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Vol. 19, Issue 4, 1295-1303, April 2008
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*Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; and Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, 577-8502, Japan
Submitted August 19, 2007;
Revised December 26, 2007;
Accepted January 8, 2008
Monitoring Editor: Benjamin Glick
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kambe et al., 2004; Cousins et al., 2006; Kambe et al., 2006). A number of zinc transporters belonging to different families have been identified. In particular, the cation diffusion facilitator (CDF) family has been implicated in the metal homeostasis of diverse organisms from prokaryotes to eukaryotes (Williams et al., 2000; Gaither and Eide, 2001; Nies, 2003; Kambe et al., 2004; Cousins et al., 2006).
Most CDF family of zinc transporters, which are ubiquitously found, have been characterized to facilitate Zn2+ efflux from the cytoplasm or to mobilize the cytoplasmic Zn2+ into the intracellular organelles. In budding yeast Saccharomyces cerevisiae, two CDF family members, Zrc1p and Cot1p, localize at the vacuolar membrane and they have been implicated in the storage of excess Zn2+ in the vacuole (Kamizono et al., 1989; Conklin et al., 1992, 1994; Li and Kaplan, 1998; MacDiarmid et al., 2000, 2002, 2003). Two other CDF family members, Msc2p and Zrg17p, form a heteromeric zinc transport complex in the endoplasmic reticulum (ER) membrane, and they have been implicated in the homeostatic maintenance of ER function. In mutant yeast deficient in these CDF proteins, the UPR induction by zinc deficiency is exacerbated (Ellis et al., 2004, 2005). In fission yeast Schizosaccharomyces pombe, Zhf1, a homolog of S. cerevisiae Zrc1p and Cot1p, localizes at the ER, mediates Zn2+ storage, and differentially affects tolerance to a range of transition metals (Clemens et al., 2002). In vertebrate cells, three CDF family members, ZnT5, ZnT6, and ZnT7, are located in the Golgi and are implicated in the entry of Zn2+ into the Golgi lumen (Kambe et al., 2002; Huang et al., 2002; Kirschke and Huang, 2003). Using DT40 cells, Kambe et al. showed that the two oligomeric zinc transport complexes, namely ZnT5/ZnT6 hetero-oligomeric complexes and ZnT7 homo-oligomeric complexes, are required for zinc incorporation into alkaline phosphatases (Suzuki et al., 2005a,b; Ishihara et al., 2006).
In this study, we identified Cis4, a new zinc transporter belonging to the CDF protein family, and we showed that its mutation affects cell wall integrity and secretion in fission yeast. In addition, we also showed that the addition of extracellular Zn2+ suppressed the phenotypes of two membrane trafficking mutants, namely ypt3-i5 and apm1-1, alleles of the genes implicated in Golgi membrane trafficking. Taken together, these results highlight the importance of zinc and a CDF zinc transporter Cis4 in Golgi membrane trafficking in fission yeast.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Standard genetic and recombinant-DNA methods (Moreno et al., 1991) were used except where noted. Tacrolimus (FK506) was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). Gene disruptions are denoted by lowercase letters representing the disrupted gene followed by two colons and the wild-type gene marker used for disruption (for example, cis4::ura4+). Gene disruptions are abbreviated by the gene preceded by 
(for example, cis4). Proteins are denoted by roman letters and only the first letter is capitalized (for example, Cis4).
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; Kita et al., 2004). The cis4-2 (KP537) mutant was obtained from a pool of mutants that showed only MgCl2 sensitivity. The original mutants isolated were backcrossed three times to wild-type strains HM123 and HM528.
