Fungal Genetics and Biology 47 (2010) 1023–1033

Contents lists available at ScienceDirect

Fungal Genetics and Biology

journal homepage: www.elsevier .com/locate /yfgbi

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

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 April 2010Accepted 8 July 2010Available online 12 July 2010

Keywords:Aspergillus nidulansUrea transportUreAUrea/H+ symporter

1087-1845/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.fgb.2010.07.004

* Corresponding author. Fax: +598 2 5258618.E-mail address: anaramon@fcien.edu.uy (A. Ramon

1 Present address: Unidad de Proteínas RecombMontevideo, Montevideo, Uruguay.

2 These authors contributed equally to this work.

We report here the characterization of UreA, a high-affinity urea/H+ symporter of Aspergillus nidulans. Thedeletion of the encoding gene abolishes urea transport at low substrate concentrations, suggesting that inthese conditions UreA is the sole transport system specific for urea in A. nidulans. The ureA gene is notinducible by urea or its precursors, but responds to nitrogen metabolite repression, necessitating forits expression the AreA GATA factor. In contrast to what was observed for other transporters in A. nidu-lans, repression by ammonium is also operative during the isotropic growth phase. The activity of UreA isdown-regulated post-translationally by ammonium-promoted endocytosis. A number of homologues ofUreA have been identified in A. nidulans and other Aspergilli, which cluster in four groups, two of whichcontain the urea transporters characterized so far in fungi and plants. This phylogeny may have arisen bygene duplication events, giving place to putative transport proteins that could have acquired novel, stillunidentified functions.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Urea occurs in nature as a byproduct of animal metabolism ofnitrogenous compounds, being the main nitrogen-containing sub-stance in the urine of mammals (Smith, 2009). On the other hand,it is the world’s most common form of nitrogen fertilizer, with asustained increase in its use during the last four decades (Glibertet al., 2006).

Bacteria, fungi and plants are able to use urea as nitrogensource, incorporating it into the cell through specific transporters.A number of transporters from fungi and plants with high speci-ficity for urea have been reported (ElBerry et al., 1993; Liu et al.,2003; Morel et al., 2008). These proteins are related to the so-dium symporter superfamily (SSS), which comprises more thana hundred membrane proteins from both prokaryotic and eukary-otic origin (Jung, 2002; Reizer et al., 1994). The first protein ofthis class to be described was ScDur3 from Saccharomyces cerevi-siae. ScDur3 incorporates urea when the external concentration is60.25 mM whereas at concentrations P0.5 mM, it enters the cellvia facilitated diffusion (Cooper and Sumrada, 1975; Sumradaet al., 1976). ScDur3 has also been reported to be involved inthe uptake of polyamines, surprisingly displaying a higher affinityfor those than for urea (Uemura et al., 2007). In S. cerevisiae urea

ll rights reserved.

).inantes, Institut Pasteur de

is one of the products of allantoin metabolism and, like othergenes of this catabolic pathway, expression of ScDUR3 is subjectto nitrogen catabolite repression and is dependent on the pres-ence of allophanate or oxaglutarate (the native and gratuitousinducers of the allantoin pathway, respectively) for high-levelexpression (ElBerry et al., 1993). The urea/H+ symporter from Ara-bidopsis thaliana, AtDur3, is able to complement a yeast dur3Ddeletion mutant and mediates the high-affinity transport (Km of3 lM) of urea at low external concentrations. AtDur3 is expressedin roots and shoots, being upregulated during early germinationand under nitrogen deficiency in roots (Liu et al., 2003; Merigoutet al., 2008). A BLAST search in ESTs databases allowed the iden-tification of putative orthologues in both vascular and non-vascu-lar plants (for a review, see Wang Wi-Hong et al., 2008). An activeurea transporter of the ectomycorrhizal basidiomycete Paxillusinvolutus has been recently characterized (Morel et al., 2008) afterfunctionally expressing the corresponding cDNA in a S. cerevisiaedur3D strain. PiDur3 shows a high-affinity for urea (Km of31.8 lM) and, similar to AtDur3, transport appears to be depen-dent on a H+ gradient. The authors focused on the regulation ofthe expression of PiDur3, demonstrating that the gene is upregu-lated under nitrogen deficiency, while being repressed by thehigh level of intracellular glutamine as a result of ammoniumavailability. Moreover, urea uptake seems to be tightly coupledto the efficiency of the urease enzyme, converting urea intoammonium and thus being inhibited by the intracellular accumu-lation of urea.

Urea can be used as a nitrogen source by Aspergillus nidulans(Darlington et al., 1965; Scazzocchio and Darlington, 1968).

1024 C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033

Evidence for a substrate concentration dependent and saturable(Km of 30 lM), active transport system for urea in this organismwas presented by Pateman and colleagues almost 30 years ago(Pateman et al., 1982). Thiourea, a toxic analogue of urea, is alsotransported by this same system, but with lower affinity and wasused to isolate a number of allelic mutants which showed impairedgrowth on urea as sole nitrogen source at concentrations lowerthan 3 mM, implying the existence of a second transport systemmediating passive or facilitated diffusion at higher urea concentra-tions, as described in S. cerevisiae. These authors also studied theregulation of this transport activity in response to nitrogen status,concluding that it is not induced by urea but is subject to nitrogenregulation, glutamine being the most likely effector of thisphenomenon.

In this article, we report the cloning and characterization of thegene encoding UreA, a specific urea transporter in A. nidulans aswell as a study of its transcriptional and post-transcriptional regu-lation. We show that this transporter appears to be the only pro-tein with high-affinity urea transport activity in A. nidulans. UreApresents putative orthologues in all other species of the genusAspergillus. We also speculate about the functionality of otherhomologous proteins found in the different members of the genus,on the basis of their phylogenetic relationships.

2. Materials and methods

2.1. Strains, media and transformation procedures

Standard complete and minimal media (MM) for A. nidulanswere employed (Cove, 1966; Scazzocchio and Arst, 1978; http://www.fgsc.net). Supplements were added when necessary at stan-dard concentrations (http://www.gla.ac.uk/ibls/molgen/aspergil-lus/supplement.html). A. nidulans strains used in this study arelisted in Table S1 (Supplementary material). Gene symbols are de-fined 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 soleN-sources. Thiourea, putrescine, spermine and spermidine wereused in concentrations of 0.6–5 mM. The Escherichia coli K-12strain used in standard protocols was DH5a. A. nidulans transfor-mation was carried out as in Tilburn et al. (1983).

2.2. Cloning of the ureA gene

ureA cloning was accomplished using a functional complemen-tation approach. A pabaA1; pyrG89; ureA1 strain (MVD101),impaired in urea transport, was transformed with a plasmid-basedgenomic library constructed in the autonomously replicating pRG3-AMA1-NotI vector (Osherov and May, 2000; www.fgsc.net). Thisplasmid carries the pyr4 gene from Neurospora crassa, which is ableto complement A. nidulans mutation pyrG89, restoring growth onmedia lacking uridine and uracil. Transformants prototrophic foruridine and uracil and able to grow on 1.25 mM urea as sole nitro-gen source were then recovered and tested for thiourea sensitivity.Plasmids pANureA-1 and pANureA-2 were rescued from two ofthese transformants, and their ability to complement growthimpairment on urea as sole nitrogen source was confirmed. UsingpANureA-1 as template and specific primers ureA-F and ureA-R(see Table S2, Supplementary material) a 4.7 Kb sequence contain-ing that corresponding to ANID_00418.1 (http://www.broad.mit.edu/annotation/fungi/aspergillus) gene was amplified and clonedin a pGEM-T-Easy vector (Promega). Its ability to restore urea trans-port was confirmed by co-transformation with pRG3AMA1 vectorin an ureA1 strain.

