Frommer, Daniel Wipf,

Legume Nitrogen Fixing Symbiosis

New PhytologistVolume 170, Issue 2 p. 401-410 Free Access Peptide uptake in the ectomycorrhizal fungus Hebeloma cylindrosporum: characterization of two di- and tripeptide transporters (HcPTR2A and B) Mariam Benjdia, Mariam Benjdia ZMBP, Plant Physiology, Auf der Morgenstelle 1, 72076 Tübingen, Germany; These authors contributed equally to this work. Search for more papers by this authorEnno Rikirsch, Enno Rikirsch ZMBP, Plant Physiology, Auf der Morgenstelle 1, 72076 Tübingen, Germany; These authors contributed equally to this work. Search for more papers by this authorTobias Müller, Tobias Müller University Bonn, IZMB, Transport in Ectomycorrhiza, 53115 Bonn, Germany; These authors contributed equally to this work. Search for more papers by this authorMélanie Morel, Mélanie Morel IFR 110, UMR INRA/UHP 1136 Interactions Arbres Microorganismes, Université Henri Poincaré– Nancy I, Faculté des Sciences et Techniques BP239, F54506 Vandoeuvre-les-Nancy Cedex, France;Search for more papers by this authorClaire Corratgé, Claire Corratgé ENSAM-INRA, Biochimie et Physiologie Moléculaire des Plantes, Place Viala, 34060 Montpellier, France;Search for more papers by this authorSabine Zimmermann, Sabine Zimmermann ENSAM-INRA, Biochimie et Physiologie Moléculaire des Plantes, Place Viala, 34060 Montpellier, France;Search for more papers by this authorMichel Chalot, Michel Chalot IFR 110, UMR INRA/UHP 1136 Interactions Arbres Microorganismes, Université Henri Poincaré– Nancy I, Faculté des Sciences et Techniques BP239, F54506 Vandoeuvre-les-Nancy Cedex, France;Search for more papers by this authorWolf B. Frommer, Wolf B. Frommer ZMBP, Plant Physiology, Auf der Morgenstelle 1, 72076 Tübingen, Germany; Carnegie Institution, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305, USASearch for more papers by this authorDaniel Wipf, Daniel Wipf ZMBP, Plant Physiology, Auf der Morgenstelle 1, 72076 Tübingen, Germany; University Bonn, IZMB, Transport in Ectomycorrhiza, 53115 Bonn, Germany;Search for more papers by this author Mariam Benjdia, Mariam Benjdia ZMBP, Plant Physiology, Auf der Morgenstelle 1, 72076 Tübingen, Germany; These authors contributed equally to this work. Search for more papers by this authorEnno Rikirsch, Enno Rikirsch ZMBP, Plant Physiology, Auf der Morgenstelle 1, 72076 Tübingen, Germany; These authors contributed equally to this work. Search for more papers by this authorTobias Müller, Tobias Müller University Bonn, IZMB, Transport in Ectomycorrhiza, 53115 Bonn, Germany; These authors contributed equally to this work. Search for more papers by this authorMélanie Morel, Mélanie Morel IFR 110, UMR INRA/UHP 1136 Interactions Arbres Microorganismes, Université Henri Poincaré– Nancy I, Faculté des Sciences et Techniques BP239, F54506 Vandoeuvre-les-Nancy Cedex, France;Search for more papers by this authorClaire Corratgé, Claire Corratgé ENSAM-INRA, Biochimie et Physiologie Moléculaire des Plantes, Place Viala, 34060 Montpellier, France;Search for more papers by this authorSabine Zimmermann, Sabine Zimmermann ENSAM-INRA, Biochimie et Physiologie Moléculaire des Plantes, Place Viala, 34060 Montpellier, France;Search for more papers by this authorMichel Chalot, Michel Chalot IFR 110, UMR INRA/UHP 1136 Interactions Arbres Microorganismes, Université Henri Poincaré– Nancy I, Faculté des Sciences et Techniques BP239, F54506 Vandoeuvre-les-Nancy Cedex, France;Search for more papers by this authorWolf B. Frommer, Wolf B. Frommer ZMBP, Plant Physiology, Auf der Morgenstelle 1, 72076 Tübingen, Germany; Carnegie Institution, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305, USASearch for more papers by this authorDaniel Wipf, Daniel Wipf ZMBP, Plant Physiology, Auf der Morgenstelle 1, 72076 Tübingen, Germany; University Bonn, IZMB, Transport in Ectomycorrhiza, 53115 Bonn, Germany;Search for more papers by this author First published: 03 March 2006 https://doi.org/10.1111/j.1469-8137.2006.01672.xCitations: 37 Author for correspondence: Daniel Wipf Tel: +49 228 736761 Fax: +49 228 736557 Email: dwipf@uni-bonn.de AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary • Constraints on plant growth imposed by low availability of nitrogen are a characteristic feature of ecosystems dominated by ectomycorrhizal plants. Ectomycorrhizal fungi play a key role in the N nutrition of plants, allowing their host plants to access decomposition products of dead plant and animal materials. Ectomycorrhizal plants are thus able to compensate for the low availability of inorganic N in forest ecosystems. • The capacity to take up peptides, as well as the transport mechanisms involved, were analysed in the ectomycorrhizal fungus Hebeloma cylindrosporum. • The present study demonstrated that H. cylindrosporum mycelium was able to take up di- and tripeptides and use them as sole N source. Two peptide transporters (HcPTR2A and B) were isolated by yeast functional complementation using an H. cylindrosporum cDNA library, and were shown to mediate dipeptide uptake. • Uptake capacities and expression regulation of both genes were analysed, indicating that HcPTR2A was involved in the high-efficiency peptide uptake under conditions of limited N availability, whereas HcPTR2B was expressed constitutively. Introduction In boreal and temperate forests of the northern-hemisphere, where ectomycorrhizal plants dominate, nitrogen is the most important growth-limiting factor (Smith & Read, 1997). In many soils a high proportion of N is present in an organic form as peptides or amino acids (Smith & Read, 1997). Ectomycorrhizas play a crucial role in the N nutrition of their host plants, but little is known about the molecular details of this interaction. Ectomycorrhizal mycelium constitutes a large proportion of the microbial biomass in many forest soils (Alexander, 1983). The ability of ectomycorrhizal fungi to degrade macromolecular N and to take up and assimilate the products of hydrolytic degradations is therefore likely to have a great influence on the dynamics of organic N utilization (Chalot & Brun, 1998). Amino acid (Nehls et al., 1999; Wipf et al., 2002), ammonium (Javelle et al., 2002) and nitrate (Jargeat et al., 2003) transporters have been identified in ectomycorrhizal fungi, but so far no peptide transporter has been isolated from mycorrhizal fungi. Hebeloma cylindrosporum is a pioneer species commonly found associated with Pinus pinaster trees in the European Atlantic sand-dune forest ecosystems. In south-west France, this species occurs in two distinct habitats (Guidot et al., 2005). In the so-called 'dune habitat' (Guidot et al., 2002), populations of H. cylindrosporum are dominated by large, perennial individuals, each producing numerous fruit bodies. The second habitat (the 'forest habitat'; Guidot et al., 2002) is more inland, within the forest, and populations are characterized by annual individuals. The soil is sandy (98–99% noncalcareous sand), slightly acidic (pH in water approx. 5.7) without any obvious horizon. The percentage of organic matter is low, ranging from < 0.1–0.5% in the dune habitat to approx. 3% in the forest habitat (Guidot et al., 2005). Such an N-poor soil has to be explored optimally by the ectomycorrhizal fungus to benefit its host plant. In a recent study, Guidot et al. (2005) illustrated the ability of H. cylindrosporum to use a wide range of amino acids and other simple (e.g. urea) or complex (e.g. proteins) compounds as N sources. In this study we investigated the capacity of H. cylindrosporum to take up 'small' peptides, and the transport mechanisms involved. Peptide transport across membranes is a widely observed phenomenon in prokaryotes and eukaryotes (Stacey et al., 2002). Genes encoding peptide transporters have been cloned and characterized from bacteria (Smid et al., 1989), fungi (Perry et al., 1994), plants (Rentsch et al., 1995), vertebrates (Fei et al., 1994) and invertebrates (Fei et al., 1998). In both eukaryotes and prokaryotes, peptide transport is carrier-mediated, energy-dependent and restricted to small peptides (two to six amino acids) (Stacey et al., 2002). Known peptide transporters fall into three superfamilies (Stacey et al., 2002): the ATP-binding cassette family (ABC; Higgins, 1992); the oligopeptide transporter family (OPT; Lubkowitz