Cloning and Knockout of cis4+ and SPBC16E9.14c (zrg17+) Genes
To clone the cis4+ gene, the cis4-1 mutant was grown at 30°C and transformed with an S. pombe genomic DNA library constructed in the vector pDB248 (Beach et al., 1982). The Leu+ transformants were replica-plated onto YPD plates containing 0.15 M MgCl2, and the plasmid DNA was recovered from the transformants that showed a plasmid-dependent rescue. These plasmids complemented both the immunosuppressant sensitivity and MgCl2 sensitivity of the cis4-1 mutant. By DNA sequencing, the suppressing plasmids fell into two classes, with one class containing the cis4+ gene (SPAC17D4.03c), and another class containing a gene distinct from the cis4+ gene. Characterization of the gene in the second class will be reported elsewhere. To investigate the relationship between the cloned SPAC17D4.03c gene and the cis4-1 mutant, linkage analysis was performed as follows. The entire SPAC17D4.03c gene was subcloned into the pUC-derived plasmid containing the S. cerevisiae LEU2 gene and was integrated by homologous recombination into the genome of the wild-type strain HM123. The integrant was mated with the cis4-1 mutant. The resulting diploid was sporulated, and tetrads were dissected. A total of 30 tetrads were dissected. In all cases, only parental ditype tetrads were found, indicating the allelism between the SPAC17D4.03c gene and the cis4-1 mutation (our unpublished data). Similarly, the allelism between the SPAC17D4.03c gene and the cis4-2 mutation was confirmed (our unpublished data). We therefore named SPAC17D4.03c as the cis4+ gene.
To knockout the cis4+ gene, a one-step gene disruption by homologous recombination was performed (Rothstein, 1983). The cis4::ura4+ disruption was constructed as follows. The 2.1-kb PstI/EcoRV genomic fragment, containing the cis4+ gene excluding the 95 base pairs from the start codon, was subcloned into the PstI/SmaI site of BlueScriptSK(+). Then, a BamHI/PstI fragment containing the ura4+ gene was inserted into the BglII/NsiI site of the previous construct. The construct containing the disrupted cis4+ gene was digested with BamHI and PstI, and the cis4::ura4+ fragment was used to transform the diploids (5A/1D, Table 1). The disruption was verified by Southern hybridization of the Ura+ heterozygous diploids and the Ura+ haploids segregants (our unpublished data).
SPBC16E9.14c gene, which we named zrg17+ gene, encodes a homolog of budding yeast Zrg17p (Score = 210, Expect = 1.2e–15). Budding yeast Zrg17p is proposed to form a heteromeric zinc transport complex with Msc2p in the ER membrane (Ellis et al., 2005). To knockout the zrg17+ gene by homologous recombination, the 2054-base pair HindIII/EcoRI genomic fragment containing the zrg17+ gene was subcloned into the HindIII/EcoRI site of BlueScriptSK(+). The NcoI site of zrg17+ gene was changed to BglII site by linker. Then, a BamHI fragment containing the LEU2 gene was inserted into the BglII site of the previous construct. The construct containing the disrupted zrg17+ gene was digested with HindIII and EcoRI, and the zrg17:: LEU2 fragment was used to transform the diploids (5A/1D, Table 1). Stable integrants were selected on medium lacking leucine. The disruption was verified by genomic Southern hybridization (our unpublished data). The cis4zrg17 double knockout mutant cells were generated by the genetic cross between cis4::ura4+ and zrg17::LEU2.
Gene Expression
Two budding yeast genes, MSC2 and ZRG17, were expressed in fission yeast as follows. The open reading frame (ORF) of MSC2 was amplified by PCR (forward primer 2149, 5'-CCG CTC GAG ATG GAT AGA GGC AGG TGG TG-3'; reverse primer 2151, 5'-GAA GAT CTT CAA TTT GCT ATA GGC TGT AGC-3') from the genomic DNA of S. cerevisiae strain W303–1A and subcloned into the XhoI/BamHI site of pDS473aL expression vector (Forsburg and Sherman, 1997) to give pKB6957. The ORF of ZRG17 was amplified by PCR (forward primer 2283, 5'-CGC GGA TCC ATG GAG ACG CCG CAA ATG AAC GC-3'; reverse primer 2284, 5'-CGC GGA TCC TTA TAT TCG GTC TAT GTC TAT TGT AGT TTC-3') from the genomic DNA of S. cerevisiae and subcloned into BamHI site of BlueScriptSK(+). The fission yeast expression vector pREP1 (Maundrell, 1993) was cut with PstI/SacI, and the fragment containing nmt1 promoter and stop codon was subcloned into the integration vector containing the asn1+ gene, pKB6864 (Ma et al., 2007), to give pKB7211. Then the above construct containing complete ZRG17 ORF was digested with BamHI, and the resulting fragment was subcloned into the BamHI site of pKB7211 to give pKB7218. These constructs were used to express the budding yeast genes in fission yeast cells.