2.3. ureA deletion

The Double-Joint PCR (DJ-PCR) method (Yu et al., 2004) usingLong PCR Enzyme Mix (Fermentas) was used to construct areplacement cassette where the riboB gene was flanked by 50 and30 non-coding regions of ureA. The primers used are included inTable S2 in Supplementary material. The upstream (1478 bp) anddownstream (1018 bp) flanking regions of ureA were amplifiedfrom genomic DNA from a wt pabaA1 strain using oligonucleotidesURE5-F and URE5-R and oligonucleotides URE3-F and URE3-R,respectively. The riboB gene, used to replace the complete ureAcoding region, was amplified using oligonucleotides Ribo-F andRibo-R. The 4.6 Kb PCR fusion product was amplified with nestedprimers Ure5-N and Ure3-N and purified with the QIAEX II GelExtraction Kit (QIAGEN). The resulting cassette was transformedin 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. Eighttransformants showed impaired growth on urea and were able togrow on thiourea. Two of them were analysed by Southern blotblotting, showing that the coding region of the ureA gene had beenreplaced by riboB.

2.4. Construction of a ureA::gfp fusion strain

Fusion PCR was used to generate the ureA::gfp transformationcassette for C-terminal tagging of proteins with green fluorescentprotein (GFP) (Yang et al., 2004). Primers used are listed inTable S2 (Supplementary material). The GFP-pyrG cassette wasamplified by PCR, using primers GFPyr-F and GFPyr-R from plasmidpl1439 containing (Gly-Ala)5-GFP plus Aspergillus fumigatus pyrG(Yang et al., 2004). The second fragment, corresponding to the up-stream targeting region (the 30 end of the ureA coding region), wasamplified using primers UAcod-F and UAcod-R and the third frag-ment, the 30-ureA untranslated region, was amplified using primersUre3-F and Ure3-R. In both cases genomic DNA of a wt pabaA1strain was used as a template. The fusion product was amplifiedwith primers UreGFP5-N and Ure3-N, purified with the QIAEX IIGel Extraction Kit (QIAGEN) and used to transform a pyrG89; pyro-A4; nkuAD::argB; riboB2 A. nidulans strain (A1145, Table S1, Supple-mentary material). Long PCR Enzyme Mix (Fermentas) was used inall cases. Two transformants capable of growing on media lackinguridine and uracyl were tested by Southern blot blotting, showingthat the ureA::gfp fusion integrated to the ureA locus.

2.5. Radiolabelled urea uptake measurements

[14C]-urea uptake in minimal media (MM) was assayed in ger-minating conidiospores of A. nidulans concentrated at 107 coni-diospores/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 incubationwith concentrations of 0.5–2.0 lV for [14C]-urea at the polaritymaintenance stage (3–4 h, 130 rpm). Time course experimentswere measured in the presence of 50 lM [14C]-urea. Km/i valueswere obtained directly by performing and analysing uptakes (typ-ical velocity/substrate concentration plots and verification byPrism 3.02: GraphPad Software, Inc.), using labelled urea at 0.5–100 lM, or various concentrations (0.5–5000 lM) of non-labelledsubstrates. Ki values were calculated from the Cheng and Prusoffequation Ki = IC50/(1 + (L/Km), in which L is the permeant concen-tration. Free Gibbs energy (DGo) was calculated from DGo = �RTln(Ki), where R is the ideal gas constant and T is the absolute tem-perature (in K). IC50 values were determined from full dose–re-sponse curves and in all cases the Hill coefficient was close to�1, consistent with the presence of one binding site. In addition,

C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033 1025

we have examined the effect of thiourea and acetamide on Km andVmax for wild-type and seen that apparent Vmax values remain unal-tered, consistent with competitive inhibition, suggesting that asimple model of competition with the binding site of the trans-porter is applicable, satisfying the criteria for use of the Chengand Prusoff equation. The H+-uncoupler carbonylcyanide chloro-phenylhydrazone (CCCP) or the H+-ATPase inhibitor N,N0-dicyclo-hexyl carbodiimide (DCCD) were added at final concentrations of30 lV and 100 lM respectively, for 10 min before initiating theuptake assay. pH dependence experiments were carried out byadjusting the MM pH value 10 min before initiating the uptake as-say. Reactions were terminated with addition of equal volumes ofice-cold MM containing 1000-fold excess of non-radiolabelled sub-strate. Background uptake values were corrected by subtractingeither values measured in the deleted mutants or values obtainedin the simultaneous presence of 1000-fold excess of non-radiola-belled substrate. Both approaches led to the same background up-take level, not exceeding 10–15% of the total counts obtained inwild-type strains. All transport assays were carried out in at leastthree independent experiments, with three replicates for eachconcentration or time point. Standard deviation was <20%. Radiola-belled [14C]-urea (55.0 mCi mmol�1) was purchased from MoravekBiochemicals, Brea, CA.

2.6. Epifluorescence microscopy

Samples for fluorescence microscopy were prepared as de-scribed previously (Valdez-Taubas et al., 2004). In brief, sampleswere incubated directly on sterile cover slips protected from lightin liquid minimal media with proline (10 mM) as nitrogen sourceand appropriate supplements, at 25 �C for 14–16 h. When indi-cated, the last incubation hour took place in MM containing10 mM ammonium L(+)-tartrate and/or cycloheximide to a finalconcentration of 20 lg ml�1. Cultures were visualized and photo-graphed in an Olympus inverted microscope CKX31 belonging tothe Cellular Biology Platform, Institut Pasteur de Montevideo, witha U-MNIBA3 filter. The microscope is equipped with a HamamatsuOrca Er camera and uses Image Pro 6.0 software for image process-ing. Vacuole staining with CMAC (7-amino-4-chloromethyl couma-rin) (Molecular Probes, Inc., USA) was according to Gournas et al.(2010). CMAC was prepared as a 5 mg ml�1 stock solution in di-methyl sulfoxide and stored frozen. Cover slips with germinatedconidia were covered with MM containing 50 lg ml�1 CMAC, incu-bated at 25 �C for 20 min, washed in 2.5 ml MM, and transferred tofresh 2.5 ml MM for 20 min chase time. Samples were observed onan Axioplan Zeiss phase contrast epifluorescent microscope withappropriate filters and the resulting images were acquired with aZeiss-MRC5 digital camera using the AxioVs40 V4.40.0 softwareand processed by Adobe Photoshop software.

2.7. Northern blot analysis

Total RNA was isolated from A. nidulans as described by Loc-kington et al. (1985) and separated on glyoxal agarose gels accord-ing to Sambrook (2001). A 657 bp PCR-amplified fragment of ureA(with primers SureA-F and SureA-R) was used as a probe in North-ern blots. To monitor the amount of loaded RNA, a 2.5-kb BamHI/KpnI fragment of plasmid pSF5 (Fidel et al., 1988) was used asprobe to detect the actin messenger (acnA). In those experimentswhere ureA expression was followed during germination, the 18SrRNA was used as control since acnA mRNA does not reach a stea-dy-state level until 4 h after germination. Probes were labelledwith [32P]-dCTP using Random primer labelling system (Amer-sham) and purified with Illustra MicroSpin G-25 column(Amersham).

