H+ symporter in Aspergillus nidulans

Fungal Genetics and Biology 47 (2010) 1023–1033

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UreA, the major urea/H+ symporter in Aspergillus nidulans Cecilia Abreu a,1,2, Manuel Sanguinetti a,2, Sotiris Amillis b, Ana Ramon a,* a b

Sección Bioquímica, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay Faculty of Biology, Department of Botany, University of Athens, Panepistimoupolis, 15781, Athens, Greece

a r t i c l e

i n f o

Article history: Received 17 April 2010 Accepted 8 July 2010 Available online 12 July 2010 Keywords: Aspergillus nidulans Urea transport UreA Urea/H+ symporter

a b s t r a c t We report here the characterization of UreA, a high-affinity urea/H+ symporter of Aspergillus nidulans. The deletion of the encoding gene abolishes urea transport at low substrate concentrations, suggesting that in these conditions UreA is the sole transport system specific for urea in A. nidulans. The ureA gene is not inducible by urea or its precursors, but responds to nitrogen metabolite repression, necessitating for its expression the AreA GATA factor. In contrast to what was observed for other transporters in A. nidulans, repression by ammonium is also operative during the isotropic growth phase. The activity of UreA is down-regulated post-translationally by ammonium-promoted endocytosis. A number of homologues of UreA have been identified in A. nidulans and other Aspergilli, which cluster in four groups, two of which contain the urea transporters characterized so far in fungi and plants. This phylogeny may have arisen by gene duplication events, giving place to putative transport proteins that could have acquired novel, still unidentified functions. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Urea occurs in nature as a byproduct of animal metabolism of nitrogenous compounds, being the main nitrogen-containing substance in the urine of mammals (Smith, 2009). On the other hand, it is the world’s most common form of nitrogen fertilizer, with a sustained increase in its use during the last four decades (Glibert et al., 2006). Bacteria, fungi and plants are able to use urea as nitrogen source, incorporating it into the cell through specific transporters. A number of transporters from fungi and plants with high specificity for urea have been reported (ElBerry et al., 1993; Liu et al., 2003; Morel et al., 2008). These proteins are related to the sodium symporter superfamily (SSS), which comprises more than a hundred membrane proteins from both prokaryotic and eukaryotic origin (Jung, 2002; Reizer et al., 1994). The first protein of this class to be described was ScDur3 from Saccharomyces cerevisiae. ScDur3 incorporates urea when the external concentration is 60.25 mM whereas at concentrations P0.5 mM, it enters the cell via facilitated diffusion (Cooper and Sumrada, 1975; Sumrada et al., 1976). ScDur3 has also been reported to be involved in the uptake of polyamines, surprisingly displaying a higher affinity for those than for urea (Uemura et al., 2007). In S. cerevisiae urea

* Corresponding author. Fax: +598 2 5258618. E-mail address: [email protected] (A. Ramon). 1 Present address: Unidad de Proteínas Recombinantes, Institut Pasteur de Montevideo, Montevideo, Uruguay. 2 These authors contributed equally to this work. 1087-1845/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2010.07.004

is one of the products of allantoin metabolism and, like other genes of this catabolic pathway, expression of ScDUR3 is subject to nitrogen catabolite repression and is dependent on the presence of allophanate or oxaglutarate (the native and gratuitous inducers of the allantoin pathway, respectively) for high-level expression (ElBerry et al., 1993). The urea/H+ symporter from Arabidopsis thaliana, AtDur3, is able to complement a yeast dur3D deletion mutant and mediates the high-affinity transport (Km of 3 lM) of urea at low external concentrations. AtDur3 is expressed in roots and shoots, being upregulated during early germination and under nitrogen deficiency in roots (Liu et al., 2003; Merigout et al., 2008). A BLAST search in ESTs databases allowed the identification of putative orthologues in both vascular and non-vascular plants (for a review, see Wang Wi-Hong et al., 2008). An active urea transporter of the ectomycorrhizal basidiomycete Paxillus involutus has been recently characterized (Morel et al., 2008) after functionally expressing the corresponding cDNA in a S. cerevisiae dur3D strain. PiDur3 shows a high-affinity for urea (Km of 31.8 lM) and, similar to AtDur3, transport appears to be dependent on a H+ gradient. The authors focused on the regulation of the expression of PiDur3, demonstrating that the gene is upregulated under nitrogen deficiency, while being repressed by the high level of intracellular glutamine as a result of ammonium availability. Moreover, urea uptake seems to be tightly coupled to the efficiency of the urease enzyme, converting urea into ammonium and thus being inhibited by the intracellular accumulation of urea. Urea can be used as a nitrogen source by Aspergillus nidulans (Darlington et al., 1965; Scazzocchio and Darlington, 1968).

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Evidence for a substrate concentration dependent and saturable (Km of 30 lM), active transport system for urea in this organism was presented by Pateman and colleagues almost 30 years ago (Pateman et al., 1982). Thiourea, a toxic analogue of urea, is also transported by this same system, but with lower affinity and was used to isolate a number of allelic mutants which showed impaired growth on urea as sole nitrogen source at concentrations lower than 3 mM, implying the existence of a second transport system mediating passive or facilitated diffusion at higher urea concentrations, as described in S. cerevisiae. These authors also studied the regulation of this transport activity in response to nitrogen status, concluding that it is not induced by urea but is subject to nitrogen regulation, glutamine being the most likely effector of this phenomenon. In this article, we report the cloning and characterization of the gene encoding UreA, a specific urea transporter in A. nidulans as well as a study of its transcriptional and post-transcriptional regulation. We show that this transporter appears to be the only protein with high-affinity urea transport activity in A. nidulans. UreA presents putative orthologues in all other species of the genus Aspergillus. We also speculate about the functionality of other homologous proteins found in the different members of the genus, on the basis of their phylogenetic relationships.

