Identification and characterization of CcCTR1, a copper uptake transporter-like gene, in Coprinopsis cinerea

ARTICLE IN PRESS Microbiological Research 165 (2010) 276—287

www.elsevier.de/micres

Identification and characterization of CcCTR1, a copper uptake transporter-like gene, in Coprinopsis cinerea Yuko Nakagawa, Sayaka Kikuchi, Yuichi Sakamoto, Akira Yano Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami-shi, Iwate 024-0003, Japan Received 19 January 2009; received in revised form 12 May 2009; accepted 21 May 2009

KEYWORDS Copper; Transporter; Basidiomycetes; Coprinopsis cinerea

Summary Copper (Cu) is an essential element for the physiological function of organisms. In basidiomycetes, Cu is necessary for the production of phenol oxidase enzymes such as laccase and tyrosinase. We isolated and characterized two genes, CcCTR1 and -2, from the model basidiomycete Coprinopsis cinerea. CcCTR1 and -2 showed similarity to the Cu transporter CTR1 in Saccharomyces cerevisiae. Both CcCTRs had a MLxxM motif that is conserved in other CTR homologs. The addition of Cu to a liquid culture of C. cinerea decreased the mRNA accumulation of CcCTR1 and -2. Heterologous expression of CcCTR1 in S. cerevisiae increased Cu sensitivity, suggesting that CcCTR1 is a Cu uptake transporter. Together, these results suggest that CcCTR1 plays an important role in Cu accumulation in C. cinerea. & 2009 Elsevier GmbH. All rights reserved.

Introduction The micronutrient Cu is an essential cofactor for numerous enzymes that participate in redox reactions. These include proteins involved in the detoxification of oxygen radicals, such as the Abbreviations: Cu, copper; CTR, Cu transporter; SOD, superoxide dismutase; MCO, multicopper oxidase; CuSE, coppersignaling element. Corresponding author. Current address: Ichinoseki National College of Technology, Takanashi, Hagisyo, Ichinoseki-shi, Iwate 021-8511, Japan. Tel.: +81 191 24 4623; fax: +81 191 24 2146. E-mail address: [email protected] (Y. Nakagawa).

cytoplasmic Cu/Zinc superoxide dismutase (SOD); electron-transport proteins, including mitochondrial cytochrome c oxidase; and proteins with oxidase activity, including fungal laccase, secreted phenol oxidase, and plant ascorbate oxidase. Some of these enzymes belong to the blue copper oxidase family, whose members contain 4 Cu atoms per molecule (Riva 2006). Cu can be toxic even at low concentrations. Cu(I) and Cu(II) ions may bind to inappropriate sites of non-copper-containing proteins with high affinity (Predki and Sarkar 1992), resulting in the generation of oxygen radicals and thus catalyzing the

0944-5013/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2009.05.004

ARTICLE IN PRESS Identification and characterization of CcCTR1 in C. cinerea auto-oxidation of biomolecules such as lipids, proteins, and nucleic acids (Halliwell and Gutteridge 1984). Organisms possess several mechanisms to maintain intracellular Cu concentrations at appropriate levels. These include various homeostasis factors, including transporters, which control the uptake, distribution, and sequestration of Cu inside the cell. Saccharomyces cerevisiae, a model organism for investigating Cu transporter mechanisms, has three Cu transporters (CTR): two high-affinity transporters (CTR1, -3; Knight et al. 1996; Dancis et al. 1994) and one vacuolar localized transporter (CTR2; Kampfenkel et al. 1995). CTR1 and CTR3 are regulated by the Cu sensor MAC1p, which is a regulatory protein related to Cu-dependent transcription factors (Pen ˜a et al. 1998; Jungmann et al. 1993). CTR homologs exist broadly in animal, plant, and fungal genomes, but have not been identified in prokaryotes (Dumay et al. 2006). Almost all CTRs have structurally conserved domains such as three transmembrane regions (Dumay et al. 2006); clustered methionine residues in the hydrophilic extracellular domain and an MxxxM motif in the second transmembrane domain, which are important for Cu uptake (Puig et al. 2002); and a number of functionally important charged amino acids at the C-terminal end (Pen ˜as et al. 2005). These conserved characteristics in CTRs suggest that CTRs play a common and essential role in eukaryotes. Basidiomycetes have many secreted enzymes that contain Cu in their active sites. Among these are the multicopper oxidases (MCOs), which include the laccase type, with three domains, and the ceruloplasmin type, with six domains (Nakamura and Go 2005). Metal affects the expression of some MCOs. For instance, laccase mRNA levels in the fungi Trametes versicolor (Collins and Dobson 1997) and Pleurotus ostreatus (Palmieri et al. 2000) are stimulated by the addition of Cu(II) to the culture medium. The addition of Cu increases the productivity of laccase in Trametes multicolor (Hess et al. 2002) and P. ostreatus (Baldrian and Gabriel 2002; Baldrian et al. 2005). Uptake of Cu from outside of the cells is necessary for the synthesis of Cucontaining proteins such as laccases and for the maintenance of Cu levels in cells. Thus, laccase function seems to be controlled at the transcriptional level or through localization, but Cu homeostasis in basidiomycetes remains largely unexplored. Uldschmid et al. (2002, 2003) reported two P-type ATPases involved in Cu trafficking in T. versicolor, and ctr1 was isolated from P. ostreatus by Pen ˜as et al. (2005), but there have not been any other reports of CTRs in basidiomycetes. In this study, we found genes that contribute to Cu homeostasis in Coprinopsis cenerea, a model

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basidiomycete. We isolated and characterized two CTRs from the cDNA of C. cinerea to investigate Cu homeostasis in basidiomycetes. CcCTR1 is a homolog of high-affinity CTR1 from S. cerevisiae, which is thought to play major roles in Cu uptake (Dancis et al. 1994). We also isolated and analyzed a homolog of CTR2 from S. cerevisiae in C. cenerea, and we will discuss the roles of both CTRs.

