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¹ Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
² Department of Life Sciences, Faculty of Agriculture, Kagawa University, 761-0795, Japan
³ Faculty of Health Science, Kobe University School of Medicine, Kobe 650-0142, Japan

	Abstract

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Schizosaccharomyces pombe pmr1⁺ gene is homologous to Saccharomyces cerevisiae PMR1 gene, which encodes the P-type Ca²⁺/Mn²⁺-ATPase. Addition of Mn²⁺, as well as Ca²⁺, to the medium induced pmr1⁺ gene expression in a calcineurin-dependent manner. The pmr1 knockout (pmr1) cells exhibited hypersensitivity to EGTA. A screen for high gene dosage-suppressors of the EGTA-hypersensitive phenotype of pmr1 led to the identification of pdt1⁺ gene, which encodes an Nramp-related metal transporter. The pmr1 cells showed round cell morphology. Although pdt1 cells appeared normal in the regular medium, it showed round cell morphology similar to that of the pmr1 cells when Mn²⁺ was removed from the medium. The removal of Mn²⁺ also exacerbated the round morphology of the pmr1 cells. The pmr1pdt1 double mutants grew very slowly and showed extremely aberrant cell morphology with round, enlarged and depolarized shape. The addition of Mn²⁺, but not Ca²⁺, to the medium completely suppressed the morphological defects, while both Mn²⁺ and Ca²⁺ markedly improved the slow growth of the double mutants. These results suggest that Pmr1 and Pdt1 cooperatively regulate cell morphogenesis through the control of Mn²⁺ homeostasis, and that calcineurin functions as a Mn²⁺ sensor as well as a Mn²⁺ homeostasis regulator.

	Introduction

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Calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, plays key roles in various Ca²⁺-mediated processes including T-cell activation (Clipstone & Crabtree 1992; O’Keefe et al. 1992), cardiac hypertrophy (Molkentin et al. 1998), memory development (Mansuy et al. 1998; Winder et al. 1998), and many other functions in mammalian cells (Sugiura et al. 2001; Rusnak & Mertz. 2000; Crabtree 2001; Hemenway & Heitman 1999). For many of these cellular events, calcineurin dephosphorylates and controls the entry of the nuclear factor of activated T cells (NF-AT) family members into the nucleus (Crabtree 2001). Furthermore, calcineurin is specifically inhibited by the immunosuppressive drugs, cyclosporin A and FK506 (Liu et al. 1991), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. In the budding yeast Saccharomyces cerevisiae, calcineurin activity is required for the recovery from pheromone-induced cell cycle arrest (Cyert & Thorner 1992; Foor et al. 1992), cell wall synthesis (Eng et al. 1994; Douglas et al. 1994), and Ca²⁺ and Na⁺ ion homeostasis (Nakamura et al. 1993; Cunningham & Fink 1994, 1996; Mendoza et al. 1994; Farcasanu et al. 1995; Pozos et al. 1996), and calcineurin regulates these cellular processes by dephosphorylating the Crz1/Tcn1, the budding yeast functional homologue of NF-AT. Crz1/Tcn1 has been shown to regulate genes encoding ion pumps and cell wall biosynthetic enzymes including Pmr1, Pmr2, Pmc1 and Fks2 (Matheos et al. 1997; Stathopoulos-Gerontides et al. 1999; Stathopoulos & Cyert 1997; Cyert 2001; Yoshimoto et al. 2002).

We have been studying the calcineurin signalling pathway in fission yeast Schizosaccharomyces pombe, because this system is amenable to genetic analysis and has many advantages in terms of its relevance to higher systems. S. pombe has a single gene encoding the catalytic subunit of calcineurin, ppb1⁺ (Yoshida et al. 1994). Analyses of its mutants (its for immunosuppressant and temperature sensitive), that require calcineurin activity for their growth, revealed that calcineurin is implicated in cytokinesis, septation initiation network, and exocytic pathway in fission yeast (Zhang et al. 2000; Yada et al. 2001; Sugiura et al. 2002; Lu et al. 2002; Cheng et al. 2002; Fujita et al. 2002). We also have shown that calcineurin acts antagonistically with the Pmk1 mitogen-activated protein (MAP) kinase in the Cl^– ion homeostasis (Sugiura et al. 1998, 1999, 2002, 2003).