2.3. ureA deletion The Double-Joint PCR (DJ-PCR) method (Yu et al., 2004) using Long PCR Enzyme Mix (Fermentas) was used to construct a replacement cassette where the riboB gene was flanked by 50 and 30 non-coding regions of ureA. The primers used are included in Table S2 in Supplementary material. The upstream (1478 bp) and downstream (1018 bp) flanking regions of ureA were amplified from genomic DNA from a wt pabaA1 strain using oligonucleotides URE5-F and URE5-R and oligonucleotides URE3-F and URE3-R, respectively. The riboB gene, used to replace the complete ureA coding region, was amplified using oligonucleotides Ribo-F and Ribo-R. The 4.6 Kb PCR fusion product was amplified with nested primers Ure5-N and Ure3-N and purified with the QIAEX II Gel Extraction Kit (QIAGEN). The resulting cassette was transformed in a pyrG89; pyroA4; nkuAD::argB; riboB2 strain (A1145, Table S1, Supplementary material). Protoplasts were plated on selective (minus riboflavin) minimal medium and incubated at 37 °C. Eight transformants showed impaired growth on urea and were able to grow on thiourea. Two of them were analysed by Southern blot blotting, showing that the coding region of the ureA gene had been replaced by riboB. 2.4. Construction of a ureA::gfp fusion strain

2. Materials and methods 2.1. Strains, media and transformation procedures Standard complete and minimal media (MM) for A. nidulans were employed (Cove, 1966; Scazzocchio and Arst, 1978; http:// www.fgsc.net). Supplements were added when necessary at standard concentrations (http://www.gla.ac.uk/ibls/molgen/aspergillus/supplement.html). A. nidulans strains used in this study are listed in Table S1 (Supplementary material). Gene symbols are defined in http://www.gla.ac.uk/ibls/molgen/aspergillus/loci.html. Urea (1–5 mM), NaNO3 (10 mM), ammonium L(+)-tartrate (5 mM), glutamine (10 mM) or proline (10 mM) were used as sole N-sources. Thiourea, putrescine, spermine and spermidine were used in concentrations of 0.6–5 mM. The Escherichia coli K-12 strain used in standard protocols was DH5a. A. nidulans transformation was carried out as in Tilburn et al. (1983).

Fusion PCR was used to generate the ureA::gfp transformation cassette for C-terminal tagging of proteins with green fluorescent protein (GFP) (Yang et al., 2004). Primers used are listed in Table S2 (Supplementary material). The GFP-pyrG cassette was amplified by PCR, using primers GFPyr-F and GFPyr-R from plasmid pl1439 containing (Gly-Ala)5-GFP plus Aspergillus fumigatus pyrG (Yang et al., 2004). The second fragment, corresponding to the upstream targeting region (the 30 end of the ureA coding region), was amplified using primers UAcod-F and UAcod-R and the third fragment, the 30 -ureA untranslated region, was amplified using primers Ure3-F and Ure3-R. In both cases genomic DNA of a wt pabaA1 strain was used as a template. The fusion product was amplified with primers UreGFP5-N and Ure3-N, purified with the QIAEX II Gel Extraction Kit (QIAGEN) and used to transform a pyrG89; pyroA4; nkuAD::argB; riboB2 A. nidulans strain (A1145, Table S1, Supplementary material). Long PCR Enzyme Mix (Fermentas) was used in all cases. Two transformants capable of growing on media lacking uridine and uracyl were tested by Southern blot blotting, showing that the ureA::gfp fusion integrated to the ureA locus.

2.2. Cloning of the ureA gene 2.5. Radiolabelled urea uptake measurements ureA cloning was accomplished using a functional complementation approach. A pabaA1; pyrG89; ureA1 strain (MVD101), impaired in urea transport, was transformed with a plasmid-based genomic library constructed in the autonomously replicating pRG3AMA1-NotI vector (Osherov and May, 2000; www.fgsc.net). This plasmid carries the pyr4 gene from Neurospora crassa, which is able to complement A. nidulans mutation pyrG89, restoring growth on media lacking uridine and uracil. Transformants prototrophic for uridine and uracil and able to grow on 1.25 mM urea as sole nitrogen source were then recovered and tested for thiourea sensitivity. Plasmids pANureA-1 and pANureA-2 were rescued from two of these transformants, and their ability to complement growth impairment on urea as sole nitrogen source was confirmed. Using pANureA-1 as template and specific primers ureA-F and ureA-R (see Table S2, Supplementary material) a 4.7 Kb sequence containing that corresponding to ANID_00418.1 (http://www.broad.mit. edu/annotation/fungi/aspergillus) gene was amplified and cloned in a pGEM-T-Easy vector (Promega). Its ability to restore urea transport was confirmed by co-transformation with pRG3AMA1 vector in an ureA1 strain.

[14C]-urea uptake in minimal media (MM) was assayed in germinating conidiospores of A. nidulans concentrated at 107 conidiospores/100 lL, at 37 °C, pH 6.8, as previously described (Amillis et al., 2007; Cecchetto et al., 2004; Papageorgiou et al., 2008). Initial velocities were measured at 1–2 min of incubation with concentrations of 0.5–2.0 lV for [14C]-urea at the polarity maintenance stage (3–4 h, 130 rpm). Time course experiments were measured in the presence of 50 lM [14C]-urea. Km/i values were obtained directly by performing and analysing uptakes (typical velocity/substrate concentration plots and verification by Prism 3.02: GraphPad Software, Inc.), using labelled urea at 0.5– 100 lM, or various concentrations (0.5–5000 lM) of non-labelled substrates. Ki values were calculated from the Cheng and Prusoff equation Ki = IC50/(1 + (L/Km), in which L is the permeant concentration. Free Gibbs energy (DGo) was calculated from DGo = RT ln(Ki), where R is the ideal gas constant and T is the absolute temperature (in K). IC50 values were determined from full dose–response curves and in all cases the Hill coefficient was close to 1, consistent with the presence of one binding site. In addition,