2.8. Determination of transcription end by 30 RACE

First-strand cDNA was synthesized from 5 lg of total RNA ex-tracted from the wild-type strain grown in proline as nitrogensource with Super Script II (Invitrogen, Carlsbad, CA, USA), usingoligo-dT primer. The 30 sequence of mRNA was amplified withCDS10 and FureA4 primers and Long PCR Enzyme Mix (Fermentas).The PCR product was purified from agarose gel with the QIAEX IIGel Extraction Kit (QIAGEN), cloned into pGEM-T-Easy vector (Pro-mega) and sequenced.

2.9. Bioinformatic tools

Sequences were obtained from the Aspergillus ComparativeDatabase, BroadInstitute (http://www.broad.mit.edu/annotation/fungi/aspergillus); the Saccharomyces genome database (http://www.yeastgenome.org); and The Arabidopsis Information Re-source (http://www.arabidopsis.org). PiDUR3 sequence was kindlyprovided by Morel et al. (2008).

Phylogeny analysis was carried out with tools available inhttp://www.phylogeny.fr (Méthodes et Algorithmes pour la Bio-informatique, of the Laboratoire d’Informatique, de Robotique etde Microélectronique de Montpellier; Dereeper et al., 2008). Multi-ple sequence alignments were carried out with Muscle, applyingcuration with G blocks. Phylogenetic trees were constructed withthe Maximum Likelihood program (PhyML) and the Bayesian Infer-ence program (Mr. Bayes) available in the site and visualized withDrawtree.

3. Results

3.1. Cloning of ureA, the gene encoding for the major urea transporterin A. nidulans

Urea transporters belonging to the same family as UreA havebeen characterized in S. cerevisiae, P. involutus and A. thaliana (seeSection 1). Early genetic and biochemical analyses have indicatedthe existence of an active urea transport system in A. nidulans (Pat-eman et al., 1982). In this work, the ureA gene was cloned by func-tional complementation of the impaired growth on urea of a ureA1strain with a genomic library constructed in the replicative plas-mid pRG3-AMA1-NotI. Transformants capable of growing on mediacontaining urea as sole nitrogen source at a concentration whereureA1 is unable to grow properly were recovered and tested fortheir sensitivity to the toxic analogue thiourea. Plasmids recoveredfrom two of the transformants (pANureA-1 and pANureA-2) werere-transformed in the original ureA1 strain, confirming their abilityto complement the growth defect on urea and to render the strainssensitive to thiourea. Restriction enzyme analysis showed that theinserts in both plasmids were partially overlapping. The insertcloned in plasmid pANureA-1 was sequenced, revealing that it con-tains the ANID_00418.1 gene (http://www.broadinstitute.org/annotation/genome/aspergillus_group). A fragment containingthe deduced coding sequence, including plus 1598 bp upstreamand 997 bp downstream from the putative initiation and termina-tion codons respectively was sub-cloned through a PCR strategy(see Materials and methods) and shown to also reestablish theability of the ureA1 strain to grow on urea and its sensitivity tothiourea.

The presence and location of the two introns predicted inANID_00418.1 (http://www.broadinstitute.org/annotation/gen-ome/aspergillus_group) was confirmed by sequencing of a cDNAclone obtained by RT-PCR. 30 RACE was performed to determinethe transcription end point at position +2082 and the position ofa polyadenylation site 90 pb downstream from the translation ter-

1026 C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033

mination codon. The sequence and all data concerning this analysiswere 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 TransportClassification Database, http://www.tcdb.org). The amino acid se-quence of ureA shows 51%, 45% and 43% identity with functionallycharacterized urea transporters from S. cerevisiae, A. thaliana and P.involutus, respectively. An alignment of the four sequences isshown in Fig. S1 (Supplementary material). The deduced proteinsequence is predicted to consist of 15 transmembrane helical do-mains (TMSs), with an extracellular N-terminus and an intracellu-lar 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 ureasestructural gene, was clustered with ureA because of the close link-age existing between these two genes. Notwithstanding, ureA andureB (ANID_10079.1) are separated by more than 30 Kb in chromo-some VIII, with more than a dozen genes deduced between them.

3.2. ureA deletion and characterization of ureA1 and ureA905 mutantalleles

A total deletion of the ureA at the locus ANID_00418.1 was car-ried out by replacing its complete coding sequence with the A.nidulans riboB gene, and thus complementing the riboflavin auxot-rophy. The double-joint PCR technique (Yu et al., 2004) was em-ployed for the construction of the replacement cassette, andsingle integration events in selected transformants were confirmedby Southern blot analysis (not shown).

Sequencing of the ureA1 and the spontaneous thiourea-resistantmutant ureA905 alleles revealed single aminoacid substitutions atpositions 168 (Gly168Asp) and 639 (Pro639Arg) respectively.According to the hydrophobicity profiles of UreA (TMPred,TMHMM-2.0), Gly168 is located immediately after transmembranehelix TMS4, facing the outside of the membrane. Pro639 is predictedto be located in the putative C-terminal domain immediately afterTMS12. The ureA deletion mutant and the single amino acid substi-tution mutants have been characterized by growth tests and up-take assays. Fig. 1 shows that the three strains are almostcompletely impaired for growth on urea as sole nitrogen source,while growing as the wild-type on minimal media (MM) supple-mented with ammonium, nitrate, proline, acetamide, arginine,hypoxanthine or uric acid as sole nitrogen sources. Resistance tothiourea is increased to similar levels in the three mutant strains.

3.3. UreA is a high-affinity, high-capacity, urea/H+ symporter activatedduring the isotropic growth phase

ureA mediated [14C]-urea uptake kinetic analysis was per-formed at 37 �C, using conidiospores germinated in the absenceof urea from the medium, at a stage prior to germ tube emergence,a stage where a number of A. nidulans transporters are significantlyexpressed (Amillis et al., 2004; Hamari et al., 2009; Tazebay et al.,1997). Fig. 2A displays a time course comparison of urea uptake inwt (ureA+), ureA-gfp, DureA and loss-of-function mutant strainsureA1 and ureA905, showing that the uptake rate for urea in germi-nating conidiospores is exclusively UreA-mediated, reaching stea-dy-state levels after 20 min of incubation, while being linear forat least 2 min. Under these conditions of linearity urea uptakeproved to have hyperbolic kinetics in relation to substrate concen-tration, as expected for a single transporter (not shown). Theapparent Km and Vm for urea were calculated to be 26.2 ± 2.1 lMand 20.9 ± 3.2 pmol min�1 � 107 conidiospores. The Km value is atthe 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 trans-porter molecules in the plasma membrane and thus is dependenton developmental and growth conditions of each experiment(see below).