To express Cis4 tagged with red fluorescent protein (RFP) from its own promoter, the cis4+ ORF was amplified by PCR (forward primer 2285, 5'-CCG CTC GAG CAC CAT GAA TGT TAA TTC TTC TGC GTT CG-3'; reverse primer 2286, 5'-ATA GTT TAG CGG CCG CCT TTT CCA CAC CAG CAA TTG TTT GGC CG-3') from the genomic DNA and was subcloned into the XhoI/NotI site of BlueScriptSK(+). The cloned ORF of the cis4+ gene was digested with HindIII/NotI and the resulting fragment (1.2 kb), which lacked half of the ORF starting from the 5' end and in addition lacked the stop codon, was inserted at the 5' end of the coding sequence for monomeric RFP gene (GenBank Accession No. AB166761) in the integration vector containing the ura4+ gene to give pKB7269. To obtain the chromosome-borne Cis4-RFP under the control of its own promoter, wild-type cells (KP456, Table 1) were transformed with pKB7269 and were integrated into the chromosome at the cis4+ gene locus of KP456. A successful integration was confirmed by PCR and Southern blot (our unpublished data). All the RFP, GFP-, or GST-tagged versions of Cis4 or Zrg17 were fully functional as demonstrated by its complementation of cis4 or zrg17 mutant phenotypes, respectively (our unpublished data).
GFP-Syb1, Rer1-GFP, and Krp1-GFP were expressed as described previously (Nakamura-Kubo et al., 2003; Kita et al., 2004; He et al., 2006).
Microscopy and Miscellaneous Methods
Acid phosphatase activity in whole cells and cell extracts was determined according to Bergman (1986), and the accumulation of acid phosphatase in cells was estimated by subtracting the cell-extract values from the whole-cell values. Methods in light microscopy, such as fluorescence microscopy which was used to observe the localization of GFP- or RFP-tagged proteins were performed as described (Kita et al., 2004). Tetrad analysis (Zhang et al., 2000), cell wall digestion by β-glucanase (Zymolyase, Seikagaku Kogyo, Tokyo, Japan, Cheng et al., 2002), GST pulldown assay (Tsutsumi et al., 2007) were performed as previously described. To quantify the GFP-Syb1 localization, at least 50 cells with GFP fluorescence were identified and scored for GFP-Syb1 localization to the cell surface. Most strains were analyzed by independent observers in a double-blind manner.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). As shown in Figure 1, the cis4-1 mutant cells grew equally well compared with the wild-type cells at 27°C. However, the cis4-1 mutant cells could not grow on YPD plate containing FK506 or 0.15 M MgCl2 at the permissive temperature, whereas wild-type cells grew normally (Figure 1). As predicted, no double mutant was obtained at any temperature by the genetic cross between cis4-1 and calcineurin deletion (ppb1; our unpublished data), indicating that cis4-1 and ppb1 are synthetically lethal.
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cis4 + vector), indicating that Cis4 is not essential for cell viability. The cis4 cells, compared with the cis4-1 cells, showed similar sensitivity to MgCl2 but were less sensitive to FK506 (Figures 1 and 2B). Meanwhile, the cis4-2 mutants, compared with the cis4 cells, showed similar MgCl2 and FK506 sensitivities (Figure 2B).