et al., 1997; Hauser et al., 2001); and the peptide transporter (PTR) or proton-coupled oligopeptide transporter family (POT), transporting di- and tripeptides but also including nitrate transporters (Paulsen & Skurray, 1994; Steiner et al., 1995). Different members of the PTR family, identified in prokaryotes and eukaryotes, have been shown to transport a wide range of N-containing substrates, including amino acids, peptides and nitrate (Williams & Miller, 2001). Heterologous expression studies of an Arabidopsis member of the PTR family (AtNTR1) demonstrated that members of this family may cotransport peptides and protons, probably di- and tripeptides (Rentsch et al., 1995). In yeast, the transport of di- and tripeptides has been well characterized both physiologically and genetically (Becker & Naider, 1995), and involves three genes: PTR1, PTR2 and PTR3 (Island et al., 1991; Perry et al., 1994; Barnes et al., 1998). ScPTR2 is an integral membrane protein that can transport peptides, while ScPTR1 and ScPTR3 are cytosolic regulators of peptide transport (Alagramam et al., 1995; Barnes et al., 1998). In the present study two peptide transporters, HcPTR2A and HcPTR2B, were isolated from H. cylindrosporum by functional complementation of a yeast peptide-uptake mutant. Both proteins shared high similarity with peptide transporters of yeast, plants and animals, and were differentially expressed in hyphae. We propose that HcPTR2B is involved in the constitutive uptake of peptides whereas HcPTR2A is responsible for peptide uptake under stress conditions such as N deficiency. Materials and Methods Strains The Hebeloma cylindrosporum Romagnes monokaryotic strain (h1) was obtained from the in vitro fruiting dikaryon HC1 (Debaud & Gay, 1987). Mycelia were grown on either cellophane-covered YMG agar medium (yeast extract 4 g l−1, malt extract 10 g l−1, glucose 4 g l−1; Rao & Niederpruem, 1969) or on NP2/2 liquid medium (pH 5.5) containing (mg l−1): CaCl2 (50), MgSO4 (150), KH2PO4 (250), NaH2PO4 (4.5), Na2HPO4 (160), (NH4)2SO4 (250) and thiamine hydrochloride (0.04); carbon was supplied as 2.5 g l−1 glucose. The Escherichia coli strain used was XL1-Blue. Classical procedures for manipulating E. coli were as described by Sambrook et al. (1989). The mutant yeast strain, LR2, was used (MATα, ura3-52, his4-401, hip1-614, ptr2ΔhisG, ino1, can1; Rentsch et al., 1995), and is deficient in histidine uptake and metabolism, as well as peptide uptake. Yeast growth, transformation and selection The yeast strain LR2 was transformed with an expression library derived from H. cylindrosporum mycelia constructed in the yeast expression plasmid pFL61 (Lambilliotte et al., 2004), according to the procedure described by Gietz et al. (1992). Eleven transformants were selected directly on solid synthetic complete (SC) medium supplemented with different concentrations of the dipeptide histidine–leucine (HisLeu) (6 mm, 0.8 mm or 0.08 mm). Colonies able to grow were tested for growth in liquid medium with 6, 0.8 or 0.08 mm HisLeu. Plasmid DNA was isolated and reintroduced into the mutant strain LR2. The cDNA clones HcPTR2A and HcPTR2B were able to restore growth of the mutant on selective conditions. To test substrate specificity, the yeast strain LR2 was transformed with each cDNA clone. Selection was carried out on N-free medium supplemented with HisLeu as the sole N source. The empty vector pFL61 served as a negative control. RNA gel-blot analysis Total RNA was isolated with the RNeasy Plant Mini Kit (Qiagen) from approx. 100 mg frozen Hebeloma mycelia. Total RNA (20 µg) was separated on 1.5% formaldehyde agarose gels (Sambrook et al., 1989). Hybridization was performed at 68°C in 0.25 m sodium phosphate pH 7.2, 7% SDS, 1 mm EDTA and 1% BSA for 16 h using the cDNA fragments of HcPTR2A, HcPTR2B or HcGAP1 as probes. Filters were washed twice with 2 × SSC and 0.1% SDS at 68°C. Transport measurements In the H. cylindrosporum mycelium-uptake experiments, discs of fungal mycelium were cut from the actively growing edge of 10-d-old colonies using a 15-mm-diameter cork borer. Discs were floated for 5 min on a solution containing 1 ml N- and glucose-free