Mfc1 Is a Novel Forespore Membrane Copper Transporter in Meiotic and Sporulating Cells*

To gain insight in the molecular basis of copper homeostasis during meiosis, we have used DNA microarrays to analyze meiotic gene expression in the model yeast Schizosaccharomyces pombe. Profiling data identified a novel meiosis-specific gene, termed mfc1+, that encodes a putative major facilitator superfamily-type transporter. Although Mfc1 does not exhibit any significant sequence homology with the copper permease Ctr4, it contains four putative copper-binding motifs that are typically found in members of the copper transporter family of copper transporters. Similarly to the ctr4+ gene, the transcription of mfc1+ was induced by low concentrations of copper. However, its temporal expression profile during meiosis was distinct to ctr4+. Whereas Ctr4 was observed at the plasma membrane shortly after induction of meiosis, Mfc1 appeared later in precursor vesicles and, subsequently, at the forespore membrane of ascospores. Using the fluorescent copper-binding tracker Coppersensor-1 (CS1), labile cellular copper was primarily detected in the forespores in an mfc1+/mfc1+ strain, whereas an mfc1Δ/mfc1Δ mutant exhibited an intracellular dispersed punctate distribution of labile copper ions. In addition, the copper amine oxidase Cao1, which localized primarily in the forespores of asci, was fully active in mfc1+/mfc1+ cells, but its activity was drastically reduced in an mfc1Δ/mfc1Δ strain. Furthermore, our data showed that meiotic cells that express the mfc1+ gene have a distinct developmental advantage over mfc1Δ/mfc1Δ mutant cells when copper is limiting. Taken together, the data reveal that Mfc1 serves to transport copper for accurate and timely meiotic differentiation under copper-limiting conditions.

Copper has a wealth of diverse functions in biological systems, making it essential to all aerobic organisms (1,2). Typically, copper serves as a catalytic and a structural cofactor for many enzymes that are intimately linked to cellular functions such as respiration, iron transport, and superoxide anion detoxification. However, because of its proclivity to change redox state, excess copper can react with hydrogen peroxide to produce the highly cytotoxic hydroxyl radical (3). It can also be deleterious by interfering with the biogenesis of Fe-S clusters (4). Consequently, it is critical that organisms use homeostatic mechanisms to acquire and maintain sufficient concentrations of copper while preventing its accumulation to protect cells from its unwanted cytotoxic effects.
Meiosis is an essential form of cell division through which sexually reproducing organisms reduce their chromosome number by half, generating haploid gametes from diploid germ cells (5,6). Meiosis shows two major differences relative to the mitotic cell cycle. First, following premeiotic DNA replication, recombination takes place between homologous chromosomes in meiotic prophase. Second, after recombination, two consecutive nuclear divisions occur without an intervening S-phase. In the first division, homologous chromosomes are separated, and in the second division, sister chromatids are segregated. Once the two nuclear divisions have been completed, a differentiation program is induced to generate haploid cells. Of significance, errors occurring in any of these steps or homeostatic micronutrient imbalances during this process can lead to a meiotic block, timely meiotic defects, or abnormal haploid cells (7,8).
Although the transport of copper across membranes is crucial to provide this metal to copper-dependent proteins, copper homeostasis has been poorly studied during meiotic differentiation. We have therefore used DNA microarrays to analyze meiotic gene expression in Schizosaccharomyces pombe to investigate the molecular determinants of the copper homeostatic machinery involved in the meiotic program. The fission yeast S. pombe is one of the best understood model systems to investigate fundamental meiotic processes (9). It is frequently used to decipher key molecular aspects of meiotic division, from its initiation through to the generation of mature spores. S. pombe follows essentially a meiotic process similar to germ line cells in higher eukaryotes, except that a fusion of two haploid cells of opposite mating type (zygote formation by conjugation) precedes entry into meiosis. A fundamental concept in the yeast cell cycle originates during G 1 , at which point the diploid cell becomes committed to either the mitotic cell cycle or the meiotic program. S. pombe is a key model because growth conditions and temperature-sensitive mutants have been developed that allow the synchronization of cells for their entry into the meiotic program (10). The three frequently analyzed types of meiosis in S. pombe are called zygotic, azygotic, and pat1-induced meiosis (11). In response to nitrogen source starvation, haploid cells of opposite mating type fuse, and the resulting zygote usually undergoes meiosis immediately by a process called zygotic meiosis. However, if the resulting zygote is returned to nitrogen-rich media before commitment to meiosis, it can resume vegetative growth and form colonies of diploid cells. Diploid cells will undergo azygotic meiosis in response to nitrogen starvation. Azygotic meiosis is more synchronous than zygotic meiosis but less synchronous than pat1induced meiosis. S. pombe cells growing mitotically carry an active Pat1 kinase that inhibits the cells from entering meiosis (12). Under conditions of nitrogen starvation, the mating type loci are activated and that enables expression of the inhibitor Mei3 of the Pat1 kinase. Cells harboring the pat1-114 mutation show temperature-sensitive growth and undergo meiosis and sporulation at the restrictive temperature (34°C), thus bypassing the Mei3-dependent inactivation pathway of Pat1. In S. pombe, meiosis is accompanied and driven by an extensive gene expression program during which several genes are either induced or repressed (13). Transcriptional profiles of the meiotic cell cycle have defined four successive waves of gene expression that coincide with major meiotic phases. These are as follows: wave 1, in response to nutrient starvation (nutrientresponsive genes); wave 2, which involves premeiotic replication and recombination (early genes); wave 3, where meiotic divisions (middle genes) occur, and wave 4, which is associated with spore formation (late genes) (13,14).
In the case of copper transport into mitotically growing S. pombe cells, it is thought that Cu 2ϩ is reduced to Cu ϩ by a putative cell surface reductase before being transported by a heteroprotein complex composed of two integral membrane proteins encoded by the ctr4 ϩ and ctr5 ϩ genes (15)(16)(17). A clear interdependence between Ctr4 and Ctr5 has been established because the trafficking of either protein to the cell surface requires the concomitant trafficking of the other (17,18). As is the case for most of the Ctr 4 transporters, Ctr4 and Ctr5 contain extracellular N-terminal methionine residues organized as MX 2 M and/or MXM motifs (denoted Mets motifs) that play an important role in copper transport when cells are grown under conditions of copper starvation (19,20). Analysis of S. pombe cells expressing Ctr4 and Ctr5 N-terminal mutated regions has revealed that, although the greatest efficiency in copper transport is achieved when both N termini are present, at least one N-terminal region provided by either protein is sufficient for copper transport (15). Previous studies have identified a third Ctr transporter, named Ctr6, which homotrimerizes in the vacuolar membrane and serves to transport intravacuolar stores of copper (21). Upon uptake into cells, copper is distributed to copper-requiring enzymes through the use of copper chaperones (22,23). A combination of observations in S. pombe and Saccharomyces cerevisiae has revealed that Atx1 delivers copper to Ccc2, a P-type ATPase on the Golgi membrane that transports copper into the secretory pathway where it can be incorporated into newly synthesized cuproproteins (24). Using mutant strains and protein-protein interaction assays, our group has reported that the fully active copper amine oxidase Cao1 is dependent on the presence of Atx1 (25). However, the mechanism by which copper is loaded into Cao1 via Atx1 is currently unknown. SOD1 receives copper from a direct interaction with Pccs (also named Ccs1) (26,27). In the mitochondrial intermembrane space, Cox17 has been demonstrated to target copper to cytochrome c oxidase via the Sco1 and Cox11 proteins (28). In S. pombe, the genes encoding proteins that are members of the copper transporter family are regulated at the level of transcription (29). They are induced under conditions of copper starvation and repressed under copper-replete conditions. Regulation of the copper transporters is mediated by cis-acting promoter elements, termed CuSEs, found as repeats in the ctr4 ϩ , ctr5 ϩ , and ctr6 ϩ promoters (21,29). The copperresponsive transcription factor of S. pombe that regulates expression of genes encoding copper transporters has been shown to be Cuf1 (30,31).