Addition of Extracellular Zn2+ Suppressed the Phenotypes of cis4 Mutant Cells
As Cis4 is structurally related to zinc transporters, we hypothesized that Cis4 may mobilize the cytoplasmic Zn2+ into the intracellular organelles. Mutation or deletion of the cis4+ gene may cause a shortage of Zn2+ in the organelles, and the shortage can be suppressed by the addition of extracellular Zn2+. Then, we examined the effect of the addition of Zn2+ on the phenotypes of cis4 mutants. As shown in Figure 2B, the addition of Zn2+ on the YPD medium dramatically suppressed the MgCl2 sensitivity of all the cis4 mutants, namely cis4-1, cis4-2, and cis4 cells. The addition of Zn2+ also suppressed the FK506 sensitivity of the cis4 mutants. The cis4 mutants showed partial suppression of their phenotypes with the addition of 0.5–1 mM Zn2+ and showed full suppression with the addition of 2 mM Zn2+. The removal of zinc from the synthetic medium recipe (a final concentration of Zn2+ in EMM was 
0.6 mM) had no effect on the phenotypes (our unpublished data).
When high levels of a metal ion are added to complex media such as YPD, the added metal can titrate other metal ions off of the metal-binding components in the media (e.g., amino acids) and make those other metals more available to the cells. Thus, the suppression of the mutant phenotype observed may not be due to the high amount of zinc per se, but rather it may be due to the increased availability of a different metal ion. Therefore, we tested if high levels of other metal ions can also suppress the mutant phenotype. Neither MnSO4 (up to 2 mM), nor FeCl3 (up to 2 mM) or CuSO4 (up to 0.2 mM), when added to the media, showed suppressive effect on the phenotypes of the cis4 mutants (our unpublished data). These results strongly suggest that Cis4 is a zinc transporter that mobilizes the cytoplasmic Zn2+ into the intracellular organelles.
Ellis et al. (2005) showed that the budding yeast zrg17 mutant exhibits the same zinc-suppressible phenotypes as the msc2 mutant and suggested that Msc2p and Zrg17p form a heteromeric zinc transport complex. Then, we examined whether the expression of these budding yeast genes could suppress the growth phenotype of cis4 cells. As shown in Figure 2C, the expression of the budding yeast MSC2 or ZRG17 alone could not suppress the growth phenotype of cis4 cells. However, when both MSC2 and ZRG17 were coexpressed, the growth defect was suppressed.
The cis4 and zrg17 Mutant Cells Shared Common and Nonadditive Zinc-suppressible Phenotypes, and Cis4 and Zrg17 Physically Interacted
The above findings prompted us to disrupt the zrg17+ gene and to examine the phenotypes of zrg17 cells and cis4zrg17 cells. As shown in Figure 3A, the zrg17 mutant also exhibited MgCl2- and FK506-sensitive growth defects, and they were also suppressible by the addition of zinc. Thus, cis4 and zrg17 mutants show very similar zinc-suppressible growth defects. As expected, the cis4zrg17 double mutant also showed MgCl2- and FK506-sensitive growth defects. The addition of similar concentrations of zinc suppressed the double mutant to the same degree as that of the single mutants, indicating that the effect of zinc on the phenotype of these two mutations are not additive.
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Intracellular Localization of Cis4
To study the intracellular localization of the Cis4 protein, we tagged the 3' end of the cis4+ gene with the sequence encoding RFP. This construct is fully functional as cells expressing Cis4-RFP complemented the phenotypes associated with the cis4 mutation, and the phenotypic analysis of the cells expressing Cis4-RFP also indicates that tagging did not abolish function (our unpublished data). Then we examined the localization of Cis4-RFP expressed from its own promoter in wild-type cells. Cis4-RFP localized to the punctate structures that were scattered throughout the cytoplasm (Figure 4). To determine whether these punctate structures indicate the Golgi-associated localization of Cis4, the colocalization of Cis4 with the cis-Golgi marker Rer1 and the trans-Golgi marker Krp1 were examined. Cis4-RFP clearly colocalized with Rer1-GFP (Figure 4A), but not with Krp1-GFP (Figure 4B), indicating that Cis4 localizes at cis-Golgi.