Materials and methods Organisms and culture conditions C. cinerea, strain Okayama7, was used throughout this study. Mycelia were maintained on 1.5% agar plates with 0.25  MYPG medium, as in Nagai et al. (2003). For liquid culture, we used the 0.25  MYPG medium without agar. For Cu treatment, 100 mM CuSO4 solution was diluted in the medium to various concentrations.

Phylogenetic analysis A similarity search was carried out using the amino acid sequence of ctr1 from Pleurotus sp. ‘Florida’ (AJ705045, Pen ˜as et al. 2005) and CTR1–3 from S. cerevisiae as a query. The amino acid sequences of the CTRs were obtained using the BLASTP programs at the Broad Institute’s Coprinus cinereus Database (http://www.broad.mit.edu/ annotation/genome/coprinus_cinereus/Home.html) first version and databases for Laccaria bicolor (Martin et al. 2008; Joint Genome Institute, http:// genome.jgi-psf.org/Lacbi1/Lacbi1.home.html), Phanerochaete chrysosporium (Joint Genome Institute, http://genome.jgi-psf.org/cgi-bin/runAlignment?db=Phchr1&advanced=1), Ustilago maydis (Broad Institute, http://www.broad.mit.edu/ annotation/genome/ustilago_maydis/Home.html), and Cryptococcus neoformans (Broad Institute, http://www.broad.mit.edu/annotation/genome/ cryptococcus_neoformans/Home.html). ScCTR1–3 from S. cerevisiae, SpCTR4–6 from Schizosaccharomyces pombe (Puig et al. 2002) and CaCTR1 from Candida albicans (Marvin et al. 2003) were obtained from NCBI (http://www.ncbi.nlm.nih. gov/). Locus numbers were also obtained from the Broad Institute, the Joint Genome Institute, and the NCBI database. Multiple alignment was performed with ClustalW, available at DDBJ (http:// clustalw.ddbj.nig.ac.jp/top-j.html), and Parallel PRRN (http://prrn.ims.u-tokyo.ac.jp/). A NeighborJoining tree was constructed with PAUP*4.0b10 (Swofford 2002), using the mean character difference

ARTICLE IN PRESS 278 as a distance measure. To assess branch support, 1000 times bootstrapping was also performed using the same settings as the original tree search.

Quantification of transcripts by quantitative reverse transcription (RT)-PCR Mycelia were cultured for 2 weeks in 100 ml liquid medium in 300-ml flasks as described previously (Sakamoto et al. 2005). The cultures were treated with no additional CuSO4 or with the addition of 10 mM CuSO4 to a final concentration of 20 or 100 mM. Mycelia were harvested after 0, 2, 6, 12, 24, 36, 48, or 72 h, or after 1 week of treatment. Total RNA was isolated as described above. Real-time PCR was performed using SYBR Green PCR Master Mix reaction solution (Applied Biosystems) and a 7500 Real-Time PCR System (Applied Biosystems). To analyze the level of CcCTRs’ mRNA accumulation, we used the primers CcCTR1-50 (30 -ATG ACC CTC CCC CTT CAA CT-50 ), CcCTR1-R202 (50 -ACA TGG CTC CTT TGG TTT TG-30 ), CcCTR2-50 (30 -CAC CAT GAG CCA CGG AGA TCA TGG50 ), and CcCTR2-R237 (50 -AGG AGA TGT GCC AGG ATT TG-30 ). As an internal control, we used btubulin (Acc no. AB000116) and the primers b-tubulin-751 (50 -AGG AAG CTC GCT GTC AAC AT-30 ) and R991 (50 -GCA TCT GCT CCT CAA CTT CC-30 ). Three real-time PCR analyses were performed for each RNA sample, and three RNA samples were independently prepared for each treatment.

Cloning of CcCTRs Total RNA was isolated from 200 mg of C. cinerea mycelia liquid cultured for 2 weeks using a MasterPure Yeast RNA purification kit (EPICENTREs Biotechnologies). After quantification with a RiboGreen RNA quantification kit (Invitrogen), 2 mg of RNA was subjected to reverse transcription using Oligo d(T)20 primers and a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The PCR reaction was carried out using one-tenth of the cDNA as a template with the primers CcCTR1-50 and 30 (50 -TTA AAT GTA CAA TCC CAC-30 ), or CcCTR2-50 and 30 (30 -TTA ATG ACA TGC CAT TCC CT-50 ) and the KOD-FX polymerase (Toyobo). The PCR conditions were 94 1C for 2 min and 35 cycles of 98 1C for 10 s, 55 1C for 30 s, and 68 1C for 1 min. The obtained cDNA fragments were cloned into pCR8 GW-TOPO (Invitrogen; for CcCTR1) or pENTR-D-TOPO (Invitrogen; for CcCTR2), and the DNA sequences were determined. The resultant plasmids were called pENT19 and pENT29, respectively (Figure 4).