Despite the essential role of copper during meiotic differentiation (8,32), the copper-dependent players and mechanisms involved in this developmental process remain poorly understood. We have used S. pombe azygotic and pat1-driven meiotic systems, as well as DNA microarrays to investigate the mechanistic aspects of copper homeostasis during the generation of spores. Here, we report the identification of a new meiosisspecific copper transporter that we denoted as Mfc1 for "Major facilitator copper transporter 1." Although the Mfc1 amino acid sequence displays no overall sequence homology with Ctr-like proteins, it harbors four putative Mets motifs. Similarly to many fungal ctr-like genes, the mfc1 ϩ gene was transcriptionally induced under low copper conditions and turned off under copper-replete conditions. However, mfc1 ϩ exhibited a distinct meiotic temporal expression profile when compared with that of ctr4 ϩ . In the presence of exogenous copper (2 M), artificial mitotic expression of Mfc1 functionally complemented ctr4⌬ ctr5⌬ cells defective in copper transport. The uptake of 64 Cu was consistently observed in ctr4⌬ ctr5⌬ cells expressing a functional mfc1 ϩ allele. In cells undergoing synchronous meiosis, microscopic analyses revealed that a functional Mfc1-Cherry protein localizes at the forespore membrane during middle to late meiosis. Interestingly, an mfc1⌬/mfc1⌬ mutant strain displayed a strong reduction of copper amine oxidase activity and an abnormal distribution of labile copper ions within the ascus. Taken together, our findings describe the discovery of a novel type of forespore membrane copper transporter that participates in the timely developmental process of meiosis under copper-limiting conditions.

EXPERIMENTAL PROCEDURES
S. pombe Strains and Media-All S. pombe strains used in this work are listed in Table 1. General methods to handle fis-sion yeast cells were performed as described previously (33). Complete medium YES (0.5% yeast extract, 2% glucose, and 225 mg/liter adenine, histidine, leucine, uracil, and lysine) and synthetic Edinburgh minimal medium (EMM) supplemented with adenine only (225 mg/liter) or 75 mg/liter adenine, histidine, leucine, uracil, and lysine were used for cultures of S. pombe strains. Haploid cells of opposite mating types were induced to conjugate on solid malt extract (ME) medium. The h ϩ /h Ϫ diploid strains used for azygotic meiosis were isolated as follows. Haploid cells of opposite mating type were fused on ME medium, and the resulting zygotes were returned to rich media (YES) before commitment to meiosis. At this point, diploid cells can undergo azygotic meiosis following a synchronized nitrogen-starvation shock. To induce azygotic meiosis, the media used were EMM lacking nitrogen (EMM-N) supplemented with 10 mg/liter of adenine or 10 mg/liter of adenine, histidine, leucine, uracil, and lysine. Diploid strains homozygous for the mating type (h ϩ /h ϩ ) were generated by protoplast fusion as described previously (34,35).
pat1-induced Meiosis-To synchronize pat1-114 diploid cells for their entry into meiosis, they were precultivated in EMM supplemented with adenine (225 mg/liter) at 25°C for 48 h. At mid-log phase (A 600 of ϳ0.5; cell titer of 1 ϫ 10 7 cells/ ml), cells were harvested, washed twice, and transferred to EMM-N supplemented with 10 mg/liter adenine. After incubation for 16 h at 25°C, 5 mg/liter of NH 4 Cl was added to the culture medium, and cells were divided and treated with either ammonium tetrathiomolybdate (TTM) (323446; Sigma) or CuSO 4 or were left untreated. At this point, the temperature was shifted to 34°C to induce meiosis. To shift all cells as rapidly as possible, each flask was shaken and warmed to 50°C in a water bath with swirling for 15 s before being transferred to a shaking water bath maintained at the restrictive temperature (34°C). Meiosis progression of zygotes was monitored by adding 5 g/ml of Hoechst 33342 stain (Invitrogen) at different times following meiotic induction.
Plasmids-Two approaches were selected to ectopically express Mfc1 in cells proliferating in mitosis. First, the constitutive mitotic cuf1 ϩ promoter up to Ϫ2092 from the start codon of the cuf1 ϩ gene was isolated by PCR and then inserted into the pSP1 vector (36) at the ApaI and PstI sites. The resulting plasmid was denoted pSP-2092cuf1 ϩ prom. The full-length coding region of mfc1 ϩ was isolated by PCR, using primers that corresponded to the initiator and stop codons of the ORF SPAPB1A11.01 from S. pombe strain FY435 genomic DNA. Because the primers contained PstI and XbaI restriction sites, the purified DNA fragment was digested with these restriction enzymes and cloned into the corresponding sites of pSP-2092cuf1 ϩ prom. The resulting plasmid was named pSPcuf1prom-mfc1 ϩ . Second, using an identical approach, we substituted the ctr4 ϩ promoter in place of the cuf1 ϩ promoter. This new plasmid was denoted pSPctr4prom-mfc1 ϩ and had the ctr4 ϩ promoter driving the heterologous mitotic expression of the mfc1 ϩ gene.
The GFP coding sequence derived from pSF-GP1 (37) was isolated by PCR, using primers designed to generate XmaI and NotI sites at the 5Ј and 3Ј termini, respectively, of the GFP gene. The resulting DNA fragment was used to clone the GFP gene into pSPcuf1prom-mfc1 ϩ and pSPctr4prom-mfc1 ϩ plasmids, in which XmaI and NotI restriction sites were previously engineered by PCR and placed in-frame immediately before the stop codon. The resulting pSPcuf1prom-mfc1 ϩ and pSPctr4prom-mfc1 ϩ plasmid derivatives were denoted pSPcuf1prom-mfc1 ϩ -GFP and pSPctr4prom-mfc1 ϩ -GFP, respectively. An ApaI-XmaI DNA fragment containing the ctr4 ϩ promoter and the ctr4 ϩ gene (without its stop codon) was isolated by PCR from the pSK-737ctr4 ϩ plasmid (29). Subsequently, this PCR product was inserted in-frame in front of the start codon of the Cherry-coding sequence (38), which had previously been inserted into pBPade6 ϩ plasmid (15) with the use of XmaI and SstI restriction enzymes. The resulting plasmid was named pBPctr4 ϩ -Cherry. To generate the pJK210ctr4 ϩ -GFP plasmid, we subcloned the ctr4 ϩ -GFP fusion allele from pBPctr4 ϩ -GFP (15) using PstI and SpeI and then inserted it into the corresponding sites of pJK210 (39). To express Mfc1 during meiosis, we ensured that the mfc1 ϩ , mfc1 ϩ -Cherry, and mfc1 ϩ -GFP genes were under the control of the mfc1 ϩ promoter. To generate the pJK148mfc1 ϩ plasmid and its derivatives, an ApaI-XmaI PCR-amplified DNA segment containing the mfc1 ϩ locus starting at Ϫ800 from the translational start codon up to the penultimate codon or the stop codon was inserted into the ApaI and XmaI sites of pJK148 (39). To create a plasmid that has the mfc1 ϩ gene fused in-frame with Cherry, an XmaI-SstI fragment containing the Cherry-coding sequence was isolated by PCR and inserted into the corresponding sites of pJK148mfc1 ϩ (which has the penultimate codon), generating the pJK148mfc1 ϩ -Cherry plasmid. Using a similar strategy, we added by PCR the restriction sites XmaI and SstI at the beginning and the end of the GFP gene, and the PCR product obtained was cloned into the corresponding sites of pJK148mfc1 ϩ (which has the penultimate codon), producing the pJK148mfc1 ϩ -GFP plasmid. Plasmid pJK210GFP-Psy1 was constructed by introducing the following DNA fragments into pJK210. The first DNA segment was an XbaI-BamHI psy1 ϩ promoter fragment up to Ϫ505 from the start codon of the psy1 