Recently, we cloned the prz1⁺ gene, encoding a Crz1/Tcn1 homologue, and suggested that calcineurin activates at least two distinct signalling branches, i.e. the Prz1-dependent branch that regulates the expression of Pmc1 Ca²⁺ pump, and an unknown pathway which functions antagonistically with the Pmk1 MAP kinase pathway (Hirayama et al. 2003).

In the present study, we showed that Mn²⁺ as well as Ca²⁺ induced the expression of the pmr1⁺ gene that encodes a putative Ca²⁺/Mn²⁺ pump in a calcineurin/Prz1-dependent manner, and that the pmr1⁺ gene had strong genetic interactions with pdt1⁺ gene that encodes the Nramp-related divalent metal transporter (Tabuchi et al. 1999). The morphological defects in the pmr1 pdt1 double knockout mutants were suppressed by the addition of Mn²⁺, but not by Ca²⁺, to the growth medium. These results indicate that Pmr1 and Pdt1 cooperatively regulate cell morphogenesis through the control of Mn²⁺ homeostasis, and that calcineurin functions as a Mn²⁺ sensor as well as a regulator of Mn²⁺ homeostasis.

	Results

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Identification of the S. pombe pmr1⁺ gene

BLAST search of the Sanger Center S. pombe database identified an open reading frame, SPBC31E1.02c, which is highly homologous to S. cerevisiae PMR1, encoding the P-type Ca²⁺/Mn²⁺-ATPase that is localized to the Golgi (BLAST at the Sanger Center database; Score = 1741 (617.9 bits), Expect = 3.2e-181, P = 3.2e-181, Identities = 382/785 (48%), Positives = 504/785 (64%)). Thus, we named the gene pmr1⁺. The pmr1⁺ is also highly homologous to the human gene, ATP2C1 causing Hailey-Hailey disease (Hu et al. 2000) (BLAST at the Sanger Center database; Score = 1942 (688.7 bits), Expect = 1.6e-202, P = 1.6e-202, Identities = 431/906 (47%), Positives = 571/906 (63%)).

Pmr1 is regulated by Ca²⁺/calcineurin/Prz1 pathway

The addition of CaCl₂ (30 mM) to the growth medium induced Pmr1 mRNA accumulation in wild-type, but not in calcineurin-knockout (ppb1) or prz1 cells, indicating that Pmr1 expression is calcineurin/Prz1 dependent (Fig. 1A,B).

View larger version (45K): [in this window] [in a new window]   Figure 1  Pmr1 is regulated by the calcineurin/Prz1 pathway. (A) Northern blot analysis of Pmr1 mRNA expression. Induction of Pmr1 mRNA by Ca2+ requires calcineurin (Ppb1) and Prz1. Wild-type, ppb1 or prz1 cells were incubated in YPD medium at 30 °C with 30 mM CaCl2 for 0, 15 or 30 min. Total RNA (20 µg) were subjected to Northern analysis using a DIG-labelled Pmr1 cRNA. Leu1 mRNA was also detected as a loading control. (B) Quantification of Pmr1 mRNA by Northern analysis. The graph shows the average of three independent experiments.

We also examined the mRNA levels of Pmr1 and Pmc1 after the addition of Mn²⁺ to the growth medium. Figure 2 shows that the mRNA levels of both Pmr1 and Pmc1 were strongly induced by the addition of 5 mM MnCl₂ in wild-type but not in ppb1 or prz1 cells, peaking at 20 min after the shift. Pre-treatment of the wild-type cells with FK506 completely blocked Pmr1 or Pmc1 mRNA accumulation induced by both Ca²⁺ and Mn²⁺, again indicating that the transient induction observed is calcineurin dependent (data not shown). It is notable that the level of Pmc1 mRNA present in either ppb1 and prz1 mutants is extremely low, suggesting that the expression of this gene, independently of the induction by MnCl₂, requires Calcineurin and Prz1 (Fig. 2A).