NP2/2 (pH 6) at 23°C supplemented with [3H]-LeuLeu, specific activity 7.66 GBq mmol−1 (Amersham, Braunschweig, Germany). Incubation times varied from 1 to 20 min. At the end of the uptake period, discs were washed with 0.1 mm CaSO4 and solubilized with 80% Soluene 350 (Packard) overnight. Uptake of [3H]-LeuLeu was determined by liquid scintillation spectrometry. For Saccharomyces cerevisiae-uptake studies, yeast cells were grown to the logarithmic phase. Cells were harvested at an OD600 of 0.5, washed twice in water, and resuspended in buffer A (0.6 m sorbitol, 50 mm potassium phosphate pH 5.5) to a final OD600 of 5. Before taking uptake measurements, cells were supplemented with 100 mm glucose and incubated for 5 min at 30°C. To start the reaction, 100 µl of the cell suspension was added to 100 µl of the same buffer containing at least 18.5 kBq [3H]-LeuLeu, specific activity 7.66 Gbq mmol−1 (Amersham), and adequate unlabelled peptide concentrations. Sample aliquots of 45 µl were removed after 15, 60, 120 and 240 s, transferred to 4 ml of ice-cold buffer A, filtered on glass fibre filters, and washed twice with 4 ml buffer A. The uptake of tritium was determined by liquid scintillation spectrometry. Competition for LeuLeu uptake was performed by adding a fivefold molar excess of the respective competitors to 120 µm LeuLeu. The influence of plasma membrane charge on the uptake rate of [3H]-LeuLeu was analysed by incubating the yeast cells for 5 min in the presence of 100 mm glucose (control), without glucose, or with glucose and 0.1 mm 2,4-dinitrophenol (DNP) or 0.1 mm carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Transport measurements were repeated independently and represent the mean of at least three experiments. Extraction of amino acids and ammonium The H. cylindrosporum h1 strain was grown for 10 d on cellophane-covered YMG agar medium (T0 treatment) and then transferred onto N-free NP2/2 liquid medium. After 12 h, colonies were transferred onto NP2/2 medium containing different N sources [(NH4)2SO4; leucine–glycine, LeuGly; NO3− and glutamine, Gln] at a concentration of 2 mm. Samples of mycelia were harvested at different times (6, 12 and 24 h) for RNA, amino acid and ammonium extractions. Concentrations of amino acids and ammonium were also analysed in culture medium samples harvested at different times (1, 3, 6, 12 and 24 h). To determine concentrations of amino acids and ammonium, 10 mg lyophilized and ground tissue (Blender RETSH model MM 300) was extracted twice with 300 µl 70% (v/v) cold ethanol. The ammonium concentration was determined according to (Botton & Chalot, 1991). To determine amino acid concentrations, samples were desiccated using a Speed Vac concentrator (Savant, Speed Vac Plus) and dissolved in 400 µl 0.1 m HCl. Amino acids and standards were then purified on a Dowex 50WX-8 cation ion-exchange column, and aliquots of purified samples were transferred to microvials, dried in a Reacti-Therm Heating Module (Pierce), and derivatized according to the method of Mawhinney et al. (1986) slightly modified by using 8.3 µl N,N-dimethylformamide and 41.7 µl N-(t-butyldimethylsilyl)-trifluoroacetamide. Gas chromatography and mass spectrometry analysis The GC–MS used was a Hewlett Packard (Palo Alto, CA, USA) 5973 HP MS engine interfaced to a model 6890 gas chromatograph and a model 7673 autosampler. The capillary column (30 m 5 0.25 mm HP5-MS; Hewlett Packard) was initially held at 110°C. After 2 min the temperature was increased linearly by 10°C min−1 to 260°C. The injector and detector temperatures were 260 and 270°C, respectively. The mass spectrometer was operated in the electrical ionization, selected ion-monitoring mode. The monitored peak was [M-57]+ for all amino acids (Mawhinney et al., 1986). Results Utilization of peptide as N source by H. cylindrosporum Uptake experiments with [3H]-LeuLeu were performed to study the ability of H. cylindrosporum to use peptides as an N source. The results demonstrated that dipeptide uptake was linear for at least 20 min (Fig. 1). As no excreted protease activity was detected in such a short time (T.M. and co-workers, unpublished results), we