ϩ gene. The second DNA segment was a BamHI-SalI PCRamplified fragment containing the GFP gene without its stop codon. Finally, the third DNA sequence was a SalI-Asp 718 PCRamplified fragment containing the psy1 ϩ locus starting at the initiator codon up to the stop codon. The resulting fluorescent protein product (GFP-Psy1) served as a forespore membrane marker. The sad1 ϩ gene containing its promoter region up to Ϫ610 from the start codon was isolated by PCR using primers designed to generate BamHI and XmaI sites and then cloned into the corresponding restriction sites of pJK210. In the design, the stop codon of sad1 ϩ was removed to make a fusion protein with the fluorescent coding fragment of Cherry. Subsequently, a copy of the Cherry gene was amplified by PCR using primers designed to generate XmaI and SstI sites at the upstream and downstream termini of the ORF. The PCR product obtained was then cloned in-frame with the sad1 ϩ gene using the XmaI and SstI sites. The resulting plasmid was denoted pJK210sad1 ϩ -Cherry and the fluorescent protein product served as a spindle pole body marker. The nmt1 ϩ pro-moter (3X or 41X) up to position Ϫ1178 from the start codon of the nmt1 ϩ gene was isolated from pREP3X or pREP41X (40) by PCR using primers that contained ApaI and PstI sites. Subsequently, the ApaI-PstI DNA fragment (either from pREP3X or pREP41X) was exchanged with the ApaI-PstI DNA fragment in plasmid pJK148mfc1 ϩ -Cherry to generate pJK148nmt1-mfc1 ϩ -Cherry, which expresses the Mfc1-Cherry fusion protein under the control of the nmt1 ϩ promoter (3X or 41X). Analysis of Gene Expression-Total RNA was extracted by a hot phenol method (41) and quantified spectrophotometrically. In the case of RNase protection assays, 15 g of RNA per reaction were used, as described previously (42). To generate pSKmfc1 ϩ , a 200-bp BamHI-EcoRI fragment from the mfc1 ϩ gene was amplified and cloned into the same sites of pBluescript SK (Stratagene, La Jolla, CA). This fragment corresponds to the region between ϩ410 and ϩ610 down to the first base of the translational start codon of mfc1 ϩ . To create pSKcuf1 ϩ -v7, a 199-bp fragment from the cuf1 ϩ gene (corresponding to the coding region between ϩ640 and ϩ839) was amplified and cloned into the BamHI and EcoRI sites of pBluescript SK. 32 P-Labeled antisense mfc1 ϩ , cuf1 ϩ , ctr4 ϩ , and act1 ϩ RNAs were produced from the BamHI-linearized plasmids pSKmfc1 ϩ , pSKcuf1 ϩ -v7, pSKctr4 ϩ (29), and pSKact1 ϩ (43), respectively, and with the use of [␣-32 P]UTP and the T7 RNA polymerase. The act1 ϩ riboprobe was used to detect act1 ϩ mRNA as an internal control for normalization during quantification of the RNase protection products.
Experimental Design and Microarray Experiments-We adopted an experimental design as follows: pat1-114 h ϩ /h ϩ mfc1 ϩ /mfc1 ϩ (WT) copper-deprived (ϪCu) versus pat1-114 h ϩ /h ϩ mfc1 ϩ /mfc1 ϩ (WT) copper replete (ϩCu). Meiotic time courses were performed as three independent biological repeats. Two of them were used in the microarray protocol for which the Cy dyes were swapped. The third independent biological repeat was used for analysis of mRNAs using the RNase protection protocol. After 5 h of meiotic induction under copper starvation (TTM) or copper repletion (CuSO 4 ), cells corresponding to 5 optical density units (ϳ1 ϫ 10 8 cells/ml) were harvested by centrifugation and snap-frozen in liquid nitrogen. Total RNA was extracted by a hot phenol method (41). Twenty g of RNA were labeled by directly incorporating Cy3-and Cy5-dCTP using Superscript (Invitrogen) reverse transcriptase as described previously (44). The resulting cDNA preparation was hybridized onto glass DNA microarrays containing probes for 99.3% of all known and predicted S. pombe genes. Microarrays were scanned using a GenePix 4000B laser scanner (Axon instruments, Foster City, CA). Data were subsequently analyzed using the GenePix pro software. Unreliable signals were filtered out, and data were normalized using a customized Per1 script (44). The script applies cutoff criteria to discard data from weak signals. Genes that did not yield reproducible results of biological repeats were eliminated. Furthermore, genes with 50% of their data points missing were also discarded. Data acquisition, processing, and normalization were further analyzed using GeneSpring GX software (Agilent Technologies, Cheshire, UK). Normalized signals were exported from Gene-Spring into Microsoft Excel. The expression ratios of biological repeat experiments were averaged. Genes were classified as copper starvation-dependent if their expression was up-regulated 1.5-fold over the average of two repeats found in the wildtype strain grown under copper-replete conditions. Gene annotations were retrieved from the GeneDB website.
Fluorescence Microscopy-When Mfc1 was artificially expressed in cells proliferating in mitosis, ctr4⌬ ctr5⌬ cells were transformed with an integrative plasmid expressing the mfc1 ϩ -Cherry or mfc1 ϩ -GFP allele under the control of a constitutive heterologous mitotic promoter. As a control, ctr4⌬ ctr5⌬ cells were transformed with an empty plasmid or were co-transformed with the functional ctr4 ϩ GFP and ctr5 ϩ -MYC 12 fusion alleles. S. pombe cells growing mitotically were viewed by direct fluorescence microscopy as described previously (15). In the case of cytological analyses throughout the meiotic program, h ϩ /h Ϫ mfc1⌬/mfc1⌬ ctr4⌬/ctr4⌬ diploid cells co-expressing mfc1 ϩ -Cherry/mfc1 ϩ -Cherry and ctr4 ϩ -GFP/ctr4 ϩ -GFP were induced in azygotic meiosis as described previously (45). An h ϩ /h Ϫ mfc1⌬/mfc1⌬ diploid strain co-expressing Mfc1-Cherry and GFP-Psy1 was also induced to undergo azygotic meiosis. Culture aliquots were taken up every 30 min and were directly examined by Nomarski optics to analyze the progression of meiosis. At the indicated times, meiotic cells were analyzed by microscopy using a ϫ1,000 magnification with the following filters: 465-495 nm (GFP) and 510 -560 nm (Cherry). Fluorescence and differential interference contrast images of the cells were obtained using a Nikon Eclipse E800 epifluorescent microscope (Nikon, Melville, NY) equipped with a Hamamatsu ORCA-ER digital cooled camera (Hamamatsu, Bridgewater, NJ). Cell fields shown in this study are representative of at least five independent experiments. The merged images were obtained using the Simple PCI software version 5.3.0.1102 (Compix, Sewickly, PA). 64 Cu Uptake Measurements-Mid-logarithmic cultures were harvested and washed twice with citrate buffer (50 mM sodium citrate, pH 6.5, 5% glucose) as described previously (46). Radioactive copper (250 Ci/g of 64 Cu in the form of 64 CuCl 2 in 0.1 M HCl) was produced at the 64 Cu production facility of the Centre d'Imagerie Moléculaire de Sherbrooke Center. 64 CuCl 2 was added to 2 ml of cell suspension to a final concentration of 2 M, and cultures were incubated for 10 min at 30 or 0°C. Uptake of 64 Cu was terminated by adding ice-cold EDTA (10 mM in PBS). Samples were collected by suction through nitrocellulose membrane filters loaded onto a 1225 Sampling Manifold (Millipore, Bedford, MA). The collected cells were washed with 25 ml of ice-cold PBS, pH 7.4, air-dried, and then counted using a ␥-counter (Canberra-Packard Cobra II). Counts obtained at 0°C were subtracted from the values at 30°C to give net uptake values. In addition, the data were normalized to culture density, as described previously (17,21).