Initial uptake rate measurements were also carried out duringgermination in the absence or presence of various nitrogensources. Fig. 2B shows that dormant and up to 2 h germinatingconidiospores display no significant uptake on any of the N-sourcestested. In the presence of urea, nitrate, proline or under N-starva-tion, [14C]-urea uptake was first detected 2 h after inoculation, dis-playing a maximum after 3 h, and then dropping to a more basallevel, or more dramatically (N-starvation) upon approaching thedevelopmental stage of late polarity establishment-young hyphae(4–6 h). In agreement with transport assays, epifluorescencemicroscopy of germlings of a strain carrying a UreA-GFP fusionshows that the appearance of fluorescence in the membrane occursalso after 2 h of culture (not shown). Interestingly and at varianceto what has been observed for other transporters (Amillis et al.,2004, 2007; Tazebay et al., 1997), uptake of urea is repressed byammonium during the period of conidial isotropic growth. No sat-urable [14C]-urea uptake was detected at any stage or N-source inthe 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 theobvious dependency of [14C]-urea uptake on the medium’s pH va-lue, 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 uptakepotentiation in the simultaneous presence of 100 mM Na+. UreAmediated [14C]-urea uptake was also subjected to inhibition exper-iments in the presence of 2 mM non-labelled NHþ4 , acetamide, thio-urea, guanidine and the polyamines spermine, spermidine andputrescine (Fig. 2C). Thiourea and acetamide showed significantinhibition. Specific Km/i and DGo values were calculated in bothcases (Fig. 2D) by using various concentrations of non-labelled sub-strates (see Section 2). Given the structural differences betweenurea, acetamide, guanidine or thiourea, these values could be takenas an indication of the contribution of each chemical group (aminoor carbonyl) of the urea molecule to the binding of the substrate tothe transporter. In view of urea transport inhibition by acetamide,we also tested whether null ureA mutations affected the utilizationof low concentrations (1.25, 2.5 and 5 mM) of this compound asnitrogen or carbon source. No effect was observed in any of thesecases.

UreA mediated [14C]-urea uptake was also inhibited by rela-tively high amounts of putrescine but not by spermine or spermi-dine (Fig. 2C), resulting in a low affinity Ki of �2.5 mM forputrescine. However, establishing if putrescine is actually trans-ported by UreA, despite the very low affinity, or simply interfereswith [14C]-urea uptake will prove difficult since the deletion ofureA leads to no effect on the putrescine utilization as sole nitrogensource, implying the existence of more than one transport system(not shown).

3.4. ureA expression is not induced by urea but is subject to nitrogenmetabolite repression

In A. nidulans and other fungi the genes that code for transport-ers and metabolic enzymes participating in the utilization of nitro-gen sources other than ammonium or glutamine are subject to avery stringent control. In A. nidulans these mechanisms involvethe GATA factor AreA, which is a transcriptional activator only ac-tive in the absence of preferred nitrogen sources such as ammo-nium or glutamine (Arst and Cove, 1973; Kudla et al., 1990;

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 thislatter 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.

C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033 1027

Wiame et al., 1985; Wilson and Arst, 1998). Additionally, alterna-tive nitrogen sources can act as inducers of the specific genes in-volved in their utilization by activating specific transcriptionalregulators (Berger et al., 2006; Cecchetto et al., 2004; Cultroneet 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 wild-type strain grown on different nitrogen sources is shown inFig. 3A. ureA expression takes place to different levels in media

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, andgermlings (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 awild-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) Structuresof urea, thiourea, acetamide and guanidine (obtained in PubChem, http://pubchem.ncbi.nlm.nih.gov/).

1028 C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033

containing urea, arginine, hypoxanthine, proline or acetamide,usually considered as non-repressive nitrogen sources. Highestexpression is reached when cells are grown under nitrogen-starva-tion conditions. These results suggest that ureA expression is notinducible by urea. In fact, when the latter is the nitrogen source,message accumulation level is the lowest achieved in non-repres-sive sources.

ureA is subject to nitrogen metabolite repression, since no ureAmessenger is detected when strains are grown in the presence ofammonium in the culture medium (Fig. 3A). Expression dependson a functional AreA factor, since in a loss-of-function areA600 mu-tant (areA�) no ureA expression is detectable even in nitrogen-starved cells (Fig. 3B), where expression is maximal in the wild-type strain. We also monitored the expression of ureA in areA102and areA30 mutant strains which affect the specificity of the acti-vator, conferring reciprocal loss-of-function, gain-of-function orwild-type phenotype depending upon the structural gene into con-sideration (Arst, 1977; Arst and Cove, 1973; Arst and Scazzocchio,1975; Gorton, 1983; Hynes, 1973a,b, 1975; Polkinghorne andHynes, 1975). The areA102 mutation (Leu683Val) shows increasedbinding to TGATAR sites and decreased binding to AGATAR andCGATAR sites, while areA30 (Leu683Met) shows a near mirror-im-age phenotype (Ravagnani et al., 1997). In the case of urea, areA30mutants show decreased utilization of this nitrogen source. This ef-

fect has been attributed to altered expression of the gene codingfor the urea permease, since areA30 have reduced urea uptake lev-els and increased resistance to thiourea. The reverse effects wereobserved for areA102 strains (Gorton, 1983; Hynes, 1973b). Inagreement with this, we observed that in non-repressive condi-tions (proline and nitrogen starvation) an areA102 mutant showsincreased expression of ureA, while in an areA30 strain messengerlevels are highly reduced with respect to the wild-type (Fig. 3B).Physiologically important AreA binding sites usually occur in pairswho promote cooperative binding of the GATA factor (Gorfinkielet al., 1993; Punt et al., 1995; Muro-Pastor et al., 1999; Gomezet al., 2003). A search for canonical GATA sites in the 1000 bp up-stream of the ATG of ureA allowed the identification of a single pos-sible pair of these sites, composed of a TGATAA and a CGATAG,separated by 9 bp (not shown), and located at approximately600 pb upstream of the ureA ATG initiation codon. The phenotypesfound for ureA expression in areA102 and areA30 mutants suggestthat at least a TGATAR site would be physiologically important. Be-cause of cooperative binding, the decrease in affinity for this sitecould be affecting binding to the CGATAR site.

The xprD1 (so called for historical reasons) allele of areA resultsin strong derepression of AreA dependent genes in the presence ofammonium (Arst and Cove, 1973; Cohen, 1973; Platt et al.,1996a,b). This is however not the case for ureA which, in an xprD1

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+ wild-type 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 mMproline, 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 dependenceon 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 (centralpanel) 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) or2.5 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 withoutnitrogen 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 ofwild-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 wasused as a control of RNA loading.

C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033 1029

background, is substantially down-regulated under non-repressingconditions (proline and nitrogen starvation cultures) and repressedin the presence of ammonium (Fig. 3B, right panel). Plate tests ofthiourea resistance of the xprD1 mutant support these results,since in the presence of ammonium the mutant exhibits normalgrowth, being indistinguishable from a wild-type strain (notshown). This is in agreement with data of Pateman et al. (1982)concerning transport activities but not with those reported by Plattand Langdon (Fig. 3 in Platt et al., 1996a) who found that an xprD1mutant shows impaired growth on 5 mM thiourea in the presenceof 10 mM NHþ4 , which would be indicative of the derepression ofthe urea permease. The strain used in our assays behaves as ex-pected for a derepressed mutant when tested for sensitivity tochlorate or 2-thioxanthine in the presence of ammonium. More-over, it was used by Apostolaki et al. (2009) who showed derepres-sion of agtA in the presence of ammonium. A similar, but evenmore extreme down-regulation in non-repressing conditions inan xprD1mutant was observed for the hxnS gene of A. nidulansencoding the purine hydroxylase enzyme II (PHII) and other genesinduced by nicotinate (R. Fernandez-Martin, Z. Hamari, A. Cultroneand 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 thecoding genes are expressed in a constitutive way, independentlyof other mechanisms of regulation acting on them in mycelialstage. ureA expression is undetectable in resting conidia, showingfurther 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 ammo-nium, since no message is detectable during germination in a wild-type strain on this nitrogen source. Transcriptional activation ofureA in germinating conidia in an areA30 mutant grown on prolinefollows the same profile than in the wild-type, but occurs at lowerlevels (Fig. 3C). This fact supports the idea that this repressibilityimplies a dependence on AreA.