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; Cheng et al., 2002; Kita et al., 2004), we hypothesized that the cis4 mutants are also defective in cell wall integrity. Defects in cell wall integrity can sometimes be compensated for by increases in the osmolarity of the growth media (Levin and Bartlett-Heubusch, 1992). Consistent with our hypothesis, all the cis4 mutant cells were capable of forming colonies on YPD plates containing both 0.15 M MgCl2 and 1.2 M sorbitol (Figure 5A).
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). As shown in Figure 5B, the cis4 cells exhibited an increased septation index that was suppressed by the addition of Zn2+. Consistently, we found that the cis4 mutants were highly sensitive to micafungin, a (1,3)-β-D-glucan synthase inhibitor (Figure 5C).
Expression of Constitutively Active Calcineurin Suppressed the Phenotypes of cis4 Mutant Cells
We then examined the effect of the overexpression of the constitutively active calcineurin on the cis4-1 mutant. The cis4-1 mutant cells were transformed with a vector containing the full-length calcineurin gene (CN full), the constitutively active truncated calcineurin gene (CNC), or a control multicopy vector (Figure 5D). The results showed that the overexpression of the constitutively active calcineurin, but not the full-length calcineurin, suppressed the MgCl2-sensitive growth defect of the cis4-1 mutant cells. Overexpression of the constitutively active calcineurin also suppressed the MgCl2-sensitive growth defect of the cis4-2 and cis4 mutant cells (our unpublished data). These results further suggest that Cis4 is implicated in cell wall integrity.
The cis4 Deletion Cells Showed Defects in Acid Phosphatase Secretion and Membrane Trafficking
In previous studies, we showed that several membrane-trafficking fission yeast mutants exhibited a defect in acid phosphatase secretion (Cheng et al., 2002; Kita et al., 2004; He et al., 2006). Here, to assess whether the cis4 mutation specifically affects acid phosphatase secretion or whether the mutation decreases the enzyme accumulation, we determined the acid phosphatase activity in whole cells and cell extracts (Bergman, 1986). As shown in Figure 5E, the acid phosphatase secretion and accumulation of cis4 cells were 25 and 55% compared with that of the wild-type cells, respectively. These results indicate that the cis4 deletion affects both the secretion and accumulation of acid phosphatase and that the secretion was more seriously impaired.
Next, we monitored the localization of the exocytic v-SNARE synaptobrevin Syb1 to assess the Golgi-to-endosome or Golgi-to-plasma membrane trafficking pathway (Edamatsu and Toyoshima, 2003; Kita et al., 2004). In wild-type cells, the fluorescence of GFP-Syb1 clearly localized to the cell membrane at the cell ends (Figure 5F, left top panel, arrows). In cis4 cells, however, the fluorescence of GFP-Syb1 at the cell membrane was markedly reduced (Figure 5F, right top panel). The addition of Zn2+ to the medium significantly increased the number of cells that showed the fluorescence of GFP-Syb1 at the cell membrane (Figure 5F, bottom panel). These results suggest that the defective membrane trafficking in the cis4 cells is due to the low intra-Golgi zinc concentration.
Genetic Interaction between cis4 and the Membrane-trafficking Mutants
The above findings led us to hypothesize that Cis4 mobilizes the cytoplasmic Zn2+ into the Golgi and that the intra-Golgi concentration of Zn2+ is important for membrane trafficking associated with the Golgi complex. To examine the genetic interaction between Cis4 and the membrane-trafficking mutants, we constructed several double mutant strains, namely ypt3-i5cis4, ryh1-i6
cis4, gdi1-i11cis4, and apm1-1cis4. Briefly describing these membrane trafficking components, first, the Rab/Ypt GTPase Ypt3 has been implicated in the membrane trafficking associated with the Golgi complex, and its mutation confers sensitivity to FK506 and defects in cell wall integrity (Cheng et al., 2002). Second, Ryh1, a homolog of mammalian Rab6 and budding yeast Ypt6p, has been implicated in retrograde traffic from endosome to the Golgi (He et al., 2006). Third, Gdi1, a Rab GDP-dissociation inhibitor, plays a role in the recycling of Rabs in the secretory pathway (Ma et al., 2006). Fourth, Apm1, a homolog of the mammalian µ1A subunit of the clathrin adaptor protein complex 1, is implicated in the Golgi/endosome function (Kita et al., 2004).