CAO Assay and CS1 Labeling of Cells-To determine the presence of CAO activity, synchronous cell cultures were harvested 8 h after the induction of meiosis. Cell lysates were prepared from each culture, and equal amounts of cellular protein were subjected to a colorimetric assay as described previously (25). In this quantitative assay, hydrogen peroxide that was released from a reaction catalyzed by CAO was determined by a method using 4-aminoantipyrine and vanillic acid to generate a quinone-imine dye, which is detectable at 498 nm. Nitrogen-starved haploid cells of opposite mating type were crossed to produce diploid zygotes, which were quickly transferred to rich YES medium to stabilize diploid cells. Azygotic meiosis of diploid cells was synchronously induced by transferring the cells in nitrogen-poor EMM as described previously (45). At zero time point when cells entered meiosis, they were maintained in nitrogen-poor EMM supplemented with 10 mg/liter adenine, histidine, leucine, uracil, and lysine as well as 5 or 10 M CS1 (47, 48) (in 1% DMSO) or left without CS1 (with 1% DMSO as a control). At various time points following CS1 treatment, aliquots of meiotic cells were viewed by direct fluorescence microscopy using ϫ1,000 magnification at 543 nm excitation to match the absorption maximum of the Cu ϩ -bound CS1 probe as described previously (47).

Copper Is Required for Normal Progression of Meiosis in S.
pombe-Although copper is a vital cofactor essential in biology, little is known about its role in meiosis. To begin to investigate whether a perturbation of intracellular copper availability would affect the progression of meiosis, haploid cells of opposite mating type were fused and returned to rich media before commitment to meiosis. Diploid cells were synchronously induced by transferring them at the same time into nitrogenpoor medium, allowing cells to undergo azygotic meiosis (45). Shortly after their entry in meiosis, the zygotes were harvested, washed, and resuspended in a nitrogen-poor medium either without treatment (basal copper conditions, 160 nM copper) or in the presence of the copper chelator TTM (200 M). Zygotes that were treated with TTM proceeded through metaphase I and stopped their progression, exhibiting a meiotic arrest (Fig.  1). To overcome the effect of TTM and reactivate progression of meiosis, TTM-treated zygotes that were blocked at metaphase I were washed, harvested, and resuspended in basal medium containing either 10 or 50 M exogenous copper. As shown in Fig. 1 (and data not shown), removal of the copper chelator TTM, followed by the addition of exogenous copper, triggered a rescue of the zygotes, overcoming the inhibitory effect of TTM. Although a delay of ϳ2 h occurred when TTMtreated zygotes were rescued by exogenous copper, supplementation with copper was able to restore the meiotic developmental program to produce normal asci with standard morphology. As positive controls, zygotes incubated under basal conditions proceeded through meiosis and formed asci containing four spores after 10 h of meiotic induction (Fig. 1). Percentages of cells with 1, 2, or 3-4 nuclei were quantitatively assessed by counting the Hoechst-stained nuclei (Fig. 1). Taken together, the results showed that copper deficiency stops meiosis, causing an arrest at metaphase I.
Copper Deficiency Affects Meiotic Gene Expression in S. pombe-Although it is currently unknown how cells acquire copper during meiosis, we have observed that the use of strong copper starvation conditions fostered a meiotic block ( Fig. 1 and data not shown). To investigate the copper deprivation response of S. pombe during meiosis, pat1-114 diploid cells were pre-synchronized in G 1 by nitrogen starvation and then incubated at 34°C to inactivate the Pat1 kinase so as to initiate and proceed through synchronous meiosis under copper-starved (100 M TTM) or copper-replete (100 M CuSO 4 ) conditions. Microarrays were hybridized with probes derived from RNA isolated from copper-limited as opposed to copper-replete meiotic cells. Five hours after meiotic induction, analysis of gene expression profiling data revealed that 304 genes were expressed at higher levels in copper-limiting cells (averaging Ն1.5-fold) (supplemental Table S1 and data not shown). Among genes that were differentially expressed, microarray data indicated a mild up-regulation of the ctr4 ϩ (2.5-fold), ctr5 ϩ (1.4-fold), and ctr6 ϩ (1.9-fold) genes, which are known to encode members of the Ctr family of copper transporters ( Table 2). As a point of comparison, under copper-limiting conditions when cells proliferate through mitotic cell cycles, the ctr4 ϩ , ctr5 ϩ , and ctr6 ϩ genes are induced ϳ10-, ϳ11-, and ϳ7-fold, respectively, as compared with their basal levels of expression in untreated (control) cells (17,21,29,31). We also noted significant changes in the transcriptional profiles of several genes, including some genes that encode uncharacterized fungus-specific proteins, unclassified orphan proteins, and meiotic proteins with diverse functions ( Table 2). As expected, because synchronous entrance in meiosis using a pat1-induced system requires a temperature shift at 34°C, several heat shock

TABLE 2 Genes expressed at higher levels in copper-starved meiotic cells (5 h after meiotic induction)
We determined that 304 genes were up-regulated (averaging Ն1.5-fold) in response to copper deficiency. Here, 66 of the most highly expressed genes are listed. genes were induced. However, our current data do not provide an explanation for the observation that heat shock genes were more highly induced in copper-starved than in copper-supplemented cells (Table 2). Data showed that the SPAPB1A11.01 gene was the most highly expressed (17.2-fold) of all of the mRNAs detected under copper-limiting conditions after 5 h of meiotic induction (Table 2). Analogous to typical copper starvation-dependent responsive genes, the SPAPB1A11.01 gene was repressed in copper-treated cells. Interestingly, the SPAPB1A11.01 gene was predicted to encode a putative transmembrane protein that was classified as a member of the major facilitator superfamily (MFS) of transporters (49). For this reason, we denoted the SPAPB1A11.01 gene as mfc1 ϩ for "major facilitator copper transporter 1." Because the mfc1 ϩ gene was induced in response to copper deficiency in a direction similar to that of the ctr copper transport genes, mfc1 ϩ was selected for further analysis. Primary Sequence and Predicted Topological Model of Mfc1-The mfc1 ϩ gene encodes a polypeptide composed of 495 amino acid residues with a predicted molecular mass of 55.3 kDa. As commonly found in a majority of MFS transporters, the Mfc1 protein was predicted to possess 12 transmembrane domains connected by short hydrophilic loops, with both its N and C termini located in the cytoplasm. A topological model, which was obtained using TOP-PRED II analysis (50), suggested that the first six putative transmembrane spans constitute the first half of Mfc1, whereas transmembrane spans 7-12 form the second half of the protein (Fig. 2). In the pre-dicted model, the two halves of Mfc1 are connected by the extended loop 6. This loop is predicted to be cytosolic and would allow interdomain movement between the N-and C-terminal halves of Mfc1. The MFS-type transporters constitute the largest group of secondary membrane transporters (49). As opposed to primary-type transporters such as P-type ATPases and ATP-binding cassette transporters that bind ATP and require ATP hydrolysis for transport activity, the MFS-mediated transport is driven by proton-motive or gradient forces (51). Interestingly, amino acid sequence analysis of Mfc1 indicated that four pairs of Met residues are present in potential copper coordination arrangements. Similar Met-rich motifs are generally found in eukaryotic copper transporters and known as Mets motifs (consisting of Met residues arranged as MXM or MX 2 M, where X is any amino acid) (Fig. 2) (19). Strikingly, Mfc1 also contains 12 other Met residues as well as 7 Cys residues scattered throughout the protein that may also represent potential copper-binding ligands. Taken together, these observations suggested that mfc1 ϩ encodes a MFS-type protein that may represent a novel meiosis-specific copper transporter. mfc1 ϩ and ctr4 ϩ Genes Displayed Distinct but Complementary Expression Profiles during Meiosis-In S. pombe, the genes encoding proteins that are members of the copper transporter family are regulated at the level of gene transcription as a function of copper availability (17,21,29), i.e. copper acquisition systems are expressed at high levels under conditions of copper scarcity, whereas