3.6. Post-translational regulation of UreA

In order to determine the subcellular localization of UreA, weconstructed a strain where the ureA wild-type allele was replacedby a ureA-gfp fusion. This strain shows normal growth on urea assole nitrogen source (Fig. 1), and Km and Vm values for urea trans-port that are comparable to those of the wild-type strain (Fig. 2A).As expected, UreA-GFP localizes to the plasma membrane of germ-lings grown on proline (Fig. 4A, left panel). The fusion protein canbe also observed in septae and in intracellular globular compart-ments that coincide with the vacuolar staining marker CMAC, atopology observed for chimeric transporters in A. nidulans studiedto date (Apostolaki et al., 2009; Gournas et al., 2010; Pantazopou-lou et al., 2007; Valdez-Taubas et al., 2004).

Upon addition of ammonium UreA-GFP disappears from theplasma membrane after 60 min and is progressively accumulatedin numerous CMAC stained compartments (Fig. 4B). This phenom-enon seems to depend on de novo protein synthesis, as indicated bythe inability of ammonium to induce internalization in the simul-taneous presence of the protein synthesis inhibitor cycloheximide(Fig. 4B). A similar phenomenon of post-translational protein turn-over has been previously described for the A. nidulans purine trans-porters UapA, UapC and FcyB (Valdez-Taubas et al., 2004,Pantazopoulou et al., 2007; Vlanti and Diallinas, 2008) and the

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 �Cfor 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. Barrepresents 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 (NHþ4 )for a period of 2 h. Arrow heads indicate vacuolar compartments labelled with both GFP and CMAC. Bar represents 5 lm.

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

Organism Accession No. Length (aa) % identity with UreA

A. nidulans ANID_00418.1 (UreA) 693 100ANID_07373.1 642 45ANID_02598.1 631 40ANID_07557.1 663 34

A. terreus ATEG_02629 649 85ATEG_07546.1 652 36ATEG_01766.1 620 35ATEG_07346.1 565 37

A. clavatus ACLA_029.180 676 82ACLA_097250 637 37ACLA_097210 616 36

N. fischeri NFIA_019890 680 81NFIA_007820 611 43NFIA_049560 636 37

A. oryzae AO090003000854 700 79AO090003001423 631 41AO090124000019 487 40AO090124000019 74 39

A. niger est_fge1_pg_C_10845 656 78fge1_pg_C_10000081 631 42fge1_pm_C_9000146 621 38fge1_pg_C_9000190 612 37

A. fumigatus Afu1g04870 680 79Afu1g17570 625 42Afu6g03200 635 37

A. flavus AFL2G_02167.2 701 74AFL2G_01655.2 596 38AFL2G_08023.2 647 35

1030 C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033

amino acid transporters AgtA and PrnB (Tazebay et al., 1997; Apos-tolaki et al., 2009).

3.7. In silico identification of UreA homologues in A. nidulans and otherAspergilli

An in silico search of putative UreA homologues in the genomesof both A. nidulans and the other seven Aspergilli whose genomesare 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 car-ried out. A summary of the sequences found is included in Table 1.In A. nidulans we identified three putative proteins, with sequencesimilarity to UreA (ANID_00418.1), while in each of the othermembers of the genus three or four putative homologues arepresent. These sequences, together with those of characterizedurea transporters from S. cerevisiae, P. involutus and A. thaliana,were used as a template for the construction of a phylogenetictree as described in Materials and methods (Fig. 5). UreA(ANID_00418.1) appears to have orthologues in all Aspergillus spe-cies. None of the other homologues is present in all of the speciesof the genus. It is interesting to note that ScDur3 cluster withUreA and its orthologues in other Aspergilli, while PiDur3 and At-Dur3 cluster with ANID_07373.1 and homologues in A. niger, A.terreus, the A. fumigatus/N. fischeri clade and A. clavatus. Notwith-standing, according to the tree ANID_07373.1 seems to have di-verged from orthologous proteins in other Aspergilli. The othertwo A. nidulans homologues, ANID_02598.1 and ANID_07557.1group in two clusters. The one including A. nidulans proteinANID_02598.1 has putative orthologues in A. oryzae and A. flavus,N. fischeri, A. fumigatus, A. niger and A. terreus. A. clavatus is absentfrom this group. The second cluster, which includesANID_07557.1, contains homologues of A. terreus, A. niger, A. clav-atus, A. oryzae and A. flavus. A. fumigatus and the closely related

species N. fischeri are absent from this cluster. A very similartopology was obtained when using Bayesian inference for con-struction of the phylogenetic tree (not shown).

Fig. 5. Phylogenetic tree of characterized urea transporters from plants (AtDur3) and fungi (UreA, PiDur3 and ScDur3) and homologues in the genus Aspergillus. Phylogenyanalysis was carried out following the ‘‘A la Carte Mode” in Phylogeny.fr (http://www.phylogeny.fr). MUSCLE was used for multiple sequence alignment, applying Gblockscuration 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 valuesfor 500 re-samplings.

C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033 1031

4. Discussion

In this study the functional characterization of UreA as an activeurea transporter of A. nidulans has been carried out. The kineticdata obtained for urea transport through this system are in agree-ment with those obtained by Pateman et al. (Km 30 lM) and arealso in the same range as those obtained for characterized ureatransporters from S. cerevisiae (ElBerry et al., 1993), A. thaliana(Liu et al., 2003) and P. involutus (Morel et al., 2008). Like most fun-gal transporters reported so far, UreA functions as a H+ symporter.Together with ScDur3, PiDur3 and AtDur3, UreA is identified as amember of the sodium:solute symporter (SSS) family of transport-ers, but all of these proteins exhibit the particularity of possessinga H+-symport mechanisms. We propose that this group of proteinswould constitute a subfamily of urea:H+ symporters included inthe SSS family of transporters. Up to now, the only other trans-porter classified into the SSS family but mechanistically involvingthe symport of protons is MctP of Rhizobium leguminosarum (Hosieet al., 2002) mediating import of alanine and other monocarboxy-lates like lactate and pyruvate.