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we have examined the effect of thiourea and acetamide on Km and Vmax for wild-type and seen that apparent Vmax values remain unaltered, consistent with competitive inhibition, suggesting that a simple model of competition with the binding site of the transporter is applicable, satisfying the criteria for use of the Cheng and Prusoff equation. The H+-uncoupler carbonylcyanide chlorophenylhydrazone (CCCP) or the H+-ATPase inhibitor N,N0 -dicyclohexyl carbodiimide (DCCD) were added at final concentrations of 30 lV and 100 lM respectively, for 10 min before initiating the uptake assay. pH dependence experiments were carried out by adjusting the MM pH value 10 min before initiating the uptake assay. Reactions were terminated with addition of equal volumes of ice-cold MM containing 1000-fold excess of non-radiolabelled substrate. Background uptake values were corrected by subtracting either values measured in the deleted mutants or values obtained in the simultaneous presence of 1000-fold excess of non-radiolabelled substrate. Both approaches led to the same background uptake level, not exceeding 10–15% of the total counts obtained in wild-type strains. All transport assays were carried out in at least three independent experiments, with three replicates for each concentration or time point. Standard deviation was <20%. Radiolabelled [14C]-urea (55.0 mCi mmol1) was purchased from Moravek Biochemicals, Brea, CA. 2.6. Epifluorescence microscopy Samples for fluorescence microscopy were prepared as described previously (Valdez-Taubas et al., 2004). In brief, samples were incubated directly on sterile cover slips protected from light in liquid minimal media with proline (10 mM) as nitrogen source and appropriate supplements, at 25 °C for 14–16 h. When indicated, the last incubation hour took place in MM containing 10 mM ammonium L(+)-tartrate and/or cycloheximide to a final concentration of 20 lg ml1. Cultures were visualized and photographed in an Olympus inverted microscope CKX31 belonging to the Cellular Biology Platform, Institut Pasteur de Montevideo, with a U-MNIBA3 filter. The microscope is equipped with a Hamamatsu Orca Er camera and uses Image Pro 6.0 software for image processing. Vacuole staining with CMAC (7-amino-4-chloromethyl coumarin) (Molecular Probes, Inc., USA) was according to Gournas et al. (2010). CMAC was prepared as a 5 mg ml1 stock solution in dimethyl sulfoxide and stored frozen. Cover slips with germinated conidia were covered with MM containing 50 lg ml1 CMAC, incubated at 25 °C for 20 min, washed in 2.5 ml MM, and transferred to fresh 2.5 ml MM for 20 min chase time. Samples were observed on an Axioplan Zeiss phase contrast epifluorescent microscope with appropriate filters and the resulting images were acquired with a Zeiss-MRC5 digital camera using the AxioVs40 V4.40.0 software and processed by Adobe Photoshop software. 2.7. Northern blot analysis Total RNA was isolated from A. nidulans as described by Lockington et al. (1985) and separated on glyoxal agarose gels according to Sambrook (2001). A 657 bp PCR-amplified fragment of ureA (with primers SureA-F and SureA-R) was used as a probe in Northern blots. To monitor the amount of loaded RNA, a 2.5-kb BamHI/ KpnI fragment of plasmid pSF5 (Fidel et al., 1988) was used as probe to detect the actin messenger (acnA). In those experiments where ureA expression was followed during germination, the 18S rRNA was used as control since acnA mRNA does not reach a steady-state level until 4 h after germination. Probes were labelled with [32P]-dCTP using Random primer labelling system (Amersham) and purified with Illustra MicroSpin G-25 column (Amersham).

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2.8. Determination of transcription end by 30 RACE First-strand cDNA was synthesized from 5 lg of total RNA extracted from the wild-type strain grown in proline as nitrogen source with Super Script II (Invitrogen, Carlsbad, CA, USA), using oligo-dT primer. The 30 sequence of mRNA was amplified with CDS10 and FureA4 primers and Long PCR Enzyme Mix (Fermentas). The PCR product was purified from agarose gel with the QIAEX II Gel Extraction Kit (QIAGEN), cloned into pGEM-T-Easy vector (Promega) and sequenced. 2.9. Bioinformatic tools Sequences were obtained from the Aspergillus Comparative Database, BroadInstitute (http://www.broad.mit.edu/annotation/ fungi/aspergillus); the Saccharomyces genome database (http:// www.yeastgenome.org); and The Arabidopsis Information Resource (http://www.arabidopsis.org). PiDUR3 sequence was kindly provided by Morel et al. (2008). Phylogeny analysis was carried out with tools available in http://www.phylogeny.fr (Méthodes et Algorithmes pour la Bioinformatique, of the Laboratoire d’Informatique, de Robotique et de Microélectronique de Montpellier; Dereeper et al., 2008). Multiple sequence alignments were carried out with Muscle, applying curation with G blocks. Phylogenetic trees were constructed with the Maximum Likelihood program (PhyML) and the Bayesian Inference program (Mr. Bayes) available in the site and visualized with Drawtree. 3. Results 3.1. Cloning of ureA, the gene encoding for the major urea transporter in A. nidulans Urea transporters belonging to the same family as UreA have been characterized in S. cerevisiae, P. involutus and A. thaliana (see Section 1). Early genetic and biochemical analyses have indicated the existence of an active urea transport system in A. nidulans (Pateman et al., 1982). In this work, the ureA gene was cloned by functional complementation of the impaired growth on urea of a ureA1 strain with a genomic library constructed in the replicative plasmid pRG3-AMA1-NotI. Transformants capable of growing on media containing urea as sole nitrogen source at a concentration where ureA1 is unable to grow properly were recovered and tested for their sensitivity to the toxic analogue thiourea. Plasmids recovered from two of the transformants (pANureA-1 and pANureA-2) were re-transformed in the original ureA1 strain, confirming their ability to complement the growth defect on urea and to render the strains sensitive to thiourea. Restriction enzyme analysis showed that the inserts in both plasmids were partially overlapping. The insert cloned in plasmid pANureA-1 was sequenced, revealing that it contains the ANID_00418.1 gene (http://www.broadinstitute.org/ annotation/genome/aspergillus_group). A fragment containing the deduced coding sequence, including plus 1598 bp upstream and 997 bp downstream from the putative initiation and termination codons respectively was sub-cloned through a PCR strategy (see Materials and methods) and shown to also reestablish the ability of the ureA1 strain to grow on urea and its sensitivity to thiourea. The presence and location of the two introns predicted in ANID_00418.1 (http://www.broadinstitute.org/annotation/genome/aspergillus_group) was confirmed by sequencing of a cDNA clone obtained by RT-PCR. 30 RACE was performed to determine the transcription end point at position +2082 and the position of a polyadenylation site 90 pb downstream from the translation ter-