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Plasmid construction Plasmid construction is illustrated in Figure 4. To construct Gal1 promoter (Gal1p):CcCTR1 or Gal1p:CcCTR2, pENT19 or -29 was used in a Gateway LR reaction, which allowed the CcCTR cDNA fragment to be fused into pYES-DEST52 (Invitrogen). The plasmid pYES is a 2 micro plasmid and the selection marker is URA3. The resulting plasmids, carrying CcCTRs under the control of the Gal1 promoter, were named pYN155 and pYN176, respectively. To generate GFP fusion constructs, the CcCTR1 cDNA fragment in pENT19 was amplified with the KpnI-CcCTR1-50 (50 -AAG GGT ACC ACC ATG GCC CTC CCC CTT CAA CT-30 ) and CcCTR1-30 -NcoI (50 -GAA ACC ATG GCT CCT CCT CCT CCT CCA TAA ATG TAC AAT CCC ACA T-30 ) primers to attach a restriction enzyme site and linker (Gly) for their expression. To create the vector, we disrupted one NcoI site of pTF478 (Takano et al. 2002). The fragment and modified pTF478 were digested with KpnI and NcoI, and ligated using T4 ligase (Promega). The resultant vector was designated pYN162. To construct Gal1p:CcCTR2-GFP, we used the Gateway LR reaction. For the destination vector, pTF478 was modified with the Gateway vector conversion system (Invitrogen) to attach the attR1 and R2, and the resultant plasmid was called pTF478GW. The pENT29 plasmid was used for the template and for the cDNA fragment, and CcCTR2-50 and -30 w/o stop (50 -ATA ATG ACA TGC CAT TCC CT-30 ) were used for primers. The cDNA fragment was cloned into pENTR-D-TOPO and the sequence was confirmed. The resultant plasmid was called pENT30. The CcCTR2 fragment was fused into pTF478GW with the Gateway LR reaction. This plasmid was designated pYN180.

Spotting assay The plasmids pYES2, pYN155, pYN176, pYN162, and pYN180 (Figure 4) were introduced into BY4849 (MATa ade2-1 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 can1-100 rad5-535). As a positive control for Cu transport, we used BY23503 (MATa MAC1-HA-901IL ade2-1 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 can1100), which over-expresses the MAC1 gene. Both yeast strains were distributed by the National BioResource Project (Yeast), Japan. The transformants were spotted on a plate containing synthetic minimum medium consisting of 2% galactose (SG plate), adenine, methionine, histidine, leucine and various concentrations of Cu. To spot BY23503, we used same plate except which was added uracil. Aliquots of a dilution series of a yeast culture were

ARTICLE IN PRESS Identification and characterization of CcCTR1 in C. cinerea spotted on these plates and incubated for 7 d at 30 1C.

database was the most similar protein to the ctr1 from P. ostreatus. We also found a CTR2- or -3-like gene, XP 001830829. From the blast search of the database of Broad Institute, XP 001830829 gave 42% identity and 82% positive versus CTR2, and a 52% identity and 81% positive versus CTR3. Both sequences of C. cinerea are annotated in the database as hypothetical proteins and contain a common domain to the CTR Cu transporter family (Figure 1a). We designated XP 001834487 as CcCTR1 and XP 001830829 as CcCTR2. We also searched the databases of other basidiomycetes, including Laccaria bicolor (Martin et al. 2008; Joint Genome Institute), Phanerochaete chrysosporium (Joint Genome Institute), Ustilago maydis (Broad Institute), and Cryptococcus neoformans (Broad Institute), and identified putative CTR genes (Table 2). CcCTR1 and -2 are predicted to encode polypeptides of 199 and 205 amino acids, respectively. An alignment of 22 residues in the conserved region of the CTRs or CTR-like amino acid sequences is shown in Figure 1a. All of the sequences, including those of CcCTR1 and -2, had an MLxxM motif, which might be important for Cu binding (Pen ˜as et al. 2005; Dumay et al. 2006; Figure 1a). We created a Neighbor-Joining tree with PAUP*4.0b10 (Swofford 2002) by comparing the same 22 residues (Figure 1b). CcCTR1 coded the most similar gene to Phchrj122324, and all of the basidiomycetes examined, except U. maydis, had a CTR1-like gene (Figure 1b). CTR2 is a vacuole-localized Cu transporter in S. cerevisiae (Rees et al. 2004), and CcCTR2 was located nearer to ScCTR2 than to

Fluorescence microscopy Fluorescence microscopy was performed using a FV1000-D BX61 microscope (Olympus) using viewer software.

Results Identification and characterization of two CcCTRs We searched the C. cinerea genome database (Broad Institute) and found more than 30 genes that are associated with Cu (Table 1). Half of these genes were laccase-like MCOs containing Cu in their structure, and the remaining were homeostatic genes, such as Cu amine oxidase, Cu/zinc superoxide dismutase, and transporters. C. cinerea had at least four genes annotated as Cu transporters. Two of these were CTR-like genes; one was a gene similar to CutC, which is a Cu transporter from Escherichia coli (Gupta et al. 1995); and one was a P-type ATPase-like gene (Table 1). CTR1 and -3 in S. cerevisiae are known to be high-affinity plasma membrane-localized Cu ion permeases (Labbe´ et al. 1997; Dancis et al. 1994). We therefore decided to analyze the high-affinity CTR-like genes in C. cinerea. The XP 001834487 protein in the C. cinerea

Table 1.