can conclude that the ectomycorrhizal fungus H. cylindrosporum possesses a constitutive capacity to take up dipeptides. Figure 1Open in figure viewerPowerPoint Peptide uptake by the ectomycorrhizal fungus Hebeloma cylindrosporum. Discs of fungal mycelium were cut from the actively growing edge of 10-d-old colonies using a 15-mm-diameter cork borer. The discs were floated for 5 min onto a solution containing 1 ml nitrogen- and glucose-free NP2/2 at 23°C, supplemented with 2 µm[3H]-LeuLeu. Uptake by the fungus was measured at various times. Values represent the mean of three independent experiments ±SD. Cloning and sequence analysis of HcPTR2A and HcPTR2B The apparent ability of H. cylindrosporum to take up peptides led us to investigate the molecular basis of peptide transport. A yeast mutant deficient in peptide uptake (LR2) (Rentsch et al., 1995) was transformed with a cDNA expression library constructed from H. cylindrosporum mRNA, under control of a yeast promoter (Lambilliotte et al., 2004). Eleven independent transformants were obtained on selective media containing 6, 0.8 and 0.08 mm HisLeu. The yeast strain LR2 was retransformed with DNA extracted from the 11 transformants to eliminate false positives. All 11 clones allowed growth of transformed LR2 on HisLeu. After detailed restriction pattern analyses (data not shown), two different profiles were observed from the 11 clones. Two cDNAs with strong homology to other fungal peptide transporter genes were identified, and were named H. cylindrosporum peptide transporter 2A and 2B (HcPTR2A and HcPTR2B). The HcPTR2A and HcPTR2B cDNAs have a length of 1770 and 1806 bp, and encode proteins of 590 and 602 amino acids with a calculated molecular mass of 65.2 and 65.9 kDa, respectively. In silico analysis of the two protein sequences with the PROSITE-scan tool (http://expasy.ch/prosite) predicts the presence of PTR-specific motifs on both sequences (Marchler-Bauer & Bryant, 2004). The HcPTR2A sequence includes the PTR2-signature 1 conserved domain (YmyFYLlINIGAL); HcPTR2B includes the PTR2-signature 2 conserved domain (GGILADtMWGrykTImifSiVcliG). The best homology for the deduced protein sequences was obtained with a cDNA from Ustilago maydis PTR2 with an identity of 61% and similarity of 75% for HcPTR2A, and an identity of 42% and similarity of 61% for HcPTR2B (blast 2.2.9). On the basis of homology to mammalian, yeast and plant peptide transporters, U. maydis PTR2 is predicted to encode a di-/tripeptide permease of the maize pathogen. Phylogenetic analysis of protein sequences of the PTR family from animals, plants, yeast and bacteria underlined the fungal origin of the isolated genes, as they were located in a cluster comprising only fungal PTRs (Fig. 2). Both HcPTR2 proteins form a distinct subgroup, closely related to the transporters of lower fungi. Hydropathy analysis of HcPTR2A and HcPTR2B predicted 10–13 putative transmembrane domains, depending on the algorithm used. Figure 2Open in figure viewerPowerPoint Phylogenetic tree of peptide transporters from the peptide transporter (PTR) family. Maximum parsimony analyses were performed using paup 4.0b10 (Swofford, 1998), with all DNA characters unweighted and gaps scored as missing characters. Heuristic tree searches were executed using 1000 random sequence additions and the tree bisection–reconnection branch-swapping algorithm with random sequence analysis. The complete alignment was based on 798 sites; 673 were phylogenetically informative. The peptide transporters can be divided into four clusters (shadowed areas). Numbers behind organisms are according to the GenBank identification accession at the National Center for Biotechnology Information (NCBI). (Af, Aspergillus fumigatus; At, Arabidopsis thaliana; Bc, Bacillus cereus; Ca, Candida albicans; Dr, Danio rerio; Ec, Escherichia coli; Ef, Enterococcus faecalis; Ef2, Enterococcus faecium; f, fungal; Gg, Gallus gallus; Hc, Hebeloma cylindrosporum; Hv, Hordeum vulgare; Lj, Lotus japonicus; Ll, Lactococcus lactis; Lm, Listeria monocytogenes; Lp, Lactobacillus plantarum; Ls, Lactobacillus sakei; Lx, Leifsonia xyli; Mf, Macaca fascicularis; Mg, Meleagris