In this work, we show that urea transport through UreA is com-petitively inhibited by thiourea and also by acetamide. These re-sults allow us to speculate about urea recognition by UreA.Specificity profiles and differences in binding energies calculatedfor each of the inhibitors in comparison to urea make evident thatthe functional groups of the urea molecule contribute nearlyequally for efficient substrate translocation, whereas the carbonyl

group appears to be the most important in either interacting di-rectly or bridging a bond with an H2O molecule, as also proposedby the model of the recently crystallized trimeric channel-like ureatransporter dvUT (Levin et al., 2009). UreA mediated [14C]-urea up-take was inhibited by putrescine but surprisingly not by spermineor spermidine. Polyamines are aliphatic amines that are positivelycharged at physiological pH values. Among the three polyamines,spermine and spermidine exhibit the highest structural flexibilityfor a theoretical binding by a bending-over conformation withinthe substrate binding pocket. On the other hand putrescine con-taining only CH2 chains in the aliphatic group and not other aminogroups as spermine and spermidine, exhibits the lowest hydrophi-licity that theoretically enables an energetically favourable accessto the substrate binding site (Weiger et al., 1998). Very recently,a high-affinity putrescine–cadaverine transporter from Trypano-soma cruzi was characterized, also not recognizing spermine orspermidine (Hasne et al., 2010), and thus demonstrating the exis-tence of specialized transporters for these polyamines.

Growth tests and kinetic experiments of the deletion and sin-gle-point mutants suggest that UreA is the only high-affinity trans-port system for urea in A. nidulans. The residual growth observedfor the three loss-of-function mutant strains when urea is utilizedas sole nitrogen source could be due to the incorporation of ureathrough a secondary energy-independent transport system operat-ing by facilitated diffusion at high urea concentrations (>3 mM), assuggested by Pateman et al. (1982) and described for S. cerevisiae(ElBerry et al., 1993) and A. thaliana (Liu et al., 2003). Another

1032 C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033

possibility would be that one of the ureA paralogues identified inthe genomes of A. nidulans could transport urea with low affinity,as suggested by Morel and coauthors (2008).

The phylogenies obtained taking into consideration UreA homo-logues in sequenced Aspergilli, as well as characterized homologuesin A. thaliana, P. involutus and S. cerevisiae suggest the occurrence ofgene duplication events before the different Aspergillus species di-verged from a common ancestor. The phylogenies determined forUreA homologues in each of the clusters are coincident with theaccepted evolutionary relationships of the Aspergilli (http://www.broad.mit.edu/annotation/genome/aspergillus_group/). Oneof the duplication events may have given origin to the two clustersof genes containing homologues of characterized urea transporters.The fact that the cluster containing UreA (ANID_00418.1) is theonly one with putative orthologues in all Aspergilli supports theidea of them being functionally relevant. The absence of homo-logues in some of the Aspergillus species in the three clusters whichdo not contain UreA (ANID_00418.1) could be interpreted as a re-sult of gene loss. It would be logical to think that the protein en-coded by ANID_7373.1, which clusters with AtDur3 and PiDur3,could be responsible for the residual growth observed on urea inthe DureA mutant. Interestingly, this protein has significantly di-verged from other Aspergillus putative orthologues in the samecluster. The other two clusters of UreA homologues in the Aspergillimay have diverged, after an earlier duplication event, to acquirenovel, unidentified specificities. It is worth mentioning that anEST corresponding to ANID_2598.1 was found (http://blas-t.ncbi.nlm.nih.gov/; Non-human, non-mouse ESTs; est others). Thispattern of duplication events with acquisition of novel specificitieshas been recently reported in the case the Fur4p-like family oftransporters in A. nidulans (Hamari et al., 2009).

Urea transport has been shown to be transcriptionally and post-translationally regulated. The transcription of ureA is dependent onAreA, strongly repressed by ammonium, but not inducible by urea.The fact that ureA is expressed in the presence of proline, acetam-ide, arginine or nitrate excludes the possibility of uric acid beingthe specific inducer, as for the other genes of the purine catabolicpathway. Moreover, strains carrying a null mutation in uaY, thegene encoding for the transcription factor mediating induction byuric acid, grow normally on urea and are sensitive to thiourea(not shown). These results are in agreement with those reportedby Pateman et al. (1982) for urea transport and by Scazzocchioand Darlington (1968) for urease activity. The different levels ofexpression in the various non-repressive sources tested could re-spond to resulting different levels of repressing nitrogen speciesproduced into the cell. The highest expression level is achieved un-der nitrogen-starved conditions.

The transcriptional activation of ureA during conidial germina-tion was found to be dependent on AreA and to respond to nitrogenmetabolite repression. A similar phenomenon has been observed inthe case of agtA (Apostolaki et al., 2009) and of uapA (Amillis et al.,2004). These results argue against the existence of a general mech-anism that triggers global expression of transporters of nitroge-nous compounds as a way of sensing solute availability, andwhich is able to by-pass those operating in mycelial stage (Amilliset al., 2004; Apostolaki et al., 2009; Momany, 2002). For sometransporters this mechanism would not be competent or nitrogenmetabolite repression could not be surpassed.

As mentioned above, post-translational regulation in responseto ammonium involves the endocytosis of the transporter. As spec-ulated for other plasma membrane transporters (Apostolaki et al.,2009; Pantazopoulou et al., 2007; Valdez-Taubas et al., 2004), thismechanism must involve the sorting into the multivesicular bodypathway of UreA molecules after internalization by endocytosis.The negative effect of cycloheximide on this phenomenon showsthat internalization requires protein synthesis.

In conclusion, UreA is the only high-affinity specific urea trans-porter of A. nidulans that together with ScDur3, AtDur3 and PiDur3would constitute a subfamily of SSS transporters, which mechanis-tically involves H+ instead of Na+ cotransport. The expression of theureA gene is not specifically induced, but is subject to nitrogenmetabolite repression, even in the isotropic growth phase whengenes coding for other transporters are usually constitutively ex-pressed. A post-translational mechanism also responding toammonium acts to regulate transport activity. The presence UreAhomologues which cluster in divergent groups suggest that theseproteins may have acquired different and unknown transportactivities.

Acknowledgments

We thank C. Scazzocchio for helpful discussion and criticalreading the manuscript. The work in Uruguay was supported bythe Comisión Sectorial de Investigación, Universidad de la Repúb-lica and by the Programa Especial de Desarrollo de las Ciencias Bás-icas. M.S. received support from the Agencia Nacional deInvestigación e Innovación (Uruguay). S.A. thanks G. Diallinas forfruitful discussions, critically reading the manuscript and for pro-viding the [14C] experimental facilities.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.fgb.2010.07.004.

References

Amillis, S., Cecchetto, G., Sophianopoulou, V., Koukaki, M., Scazzocchio, C., Diallinas,G., 2004. Transcription of purine transporter genes is activated during theisotropic growth phase of Aspergillus nidulans conidia. Mol. Microbiol. 52, 205–216.

Amillis, S., Hamari, Z., Roumelioti, K., Scazzocchio, C., Diallinas, G., 2007. Regulationof expression and kinetic modeling of substrate interactions of a uraciltransporter in Aspergillus nidulans. Mol. Membr. Biol. 24, 206–214.

Apostolaki, A., Erpapazoglou, Z., Harispe, L., Billini, M., Kafasla, P., Kizis, D., Penalva,M.A., Scazzocchio, C., Sophianopoulou, V., 2009. AgtA, the dicarboxylic aminoacid transporter of Aspergillus nidulans, is concertedly down-regulated byexquisite sensitivity to nitrogen metabolite repression and ammonium-elicitedendocytosis. Eukaryot. Cell 8, 339–352.