279

Cu-related genes in C. cinerea.

Gene annotation

Number of genes

Locus

Multicopper oxidase

18

Cu amine oxidase, enzyme domain

5

Cu/zinc superoxide dismutase CTR family P-type ATPase, heavy metal-associated, haloacid dehalogenase-like hydrolase CutC family Cu fist binding SCO1/SenC S-adenosyl-L-homocysteine hydrolase and NAD binding

2 2 1

Cc1G_03936.1, Cc1G_00686.1, Cc1G_00690.1, Cc1G_09610.1, Cc1G_09609.1, Cc1G_09611.1, Cc1G_02533.1, Cc1G_09263.1, Cc1G_05972.1, Cc1G_02449.1, Cc1G_05965.1, Cc1G_09614.1, Cc1G_03946.1, Cc1G_03940.1, Cc1G_06019, Cc1G_05998, Cc1G_08591.1, Cc1G_08587.1 Cc1G_06947.1, Cc1G_00597.1, Cc1G_07935.1, Cc1G_11177.1, Cc1g_07936.1 Cc1G_07168.1, Cc1G_07167.1 Cc1G_02223.1, Cc1G_02280.1 Cc1G_00830.1

1 1 1 1

Cc1G_06578.1 Cc1G_08665.1 Cc1G_08218.1 Cc1G_11614.1

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:hydrophobic,

:MLX2M motif

ctr1 ScCTR1

50.4% CaCTR1 CcCTR2 96.5%

LbCTR-B LbCTR-C Phchr|134019 ScCTR2 CNGC01872 Um03219 SpCTR6 ScCTR3 Um05980 CNGC00979 SpCTR4 SpCTR5 CNGC05959 CcCTR1

57.2% 76.3%

LbCTR-A Phchr|122324

2 amino acid differences

Figure 1. (a) Alignment of the predicted amino acid sequences of the C-terminal conserved region of CTR-like proteins. The details of these sequences are presented in Table 2. CcCTR1 and -3 from C. cinerea; LbCTR-A, B, and C from L. bicolor; Phchrj122324 and Phchrj134019 from P. chrysosporium; CNGC05959, CNGC00979, and CNGC01872 from C. neoformans; SpCTR4–6 from S. pombe; ScCTR1–3 from S. cerevisiae; Um05980 and Um03219 from U. maydis; CaCTR1 from C. albicans (Marvin et al. 2003); and ctr1 amino acid from P. ostreatus sp. ‘Florida’ (Pen ˜as et al. 2005). Hydrophobic residues are indicated by shaded boxes, and the conserved residues of the MLxxM motif are boxed. Asterisks indicate fully conserved single residues; colons indicate strongly similar conserved residues for all sequences; and dots indicate weakly similar conserved residues for all sequences. (b) Neighbor-Joining tree of the 20 CTR or CTRlike genes shown in panel (a) from yeasts and five kinds of basidiomycetes. Bootstrap values (over 50%) are represented as percentages, and the bar length indicates a two amino acid difference.

ARTICLE IN PRESS Identification and characterization of CcCTR1 in C. cinerea Table 2.

281

Nomenclature of CTR1-like genes in yeasts and basidiomycete.

Gene name

Organisms

Locus number from database

NCBI locus number

ScCTR1 ScCTR2 ScCTR3 SpCTR4 SpCTR5 SpCTR6 CaCTR1 ctr1 CcCTR1 CcCTR2 LbCTR-A LbCTR-B LbCTR-C Phchrj122324 Phchrj134019 Um05980 Um03219 CNGC05959 CNGC00979 CNGC01872

Saccharomyces cerevisiae

P49573 EDN62413 Q06686 O94722 Q9P7F9 Q9USV7

NP_015449 NP_012045 NP_013515 NP_587968 NP_594269 NP_595861 AJ277398 CAG_29170 XP_001834487 XP_001830829 XP_001879962 XP_001877928 XP_001877591

Saccharomyces pombe

Candida albicans Pleurotus ostreatus Coprinopsis cinerea Laccaria bicolor

Phanerochaete chrysosporium Ustilago maydis Cryptococcus neoformans

ScCTR3 in the tree. LbCTR-B and C and Phchrj134019 were located on the same branch (Figure 1b). The NBRC blast search gave following scores versus CTR2: LbCTR-B: identity 33%, positive 44%; LbCTR-C: identity 36%, positive 45%; Phchrj134019: identity 35%, positive 47%; and CNGC01872: identity 28%, positive 42%. The SMART program (http://smart.embl-heidelberg.de/ Schultz et al. 1998) predicted that CcCTR1 contains two transmembrane domains, as does ctr1 from P. ostreatus, and that CcCTR2 has three transmembrane domains, as do CTR1, CTR3, SpCTR4, and Um05980. Therefore, like all CTR1and -2-like genes (Dumay et al. 2006), CcCTR1 and -2 have two and three transmembrane domains, respectively, and are thus likely to function as Cu transporters in C. cenerea. To estimate the subcellular localization of CcCTR, we analyzed the amino acid sequences with WoLF PSORT (Horton et al. 2007). CTR1, ctr1, CcCTR1, and LbCTR were predicted to be plasma membrane-localized proteins. CcCTR2 was also predicted to localize to the plasma membrane, as were LbCTR-B and C, Phchrlj134019, and ScCTR2. We also studied the predicted promoter region of CcCTR1 using the Genetyx-Mac v.14 Motif finder. The 2 kb upstream of the genomic sequence of CcCTR1 contained two TATA boxes (at 1625 and 714), seven CAAT boxes (at 1549, 1026, 783, 708, 300, 171, and 25), and three Myb domains (at 1710, 1150, and 457) (Figure 2a). The