Interestingly, results showed that there is a genetic interaction between Cis4 and all of the membrane-trafficking mutants tested. As shown in Figure 6A, the ypt3-i5cis4 double mutants were viable but showed more marked temperature sensitivity than that of the ypt3-i5 single mutants. Likewise, other double mutants showed more marked temperature sensitivity compared with that of the single mutants (Figure 6A).
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). The ypt3-i5cis4 double mutants showed more marked sensitivity to Zymolyase than that of the ypt3-i5 cells, whereas the sensitivity of the cis4 single mutant to Zymolyase was not distinctly different from that of the wild-type cells (Figure 6B). Likewise, the gdi1-i11cis4 double mutants showed more marked sensitivity to Zymolyase than that of the gdi1-i11 cells (our unpublished data).
The Addition of Extracellular Zn2+ Suppressed the Phenotypes of ypt3-i5 and apm1-1 Mutant Cells
As the phenotypes of the membrane-trafficking mutants were exacerbated by the cis4 deletion, we next examined the effect of the addition of extracellular Zn2+ on various membrane-trafficking mutants. As shown in Figure 7A, the addition of extracellular Zn2+ significantly attenuated the temperature sensitivity of the ypt3-i5 mutant and that of the apm1-1 mutant, while only slightly affecting that of the ryh1-i6 and gdi1-i11 mutants. The FK506 sensitivity of the ypt3-i5 and apm1-1 mutants was also suppressed by the addition of Zn2+ (Figure 7B). Consistently, the addition of Zn2+ also suppressed the defective septation of the ypt3-i5 and apm1-1 mutant cells, while not affecting that of the ryh1-i6 and gdi1-i11 mutants (Figure 7C).
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C) or a control multicopy vector (Figure 7D). The results showed that the overexpression of the constitutively active calcineurin significantly suppressed the temperature-sensitive growth defect of the ryh1-i6 and gdi1-i11 mutant cells while not affecting that of the ypt3-i5 and apm1-1 mutants. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,b; Ishihara et al., 2006); however, evidence on the role of zinc or zinc transporters in membrane-trafficking events remains lacking. Here in fission yeast, we report that Cis4 is implicated in the Golgi membrane trafficking through the regulation of zinc homeostasis in fission yeast. To our knowledge, this is the first report of the involvement of zinc or a zinc transporter in the regulation of membrane trafficking. Three lines of evidence support the hypothesis that Cis4 is a zinc transporter. First, Cis4 is structurally similar to the budding yeast and human CDF family zinc transporters. Second, the mutations in the cis4+ gene resulted in several phenotypes that are zinc suppressible. Third, the simultaneous expression of MSC2 and ZRG17 that encode the components of the budding yeast zinc transporter complex suppressed the phenotypes of cis4 mutant cells.
Furthermore, our results suggest that Cis4 delivers Zn2+ to the lumen of the Golgi to maintain membrane-trafficking function of the cellular compartment. First, Cis4 localizes to the cis-Golgi. Second, the cis4 mutation affects the membrane-trafficking events related to the Golgi function. Third, the cis4 mutants show a genetic interaction with several membrane trafficking mutants. These results suggest that there exists in the Golgi as yet unknown zinc-requiring components that are involved in the membrane trafficking function and that these zinc-requiring components and Ypt3, Ryh1, Gdi1, or Apm1 are required in parallel pathways with a common essential function in membrane trafficking (Finger and Novick, 2000). In addition, it was observed that the phenotypes of ypt3-i5 and apm1-1 mutants were suppressed by the addition of Zn2+, whereas the phenotypes of ryh1-i6 and gdi1-i11 mutants were not affected. The results suggest that the ypt3-i5 or apm1-1 mutation caused a zinc deficiency that is presumably due to the impairment of the function of unknown zinc transporter(s) or zinc-requiring protein(s) and that zinc is required at multiple pathways in membrane trafficking. These results also suggest a previously undetected interdependency between membrane trafficking and zinc homeostasis in fission yeast.