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mination codon. The sequence and all data concerning this analysis were submitted to GeneBank, under accession No. GQ409504. ureA codes for a putative polypeptide chain of 693 aminoacids, which is classified as a member of the sodium symporter family (SSS, TC 2.A.21; PFAM, http://pfam.sanger.ac.uk and the Transport Classification Database, http://www.tcdb.org). The amino acid sequence of ureA shows 51%, 45% and 43% identity with functionally characterized urea transporters from S. cerevisiae, A. thaliana and P. involutus, respectively. An alignment of the four sequences is shown in Fig. S1 (Supplementary material). The deduced protein sequence is predicted to consist of 15 transmembrane helical domains (TMSs), with an extracellular N-terminus and an intracellular C-terminus (TMHMM v2.0, http://www.cbs.dtu.dk/services/ TMHMM-2.0), and TMPred, http://www.ch.embnet.org/software/ TMPRED_form.html. Mackay and Pateman (1982) proposed that ureB, the urease structural gene, was clustered with ureA because of the close linkage existing between these two genes. Notwithstanding, ureA and ureB (ANID_10079.1) are separated by more than 30 Kb in chromosome VIII, with more than a dozen genes deduced between them. 3.2. ureA deletion and characterization of ureA1 and ureA905 mutant alleles A total deletion of the ureA at the locus ANID_00418.1 was carried out by replacing its complete coding sequence with the A. nidulans riboB gene, and thus complementing the riboflavin auxotrophy. The double-joint PCR technique (Yu et al., 2004) was employed for the construction of the replacement cassette, and single integration events in selected transformants were confirmed by Southern blot analysis (not shown). Sequencing of the ureA1 and the spontaneous thiourea-resistant mutant ureA905 alleles revealed single aminoacid substitutions at positions 168 (Gly168Asp) and 639 (Pro639Arg) respectively. According to the hydrophobicity profiles of UreA (TMPred, TMHMM-2.0), Gly168 is located immediately after transmembrane helix TMS4, facing the outside of the membrane. Pro639 is predicted to be located in the putative C-terminal domain immediately after TMS12. The ureA deletion mutant and the single amino acid substitution mutants have been characterized by growth tests and uptake assays. Fig. 1 shows that the three strains are almost completely impaired for growth on urea as sole nitrogen source, while growing as the wild-type on minimal media (MM) supplemented with ammonium, nitrate, proline, acetamide, arginine, hypoxanthine or uric acid as sole nitrogen sources. Resistance to thiourea is increased to similar levels in the three mutant strains. 3.3. UreA is a high-affinity, high-capacity, urea/H+ symporter activated during the isotropic growth phase ureA mediated [14C]-urea uptake kinetic analysis was performed at 37 °C, using conidiospores germinated in the absence of urea from the medium, at a stage prior to germ tube emergence, a stage where a number of A. nidulans transporters are significantly expressed (Amillis et al., 2004; Hamari et al., 2009; Tazebay et al., 1997). Fig. 2A displays a time course comparison of urea uptake in wt (ureA+), ureA-gfp, DureA and loss-of-function mutant strains ureA1 and ureA905, showing that the uptake rate for urea in germinating conidiospores is exclusively UreA-mediated, reaching steady-state levels after 20 min of incubation, while being linear for at least 2 min. Under these conditions of linearity urea uptake proved to have hyperbolic kinetics in relation to substrate concentration, as expected for a single transporter (not shown). The apparent Km and Vm for urea were calculated to be 26.2 ± 2.1 lM and 20.9 ± 3.2 pmol min1  107 conidiospores. The Km value is at the same range as for the other urea transporters characterized

so far (ElBerry et al., 1993; Liu et al., 2003; Morel et al., 2008). The Vm value, however, depends on the total quantity of transporter molecules in the plasma membrane and thus is dependent on developmental and growth conditions of each experiment (see below). Initial uptake rate measurements were also carried out during germination in the absence or presence of various nitrogen sources. Fig. 2B shows that dormant and up to 2 h germinating conidiospores display no significant uptake on any of the N-sources tested. In the presence of urea, nitrate, proline or under N-starvation, [14C]-urea uptake was first detected 2 h after inoculation, displaying a maximum after 3 h, and then dropping to a more basal level, or more dramatically (N-starvation) upon approaching the developmental stage of late polarity establishment-young hyphae (4–6 h). In agreement with transport assays, epifluorescence microscopy of germlings of a strain carrying a UreA-GFP fusion shows that the appearance of fluorescence in the membrane occurs also after 2 h of culture (not shown). Interestingly and at variance to what has been observed for other transporters (Amillis et al., 2004, 2007; Tazebay et al., 1997), uptake of urea is repressed by ammonium during the period of conidial isotropic growth. No saturable [14C]-urea uptake was detected at any stage or N-source in the DureA strain. Inhibition experiments with the H+-uncoupler CCCP and the H+ATPase inhibitor DCCD suggested that as most fungal transporters, UreA functions as a H+ symporter. This fact is supported by the obvious dependency of [14C]-urea uptake on the medium’s pH value, since at pH 9 urea transport is substantially impaired (Fig. 2C). This feature was also observed for the A. thaliana urea transporter (Liu et al., 2003) and also further supported by the lack of uptake potentiation in the simultaneous presence of 100 mM Na+. UreA mediated [14C]-urea uptake was also subjected to inhibition experiments in the presence of 2 mM non-labelled NHþ 4 , acetamide, thiourea, guanidine and the polyamines spermine, spermidine and putrescine (Fig. 2C). Thiourea and acetamide showed significant inhibition. Specific Km/i and DGo values were calculated in both cases (Fig. 2D) by using various concentrations of non-labelled substrates (see Section 2). Given the structural differences between urea, acetamide, guanidine or thiourea, these values could be taken as an indication of the contribution of each chemical group (amino or carbonyl) of the urea molecule to the binding of the substrate to the transporter. In view of urea transport inhibition by acetamide, we also tested whether null ureA mutations affected the utilization of low concentrations (1.25, 2.5 and 5 mM) of this compound as nitrogen or carbon source. No effect was observed in any of these cases. UreA mediated [14C]-urea uptake was also inhibited by relatively high amounts of putrescine but not by spermine or spermidine (Fig. 2C), resulting in a low affinity Ki of 2.5 mM for putrescine. However, establishing if putrescine is actually transported by UreA, despite the very low affinity, or simply interferes with [14C]-urea uptake will prove difficult since the deletion of ureA leads to no effect on the putrescine utilization as sole nitrogen source, implying the existence of more than one transport system (not shown). 3.4. ureA expression is not induced by urea but is subject to nitrogen metabolite repression In A. nidulans and other fungi the genes that code for transporters and metabolic enzymes participating in the utilization of nitrogen sources other than ammonium or glutamine are subject to a very stringent control. In A. nidulans these mechanisms involve the GATA factor AreA, which is a transcriptional activator only active in the absence of preferred nitrogen sources such as ammonium or glutamine (Arst and Cove, 1973; Kudla et al., 1990;