these systems are repressed or expressed at very low levels under conditions of copper abundance. Further- more, the transcriptional activation of the copper transport genes is under the control of the copper-dependent transcription factor Cuf1 (17,30,31). Because previous studies had been performed with cells proliferating in mitosis, little is known about the regulation of copper-responsive genes during meiosis. Because of the fact that gene expression profiling data suggested that mfc1 ϩ and ctr4 ϩ genes were induced in copperstarved meiotic cells, we further investigated their profiles of expression during meiosis as a function of copper and Cuf1 availability (Fig. 3). Diploid strains pat1-114 cuf1 ϩ/ϩ and pat1-114 cuf1⌬/⌬ were synchronized through meiosis in the absence or the presence of either 100 M TTM or 100 M CuSO 4 . In response to 100 M added TTM, ctr4 ϩ mRNA levels were quickly induced (ϳ32-fold compared with the basal levels observed in untreated or copper-replete cells), exhibiting maximum levels of ctr4 ϩ mRNA after 30 min of meiotic induction (Fig. 3, C and E). This was followed by a strong reduction of ctr4 ϩ mRNA levels within ϳ5-6 h (Fig. 3, C and E). There was a complete lack of induction of ctr4 ϩ mRNA under basal and copper-replete conditions (Fig. 3C). RNA samples prepared from a pat1-114 cuf1⌬/⌬ deletion strain showed loss of copper starvation-dependent induction of ctr4 ϩ gene expression, indicating that the copper-dependent regulation of ctr4 ϩ mRNA required Cuf1 in early meiosis (Fig. 3D). Expression of mfc1 ϩ was also analyzed as a function of time in meiosis (Fig. 3). Although the mfc1 ϩ gene exhibited a distinct temporal expression profile when compared with that of ctr4 ϩ , mfc1 ϩ expression was primarily detected in cells treated with TTM (induced ϳ22-fold compared with levels observed in untreated or copper-replete cells), peaking at middle meiosis. Throughout the meiotic program, only very weak mfc1 ϩ mRNA levels were detected in cells incubated under both standard (basal) and copper-replete conditions (Fig. 3A). After 3 h of meiotic induction, at which point ctr4 ϩ mRNA levels were strongly reduced, the steady-state levels of mfc1 ϩ mRNA increased ϳ14-fold above the levels observed in untreated cells (Fig. 3A). Moreover, under low copper conditions, the transcript levels of mfc1 ϩ remained up-regulated throughout the meiotic division and spore maturation processes, being detected 9 h after meiotic induction (Fig. 3, A and E, and data not shown). To further examine whether mfc1 ϩ transcription was controlled by Cuf1, a pat1-114 cuf1⌬/⌬ mutant strain was incubated in the absence or presence of 100 M TTM or 100 M CuSO 4 . Although deletion of cuf1 ϩ impaired the induction of ctr4 ϩ , the cuf1⌬/⌬ mutant did not show any significant effect on the copper starvation-dependent induction of mfc1 ϩ during meiosis (Fig. 3B). We therefore concluded that Cuf1 was not required for the induction of mfc1 ϩ in response to copper deprivation. Furthermore, a second difference between mfc1 ϩ and ctr4 ϩ was the fact that the mfc1 ϩ gene was exclusively expressed during meiosis (when under the control of its own promoter). There was no detection of the mfc1 ϩ transcript in mitotic cells grown in the absence or presence of TTM or CuSO 4 (Fig. 3F). In contrast, the ctr4 ϩ transcript was clearly detected in both mitosis and meiosis (Fig. 3, C, E, and F). Collectively, these results showed that mfc1 ϩ exhibited a distinct meiotic temporal expression profile when compared with that of ctr4 ϩ and that the mfc1 ϩ transcript was meiosis-specific.

Mfc1 Expressed in ctr4⌬ ctr5⌬ Cells Proliferating in Mitosis Localized to the Plasma Membrane and Rescued the Respiratory
Deficiency in the Presence of Copper-Because of the presence of common copper-binding-like motifs between the Mfc1 protein and different members of the Ctr family of copper transporters, we assessed whether Mfc1 could complement the inability of a ctr4⌬ ctr5⌬ mutant strain to grow on nonfermentable carbon sources due to copper deficiency. As an initial functional assay to test this possibility, we expressed the mfc1 ϩ gene in ctr4⌬ ctr5⌬ cells proliferating in mitosis. The mfc1 ϩ and mfc1 ϩ -GFP open reading frames were placed under the control of the heterologous mitotic cuf1 ϩ promoter, which is a cell cycle constitutive and medium strength promoter (31,52). Mitotic ctr4⌬ ctr5⌬ cells expressing Mfc1-GFP were analyzed for protein cellular localization, whereas these same transformed cells expressing Mfc1 or Mfc1-GFP were tested for growth on the respiratory carbon sources glycerol/ethanol, for which copper transport is required for delivery of copper to cytochrome c oxidase (53,54). Results showed that the Mfc1-GFP fusion protein localized to the plasma membrane (Fig. 4A). As a positive control, co-expression of two functional versions of Ctr4-GFP and Ctr5-Myc 12 fusion proteins showed the presence of Ctr4-GFP in the plasma membrane (Fig. 4A) (15). To evaluate the potential contribution of Mfc1 in copper acquisition, cells expressing the Mfc1 and Mfc1-GFP proteins were analyzed for respiratory competency as compared with cells co-transformed with the functional Ctr4-GFP and Ctr5-Myc 12 proteins. As shown in Fig. 4B, the GFP-tagged Ctr4-and Myc 12tagged Ctr5 proteins used here as positive controls functionally complemented the respiratory deficiency of a ctr4⌬ ctr5⌬ strain in a manner indistinguishable from the ctr4 ϩ and ctr5 ϩ wildtype alleles. In a distinctive manner, ctr4⌬ ctr5⌬ cells expressing the mfc1 ϩ or mfc1 ϩ -GFP allele exhibited only a negligible growth on glycerol/ethanol medium compared with cells coexpressing Ctr4-GFP and Ctr5-Myc 12 (Fig. 4B). Based on these results and to further evaluate the function of Mfc1 in copper acquisition, CuSO 4 (1 and 2 M) was added to the glycerol/ ethanol medium. Although the ability of the cells to grow on glycerol/ethanol was still extensively compromised in the presence of 1 M CuSO 4 , ctr4⌬ ctr5⌬ cells harboring Mfc1 or Mfc1-GFP exhibited a robust growth on glycerol/ethanol medium containing 2 M CuSO 4 that was comparable with that observed for cells co-expressing Ctr4-GFP and Ctr5-Myc 12 (Fig. 4B).
To further analyze the relative contribution of Mfc1 compared with the heteromeric Ctr4-Ctr5 complex for copper acquisition in glycerol/ethanol medium, we measured the rate of growth in liquid medium for the isogenic parent strain (ctr4 ϩ ctr5 ϩ ), the ctr4⌬ ctr5⌬ double mutant strain harboring an empty vector or expressing Mfc1, Mfc1-GFP, or the heteromeric complex Ctr4-Ctr5. In the absence of exogenous copper, ctr4⌬ ctr5⌬ cells expressing the mfc1 ϩ and mfc1 ϩ -GFP alleles failed to complement the respiratory deficiency and to grow. Similarly, the ctr4⌬ ctr5⌬ cells did not grow when they contained an empty vector (Fig. 4D). In liquid assays, ctr4⌬ ctr5⌬ cells co-expressing the ctr4 ϩ -GFP and ctr5 ϩ -myc 12 alleles were able to grow at the same rate as the wild-type parental strain in a glycerol/ethanol medium (Fig. 4D). Expression of individual  . Copper starvation induces mfc1 ؉ and ctr4 ؉ transcripts at distinct times following meiotic induction. pat1-114 cuf1 ϩ/ϩ and pat1-114 cuf1⌬/⌬ strains were pre-synchronized by nitrogen starvation at 25°C and then induced to undergo synchronous meiosis at 34°C, a temperature that inactivates the temperature-sensitive protein kinase Pat1-114. Cells underwent meiosis in the presence of TTM (100 M), CuSO 4 (100 M), or without treatment (basal). At the indicated time points, mfc1 ϩ (A and B) and ctr4 ϩ (C and D) mRNAs were analyzed in a diploid pat1-114 cuf1 ϩ/ϩ strain and an isogenic strain lacking the cuf1 ϩ alleles. As controls, untreated cultures (basal) were maintained in mitosis at 25°C. Total RNA was analyzed by RNase protection assays. As a control, RNA preparations were probed for act1 ϩ steady-state mRNA levels. E, graphic representation of expression profiles of the mfc1 ϩ and ctr4 ϩ genes during meiosis. The peak of ctr4 ϩ mRNA expression was observed at 0.5 h, whereas the maximal induction of mfc1 ϩ mRNA was detected 5 h after meiotic induction. Meiosis was divided into four temporal phases that were termed induction, early, middle, and late meiosis. F, representative RNase protection assays of mfc1 ϩ and ctr4 ϩ , indicating steady-state mRNA levels in mitosis or after 5 h of meiotic induction. Mid-logarithmic cultures (in mitosis) or pat1-synchronized diploid cells (in meiosis) were incubated in the absence (Ϫ) or presence of TTM (100 M) or CuSO 4 (100 M). Actin (act1 ϩ ) mRNA levels were detected as an internal control. glycerol/ethanol medium was restored 85-88% of the wildtype strain (Fig. 4F).