View larger version (51K): [in this window] [in a new window]   Figure 2  Mn2+ induces accumulation of Pmr1 and Pmc1 mRNAs in a calcineurin/Prz1-dependent manner. (A) Northern blot analysis of Pmr1 and Pmc1 mRNA expression. Wild-type, ppb1 or prz1 cells were incubated in YPD medium at 30 °C with 5 mM MnCl2 for 0, 20 or 40 min. Total RNA (20 µg) were subjected to Northern analysis using DIG-labelled Pmr1 cRNA (upper panel) or Pmc1 cRNA (lower panel) probes. Leu1 mRNA was also detected as a loading control. (B) Quantification of Pmr1 and Pmc1 mRNAs by Northern analysis. The graphs show the averages of three independent experiments.

pmr1 showed hypersensitivity to EGTA

pmr1 cells were hypersensitive to the metal chelator EGTA and failed to grow when EGTA was added to EMM plate at a final concentration of 10 mM EGTA (Fig. 3), while the wild-type cells could grow in EMM plate containing 20 mM EGTA (data not shown). It was previously reported that null mutations in the S. cerevisiae PMR1 gene caused hypersensitivity to EGTA (Durr et al. 1998), and this is consistent with the functional conservation of Pmr1 in S. cerevisiae and S. pombe.

View larger version (87K): [in this window] [in a new window]   Figure 3  Over-expression of pdt1+ gene suppressed the EGTA-sensitive growth defects of pmr1 cells. Serial 10-fold dilutions starting with OD660 = 0.3 of log-phase pmr1 cells carrying a multicopy vector containing pmr1+, pdt1+ or an empty vector were spotted (5 µL) on EMM medium without or with EGTA as indicated. Wild-type cells carrying the vector alone were also spotted on the plates as a control. Plates were incubated at 30 °C and photographed on the 3rd day.

Over-expression of pdt1⁺ gene that encodes the Nramp-related divalent metal transporter, suppressed the EGTA-hypersensitive phenotype of pmr1

To identify genes that are functionally connected to the pmr1⁺ gene, we screened an S. pombe genome library constructed in the multiple copy vector pDB248 (Beach et al. 1982) for genes that, when over-expressed, could suppress the EGTA-sensitive growth defect of the pmr1 cells. Transformants were obtained and subsequently screened for the ability to grow on EMM plates containing 10 mM EGTA at 30 °C. The suppressing plasmids fell into two classes by sequencing. As expected, one class contained the pmr1⁺ gene. Another class contained the pdt1⁺ gene, encoding the Nramp-related divalent metal transporter (Tabuchi et al. 1999). As shown in Fig. 3, the pmr1 cells over-expressing pdt1⁺ gene grew in EMM containing 14 mM EGTA, but the pmr1 cells carrying the vector alone failed to grow.

Consistent with the above result and the previous study by Tabuchi et al. (1999), pdt1 cells were also hypersensitive to EGTA, and their sensitivity to EGTA was lower than that of pmr1 cells (Fig. 6A). Over-expression of pmr1⁺ gene could not suppress the EGTA-hypersensitivity of pdt1 cells (data not shown).