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Fig. 1. Growth tests of A. nidulans ureA::gfp, ureAD, ureA905 and ureA1 strains on media with ammonium or urea as nitrogen sources or thiourea to test resistance. In this latter case, nitrate 5 mM was used as nitrogen source. For complete genotypes see Table S1 (Supplementary material). Strains carrying the ureA::gfp fusion grow as the wt. ureAD, ureA905 and ureA1 show impaired growth on urea an augmented resistance to thiourea.

Wiame et al., 1985; Wilson and Arst, 1998). Additionally, alternative nitrogen sources can act as inducers of the specific genes involved in their utilization by activating specific transcriptional regulators (Berger et al., 2006; Cecchetto et al., 2004; Cultrone et al., 2007; Diallinas et al., 1995; Gomez et al., 2003; Gorfinkiel

et al., 1993; Hutchings et al., 1999; Muro-Pastor et al., 1999, 2004; Unkles et al., 1991). Northern blot analysis of ureA mRNA accumulation in a wildtype strain grown on different nitrogen sources is shown in Fig. 3A. ureA expression takes place to different levels in media

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Fig. 2. (A) Time course of [14C]-urea uptake in a wild-type strain (UreA), in a strain expressing the ureA::gfp fusion (UreA-GFP), in a strain lacking the UreA transporter (UreAD) and in two loss-of-function UreA mutant strains (UreA1, UreA905). (B) [14C]-urea uptake rates in dormant (0 h) and germinating (1–4 h) conidiospores, and germlings (5–6 h) in MM supplemented with various sole nitrogen sources or in nitrogen-starvation conditions (see Section 2). (C) Comparison of [14C]-urea initial uptake in a wild-type strain in the presence of the H+-uncoupler CCCP and the H+-ATPase inhibitor DCCD, of NaCl (100 mM) and at different pH values. Ammonium tartrate, acetamide, thiourea, guanidine, spermine, spermidine and putrescine were used at concentrations of 2 mM. Standard uptake measurements were on MM, at 37 °C, pH 6.8. (D) Structures of urea, thiourea, acetamide and guanidine (obtained in PubChem, http://pubchem.ncbi.nlm.nih.gov/).

containing urea, arginine, hypoxanthine, proline or acetamide, usually considered as non-repressive nitrogen sources. Highest expression is reached when cells are grown under nitrogen-starvation conditions. These results suggest that ureA expression is not inducible by urea. In fact, when the latter is the nitrogen source, message accumulation level is the lowest achieved in non-repressive sources. ureA is subject to nitrogen metabolite repression, since no ureA messenger is detected when strains are grown in the presence of ammonium in the culture medium (Fig. 3A). Expression depends on a functional AreA factor, since in a loss-of-function areA600 mutant (areA) no ureA expression is detectable even in nitrogenstarved cells (Fig. 3B), where expression is maximal in the wildtype strain. We also monitored the expression of ureA in areA102 and areA30 mutant strains which affect the specificity of the activator, conferring reciprocal loss-of-function, gain-of-function or wild-type phenotype depending upon the structural gene into consideration (Arst, 1977; Arst and Cove, 1973; Arst and Scazzocchio, 1975; Gorton, 1983; Hynes, 1973a,b, 1975; Polkinghorne and Hynes, 1975). The areA102 mutation (Leu683Val) shows increased binding to TGATAR sites and decreased binding to AGATAR and CGATAR sites, while areA30 (Leu683Met) shows a near mirror-image phenotype (Ravagnani et al., 1997). In the case of urea, areA30 mutants show decreased utilization of this nitrogen source. This ef-

fect has been attributed to altered expression of the gene coding for the urea permease, since areA30 have reduced urea uptake levels and increased resistance to thiourea. The reverse effects were observed for areA102 strains (Gorton, 1983; Hynes, 1973b). In agreement with this, we observed that in non-repressive conditions (proline and nitrogen starvation) an areA102 mutant shows increased expression of ureA, while in an areA30 strain messenger levels are highly reduced with respect to the wild-type (Fig. 3B). Physiologically important AreA binding sites usually occur in pairs who promote cooperative binding of the GATA factor (Gorfinkiel et al., 1993; Punt et al., 1995; Muro-Pastor et al., 1999; Gomez et al., 2003). A search for canonical GATA sites in the 1000 bp upstream of the ATG of ureA allowed the identification of a single possible pair of these sites, composed of a TGATAA and a CGATAG, separated by 9 bp (not shown), and located at approximately 600 pb upstream of the ureA ATG initiation codon. The phenotypes found for ureA expression in areA102 and areA30 mutants suggest that at least a TGATAR site would be physiologically important. Because of cooperative binding, the decrease in affinity for this site could be affecting binding to the CGATAR site. The xprD1 (so called for historical reasons) allele of areA results in strong derepression of AreA dependent genes in the presence of ammonium (Arst and Cove, 1973; Cohen, 1973; Platt et al., 1996a,b). This is however not the case for ureA which, in an xprD1