Based on these results, we next sought to determine the relative contribution of Mfc1 to copper transport activity in 64 Cu uptake assays. In these experiments, the mfc1 ϩ and mfc1 ϩ -GFP alleles were cloned under the control of the thiamine-regulatable 41X promoter. This strategy allowed the induction of the biosynthesis of Mfc1 and Mfc1-GFP during mitosis when cells reached the mid-logarithmic phase. The nmt ϩ 41X promoter gave regulatable levels of mfc1 ϩ mRNA comparable with those observed with the constitutive cuf1 ϩ promoter (data not shown). Parental wild-type strain transported 64 Cu with high affinity in a manner that was consistent with its capacity to utilize nonfermentable carbon sources for growth (Fig. 4G). In contrast, a ctr4⌬ ctr5⌬ double mutant strain exhibited a striking loss of 64 Cu transport activity (Fig. 4G). ctr4⌬ ctr5⌬ cells expressing Mfc1 and Mfc1-GFP proteins showed a 61-65% reduction in the rate of 64 Cu uptake as compared with that of the starting parental strain (ctr4 ϩ ctr5 ϩ ) (Fig. 4G). As a positive control, 64 Cu uptake was restored to approximately wild-type levels (97%) when ctr4⌬ ctr5⌬ cells were co-transformed with integrative plasmids expressing the Ctr4-GFP and Ctr5-Myc 12 proteins (Fig. 4G). To verify that the Mfc1-GFP and Ctr5-Myc 12 proteins were expressed in ctr4⌬ ctr5⌬ cells, total protein FIGURE 4. Mitotic expression of Mfc1-GFP produces a fluorescent signal at the plasma membrane and functionally complements the respiratory deficiency of a ctr4⌬ ctr5⌬ mutant in the presence of copper. A, ctr4⌬ ctr5⌬ cells were transformed with an empty vector or integrative plasmids expressing the mfc1 ϩ -GFP allele (under the control of a mitotic constitutive gene promoter) or both the ctr4 ϩ -GFP and ctr5 ϩ -MYC 12 fusion alleles. The cells were then visualized by fluorescence microscopy (GFP). Ctr4-GFP was used as a control for plasma membrane localization. Cell morphology was examined by Nomarski optics. B, cells harboring a ctr4⌬ ctr5⌬ double deletion were transformed with an empty vector, mfc1 ϩ , mfc1 ϩ -GFP, or ctr4 ϩ -GFP and ctr5 ϩ -MYC 12 . Cultures were spotted onto YES media containing glucose or glycerol/ethanol (Gly/EtOH) and CuSO 4 (0, 1, and 2 M). WT, isogenic parental strain FY435 (ctr4 ϩ ctr5 ϩ ). C, whole cell extracts were prepared from aliquots of cultures used in G and analyzed by immunoblotting. D-F, growth of ctr4⌬ ctr5⌬ cells transformed with the indicated alleles (empty vector, black; mfc1 ϩ , green; mfc1 ϩ -GFP, blue; ctr4 ϩ -GFP, and ctr5 ϩ -MYC 12 , red) was assessed in glycerol/ethanol (Gly/EtOH) liquid cultures in the absence (C, 0 M) or presence of CuSO 4 (D, 1 M; E, 2 M) for 8 days. G, indicated transformed ctr4⌬ ctr5⌬ cells were incubated with 2 M 64 CuCl 2 for 10 min. Absorption of 64 CuCl 2 was carried out at 30°C, and the values were corrected with respect to culture density and temperature (i.e. uptake at 30°C subtracted from uptake at 0°C). Data are the averages of triplicate samples from three independent cultures. Error bars indicate the averages Ϯ S.D. extracts were analyzed by immunoblotting (Fig. 4C). The results showed that the Mfc1-GFP and Ctr5-Myc 12 proteins were both produced at comparable levels. Taken together, these results revealed that the Mfc1 protein could mediate the transport of copper in medium containing very low micromolar copper concentrations, although the Ctr4-Ctr5 heteromeric complex had the highest efficacy for high affinity copper transport.
Mfc1 Localizes at the Forespore Membrane-Based on the fact that Mfc1 is a meiosis-specific protein, we next sought to determine its subcellular location during the meiotic program. The Cherry fluorescent coding sequence was fused in-frame with the 3Ј-terminal end of the mfc1 ϩ gene. When the Mfc1-Cherry fusion protein was artificially expressed during the mitotic growth phase, it complemented the respiratory deficiency and triggered 64 Cu transport activity in a manner comparable with that of the wild-type (untagged) or the GFP epitope-tagged Mfc1 protein (data not shown). The experiments were set by integrating a functional ctr4 ϩ -GFP allele into h Ϫ ctr4⌬ and h ϩ ctr4⌬ cells, whereas a functional mfc1 ϩ -Cherry allele was integrated into h Ϫ mfc1⌬ and h ϩ mfc1⌬ cells. After mating, either h Ϫ /h ϩ ctr4⌬/⌬ ctr4 ϩ -GFP/ctr4 ϩ -GFP or h Ϫ /h ϩ mfc1⌬/⌬ mfc1 ϩ -Cherry/mfc1 ϩ -Cherry diploid cells were cultured to undergo azygotic synchronous meiosis. In the presence of 100 M TTM, the Ctr4-GFP fluorescent protein was observed at the cell surface 1 and 3 h following induction of meiosis (Fig. 5A). However, Ctr4-GFP-associated fluorescence disappeared after 3 h, an observation that was consistent with the dramatic decrease of ctr4 ϩ mRNA levels at this time (Figs. 3 and 5A). Under the same conditions (100 M TTM), the Mfc1-Cherry fluorescent protein was first detected at the 3-h time point. Its intracellular distribution had the appearance of cytoplasmic vesicular structures within the zygote (Fig. 5B). At 6 h, the zygotic cells displayed Mfc1-Cherry fluorescence as vesicles that appeared to fuse to subsequently form the forespore membrane (FSM) (Fig. 5B). To confirm that Mfc1-Cherry localized to the FSM, we used the GFP-tagged Psy1 protein, because Psy1 is a well established component of the FSM (55). Mfc1-Cherry and GFP-Psy1 fluorescent signals co-localized at the FSM following azygotic meiosis induction (Fig. 5C). Interestingly, when spores were released from the asci, the Mfc1-Cherry fusion protein generated a fluorescent signal at the spore membrane (Fig.  5), suggesting a role as a copper transporter at the spore surface. Taken together, the results revealed that the Mfc1 protein appears during the fusion of precursor membrane vesicles that, once expanded, form the FSM. After FSM closure, Mfc1 resides at the FSM where it persists until matured spores are released from the asci.
Inactivation of the mfc1 ϩ Gene Alters Cellular Copper Distribution as Well as Activity of Cuproenzyme-We reasoned that if Mfc1 acts as a copper transporter, its deletion would result in alteration of intracellular copper distribution. To investigate this possibility, wild-type h Ϫ /h ϩ mfc1 ϩ/ϩ and h Ϫ /h ϩ mfc1⌬/⌬ mutant diploid cells were induced to undergo azygotic meiosis in the presence of the Coppersensor-1 (CS1), a selective Cu ϩ fluorescent probe (47,48), which was used for imaging copper pools in living meiotic cells. Before meiosis-specific induction, cells were precultivated in the presence of 5 M copper, thereby ensuring intracellular copper sequestration. Subsequently, cells were harvested, washed, and resuspended in low nitrogen-and low copper (ϳ0.16 nM CuSO 4 ; 100 times less than basal conditions)-containing media, allowing their preparation for azygotic meiosis induction. As shown in Fig. 6, A and B, 8 h after induction of meiosis, wild-type cells displayed fluorescent CS1copper complexes that were mainly detected within the forespores. In contrast, in the absence of Mfc1 (h Ϫ /h ϩ mfc1⌬/⌬ mutant), cells displayed an intracellular dispersed punctate pattern of CS1-Cu complexes that were distributed throughout the ascospores without any marked preference for the forespore (Fig. 6, A and B).