View larger version (114K): [in this window] [in a new window]   Figure 6  Effects of EGTA, CaCl2, MnCl2, FK506, and calcineurin over-expression on the growth of wild-type, pmr1, pdt1, and pmr1pdt1 cells. (A) Effects of EGTA, CaCl2 and MnCl2 on the cell growth. (B) Effects of FK506 on the cell growth. (C) Over-expression of constitutively active calcineurin stimulated the growth of pmr1pdt1 cells. Cells were spotted on the EMM medium without or with the indicated additives and analysed as described in Fig. 3. Plates were incubated at 30 °C for 3 days (A, B), or 4 days (C). (D) Effect of high concentration of MnCl2 on the cell growth. Log phase cells were resuspended at OD660= 0.2 in fresh EMM and were cultured for 6 h in the presence or absence of 50 mM MnCl2. Cell growth was quantified by measuring the increase in OD660. The graphs show the averages of three independent experiments.

pmr1 andpdt1 cells showed round cell morphology

A comparison of the morphology of pmr1, pdt1, and wild-type cells is shown in Fig. 4. A typical cylindrical shape was evident in pdt1 and wild-type cells when they were cultured in normal EMM medium. On the other hand, more than 50% of the pmr1 mutant cells were somewhat round or pear-shaped wherein the diameter was larger and cell length was shorter (Fig. 4). The removal of Mn²⁺ from the medium exacerbated the aberrant morphology causing many of the pmr1 cells to become completely round (Fig. 4). Interestingly, pdt1 cells showed a round cell morphology similar to that of the pmr1 knockout cells when Mn²⁺ was removed from the medium, while the wild-type cells were unaffected by the removal of Mn²⁺ (Fig. 4). Consistently, the addition of Mn²⁺ to the medium suppressed the round cell morphology of pmr1 cells (Fig. 4).

View larger version (78K): [in this window] [in a new window]   Figure 4  Effects of the removal and the addtion of Mn2+ to the medium on the morphology of wild-type, pmr1, and pdt1 cells. Cells were grown to mid-log phase in the indicated medium and were examined by DIC microscopy. ‘EMM - Mn2+’ indicates the Mn2+-deprived EMM medium. The bar indicates 10 µm.

The pmr1pdt1 double mutants showed extremely aberrant morphology, which was suppressed by Mn²⁺ but not by Ca²⁺

Microscopic observation revealed that the pmr1pdt1 double mutant cells exhibited extremely aberrant morphology with round, enlarged and depolarized shape (Fig. 5). Calcoflour staining also revealed that some of these cells had multiple and irregular septa, indicating an impaired cytokinesis (Fig. 5). Surprisingly, the aberrant cell morphology was completely suppressed by the addition of Mn²⁺, and cell shape was almost identical to that of wild-type when the double mutant cells were cultured in EMM medium supplemented with 2 mM MnCl₂ (Fig. 5). On the other hand, supplementation of the medium with 10 mM CaCl₂ had no effect on the aberrant morphology of the double mutants (Fig. 5).

View larger version (77K): [in this window] [in a new window]   Figure 5  Effects of the addition of Mn2+ or Ca2+ on the aberrant morphology of pmr1pdt1 double mutant cells. pmr1pdt1 cells were grown to mid-log phase at 30 °C in the indicated medium. Cells were then fixed, and washed with phosphate buffered saline (pH 7.0), and stained with Calcofluor to visualize the septum, and were examined by differential interference contrast (DIC) and fluorescent microscopy. The bar indicates 10 µm.

Growth of the pmr1pdt1 double mutants was affected by Mn²⁺, Ca²⁺, FK506 and calcineurin over-expression

pmr1pdt1 cells grew very slowly in normal EMM plate, and they could not grow in the presence of very low concentration (0.3 mM) of EGTA (Fig. 6A,C). In contrast, the addition of Mn²⁺ or Ca²⁺ to the medium significantly improved the growth of pmr1pdt1 cells, and the maximum effects were observed in EMM plate containing 2 mM MnCl₂ or 10 mM CaCl₂, respectively (Fig. 6A). These results are in good agreement with the studies on S. cerevisiae PMR1 mutants suggesting that Mn²⁺ and Ca²⁺ are interchangeable for the cell cycle progression (Loukin & Kung 1995; Durr et al. 1998). Knockout of the pmr1⁺ gene caused a hypersensitivity to MnCl₂ concentration, but did not affect the CaCl₂ sensitivity (Fig. 6A,D). The pdt1⁺ knockout cells were resistant to high MnCl₂ concentration, but were hypersensitive to high CaCl₂ concentration (Fig. 6A,D).