AJ705045 Cc1g_02223 Cc1g_02280 Lacbij248943 Lacbij314154 Lacbilj324430 Phchrj122324 Phchrj134019 Um_05980 Um_03219 CNGC_05959 CNGC_00979 CNGC_01872

XP_762127 XP_759366 XP_571290 XP_570353 XP_772768

Cu-signaling element (CuSE) with the consensus sequence 50 -DWDDHGCTGD-30 (D ¼ A, G, or T; H ¼ A, C, or T; and W ¼ T or A) is defined in S. pombe as a cis-acting element found in the promoter of genes regulated by Cu (Beaudoin and Labbe ´ 2001). CuSEs were located at positions 1673, 1166, and 745 upstream of CcCTR1 (Figure 2a). In the 2 kb upstream of genomic CcCTR2 there were five TATA boxes (at 1902, 1647, 550, 482, and 186), six CAAT boxes (at 1790, 751, 201, 179, 136, and 105), and three Myb domains (at 1352, 1161, and 374), as well as a CuSE (at 874) and an Ace1 element that is a Cudependent transcription factor in S. pombe (Beaudoin et al. 2003) at (1925) (Canessa et al. 2008; Figure 2b).

Quantitative RT-PCR To investigate how C. cinerea reacts to Cu, we carried out a time course experiment. Mycelia cultured for 2 weeks were treated with 20 or 100 mM CuSO4 and harvested after 0, 2, 6, 12, 24, 36, 48, or 72 h, or after 1 week of treatment. We used quantitative RT-PCR with gene-specific primers to analyze the level of CcCTR1 and -2 transcripts. At 36 h, the amount of CcCTR1 transcripts from untreated mycelia had not changed from the levels at 0 h (Figure 3). The level of CcCTR1 transcripts only decreased 36 h after

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TGAGGCTCTA CCACAGGAGC ACTGACAAGG TTGGCAACTT CTAGTTCCGC TACATCAGCC TCTCGATGAC CGGCGGATCA ATGAAGCGGA CCGGCTCGAC GTCAAGAACT TGCGTCGCAG TGAAGTGTTC TTTCTGTCTC CAAAGCTCCT GCTATCCGTA TGGGTGGCCT ACGAAGATCG CTGGTGCCCA GTCGCATGGA CCCCACACCA TAATTATTCG ACCAGTTGCG CCCCTGGCTT GTACCCGATG TCAAACATGC AGCACCGTCA CGCCTGACGA ACGACCTGAC

GGTCCAGGGA GGTCTCCGAG TATGTGCGAA TGCGCATGTC AACGGCGTAG ATGGATCGAG GATGAGCCAC TTGAAGACGA TGAAGCAAAT TACGTCGGGG CGAGAGAAGG CGAGCAGTTT CAACGGGCTG CTCTGCGCCT CCCAGTATGC CTCGTGTTCA ATCGGCAAAT AAGATATTGG GCGTTCATTC CGCAGTCTGA GGACCTCACT GCGATCAAAC TCGCATTCGT GCTGTTCTGG CCCTCTCGCC AAAGCCTCTT ATTTGCCTCT TCCCCGTTTC GACCCAATGA

TGGAGCGTCC TGAGGCTGAG GTTGCGGAGT CTCCGTGGTC CTTCCAGGAG GTGATATACG TGCTGCCCTC TGACTGGGTT CTTGTTGCAA AATGGCATAG GCGTTGGTTG TCGTCTCGCA GGAGGTGTTC TTGTTAATCC TTCGTCGTTG CAGATTATCG CGCGCGAACA AGATCTCAAT CCTGATATAG GCAGTACTTA GATGTGACCA GGCAGACGAT TCTTGAATCC CTCTGAGGTT AATCCCTTCG GTCGCGATGA CTTGTGTTGC TCCCAAGGGC CTCTCCCACA

ACGTATTCAT GGCACGACAG GGTCCAAATC TTGACCTCTT TGGGATGTAC GAAGGTACTT CAATAAAGGG GCTGCTCCCG ATGGTGATCA AGTTACTCTT ACGGTCGGAA AGGTCAACCA TGAGAGACGA GTAGCCTGGA TCTTCAACGC CAAGTCAACA CGTTTTCACT GACGCATACC GCAATGGTAA CTCTGAATCA TCGCAAGGGA TTAGGAACGT TTGAACTTGC GCGCATTCTG TCATCATCAT CTGGGAAGCT TTCTAAATGG TTGAATCCCC ACGACTCAAG

CGTTTCCCTC AGGGAAGTCA AACTTGTATC TAACCACTTC TACATCGTAA ACGCAGGGGA GGCCAGATAC GTCGCAGACT CGGCGTGGCC GTGACCACCA AAAATGGGTT AACGGAAGGA GACTGCAAAG AGCTGTGAGC TGTGCGCCTA AAGGCCAGAT TTAGCCTTTG ACTTTAGCGT AGATGCAACA AGTTAGGGGC CGAGGGAGTG TATTTCGCAC GCATTGCTCT CTCTGGGGTC GAACGCCAGC TCCTCGGATA CTCAAAAACG CCACTTTACT