Arst Jr., H.N., 1977. Some genetical aspects of ornithine metabolism in Aspergillusnidulans. Mol. Gen. Genet. 151, 105–110.

Arst Jr., H.N., Cove, D.J., 1973. Nitrogen metabolite repression in Aspergillus nidulans.Mol. Gen. Genet. 126, 111–141.

Arst Jr., H.N., Scazzocchio, C., 1975. Initiator constitutive mutation with an ‘up-promoter’ effect in Aspergillus nidulans. Nature 254, 31–34.

Berger, H., Pachlinger, R., Morozov, I., Goller, S., Narendja, F., Caddick, M., Strauss, J.,2006. The GATA factor AreA regulates localization and in vivo binding siteoccupancy of the nitrate activator NirA. Mol. Microbiol. 59, 433–446.

Cecchetto, G., Amillis, S., Diallinas, G., Scazzocchio, C., Drevet, C., 2004. The AzgApurine transporter of Aspergillus nidulans. Characterization of a proteinbelonging to a new phylogenetic cluster. J. Biol. Chem. 279, 3132–3141.

Cohen, B.L., 1973. Regulation of intracellular and extracellular neutral and alkalineproteases in Aspergillus nidulans. J. Gen. Microbiol. 79, 311–320.

Cooper, T.G., Sumrada, R., 1975. Urea transport in Saccharomyces cerevisiae. J.Bacteriol. 121, 571–576.

Cove, D.J., 1966. The induction and repression of nitrate reductase in the fungusAspergillus nidulans. Biochim. Biophys. Acta 113, 51–56.

Cultrone, A., Dominguez, Y.R., Drevet, C., Scazzocchio, C., Fernandez-Martin, R.,2007. The tightly regulated promoter of the xanA gene of Aspergillus nidulans isincluded in a helitron. Mol. Microbiol. 63, 1577–1587.

Darlington, A.J., Scazzocchio, C., Pateman, J.A., 1965. Biochemical and geneticalstudies of purine breakdown in Aspergillus. Nature 206, 599–600.

Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F.,Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., Gascuel, O., 2008. Phylogeny.fr:robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36,W465–469.

Diallinas, G., Gorfinkiel, L., Arst Jr., H.N., Cecchetto, G., Scazzocchio, C., 1995. Geneticand molecular characterization of a gene encoding a wide specificity purinepermease of Aspergillus nidulans reveals a novel family of transportersconserved in prokaryotes and eukaryotes. J. Biol. Chem. 270, 8610–8622.

C. Abreu et al. / Fungal Genetics and Biology 47 (2010) 1023–1033 1033

ElBerry, H.M., Majumdar, M.L., Cunningham, T.S., Sumrada, R.A., Cooper, T.G., 1993.Regulation of the urea active transporter gene (DUR3) in Saccharomycescerevisiae. J. Bacteriol. 175, 4688–4698.

Fidel, S., Doonan, J.H., Morris, N.R., 1988. Aspergillus nidulans contains a single actingene which has unique intron locations and encodes a gamma-actin. Gene 70,283–293.

Glibert, P.M., Harrison, J., Heil, C., Seitzinger, S., 2006. Escalating worldwide use ofurea – a global change contributing to coastal eutrophication. Biogeochemistry77, 441–463.

Gomez, D., Garcia, I., Scazzocchio, C., Cubero, B., 2003. Multiple GATA sites: proteinbinding and physiological relevance for the regulation of the proline transportergene of Aspergillus nidulans. Mol. Microbiol. 50, 277–289.

Gorfinkiel, L., Diallinas, G., Scazzocchio, C., 1993. Sequence and regulation of theuapA gene encoding a uric acid-xanthine permease in the fungus Aspergillusnidulans. J. Biol. Chem. 268, 23376–23381.

Gorton, D.J., 1983. Genetic and biochemical studies of the uptake of purines andtheir degradation products in Aspergillus nidulans. Ph.D., University of Essex.

Gournas, C., Amillis, S., Vlanti, A., Diallinas, G., 2010. Transport-dependentendocytosis and turnover of a uric acid-xanthine permease. Mol. Microbiol.75, 246–260.

Hamari, Z., Amillis, S., Drevet, C., Apostolaki, A., Vagvolgyi, C., Diallinas, G.,Scazzocchio, C., 2009. Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter inAspergillus nidulans. Mol. Microbiol. 73, 43–57.

Hasne, M.P., Coppens, I., Soysa, R., Ullman, B., 2010. A high-affinity putrescine–cadaverine transporter from Trypanosoma cruzi. Mol. Microbiol. 76, 78–91.

Hosie, A.H., Allaway, D., Poole, P.S., 2002. A monocarboxylate permease ofRhizobium leguminosarum is the first member of a new subfamily oftransporters. J. Bacteriol. 184, 5436–5448.

Hutchings, H., Stahmann, K.P., Roels, S., Espeso, E.A., Timberlake, W.E., Arst Jr., H.N.,Tilburn, J., 1999. The multiply-regulated gabA gene encoding the GABApermease of Aspergillus nidulans: a score of exons. Mol. Microbiol. 32, 557–568.

Hynes, M.J., 1973a. Alterations in the control of glutamate uptake in mutants ofAspergillus nidulans. Biochem. Biophys. Res. Commun. 54, 685–689.

Hynes, M.J., 1973b. Pleiotropic mutants affecting the control of nitrogenmetabolism in Aspergillus nidulans. Mol. Gen. Genet. 125, 99–107.

Hynes, M.J., 1975. Studies on the role of the areA gene in the regulation of nitrogencatabolism in Aspergillus nidulans. Aust. J. Biol Sci. 28, 301–313.

Jung, H., 2002. The sodium/substrate symporter family: structural and functionalfeatures. FEBS Lett. 529, 73–77.

Kudla, B., Caddick, M.X., Langdon, T., Martinez-Rossi, N.M., Bennett, C.F., Sibley, S.,Davies, R.W., Arst Jr., H.N., 1990. The regulatory gene areA mediating nitrogenmetabolite repression in Aspergillus nidulans. Mutations affecting specificity ofgene activation alter a loop residue of a putative zinc finger. EMBO J. 9, 1355–1364.

Levin, E.J., Quick, M., Zhou, M., 2009. Crystal structure of a bacterial homologue ofthe kidney urea transporter. Nature 462, 757–761.

Liu, L.H., Ludewig, U., Frommer, W.B., von Wiren, N., 2003. AtDUR3 encodes a newtype of high-affinity urea/H+ symporter in Arabidopsis. Plant Cell 15, 790–800.

Lockington, R.A., Sealy-Lewis, H.M., Scazzocchio, C., Davies, R.W., 1985. Cloning andcharacterization of the ethanol utilization regulon in Aspergillus nidulans. Gene33, 137–149.

Merigout, P., Lelandais, M., Bitton, F., Renou, J.P., Briand, X., Meyer, C., Daniel-Vedele,F., 2008. Physiological and transcriptomic aspects of urea uptake andassimilation in Arabidopsis plants. Plant Physiol. 147, 1225–1238.

Momany, M., 2002. Polarity in filamentous fungi: establishment, maintenance andnew axes. Curr. Opin. Microbiol. 5, 580–585.