gallopavo; Mm, Macaca mulatta; Mmus, Mus musculus; Oa, Ovis aries; Os, Oryza sativa; Pd, Prunus dulcis; Pn, Phaeosphaeria nodorum; Rn, Rattus norvegicus; Sa, Streptococcus agalactiae; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Ss, Sus scrofa; Scoe, Streptomyces coelicolor; Vf, Vicia faba; Xa, Xanthomonas axonopodis; Yp, Yersinia pestis). The transporters are shortened as follows: PTR, PTP or PEPT, peptide transporter; pPTR, putative peptide transporter; dtpT, di-/tripeptide transporter; dPcT, proton-coupled dipeptide cotransporter; pTIP, peptide transporter-like protein; dtPer, di-/tripeptide permease; ppSP, proton/peptide symporter family protein; pTR, putative transport protein; pdPTR, putative proton-dependent peptide transporter. All names and following numbers are original NCBI-published transporter names. Kinetics of LeuLeu uptake in a yeast mutant deficient in peptide-uptake systems To determine the distinct transport properties of both peptide transporters, radiotracer-uptake studies were performed using [3H]-labelled LeuLeu. LR2 yeast cells expressing HcPTR2A (Fig. 3a) or HcPTR2B were able to take up [3H]-LeuLeu. HcPTR2B-mediated uptake was twofold higher compared with yeast cells transformed by the empty pFL61 vector (data not shown), whereas yeast cells expressing HcPTR2A showed a 15-fold increased uptake rate of [3H]-LeuLeu compared with the control (Fig. 3a). Under standard assay conditions, HcPTR2A- and HcPTR2B-mediated uptake of [3H]-LeuLeu was linear for at least 4 min. As the HcPTR2B-uptake rate was comparatively low, further kinetic studies could not be performed (KM for LeuLeu could be estimated in the range 100–250 µm), while its close homologue, HcPTR2A, was characterized in further detail. Figure 3Open in figure viewerPowerPoint Kinetics of [3H]-LeuLeu uptake by the yeast mutant LR2 expressing HcPTR2A. (a) Time-course of [3H]-LeuLeu uptake. Mutants were transformed with the empty vector pFL61 (triangles) or with pFL61 expressing HcPTR2A (circles). Yeast cells were assayed for [3H]-LeuLeu uptake at 1.5 µm and pH 5. (b) HcPTR2A-mediated [3H]-LeuLeu uptake at different substrate concentrations. HcPTR2A-uptake rate was concentration-dependent and displayed saturation kinetics (Fig. 3b). The KM value for LeuLeu transport was approx. 1.5 µm at pH 5. The HcPTR2A activity was pH-dependent with an optimum at around pH 5 (data not shown). [3H]-LeuLeu uptake was dependent on the presence of glucose and was sensitive to the protonophores 2,4 DNP and CCCP (Fig. 4), indicating a proton-cotransport mechanism similar to the mammalian homologues. The strong dependence on glucose and proton gradient indicates that HcPTR2A transport is mediated by a secondary active transport mechanism similar to its yeast homologues (Perry et al., 1994). Competition studies (Fig. 5) showed that HcPTR2A binds di- and tripeptides. However, it is interesting to note that when dipeptides were used as competitors, HcPTR2A appeared to prefer hydrophobic residues at the C-terminus (Leu, Val and Phe). When the C-terminal residue was Gly (smaller and less hydrophobic) the affinity for the dipeptide decreased. Figure 4Open in figure viewerPowerPoint Influence of plasma-membrane energization on the uptake rate of [3H]-LeuLeu uptake by yeast cells expressing HcPTR2A. Yeast cells were preincubated for 5 min in the presence of 100 mm glucose, without glucose, or with glucose and 0.1 mm dinitrophenol (DNP) or 0.1 mm carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Figure 5Open in figure viewerPowerPoint Substrate specificity of HcPTR2A. Inhibition of 1.5 µm[3H]-LeuLeu uptake by a fivefold molar excess of competing nitrogen source. Data are expressed as percentage of uptake rate in the presence of 1.5 µm[3H]-LeuLeu. Values represent the mean of three independent experiments ±SD. Expression and regulation of H. cylindrosporum peptide transporters To understand the respective functions of HcPTR2A and HcPTR2B in peptide uptake under different nutritive conditions, their transcriptional regulation was analysed by RNA gel-blot hybridization. To obtain hints on the regulation of organic N import, the expression of the recently