CTCCTCCGCG AGCTCCAGGT CCGGTCTGGC CACCAGGAAC GCTGGAGTAG GTCCACTTCA TGTCCAACGC GGTTCGGGTT ACGCTCGCAG TCCGAGTCGG TAATACCCTT TTCGCTCATG CACTGCTCGT CGTCCTCCTC CATATTTCTC CAGGCGGCTG TCCAGGAAAC GCTGTGGGTC AAGTTGCGTC CGAAGTCTTC TGATTAGCCC GTTCTTATTG CCATTGGCCA TTATTGAACG TGCTTAAAAG ATCTGAGTTC TTGATAATCC CGCACTCGAC

TCGAACTTGG CTCGGTGGGG GGAAAGGACT TGGCGCACGC CTCTGATGAA CCGAGGATGT TGGGACTCGG CAGCTGAAGG GGTCGGAGAA CTGAGGAGAT GGGTGAGGAC ACGTTGGTGC TTTTATGCAT AAACAATTGT TGCGGCAGGC CTGTCGATTG AGGCGCCCAC ACACGAAGTG GCAGAATCAT GGAGGGTTTT AAGGGGTCAG GTCCGGAGGG CTTGCGCTGC CGCTGAAGGT TCGGAACAAC TTTTCATCTC GGGGCATGTA GACTTTACGA

AACGTCTCTG AGTCCTCGAG GCACCCACCC AATACTTGGA ACATTTGCCC AATATACGTA CTTGGAGAGT CTTTCCTAGT CGTAGGGTAC ACCATCGAAC GGTAAATCCA GTGCTGGACG ACTGAGGCCG CGACTGTGAA TTAGTCTACA GCAGACTTAC CTGTTAAAAC AGCAAAGCCT CTCCACGTCT ACCGTTCTCT TCTTCTCGTG ATAGACTGGG GTCTTCCTCT GCTGATCAAA TTGTTGACTG ACCCTGGCCT CAATATCCCA CCATCAATCT TTTACTTCAA

AGAGTTGCTT CTGGGTCAAC AACATCTGCA TGTCCTTGGT TAACAAATCC CAGAAATCAT GTCGATTTCG GATGCCTCTA GAACGGACCA GGGTCGTCCG TCCTGGGCGG ATGCCAAGGC GCGGACTCGG CTCTGGACCG TAGCAGACGA GAAGGTCATC GCTGAGCTTT TCTCTGGGAG CTGACTATTT CTTGTAAGCC CATGATGTTG GTGGTTCTAC GTGAAACCCT GAGAACAACT AGACGATGAA AACCGGGTCT TACCACCGGA CGCTTGGCAC AGGTCTCCAC

GACTGCGGAC GCGTTCACTA ACCTCTTCAA GATATTCCTA TTCCCTCGTT TTCCTGGATT GCTTGGATTT TTTTCGCTCG TCGGGGAAGA TTGTAGGTCT GGCGGCTTTG TCGGTGGGCT AGGCGAGCCT CGACTACGAC TTGAAGAGCA CGACATACTC ATGTCTCCTA AGACGGTCGA GCACAGTTTA TTAAAAGACC TAGGACCGGA CATAAAGCCG GACTGAATTC GGACTCTGGA ATTGTCGGTC GTACTTTTAC GAGGTACGAC GCTGCTCGGC ATAAGTAGAA

AACGTCTGCG TACGGGTATC CATCGTCATA ACCACAGCCT ACTTTAAAGA ACAGACATCC GTTTCTGCGT TCAGTCTCAT ACTCATTGAG GGCATCTAGG CTCGTCGTCG GAGTTTGAAC GGTTGACCTC TGGTGGATGA TGGCTAGATC TTGTGCAAAG ATATCCCTGA GGATTGTGAG ATGCAGTCAG CCTTCTCGAG GTGCACACCG CAGATAACTC ACCTTGTGTT CGGGGAGTAG ACCCTGTTTC ATTTCCTGTA CAGACAAAGG TACCGCAGCC TTAACTAAAG

CGACATCAGC AGCACTCTCT CTCATAATCA CGCTCTCCGT CGCGAAAAAA GTTCAGGATC TTCTGCGAGT CTGTTTGGGC GATGGAGACG ATCCTCTGAA GACACGATGA TTGCGATCAG ATCGTTTTGG ACCTCTAGAC GAATTCGGAG TGTTCTGCTG CGAATACAGA TTCATCCATC CAACGAATGT CTAAACGGTA TCGCCGATGT CCAGAAGTAT AACAGGAAAG TGAGACGTGG CCATCTCCAA TCATAGTCCA TCCCAATCAC CGCACCTCTT

GTACTTTCGC TACGACGCTC AATTCTTCTT CGCCTTCTCC CCCAGATTTT CTGGTTCGTA TGGGCGTTGA TTGTGAAGCG GTGCTAAACT TGGCGAGTAC GGATGACACG CGCGATCAGG AAGTCTGGGG GGCGACTACG GATACCAAGG TTATCGTGCG GCCTCCAAGA TGAATCTACA CAAGTTGGGA GACAAGAGTG ATACTGCGTT AAAGAAGGTG TACTTGCTGG TCAAGCTGGC ATATTACCTT ATAGCACTCT GGCTACCAAA CATCTCCTCC