Morel, M., Jacob, C., Fitz, M., Wipf, D., Chalot, M., Brun, A., 2008. Characterizationand regulation of PiDur3, a permease involved in the acquisition of urea by theectomycorrhizal fungus Paxillus involutus. Fungal Genet. Biol. 45, 912–921.

Muro-Pastor, M.I., Gonzalez, R., Strauss, J., Narendja, F., Scazzocchio, C., 1999. TheGATA factor AreA is essential for chromatin remodelling in a eukaryoticbidirectional promoter. EMBO J. 18, 1584–1597.

Muro-Pastor, M.I., Strauss, J., Ramon, A., Scazzocchio, C., 2004. A paradoxical mutantGATA factor. Eukaryot. Cell 3, 393–405.

Osherov, N., May, G., 2000. Conidial germination in Aspergillus nidulans requires RASsignaling and protein synthesis. Genetics 155, 647–656.

Pantazopoulou, A., Lemuh, N.D., Hatzinikolaou, D.G., Drevet, C., Cecchetto, G.,Scazzocchio, C., Diallinas, G., 2007. Differential physiological and developmentalexpression of the UapA and AzgA purine transporters in Aspergillus nidulans.Fungal Genet. Biol. 44, 627–640.

Papageorgiou, I., Gournas, C., Vlanti, A., Amillis, S., Pantazopoulou, A., Diallinas, G.,2008. Specific interdomain synergy in the UapA transporter determines itsunique specificity for uric acid among NAT carriers. J. Mol. Biol. 382, 1121–1135.

Pateman, J.A., Dunn, E., Mackay, E.M., 1982. Urea and thiourea transport inAspergillus nidulans. Biochem. Genet. 20, 777–790.

Platt, A., Langdon, T., Arst Jr., H.N., Kirk, D., Tollervey, D., Sanchez, J.M., Caddick, M.X.,1996a. Nitrogen metabolite signalling involves the C-terminus and the GATAdomain of the Aspergillus transcription factor AREA and the 30 untranslatedregion of its mRNA. EMBO J. 15, 2791–2801.

Platt, A., Ravagnani, A., Arst Jr., H., Kirk, D., Langdon, T., Caddick, M.X., 1996b.Mutational analysis of the C-terminal region of AREA, the transcription factormediating nitrogen metabolite repression in Aspergillus nidulans. Mol. Gen.Genet. 250, 106–114.

Polkinghorne, M., Hynes, M.J., 1975. Mutants affecting histidine utilization inAspergillus nidulans. Genet Res. 25, 119–135.

Punt, P.J., Strauss, J., Smit, R., Kinghorn, J.R., van den Hondel, C.A.M.J.J., Scazzocchio,C., 1995. The intergenic region between the divergently transcribed niiA andniaD genes of Aspergillus nidulans contains multiple NirA binding sites which actbidirectionally. Mol. Cell. Biol. 15, 5688–5699.

Ravagnani, A., Gorfinkiel, L., Langdon, T., Diallinas, G., Adjadj, E., Demais, S., Gorton,D., Arst Jr., H.N., Scazzocchio, C., 1997. Subtle hydrophobic interactions betweenthe seventh residue of the zinc finger loop and the first base of an HGATARsequence determine promoter-specific recognition by the Aspergillus nidulansGATA factor AreA. EMBO J. 16, 3974–3986.

Reizer, J., Reizer, A., Saier Jr., M.H., 1994. A functional superfamily of sodium/solutesymporters. Biochim. Biophys. Acta 1197, 133–166.

Sambrook, J.R.D. (Ed.), 2001. Molecular Cloning: A Laboratory Manual Cold SpringHarbour Laboratory Press. Cold Spring Harbour, NY, USA.

Scazzocchio, C., Arst Jr., H.N., 1978. The nature of an initiator constitutive mutationin Aspergillus nidulans. Nature 274, 177–179.

Scazzocchio, C., Darlington, A.J., 1968. The induction and repression of the enzymesof purine breakdown in Aspergillus nidulans. Biochim. Biophys. Acta 166, 557–568.

Smith, C.P., 2009. Mammalian urea transporters. Exp. Physiol. 94, 180–185.Sumrada, R., Gorski, M., Cooper, T., 1976. Urea transport-defective strains of

Saccharomyces cerevisiae. J. Bacteriol. 125, 1048–1056.Tazebay, U.H., Sophianopoulou, V., Scazzocchio, C., Diallinas, G., 1997. The gene

encoding the major proline transporter of Aspergillus nidulans is upregulatedduring conidiospore germination and in response to proline induction andamino acid starvation. Mol. Microbiol. 24, 105–117.

Tilburn, J., Scazzocchio, C., Taylor, G.G., Zabicky-Zissman, J.H., Lockington, R.A.,Davies, R.W., 1983. Transformation by integration in Aspergillus nidulans. Gene26, 205–221.

Uemura, T., Kashiwagi, K., Igarashi, K., 2007. Polyamine uptake by DUR3 and SAM3in Saccharomyces cerevisiae. J. Biol. Chem. 282, 7733–7741.

Unkles, S.E., Hawker, K.L., Grieve, C., Campbell, E.I., Montague, P., Kinghorn, J.R.,1991. CrnA encodes a nitrate transporter in Aspergillus nidulans. Proc. Natl. Acad.Sci. USA 88, 204–208.

Valdez-Taubas, J., Harispe, L., Scazzocchio, C., Gorfinkiel, L., Rosa, A.L., 2004.Ammonium-induced internalisation of UapC, the general purine permease fromAspergillus nidulans. Fungal Genet. Biol. 41, 42–51.

Vlanti, A., Diallinas, G., 2008. The Aspergillus nidulans FcyB cytosine-purine scavengeris highly expressed during germination and in reproductive compartments andis downregulated by endocytosis. Mol. Microbiol. 68, 959–977.

Wang Wi-Hong, B.K., Cao, Feng.-Qiu., Liu, Lai.-Hua., 2008. Molecular andphysiological aspects of urea transport in higher plants. Plant Sci. 175, 467–477.

Weiger, T.M., Langer, T., Hermann, A., 1998. External action of di- and polyamineson maxi calcium-activated potassium channels: an electrophysiological andmolecular modeling study. Biophys. J. 74, 722–730.

Wiame, J.M., Grenson, M., Arst Jr., H.N., 1985. Nitrogen catabolite repression inyeasts and filamentous fungi. Adv. Microb. Physiol. 26, 1–88.

Wilson, R.A., Arst Jr., H.N., 1998. Mutational analysis of AREA, a transcriptionalactivator mediating nitrogen metabolite repression in Aspergillus nidulans and amember of the ‘‘streetwise” GATA family of transcription factors. Microbiol.Mol. Biol. Rev. 62, 586–596.

Yang, L., Ukil, L., Osmani, A., Nahm, F., Davies, J., De Souza, C.P., Dou, X., Perez-Balaguer, A., Osmani, S.A., 2004. Rapid production of gene replacementconstructs and generation of a green fluorescent protein-tagged centromericmarker in Aspergillus nidulans. Eukaryot. Cell 3, 1359–1362.

Yu, J.H., Hamari, Z., Han, K.H., Seo, J.A., Reyes-Dominguez, Y., Scazzocchio, C., 2004.Double-joint PCR: a PCR-based molecular tool for gene manipulations infilamentous fungi. Fungal Genet. Biol. 41, 973–981.