Mfc1 is a novel forespore membrane copper transporter in meiotic and sporulating cells

Abstract

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.

FIGURE 1.

Arrest of the meiotic program at metaphase I in zygotes grown under copper starvation conditions. A, representative vegetative diploid cells that were transferred to nitrogen-poor media (T0) and then left untreated. Shown are three representative stages of meiosis after 3, 6, and 10 h of meiotic induction. Spindle pole bodies were monitored by expressing the Sad1-Cherry fusion protein (center left). The chromosomal material was probed by Hoechst 33342 staining (center right). Fluorescence microscopy images show DNA (blue) and Sad1-Cherry (red) and the corresponding merged images. Schematically depicted images are illustrated at the far right. Nomarski images are shown at the far left. B, at the onset of the experiment (zero time point), cells were treated with TTM (200 μm). Zygotes incubated in the presence of TTM underwent a meiotic block at metaphase I (even after 6 and 10 h of meiotic induction). C, aliquot of TTM-treated zygotes (that were blocked for 2 h) was washed and incubated in the presence of exogenous copper (10 μm), which resulted in release from metaphase I. The graphics (right) indicate the meiotic profiles of the cells. The cells with 1, 2, or 3–4 nuclei were determined by counting the Hoechst 33342-stained nuclei after meiotic induction. At least 100 cells were counted under each above-mentioned condition. The reported values of cells are the means of three replicates ± S.D.

FIGURE 2.

Predicted topological model of Mfc1. A, primary amino acid sequence of Mfc1. The Cys, Met, and His residues are indicated in bold. Putative copper-binding motifs, called Mets motifs (MX₂M and/or MXM; letters a–d), are underlined. The gray boxes indicate the 12 predicted transmembrane domains (TMDs 1–12). B, according to the predicted topology, both the N and C termini of Mfc1 are located in the cytosol. The locations of the Cys, Met, and His residues are indicated. The 12 predicted transmembrane domains (ovals) are depicted, and their positions are indicated with numbers. The amino acid sequence numbers refer to the position relative to the first amino acid of Mfc1.

FIGURE 3.

Copper starvation induces mfc1⁺ and ctr4⁺ transcripts at distinct times following meiotic induction. pat1-114 cuf1^+/+ and pat1-114 cuf1Δ/Δ 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 μm), CuSO₄ (100 μm), or without treatment (basal). At the indicated time points, mfc1⁺ (A and B) and ctr4⁺ (C and D) mRNAs were analyzed in a diploid pat1-114 cuf1^+/+ strain and an isogenic strain lacking the cuf1⁺ 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⁺ steady-state mRNA levels. E, graphic representation of expression profiles of the mfc1⁺ and ctr4⁺ genes during meiosis. The peak of ctr4⁺ mRNA expression was observed at 0.5 h, whereas the maximal induction of mfc1⁺ 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⁺ and ctr4⁺, 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 (−) or presence of TTM (100 μm) or CuSO₄ (100 μm). Actin (act1⁺) mRNA levels were detected as an internal control.

FIGURE 4.

Mitotic expression of Mfc1-GFP produces a fluorescent signal at the plasma membrane and functionally complements the respiratory deficiency of a ctr4Δ ctr5Δ mutant in the presence of copper. A, ctr4Δ ctr5Δ cells were transformed with an empty vector or integrative plasmids expressing the mfc1⁺-GFP allele (under the control of a mitotic constitutive gene promoter) or both the ctr4⁺-GFP and ctr5⁺-MYC₁₂ 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 ctr4Δ ctr5Δ double deletion were transformed with an empty vector, mfc1⁺, mfc1⁺-GFP, or ctr4⁺-GFP and ctr5⁺-MYC₁₂. Cultures were spotted onto YES media containing glucose or glycerol/ethanol (Gly/EtOH) and CuSO₄ (0, 1, and 2 μm). WT, isogenic parental strain FY435 (ctr4⁺ ctr5⁺). C, whole cell extracts were prepared from aliquots of cultures used in G and analyzed by immunoblotting. D–F, growth of ctr4Δ ctr5Δ cells transformed with the indicated alleles (empty vector, black; mfc1⁺, green; mfc1⁺-GFP, blue; ctr4⁺-GFP, and ctr5⁺-MYC₁₂, red) was assessed in glycerol/ethanol (Gly/EtOH) liquid cultures in the absence (C, 0 μm) or presence of CuSO₄ (D, 1 μm; E, 2 μm) for 8 days. G, indicated transformed ctr4Δ ctr5Δ cells were incubated with 2 μm ⁶⁴CuCl₂ for 10 min. Absorption of ⁶⁴CuCl₂ 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 ± S.D.

FIGURE 5.

Analysis of Ctr4-GFP and Mfc1-Cherry localization during meiosis and sporulation. Fluorescence signals from Ctr4-GFP (A) and Mfc1-Cherry (B) were observed at different stages of meiosis after azygotic meiotic induction of h⁺/h⁻ ctr4Δ/Δ ctr4⁺-GFP/ctr4⁺-GFP and h⁺/h⁻ mfc1Δ/Δ mfc1⁺-Cherry/mfc1⁺-Cherry strains, respectively. Once induced, azygotic meiotic cells were differentiated in the presence of 100 μm TTM. C, h⁺/h⁻ mfc1Δ/Δ diploid strain co-expressing Mfc1-Cherry and GFP-Psy1 was induced to undergo azygotic meiosis. At the 8-h time point, in the presence of 100 μm TTM, the Mfc1-Cherry fluorescent signal was detected at the FSM (center top) where the GFP-Psy1 fluorescent protein was also found (center bottom). GFP-Psy1 was used as a FSM-resident marker. The merged image of both fluorescent proteins is shown in the bottom panel. Nomarski microscopy was used to ascertain cell morphology (top).

FIGURE 6.

Mfc1 is required for copper accumulation into forespores and the production of fully active CAOs. A, live cell copper imaging with the fluorescent copper-binding tracker CS1 (10 μm) after 8 h of meiotic induction in wild-type (mfc1⁺/mfc1⁺) versus mfc1Δ/mfc1Δ mutant cells. The fluorescent CS1-Cu complexes (red) detected into the forespores are indicated by white arrowheads (top left). In contrast, the mfc1Δ/mfc1Δ 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 μm CS1 was carried out in wild-type (mfc1⁺/mfc1⁺) and mfc1Δ/mfc1Δ 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⁻/h⁺ cao1Δ/cao1Δ and h⁻/h⁺ cao1Δ/cao1Δ mfc1Δ/mfc1Δ diploid mutant strains were transformed with an integrative plasmid expressing cao1⁺-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^+/+ and mfc1Δ/mfc1Δ strains were cultured to undergo synchronous azygotic meiosis under copper-limiting conditions (100 μm 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 peroxide-mediated colorimetric assay for CAO activity. The reported values of total CAO activity are the means of three replicates ± S.D.

FIGURE 7.

Normal meiotic progression requires Mfc1 under low copper conditions. Cultures of pat1-114 mfc1^+/+ and pat1-114 mfc1Δ/mfc1Δ strains were presynchronized in G₁ 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 μm), the meiotic maturation of a strain lacking Mfc1 (mfc1Δ/mfc1Δ) was significantly delayed as compared with control strain. Under these same conditions, an mfc1Δ/mfc1Δ 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 ≥50%.