isolated HcGAP1 (general amino acid permease 1; Wipf et al., 2002) was also investigated. Fungal colonies were grown for 10 d on rich medium (YMG), then transferred for 12 h on liquid NP2/2 medium without N, and finally grown on liquid NP2/2 medium containing different N sources. Transcript levels for HcPTR2A, HcPTR2B and HcGAP1 (Fig. 6a) as well as the intracellular concentration of amino acids (Fig. 6b) and ammonium content in the medium (data not shown) were determined. Figure 6Open in figure viewerPowerPoint Regulation of HcPTR2A, HcPTR2B and HcGAP1 expression by nitrogen sources. (a) Expression level of HcPTR2A, HcPTR2B and HcGAP1 according to N source in the medium and incubation time. Fungal colonies were grown for 10 d on rich medium (yeast-malt-glucose, YMG), then transferred for 12 h onto a minimal medium without N, and finally grown on minimal medium containing different N sources. Total RNA was extracted for each growth condition. Hebeloma cylindrosporum 5.8S rRNA probe was used as a loading control. The nylon membrane was successively hybridized with 32P-HcPTR2A cDNA, 32P-HcPTR2B cDNA, 32P-HcGAP1 cDNA and the 32P-Hc 5.8S region. (b) Intracellular concentrations of amino acid content. Amino acid concentration was measured by GC–MS. Values represent the mean of three independent experiments ±SD. HcPTR2B was constitutively expressed, independently of N source and incubation time. In contrast, HcPTR2A was strongly expressed in mycelia grown on nitrate (NO3−) or under N starvation (–N). Interestingly, under these growth conditions (NO3− and –N), the intracellular amino acid levels were comparable (Fig. 7). When mycelia were fed with NH4+, HcPTR2A was initially weakly expressed (after 6 and 12 h), but increased after 24 h. After 6 h ammonium feeding, the intracellular amino acid pools were partially restored when compared with the rich medium condition, and further decreased (Javelle et al., 2003). The expression level of HcPTR2A was low when the mycelia were grown on the glutamine or dipeptide media. The weak expression of HcPTR2A, in mycelia grown on Gln, was correlated with a complete restoration of the amino acid pools after 12 h, which then decreased. When mycelia were grown on LeuGly, the amino acid pool was partially restored after 6 h when compared with the rich medium condition, and then declined. Figure 7Open in figure viewerPowerPoint Intracellular concentrations of glutamine, leucine and glycine in mycelium grown on different nitrogen sources (NH4+, Gln, LeuGly and NO3−) or without N. Gln (dark grey bars); Leu (light grey bars); and Gly (black bars) concentrations were measured by GC–MS. The HcGAP1-expression pattern was similar to HcPTR2A, with the exception of the expression level observed after 10 d culture on rich medium. Discussion Utilization of peptides as N source by H. cylindrosporum The ectomycorrhizal fungus H. cylindrosporum can take up dipeptides, as shown by radiotracer-uptake studies using [3H]-labelled dipeptide. Dipeptide uptake was confirmed by analysing Leu and Gly intracellular concentrations in fungal colonies, after the mycelium was grown on different N sources including the LeuGly dipeptide. Under the latter growth condition, increased intracellular Leu and Gly concentrations were observed (Fig. 7). No significant increase in the amino acid concentration in the feeding solution was observed (Fig. 7). Taken together, these results suggest that H. cylindrosporum can take up and use dipeptides as the sole N source. Biochemical properties of peptide transporters Tritium-labelled LeuLeu-uptake experiments showed that yeast expressing HcPTR2A and HcPTR2B could transport [3H]-LeuLeu, although HcPTR2A-mediated uptake was higher than HcPTR2B-mediated uptake. As the HcPTR2B-uptake rate was low, further kinetic studies could not be performed and only HcPTR2A was characterized. HcPTR2A encodes a high-affinity H+/peptide transporter, which mediates uptake of di- and tripeptides, as already found for the peptide transporters rabbit PepT1 and PepT2 (Fei et al., 1994), Arabidopsis AtNTR1 (Rentsch et al., 1995), yeast ScPTR2 (Perry et al., 1994) and fungus (Chiang et al., 2004). Competition