Previous studies have shown that S. pombe possesses two copper-dependent amine oxidases, denoted Cao1 and Cao2 (25). It has been reported that Cao1 requires copper for its activity when expressed in mitotically growing cells (25). Because Cao1 molecular and cellular characterization has not yet been ascertained in meiosis, we have examined the subcellular location of an active Cao1-GFP fusion protein into an S. pombe h Ϫ /h ϩ cao1⌬/⌬ mutant strain that was induced to undergo azygotic meiosis. After 8 h of meiotic induction, Cao1-GFP displayed fluorescent staining that was mainly observed within the forespores (Fig. 6C). Given this common cellular localization of fluorescent CS1-copper complexes (in wild-type zygotic cells) and Cao1-GFP protein, we tested whether h Ϫ /h ϩ mfc1⌬/⌬ diploid mutant cells displayed a lower efficiency in making Cao1-GFP active. As shown in Fig. 6D, diploid cells carrying disrupted mfc1⌬/⌬ alleles displayed 89% less CAO activity as compared with the wild-type mfc1 ϩ/ϩ cells. Taken together, the results strongly suggested that Mfc1 activity may be required to mobilize copper into the forespores, thereby providing copper to copper-requiring enzymes that localize into the spore precursor.
Mfc1 Contributes to the Timely Process of Meiosis under Copper-limited Conditions-Our results revealed that Mfc1 appeared at the membrane of forespores during the second meiotic division. One possible explanation is that Mfc1 may participate in providing copper (as a co-factor) to cuproproteins to ensure timely meiotic development. To test this hypothesis, h ϩ /h ϩ pat1-114 mfc1⌬/mfc1⌬ diploid cells were used and compared with h ϩ /h ϩ pat1-114 mfc1 ϩ/ϩ control cells (Fig. 7). Both strains were pre-synchronized in G 1 by nitrogen starvation at 25°C. Temperature was then shifted to 34°C to inactivate the Pat1 kinase, allowing the strains to undergo synchronous meiosis. At this induction time point (zero time point), cells were left untreated or were treated with 100 M TTM. For control cells, meiosis I occurred mainly between 4 and 6.5 h, meiosis II between 6 and 8 h, and spore formation after 8.5 h of meiotic induction under basal conditions (Fig. 7). Moreover, in the case of control cells whereby meiosis was induced in the presence of TTM (100 M), the first meiotic division was delayed by ϳ1 h (Fig. 7). Under basal conditions, a In contrast, the mfc1⌬/mfc1⌬ mutant strain displayed an intracellular dispersed punctate pattern of CS1-copper complexes (red) (bottom left). Nomarski microscopy was used to examine asci morphology. B, at the 8-h time point, live cell copper imaging using 5 M CS1 was carried out in wild-type (mfc1 ϩ /mfc1 ϩ ) and mfc1⌬/mfc1⌬ mutant cells. Each strain had previously been transformed with pJK210GFP-Psy1, which encodes GFP-Psy1 used as an FSM resident marker. The merged image of CS1 dye and GFP-Psy1 is shown in the bottom panel.
Nomarski microscopy was used to verify cell morphology. C, isogenic h Ϫ /h ϩ cao1⌬/cao1⌬ and h Ϫ /h ϩ cao1⌬/cao1⌬ mfc1⌬/mfc1⌬ diploid mutant strains were transformed with an integrative plasmid expressing cao1 ϩ -GFP. Strains were cultivated to undergo azygotic meiosis and then analyzed by fluorescence microscopy for GFP (center left) after 8 h of meiotic induction. Hoechst staining was used to visualize DNA (center right), and ascus morphology was examined using Nomarski optics (far left). Merged images are shown in the far right panels. D, diploid mfc1 ϩ/ϩ and mfc1⌬/mfc1⌬ strains were cultured to undergo synchronous azygotic meiosis under copper-limiting conditions (100 M TTM). Eight hours after the induction of meiosis, cell lysates were prepared from each culture, and equal amounts of cellular protein were subjected to a hydrogen peroxidemediated colorimetric assay for CAO activity. The reported values of total CAO activity are the means of three replicates Ϯ S.D. similar progression through meiosis was observed in the case of control and mutant strain lacking Mfc1 (mfc1⌬/⌬) (Fig. 7). However, when meiosis was induced in the presence of 100 M TTM, the meiotic progression of a strain lacking Mfc1 (mfc1⌬/⌬) was significantly delayed as compared with a control strain. As shown in Fig. 7, the first meiotic division (2 N peak) was only observed 8 h after meiotic induction, although it occurred normally between 5 and 7.5 h in control starved cells. Furthermore, at 9 h, mfc1⌬/⌬ mutant cells were still largely at the stage of 2 N chromosomes, whereas more than 55% of control cells were already sporulated (Fig. 7). To validate that the meiotic maturation defect was attributable to the inactivation of mfc1 ϩ , we created a h ϩ /h ϩ pat1-114 mfc1⌬/mfc1⌬ strain in which wild-type mfc1 ϩ/ϩ alleles were returned by integration and expressed under the control of the nmt1 ϩ 41X promoter (40,56). In these experiments, when the strain underwent synchronous meiosis in the presence of 100 M TTM and 5 M thiamine, we observed the absence of spore formation after 9 h of meiotic induction (data not shown). The phenotype was similar to that observed in mfc1⌬/⌬ null cells (Fig. 7). In contrast, when the transformed strain (nmt1 ϩ -mfc1 ϩ /nmt1 ϩ -mfc1 ϩ ) was synchronized in the presence of TTM (100 M) but in the absence of thiamine, diploid cells expressing mfc1 ϩ/ϩ alleles underwent normal meiotic maturation over time similarly to that observed in control cells (data not shown). Taken together, the results showed that, under copper-limiting conditions, Mfc1 plays an important role to ensure timely meiotic development because cells carrying inactivated mfc1⌬/⌬ alleles displayed delayed and prolonged meiosis.

DISCUSSION
Copper is essential to several biological processes, including free radical detoxification, mitochondrial respiration, and meiosis. Copper insufficiency leads to a block in meiosis at metaphase I in S. pombe (Fig. 1), an observation reminiscent of that seen in mouse oocytes regarding zinc availability (8,57). In this FIGURE 7. Normal meiotic progression requires Mfc1 under low copper conditions. Cultures of pat1-114 mfc1 ϩ/ϩ and pat1-114 mfc1⌬/mfc1⌬ strains were presynchronized in G 1 by nitrogen starvation at 25°C. The cultures were then incubated at 34°C to initiate (T0, zero time point) and proceed through synchronous meiosis under both basal and copper-limiting conditions. After 9 h (T9) of meiotic induction, Nomarski microscopy was used to examine cell or ascus morphology (center). In each strain and growth condition, samples were taken every 60 min, and percentage of cells with 1 (1N), 2 (2N), and 3-4 (3-4 N) nuclei and percentage of asci (asci) were calculated (graphics on right). In the presence of TTM (100 M), the meiotic maturation of a strain lacking Mfc1 (mfc1⌬/mfc1⌬) was significantly delayed as compared with control strain. Under these same conditions, an mfc1⌬/mfc1⌬ mutant strain was still at the stage of 2 N chromosomes at the time point 9 h, whereas the control strain was already at the tetranucleated (4 N) stage in a proportion of Ն50%.
latter case, the zinc ions acquired during mouse meiosis are critical for egg development. Any zinc-insufficient oocytes undergo meiotic arrest at metaphase II (8). Furthermore, copper and zinc levels rise significantly during meiotic maturation in mice (8). Based on these observations, we applied DNA microarrays in fission yeast to investigate the copper-dependent transcriptional program during meiosis. Many genes were induced in response to copper starvation, with mfc1 ϩ being the most highly induced gene. The mfc1 ϩ -encoded protein was predicted to be a MFS-type transporter, which is to say that it is a member of the largest group of secondary membrane transporters found in eukaryotes (49).