Interestingly, in the presence of FK506, the specific calcineurin inhibitor, the growth of the pmr1pdt1 cells were completely blocked. FK506 also abolished the growth stimulatory effect of Ca²⁺ but did not affect the growth stimulatory effect of Mn²⁺ (Fig. 6B). FK506 had no or little effect on the growth of each single mutant (Fig. 6B). These results suggest that the growth stimulatory effect of Ca²⁺ is the secondary effect due to altered calcineurin activity (Park et al. 2001). Consistently, pmr1pdt1 cells expressing the activated mutant of calcineurin (Sugiura et al. 1998) grew faster than pmr1pdt1 cells harbouring a control vector (Fig. 6C). Furthermore, expression of the activated mutant of calcineurin enabled pmr1pdt1 cells to grow in EMM containing 0.3 mM EGTA (Fig. 6C). However, it had no effect on the aberrant morphology of the double mutants (data not shown).

The pmr1pdt1 double mutants missort and secrete Cpy1

To investigate the biochemical changes associated with the morphological phenotype, protein sorting in pmr1, pdt1 and pmr1pdt1 mutants was examined. The previous study in S. cerevisiae showed that PMR1 knockout cells missort and secrete a vacuolar protein carboxypeptidase Y (CPY) (Durr et al. 1998). Thus, immunoblot analysis using anti-Cpy1 antibodies was performed to detect the secreted Cpy1 in the single and double knockout mutants in S. pombe. Contrary to our expectation, immunoblot analysis did not detect the secreted CPY from pmr1 cells or from pdt1 cells (Fig. 7A). Interestingly, the pmr1pdt1 cells secreted considerable amount of CPY when the cells were cultured in normal EMM medium. Addition of Mn²⁺ (2 mM) as well as Ca²⁺ (10 mM) to the medium significantly reduced the secreted CPY (Fig. 7A).

View larger version (58K): [in this window] [in a new window]   Figure 7  Vacuolar CPY sorting (A) and acid phosphatase glycosylation (B) in wild-type, pmr1, pdt1 and pmr1pdt1 cells. Effects of the addition of 2 mM MnCl2 or 10 mM CaCl2 to the media on these cellular processes were also examined. Immunoblot analysis and acid phosphatase staining were performed as described in Experimental procedures.

Impaired glycosylation of acid phosphatase in pmr1, pdt1 and pmr1pdt1 mutants

On native gels, acid phosphatase isolated from pmr1, pdt1 andpmr1pdt1 mutants migrated significantly faster than acid phosphatase isolated from a wild-type strain (Fig. 7B), suggesting impaired protein glycosylation in these mutants. The enzyme from the pmr1pdt1 double mutant cells showed a narrower band with a marked lower molecular mass, suggesting that its glycosylation is severely impaired (Fig. 7B). The presence of Mn²⁺ ions (2 mM) during incubation in EMM strongly stimulated acid phosphatase glycosylation in the double mutant cells, and acid phosphatase from the double mutant cells in Mn²⁺-supplemented medium migrated almost similarly to the enzyme from the wild-type cells. In contrast, Ca²⁺ ions (10 mM) exerted a modest effect on acid phosphatase glycosylation in the double mutant cells. On the other hand, both Mn²⁺ and Ca²⁺ stimulated acid phosphatase glycosylation in the pmr1 cells (Fig. 7B).

	Discussion

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We identified pmr1⁺, the S. pombe homologue of PMR1, which encodes the P-type Ca²⁺/Mn²⁺-ATPase localized to the Golgi membrane in S. cerevisiae (Antebi & Fink 1992). Similar to its budding yeast homologue, the S. pombe Pmr1 is regulated by calcineurin and Prz1, a zinc-finger transcription factor (Hirayama et al. 2003). It is known that Mn²⁺in vitro efficiently activates the calcineurin/calmodulin complex (Pallen & Wang 1984). It is noteworthy that in this study the addition of Mn²⁺ to the growth medium stimulated the accumulation of Pmr1 and Pmc1 mRNAs in a calcineurin/Prz1-dependent manner. This suggests that calcineurin is a physiological target of Mn²⁺, and that calcineurin acts as a Mn²⁺ sensor in fission yeast.