TCCTTGATCA CGCTACGAGA CGCCAAATCC CGAATCTTCG ACACAAGAAG CGCAGTTCAG CTTCGTCTAG GCGATGGGCG CCTCCACGAG GACTTCTTGC GCGCTAAGCA GTCAGGTCCA CCATGTACTG ACGACGAGAA CGCGGGTTGT TAGTTGCTCA TGAACTCATT ATATCTGGCA AAGCGGGAAG CTAATCCATC GGATGTGGCG AGTCCATTAT AACGTGAAAG GAAGCTGTGG GGATATCAAC CTCTATACCC TAATAACCGT CTCCACGACC

Figure 2. Predicted promoter region of CcCTR1 (a) and -2 (b). TATA boxes, underlined and italic; CAAT boxes, underlined; Myb domains, boxed; CuSE or ACE1 element, shaded boxes.

treatment with 100 mM Cu (Figure 3). In contrast, the mRNA accumulation of CcCTR2 was decreased by only 6 h after treatment with 100 mM Cu and recovered the same level as the other samples after 12 h (Figure 3).

Functional analysis of CcCTR1 and -2 To investigate the roles of CcCTR1 and -2 in Cu transport, we obtained cDNA from the total RNA isolated from C. cinerea mycelia cultured for

ARTICLE IN PRESS Identification and characterization of CcCTR1 in C. cinerea 36 h

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Figure 3. Relative accumulation of CcCTR1 and -2 transcripts after treatment of mycelia with Cu. Mycelia were treated with 20 or 100 mM of CuSO4, or not treated. The level of transcripts from CcCTR genes were analyzed by quantitative RTPCR. The results were normalized to b-tubulin. Means and standard deviations are shown (n ¼ 3).

2 weeks in liquid medium. Using the cDNA as a template, we amplified CcCTR1 and -2 with primers based on their predicted open reading frame (ORF) sequences. The cDNAs of the two CcCTRs were cloned into a pYES2-based expression vector, as described in the Materials and Methods section and illustrated in Figure 4. The plasmids were introduced into S. cerevisiae BY4849 cells. All transformants showed similar growth on SG plates without additional Cu. Although the growth of transformants carrying the empty vector pYES2 was not severely inhibited by the addition of 500 mM Cu, the growth of CcCTR1-, CcCTR1-GFP-, and MAC1expressing cells were inhibited (Figure 5a). The growth of CcCTR2- and CcCTR2-GFP-expressing cells was not different from that of the vector control (Figure 5a). MAC1 (BY23503) is a cell strain that over-expresses the MAC1 gene and is obtained from NBRP, which is a transcription factor that regulates CTR1 and CTR3 in S. cerevisiae (Gross et al. 2000). In MAC1 cells, CTR1 and CTR3 genes are highly expressed and are hypersensitive to elevated levels of Cu (Jungmann et al. 1993). The expression of the CcCTRs was confirmed by GFP fluorescence from [Gal1p:CcCTR1 or -2-GFP] transformants. The GFP fluorescence did not localize to the plasma membrane (Figure 5b). The fluorescence of CcCTR1-GFP was observed as patches, and that of CcCTR2-GFP was faint and rare (Figure 5b). The sensitivities of the transformants to Cu concentration were the same in [Gal1p:CcCTR-GFP] as in [Gal1p:CcCTR], indicating that the presence of the GFP did not affect the sensitivity (Figure 5a). We tested several

concentrations of Cu in the medium, and growth inhibition of the CcCTR1-expressing cells was observed when the Cu concentration was 4300 mM (data not shown). Transformants carrying [Gal1p:CcCTR1] were less sensitive than MAC1 cells (Figure 5a).

Discussion We isolated and characterized CcCTR1 and -2 as being likely Cu uptake transporters in C. cinerea. The expression of these two CcCTRs in mycelia was regulated by the concentration of Cu in the medium (Figure 3), suggesting that both CcCTRs were related to Cu homeostasis. Heterologous expression of CcCTR1 in S. cerevisiae changed the Cu sensitivity (Figure 5a), indicating that CcCTR1 is especially important for Cu uptake into cells. From a phylogenic analysis of the CcCTRs, CcCTR2 was located on the same branch as CTR2 (Figure 1b). CTR2 has been suggested to be a lowaffinity Cu importer (Kampfenkel et al., 1995), but Rees et al. (2004) showed that CTR2 is localized at the vacuole membrane, where it mobilizes vacuolar Cu stores to cytosolic Cu chaperones. Since the blast search gave a higher similarity score for CcCTR2 versus CTR3 than versus CTR2, it was difficult to predict the role of CcCTR2. We therefore carried out promoter sequence analysis of the two CcCTRs (Figure 2) and also made a cDNA expression series in yeast cells (Figure 5). With a sequence analysis of the putative promoter region of the CcCTRs, we found three Myb domains

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Figure 4. Schematic diagrams of vector construction. attL1 and L2: Bacteriophage l-derived recombination sequences, pUC ori: pUC origin, Spectinomycin: spectinomycin resistance gene, Kanamycin: kanamycin resistance gene, attR1 and R2: recombination sites that allow recombinational cloning of the gene of interest from an entry clone, CmR: chloramphenicol resistance gene, ccdB: allows negative selection of expression clones, 6  His: polyhistidine region, Gal1p: Gal1 promoter, f1 ori: f1 origin, 2m ori: 2m origin, URA3: URA3 gene, Ampicillin: ampicillin resistance gene, and sGFP: S65 T GFP gene.