The overexpression of the constitutively active calcineurin suppressed the temperature-sensitive growth defect of the ryh1-i6 and gdi1-i11 mutant cells, whereas it did not affect that of the ypt3-i5 and apm1-1 mutants (Figure 7D). Together with above findings, these results suggest that calcineurin also plays a role in membrane trafficking at multiple pathways and that calcineurin-related pathways are distinct from those related to zinc (Figure 8). As shown in Figure 2B, the cis4-1 allele was more sensitive to the calcineurin inhibitor compared with that of the cis4-2 or cis4 alleles. The cis4-1 allele might somehow interfere with other mechanisms of transporting zinc into the Golgi or that is related to calcineurin.
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; Borrelly et al., 2002) and SPBC16E9.14c, that encode CDF proteins, which are structurally related to zinc transporters in other species. On Zhf1, the protein localizes to the ER membrane (Clemens et al., 2002) as shown by the immunoelectron microscopic analysis and appears to be required for zinc storage and detoxification, similar to its budding yeast homolog Zrc1p and Cot1p. Consistently, Zhf1 had no effect on the phenotypes of the cis4 mutants (our unpublished data). On SPBC16E9.14c gene that was named zrg17+ because the gene encodes a homolog of the budding yeast Zrg17p, we showed that cis4 and zrg17 mutant cells shared common and nonadditive zinc-suppressible phenotypes. We also showed that Cis4 and Zrg17 physically interacted. These results suggest that Cis4 and Zrg17 form a heteromeric functional complex for zinc transport. The formation of an oligomeric functional complex seems to be an evolutionarily conserved mechanism for zinc transport in the eukaryotic secretory pathway (Ellis et al., 2005; Suzuki et al., 2005a,b; Ishihara et al., 2006).
The cis4 cells showed defects in cell wall integrity (Figure 6). Similar phenotypes were reported by Kumanovics et al. (2006). In the same study, it was suggested that the cell wall defect is attributed to the phospholipids biosynthetic pathway because lower concentrations of zinc decreased the phospholipid activity by up to 50%. To test this possibility, we examined the effect of ethanolamine in the growth medium to increase the cellular phosphatidylethanolamine as described by Kumanovics et al. However, the phenotypes of the cis4 mutants were unaffected by the addition of ethanolamine (our unpublished data), suggesting that other enzymatic steps in the phospholipid pathway may also be zinc-dependent.
The cis4-mediated MgCl2 sensitivity was suppressed by the expression of the constitutively active calcineurin (Figure 5D). This suggests that calcineurin regulates zinc homeostasis in fission yeast. Alternatively, calcineurin may regulate other cellular events and may indirectly counteract the toxic effects of a high Mg2+ concentration. Calcineurin contains a binuclear Fe-Zn center that is required for its activity (Namgaladze et al., 2002), and a likely reason for the effects observed is that Cis4 is required to supply Zn directly to calcineurin.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present addresses: 
Sysmex Corporation, Kobe 651-0073, Japan; 
Chugai Pharmaceutical Co., Tokyo 103-8324, Japan.
Address correspondence to: Takayoshi Kuno (tkuno{at}med.kobe-u.ac.jp)
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, percentage of cells with a septum) of log phase cells was monitored. At least 50 cells were observed at one time in each experiment. The data represent mean ± SD of 10 determinations. The reduction in the septation index by the addition of Zn2+ was statistically significant (p < 0.005). (C) The cis4 mutants are hypersensitive to micafungin. The cis4 mutants showed hypersensitivity to micafungin, a (1,3)-β-D-glucan synthase inhibitor. Cells were spotted onto YPD plates and YPD plus 0.9 µg/ml micafungin and incubated at 27°C for 4 d. (D) MgCl2-sensitive phenotype of cis4 mutants were suppressed by the expression of constitutively active calcineurin. Wild-type cells or cis4-1 mutant cells transformed with a control vector, the vector containing the full-length ppb1+ gene (CN full), or the vector containing the constitutively active truncated calcineurin gene (CN