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Fig. 3. (A) ureA expression is not inducible by urea but is subject to ammonium repression. Northern blot analysis of ureA mRNA steady-state levels in mycelia of a areA+ wildtype strain grown in 2.5 mM NH4 and transferred by 1 h to fresh MM supplemented with 5 mM urea, 10 mM sodium nitrate, 30 mM arginine, 73 mM hypoxantine, 4 mM proline, 10 mM acetamide, 10 mM ammonium tartrate or no nitrogen source (N-free). The acnA mRNA was used as a control of RNA loading. (B) ureA expression dependence on AreA GATA factor. Northern blot analysis of ureA mRNA steady-state levels in different areA mutant strains: areA600 null mutant (left panel); areA30 and areA102 (central panel) and xprD1 (left panel). For description of areA30, areA102 and xprD1 mutations refer to main text. Strains were grown in 4 mM proline (areA+, areA30 and areA102) or 2.5 mM ammonium tartrate (areA600 and xprD1) and transferred for 1 h to fresh MM supplemented with 4 mM proline (Pro), 10 mM ammonium tartrate (NH4) or without nitrogen source (NF). The acnA mRNA was used as a control of RNA loading, except for xprD1, in which case ribosomal RNAs stained with methylene blue are shown. (C) Expression of ureA mRNA during germination and early mycelial development. Northern blot analyses of ureA mRNA extracted at different times (0–5 h) from cultures of wild-type (areA+) and an areA30 mutant strain grown in MM supplemented with 4 mM proline or 10 mM ammonium tartrate as nitrogen sources. The 18S rRNA probe was used as a control of RNA loading.

background, is substantially down-regulated under non-repressing conditions (proline and nitrogen starvation cultures) and repressed in the presence of ammonium (Fig. 3B, right panel). Plate tests of thiourea resistance of the xprD1 mutant support these results, since in the presence of ammonium the mutant exhibits normal growth, being indistinguishable from a wild-type strain (not shown). This is in agreement with data of Pateman et al. (1982) concerning transport activities but not with those reported by Platt and Langdon (Fig. 3 in Platt et al., 1996a) who found that an xprD1 mutant shows impaired growth on 5 mM thiourea in the presence of 10 mM NHþ 4 , which would be indicative of the derepression of the urea permease. The strain used in our assays behaves as expected for a derepressed mutant when tested for sensitivity to chlorate or 2-thioxanthine in the presence of ammonium. Moreover, it was used by Apostolaki et al. (2009) who showed derepression of agtA in the presence of ammonium. A similar, but even more extreme down-regulation in non-repressing conditions in an xprD1mutant was observed for the hxnS gene of A. nidulans encoding the purine hydroxylase enzyme II (PHII) and other genes induced by nicotinate (R. Fernandez-Martin, Z. Hamari, A. Cultrone and C. Scazzocchio, unpublished results).

3.5. Expression of ureA during conidial isotropic growth It has been shown for many permeases, including those from A. nidulans specific for proline PrnB (Tazebay et al., 1997), for purines, UapC, FcyB and AzgA (Amillis et al., 2004; Cecchetto et al., 2004; Vlanti and Diallinas, 2008), and dicarboxylic aminoacids, AgtA (Apostolaki et al., 2009) that during conidial isotropic growth the coding genes are expressed in a constitutive way, independently of other mechanisms of regulation acting on them in mycelial stage. ureA expression is undetectable in resting conidia, showing further a sharp increase during the isotropic growth phase and

reaching a maximum level in approximately 2 h. Unexpectedly, ureA transcription during germination is still repressible by ammonium, since no message is detectable during germination in a wildtype strain on this nitrogen source. Transcriptional activation of ureA in germinating conidia in an areA30 mutant grown on proline follows the same profile than in the wild-type, but occurs at lower levels (Fig. 3C). This fact supports the idea that this repressibility implies a dependence on AreA.

3.6. Post-translational regulation of UreA In order to determine the subcellular localization of UreA, we constructed a strain where the ureA wild-type allele was replaced by a ureA-gfp fusion. This strain shows normal growth on urea as sole nitrogen source (Fig. 1), and Km and Vm values for urea transport that are comparable to those of the wild-type strain (Fig. 2A). As expected, UreA-GFP localizes to the plasma membrane of germlings grown on proline (Fig. 4A, left panel). The fusion protein can be also observed in septae and in intracellular globular compartments that coincide with the vacuolar staining marker CMAC, a topology observed for chimeric transporters in A. nidulans studied to date (Apostolaki et al., 2009; Gournas et al., 2010; Pantazopoulou et al., 2007; Valdez-Taubas et al., 2004). Upon addition of ammonium UreA-GFP disappears from the plasma membrane after 60 min and is progressively accumulated in numerous CMAC stained compartments (Fig. 4B). This phenomenon seems to depend on de novo protein synthesis, as indicated by the inability of ammonium to induce internalization in the simultaneous presence of the protein synthesis inhibitor cycloheximide (Fig. 4B). A similar phenomenon of post-translational protein turnover has been previously described for the A. nidulans purine transporters UapA, UapC and FcyB (Valdez-Taubas et al., 2004, Pantazopoulou et al., 2007; Vlanti and Diallinas, 2008) and the