Over the past years, various studies have suggested the presence of independent means for the transport of copper into cells (1,2,58). Furthermore, cells frequently possess both primary and secondary active transporters for the same substrate. It is known that copper can be transported across membranes by at least one type of primary transporters, the P-type copper ATPases (1). The Ctr family constitutes an important second group of copper transporters (19). Although the Ctr-like proteins lack an identifiable ATP hydrolysis domain, they are able to transport copper in the low micromolar range and in a highly specific manner (59). The Ctr family of transporters is structurally similar to channel proteins as they are generally assembled as symmetrical homotrimers possessing a cone-shaped pore in the center (60,61). Met residues located at the narrowest end of the pore are thought to mediate the passage of copper ions through the pore.
Here, we report the first example of a secondary-type transporter for copper ions in eukaryotes, which we named Mfc1. The Mfc1 protein is strictly expressed during meiosis. The fact that Mfc1 is a secondary copper transporter possessing ϳ61-65% less efficacy than the heteromeric Ctr4-Ctr5 complex may represent an advantage as meiotic S. pombe cells are sensitive to copper, exhibiting meiosis-specific timing defects in the presence of Ն75 M copper (data not shown).
Based on computer analysis, the N and C termini of Mfc1 were predicted to be located inside the forespore membrane, which correlates with the general structure of MFS-type proteins (49). Furthermore, the primary sequence of Mfc1 encompassed 495 amino acids, a number typical of MFS-type proteins which are usually composed of 400 -600 amino acids (49). Typically, MFS-type transporters contain 12 transmembrane domains, the number of membrane spanning domains predicted to be present in Mfc1. In contrast, Mfc1 had one distinctive feature, namely the presence of 19 Met and 7 Cys residues scattered throughout the protein. Interestingly, transmembrane domains 1 and 9 of Mfc1 contained sequences rich in Met residues arranged in both MXM and MX 2 M clusters (denoted Mets motifs), which are thought to either capture copper or to translocate it across the membrane. A third putative Mets motif, 413 MXM 415 that is located after transmembrane domain 10, might also play a role in copper handling. Based on structural, computational, and biochemical analyses of MFS proteins, it has been proposed that MFS-type transporters operate via an alternating-access mechanism involving a rocker-switch type movement of the transporter (49). In this model, the transmembrane domain 1 of a MFS-type transporter, which in the case of Mfc1 contains two copies of partially overlapping Mets motifs, is predicted to play a key role in the rocker-switch mechanism by allowing translocation of the substrate after its binding with the transporter. This 48 MTMLCM 53 motif may play an important function in copper transport by Mfc1.
The localization of Mfc1 in meiosis was determined by comparing the fluorescence pattern of a functional Mfc1-Cherry fusion protein to that of GFP-tagged Psy1, a protein known to function in the assembly of the forespore membrane (55). Fluorescence microscopy revealed that Mfc1 is localized on the membrane of forespores in a manner identical to that observed for GFP-Psy1. When expressed in cells proliferating in mitosis, Mfc1 localized to the plasma membrane where it restored the inability to grow on nonfermentable carbon sources, a phenotype previously associated with the loss of the heteromeric Ctr4-Ctr5 complex (15). Importantly, a similar subcellular localization has been reported for the Psy1 protein when it was expressed under the control of a heterologous mitotic promoter (62). Thus, in a manner analogous to Psy1, Mfc1 was observed to surround the cell surface during mitosis. That said, it is clear that under physiological conditions in which both genes are exclusively expressed during meiosis, Mfc1 and Psy1 localize to the forespore membrane of developing asci.
The novel S. pombe copper-responsive gene mfc1 ϩ identified in this study encodes a meiosis-specific forespore membrane protein. In a manner similar to the ctr4 ϩ and ctr5 ϩ genes that encode the high affinity copper heteromeric transport complex located at the cell surface, mfc1 ϩ is induced at the transcriptional level in response to copper starvation. In contrast to ctr4 ϩ and ctr5 ϩ , for which the transcription factor Cuf1 is essential for their induction under conditions of copper starvation, the inactivation of the cuf1 ϩ locus did not affect the transcription of mfc1 ϩ . This result was also consistent with the fact that only a single, inverted weak putative CuSE element was identified in the promoter region of mfc1 ϩ (data not shown). We conclude that a distinct regulator of copper limitation-dependent gene expression must exist for mfc1 ϩ induction.
The observation that mfc1 ϩ was transcriptionally regulated by copper in the same way as the genes encoding the components of the high affinity copper uptake machinery suggests that Mfc1 is involved in copper utilization, rather than in copper detoxification. The subcellular localization of Mfc1 at the forespore membrane suggests that Mfc1 is an intracellular transporter that mediates copper uptake into the forespore, thus indicating a pathway by which copper could be transported into this compartment where copper is required for copper-dependent enzyme activities. This proposed function of Mfc1 is supported by the fact that deletion of the mfc1 ϩ gene (mfc1⌬/⌬) resulted in a strong reduction in CAO activity, a cuproenzyme found in the forespore. Furthermore, as shown by experiments using Coppersensor-1 (CS1), analysis of live cell labile copper in an mfc1⌬/mfc1⌬ mutant revealed that labile copper pools were mainly detected outside of the forespores. In contrast, wild-type cells (mfc1 ϩ /mfc1 ϩ ) exhibited an intense punctate fluorescence corresponding to the presence of labile copper pools within the forespores (Fig. 6). The fluorescent detection of labile copper pools in forespores in mfc1 ϩ /mfc1 ϩ cells adds further weight to the argument favoring a role for Mfc1 in mediating copper uptake into forespores.
The essential yet toxic nature of copper ions in biological systems demands a tight regulation of the expression of genes involved in copper acquisition. A fine-tuned regulation is critical to maintaining the appropriate intracellular levels of copper in cells at all times. Consistent with this fine regulation, under copper-deficient conditions and upon entry to meiosis, the ctr4 ϩ gene that encodes one of the two components of the Ctr4-Ctr5 complex was initially transcriptionally induced within 1 h, followed by its down-regulation to complete repression within ϳ5-6 h. The Ctr4-Ctr5 complex showed a peak level of expression that coincided primarily with the start of meiosis and the premeiotic S-phase and recombination. As the levels of ctr4 ϩ mRNA were reduced, the mfc1 ϩ mRNA levels increased, with a short window of overlap between the Ctr4-Ctr5 and Mfc1 copper-transporting systems. The mfc1 ϩ mRNA levels reached a maximum within 5 h, coinciding with the meiotic divisions. Thereafter, the expression profile of mfc1 ϩ was sustained, with only a slight decrease over time being observed. Clearly, Mfc1 was also produced during the sporulation process, including during forespore membrane formation. This is consistent with the fact that Mfc1 was localized to the forespore membrane after its closure. Because the two copper transport machineries (Ctr4-Ctr5 complex and Mfc1) function in meiosis, these results suggest the existence of a dynamic interplay between the two copper uptake systems and that they are regulated in an ordered and timely succession of transcriptional waves that occur during the meiotic developmental program. Consistent with their function in copper transport, both the ctr4 ϩ and mfc1 ϩ genes were repressed by copper regardless of the meiotic stage. The results presented here are compatible with those showing an important role for copper in germ line differentiation in Drosophila (32). It has been shown that a third Ctr-like protein, denoted Ctr1C, is required for copper transport in the male germ line, participating in both spermatocyte and sperm maturation. Although their roles have not yet been ascertained, high levels of the mouse Ctr1 and Ctr2 proteins have been also detected in spermatozoa and testes in mice (32,63). Given these observations, it is tempting to suggest that the requirement for the presence of copper transporters in sexual differentiation is conserved from yeast to insects and mammals.