(Figure 2) and a CuSE element that bore a strong sequence similarity to the recognition site, denoted the metal response element (Beaudoin and Labbe´ 2001) (Figure 2). Probably, CcCTRs are stressinducible genes whose expression is regulated by metals in the environment. The CuSE element controls the expression of Cu transport genes in S. pombe and is necessary for their activation in response to Cu deprivation conditions (Beaudoin and Labbe ´ 2001). CuSE is the binding site for Cuf1, which is a transcription factor that regulates the high-affinity CTR family (Beaudoin et al. 2003). In the C. cinerea genomic database, we could not find

a complete Cuf1 homolog. However, the N-terminus of Cuf1 is highly similar to Ace1, which is a Cudependent transcription factor in S. pombe (Beaudoin et al. 2003), and an Ace1 homolog is present in the C. cinerea database (Cc1g_08665: XM_001837600). Furthermore, an Ace1 element exists in the predicted promoter region of CcCTR2 (Figure 2b). We could not find a MAC1-binding site such as in CTR1, -3, or CaCTR1 (Marvin et al. 2003), but the existence of CuSE in the promoter regions of both CcCTRs indicated that they are required for Cu homeostasis. The heterologous expression of both CcCTRs in S. cerevisiae showed the function of CcCTR1 as a Cu

ARTICLE IN PRESS Identification and characterization of CcCTR1 in C. cinerea

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Figure 5. Heterologous expression of the CcCTR genes in S. cerevisiae. a, Aliquots (7 ml) of a 51 to 54 dilution series of a yeast culture were spotted on selective agar plates and incubated for 7 d at 30 1C. BY4849 cells containing pYES2 vector control: Vec, [Gal1p:CcCTR1]: CcCTR1, [Gal1p:CcCTR1-GFP]: CcCTR1-GFP, [Gal1p:CcCTR2]: CcCTR2, [Gal1p:CcCTR2-GFP]: CcCTR2-GFP, and BY23503 cell strain overexpressing the MAC1 gene: MAC1. Arrows indicate the concentration of cells. (b) Localization of the GFP-fusion protein in S. cerevisiae. Fluorescent and bright-field images are merged. White bars represent the scale (20 mm).

importer (Figure 5). In S. cerevisiae, under conditions of sufficient Cu, Ace1 acts as a toxic Cu sensor to regulate the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to Cu (Pen ˜a et al. 1998). In contrast, the high-affinity Cu(I) uptake genes CTR1, -3, and FRE1 are transcriptionally downregulated by Cu ions (Pen ˜a et al. 1998). This Cu ion-dependent regulation requires a wild-type MAC1 Cu metalloregulatory transcription factor (Jungmann et al. 1993; Gross et al. 2000). We transformed the delta-mac1 S. cerevisiae strain (BY23481) with [Gal1p:CcCTR1] and observed the phenotype in low-Cu medium (Keller et al. 2005), but did not observe significant growth inhibition in either the vector control or the CcCTR1 expression strains (data not shown). Overexpression of CcCTR1 in wild-type S. cerevisiae likely increased the Cu sensitivity by activating Cu transport, leading to its accumulation (Figure 5a). We tried to measure the difference in Cu accumulation in these strains after short-term (o60 min) Cu treatment, but the Cu contents in the transgenic

strains were similar, and we did not detect a difference between the vector control and [Gal1p:CcCTR1] (data not shown). This result and the reduction of mRNA accumulation in C. cinerea after 36 h of treatment with 100 mM Cu (Figure 3) suggest that the transport activity of CcCTR1 is not very high, but can change the Cu sensitivity (Figure 5a), albeit with a slower response than in S. cerevisiae (Keller et al. 2005). Although we could not detect the Cu transport activity of CcCTR2 in this study, the mRNA accumulation level of CcCTR2 was affected by Cu treatment and was higher than that of CcCTR1 (Figure 3). We therefore propose two possibilities about the role(s) of CcCTR2. One possibility is that CcCTR2 localizes to the tonoplasts in C. cinerea and helps maintain Cu homeostasis in the cytosol, similar to CTR2 in S. cerevisiae. Another possibility is that CcCTR2 localizes to the plasma membrane as a prediction of the pSORT program, and acts as a low-affinity Cu transporter. In heterologous expression, sometimes the proteins were miss-localized

ARTICLE IN PRESS 286 and their function could not be detected in vivo. The localization of the CcCTR-GFP fusion protein in S. cerevisiae (Figure 5b) suggests that the two CTRs could be expressed in this heterologous system, but probably were not localized correctly. Transgenic or knock-out lines would be helpful for further study, in order to reveal the role of CTRs in C. cinerea. We are now analyzing Cu-dependent transcription factors, such as Ace1 and MAC1, which act upstream of CTRs and control Cu movement in basidiomycetes.

Acknowledgements BY4849, BY23503 and BY23481 were obtained from the National BioResource Project (Yeast), Japan (NBRP/YGRC). We thank Dr. Y. Okuyama for producing the NJ tree and Dr. M. Sakamoto for taking the fluorescence pictures.

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