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Fig. 4. (A) The UreA-GFP fusion localizes to the plasma membrane in non-repressive conditions and is internalized upon addition of ammonium. Strains were grown at 25 °C for 16 h on minimal medium containing 4 mM proline as nitrogen source. Ammonium effect was evaluated by addition of 10 mM ammonium tartrate for the last 30 min. Bar represents 5 lm. (B) Protein synthesis is necessary for ammonium-dependent UreA internalization. Epifluorescence of UreA-GFP cellular expression in young hyphae (14– 16 h on MM at 25 °C) of A. nidulans strains, in the presence of proline (Pro), and after treatment with cycloheximide (CHX) 15 min prior addition of ammonium tartrate (NHþ 4) for a period of 2 h. Arrow heads indicate vacuolar compartments labelled with both GFP and CMAC. Bar represents 5 lm.

amino acid transporters AgtA and PrnB (Tazebay et al., 1997; Apostolaki et al., 2009). 3.7. In silico identification of UreA homologues in A. nidulans and other Aspergilli An in silico search of putative UreA homologues in the genomes of both A. nidulans and the other seven Aspergilli whose genomes are available (A. fumigatus, A. oryzae, A. terreus, A. niger, A. clavatus, A. flavus and Neosartorya fischeri, http://www.broadinstitute.org/ annotation/genome/aspergillus_group/MultiHome.html) was carried out. A summary of the sequences found is included in Table 1. In A. nidulans we identified three putative proteins, with sequence similarity to UreA (ANID_00418.1), while in each of the other members of the genus three or four putative homologues are present. These sequences, together with those of characterized urea transporters from S. cerevisiae, P. involutus and A. thaliana, were used as a template for the construction of a phylogenetic tree as described in Materials and methods (Fig. 5). UreA (ANID_00418.1) appears to have orthologues in all Aspergillus species. None of the other homologues is present in all of the species of the genus. It is interesting to note that ScDur3 cluster with UreA and its orthologues in other Aspergilli, while PiDur3 and AtDur3 cluster with ANID_07373.1 and homologues in A. niger, A. terreus, the A. fumigatus/N. fischeri clade and A. clavatus. Notwithstanding, according to the tree ANID_07373.1 seems to have diverged from orthologous proteins in other Aspergilli. The other two A. nidulans homologues, ANID_02598.1 and ANID_07557.1 group in two clusters. The one including A. nidulans protein ANID_02598.1 has putative orthologues in A. oryzae and A. flavus, N. fischeri, A. fumigatus, A. niger and A. terreus. A. clavatus is absent from this group. The second cluster, which includes ANID_07557.1, contains homologues of A. terreus, A. niger, A. clavatus, A. oryzae and A. flavus. A. fumigatus and the closely related

Table 1 Putative UreA homologues A. nidulans and other Aspergilli. Organism

Accession No.

Length (aa)

% identity with UreA

A. nidulans

ANID_00418.1 (UreA) ANID_07373.1 ANID_02598.1 ANID_07557.1

693 642 631 663

100 45 40 34

A. terreus

ATEG_02629 ATEG_07546.1 ATEG_01766.1 ATEG_07346.1

649 652 620 565

85 36 35 37

A. clavatus

ACLA_029.180 ACLA_097250 ACLA_097210

676 637 616

82 37 36

N. fischeri

NFIA_019890 NFIA_007820 NFIA_049560

680 611 636

81 43 37

A. oryzae

AO090003000854 AO090003001423 AO090124000019 AO090124000019

700 631 487 74

79 41 40 39

A. niger

est_fge1_pg_C_10845 fge1_pg_C_10000081 fge1_pm_C_9000146 fge1_pg_C_9000190

656 631 621 612

78 42 38 37

A. fumigatus

Afu1g04870 Afu1g17570 Afu6g03200

680 625 635

79 42 37

A. flavus

AFL2G_02167.2 AFL2G_01655.2 AFL2G_08023.2

701 596 647

74 38 35

species N. fischeri are absent from this cluster. A very similar topology was obtained when using Bayesian inference for construction of the phylogenetic tree (not shown).

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Fig. 5. Phylogenetic tree of characterized urea transporters from plants (AtDur3) and fungi (UreA, PiDur3 and ScDur3) and homologues in the genus Aspergillus. Phylogeny analysis was carried out following the ‘‘A la Carte Mode” in Phylogeny.fr (http://www.phylogeny.fr). MUSCLE was used for multiple sequence alignment, applying Gblocks curation prior to obtaining a maximum likelihood tree visualized with Drawtree. The tree was redrawn on the image obtained. The digits at the nodes are the bootstrap values for 500 re-samplings.

4. Discussion In this study the functional characterization of UreA as an active urea transporter of A. nidulans has been carried out. The kinetic data obtained for urea transport through this system are in agreement with those obtained by Pateman et al. (Km 30 lM) and are also in the same range as those obtained for characterized urea transporters from S. cerevisiae (ElBerry et al., 1993), A. thaliana (Liu et al., 2003) and P. involutus (Morel et al., 2008). Like most fungal transporters reported so far, UreA functions as a H+ symporter. Together with ScDur3, PiDur3 and AtDur3, UreA is identified as a member of the sodium:solute symporter (SSS) family of transporters, but all of these proteins exhibit the particularity of possessing a H+-symport mechanisms. We propose that this group of proteins would constitute a subfamily of urea:H+ symporters included in the SSS family of transporters. Up to now, the only other transporter classified into the SSS family but mechanistically involving the symport of protons is MctP of Rhizobium leguminosarum (Hosie et al., 2002) mediating import of alanine and other monocarboxylates like lactate and pyruvate. In this work, we show that urea transport through UreA is competitively inhibited by thiourea and also by acetamide. These results allow us to speculate about urea recognition by UreA. Specificity profiles and differences in binding energies calculated for each of the inhibitors in comparison to urea make evident that the functional groups of the urea molecule contribute nearly equally for efficient substrate translocation, whereas the carbonyl

group appears to be the most important in either interacting directly or bridging a bond with an H2O molecule, as also proposed by the model of the recently crystallized trimeric channel-like urea transporter dvUT (Levin et al., 2009). UreA mediated [14C]-urea uptake was inhibited by putrescine but surprisingly not by spermine or spermidine. Polyamines are aliphatic amines that are positively charged at physiological pH values. Among the three polyamines, spermine and spermidine exhibit the highest structural flexibility for a theoretical binding by a bending-over conformation within the substrate binding pocket. On the other hand putrescine containing only CH2 chains in the aliphatic group and not other amino groups as spermine and spermidine, exhibits the lowest hydrophilicity that theoretically enables an energetically favourable access to the substrate binding site (Weiger et al., 1998). Very recently, a high-affinity putrescine–cadaverine transporter from Trypanosoma cruzi was characterized, also not recognizing spermine or spermidine (Hasne et al., 2010), and thus demonstrating the existence of specialized transporters for these polyamines. Growth tests and kinetic experiments of the deletion and single-point mutants suggest that UreA is the only high-affinity transport system for urea in A. nidulans. The residual growth observed for the three loss-of-function mutant strains when urea is utilized as sole nitrogen source could be due to the incorporation of urea through a secondary energy-independent transport system operating by facilitated diffusion at high urea concentrations (>3 mM), as suggested by Pateman et al. (1982) and described for S. cerevisiae (ElBerry et al., 1993) and A. thaliana (Liu et al., 2003). Another

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possibility would be that one of the ureA paralogues identified in the genomes of A. nidulans could transport urea with low affinity, as suggested by Morel and coauthors (2008). The phylogenies obtained taking into consideration UreA homologues in sequenced Aspergilli, as well as characterized homologues in A. thaliana, P. involutus and S. cerevisiae suggest the occurrence of gene duplication events before the different Aspergillus species diverged from a common ancestor. The phylogenies determined for UreA homologues in each of the clusters are coincident with the accepted evolutionary relationships of the Aspergilli (http:// www.broad.mit.edu/annotation/genome/aspergillus_group/). One of the duplication events may have given origin to the two clusters of genes containing homologues of characterized urea transporters. The fact that the cluster containing UreA (ANID_00418.1) is the only one with putative orthologues in all Aspergilli supports the idea of them being functionally relevant. The absence of homologues in some of the Aspergillus species in the three clusters which do not contain UreA (ANID_00418.1) could be interpreted as a result of gene loss. It would be logical to think that the protein encoded by ANID_7373.1, which clusters with AtDur3 and PiDur3, could be responsible for the residual growth observed on urea in the DureA mutant. Interestingly, this protein has significantly diverged from other Aspergillus putative orthologues in the same cluster. The other two clusters of UreA homologues in the Aspergilli may have diverged, after an earlier duplication event, to acquire novel, unidentified specificities. It is worth mentioning that an EST corresponding to ANID_2598.1 was found (http://blast.ncbi.nlm.nih.gov/; Non-human, non-mouse ESTs; est others). This pattern of duplication events with acquisition of novel specificities has been recently reported in the case the Fur4p-like family of transporters in A. nidulans (Hamari et al., 2009). Urea transport has been shown to be transcriptionally and posttranslationally regulated. The transcription of ureA is dependent on AreA, strongly repressed by ammonium, but not inducible by urea. The fact that ureA is expressed in the presence of proline, acetamide, arginine or nitrate excludes the possibility of uric acid being the specific inducer, as for the other genes of the purine catabolic pathway. Moreover, strains carrying a null mutation in uaY, the gene encoding for the transcription factor mediating induction by uric acid, grow normally on urea and are sensitive to thiourea (not shown). These results are in agreement with those reported by Pateman et al. (1982) for urea transport and by Scazzocchio and Darlington (1968) for urease activity. The different levels of expression in the various non-repressive sources tested could respond to resulting different levels of repressing nitrogen species produced into the cell. The highest expression level is achieved under nitrogen-starved conditions. The transcriptional activation of ureA during conidial germination was found to be dependent on AreA and to respond to nitrogen metabolite repression. A similar phenomenon has been observed in the case of agtA (Apostolaki et al., 2009) and of uapA (Amillis et al., 2004). These results argue against the existence of a general mechanism that triggers global expression of transporters of nitrogenous compounds as a way of sensing solute availability, and which is able to by-pass those operating in mycelial stage (Amillis et al., 2004; Apostolaki et al., 2009; Momany, 2002). For some transporters this mechanism would not be competent or nitrogen metabolite repression could not be surpassed. As mentioned above, post-translational regulation in response to ammonium involves the endocytosis of the transporter. As speculated for other plasma membrane transporters (Apostolaki et al., 2009; Pantazopoulou et al., 2007; Valdez-Taubas et al., 2004), this mechanism must involve the sorting into the multivesicular body pathway of UreA molecules after internalization by endocytosis. The negative effect of cycloheximide on this phenomenon shows that internalization requires protein synthesis.

In conclusion, UreA is the only high-affinity specific urea transporter of A. nidulans that together with ScDur3, AtDur3 and PiDur3 would constitute a subfamily of SSS transporters, which mechanistically involves H+ instead of Na+ cotransport. The expression of the ureA gene is not specifically induced, but is subject to nitrogen metabolite repression, even in the isotropic growth phase when genes coding for other transporters are usually constitutively expressed. A post-translational mechanism also responding to ammonium acts to regulate transport activity. The presence UreA homologues which cluster in divergent groups suggest that these proteins may have acquired different and unknown transport activities.

Acknowledgments We thank C. Scazzocchio for helpful discussion and critical reading the manuscript. The work in Uruguay was supported by the Comisión Sectorial de Investigación, Universidad de la República and by the Programa Especial de Desarrollo de las Ciencias Básicas. M.S. received support from the Agencia Nacional de Investigación e Innovación (Uruguay). S.A. thanks G. Diallinas for fruitful discussions, critically reading the manuscript and for providing the [14C] experimental facilities.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fgb.2010.07.004.

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