Also identified was the pdt1⁺ that encodes an Nramp-related divalent metal transporter. The pdt1⁺ was identified as a high gene-dosage suppressor of the EGTA-sensitive phenotype of the pmr1⁺ knockout cells. The pmr1pdt1 double knockout cells failed to grow in the presence of 0.3 mM EGTA, while each single knockout cells grew well in the presence of 3 mM EGTA, suggesting strong cooperativity between these two gene products.

The aberrant round cell shape caused by each single knockout gene, together with the synthetic morphological defects caused by the double knockout of pmr1⁺ and pdt1⁺ genes suggest that the two genes cooperatively regulate cell morphogenesis and maintain the cylindrical shape of S. pombe. Several lines of evidence suggest that Mn²⁺ homeostasis is mainly involved in the regulation of cell morphologenesis. First, the addition of Mn²⁺ to the growth medium completely suppressed the aberrant morphological phenotypes of pmr1 and pmr1pdt1 cells. Second, the removal of Mn²⁺ from the medium exacerbated the morphological defect of pmr1 cells, and caused pdt1 cells to acquire round cell morphology that is very similar to those seen in pmr1 cells.

It should be noted that Mn²⁺, but not Ca²⁺, suppressed the aberrant morphological phenotype and improved the glycosylation of acid phosphates in the pmr1pdt1 double mutant cells. On the other hand, both Mn²⁺ and Ca²⁺ suppressed the slow growth phenotype and the missorting phenotype of the double mutant cells. These results indicate that Mn²⁺ and Ca²⁺ are interchangeable for not all the cellular processes in S. pombe.

It is interesting that the morphological phenotype of pmr1 cells is suppressed using 2 mM MnCl₂ (Fig. 4) and glycosylation is considerably restored (Fig. 7B), but the cells did not grow very well in that concentration of MnCl₂ (Fig. 6). Aberrant intracellular distribution of Mn²⁺ caused by pmr1 deletion might be the reason for this apparent discrepancy.

It has been described that defects in glycosylation can affects S. pombe morphology (de Mora et al. 1990; Ribas et al. 1991). It is possible that pmr1 defects in glycosylation might causes alteration on the cell wall glycoproteins, and as consequence, a defective cell wall alters the morphology. However, osmotic stabilization of the cells (1.2 M sorbitol) failed to correct the morphological alterations, suggesting that cell wall weakness alone cannot explain the aberrant morphology (data not shown).

The supersensitivity of pmr1 cells to a high concentration of Mn²⁺ revealed in the present study (Fig. 6A,D) suggested that Pmr1 localizes to the Golgi membrane, transport Mn²⁺ into the Golgi apparatus and removes excess Mn²⁺ by delivery into the secretory pathway similarly to its homologue in S. cerevisiae (Lapinskas et al. 1995).

S. cerevisiae expresses three homologues of the Nramp family of proton-driven metal transporters namely Smf1, Smf2 and Smf3 (Portnoy et al. 2000). Smf1 and Smf2 function in cellular accumulation of Mn²⁺, wherein Smf1 localizes at the cell surface while Smf2 is restricted to intracellular vesicles (Portnoy et al. 2000). Judging from the information on the Sanger Center S. pombe database (http://www.sanger.ac.uk), pdt1⁺ seems to be the only gene that encodes the member of the Nramp family in the S. pombe genome. It was previously reported that GFP-Pdt1 is localized to the cell surface (Tabuchi et al. 1999). In this study, knockout of pdt1⁺ gene caused an increased resistance to high concentration of MnCl₂ (Fig. 6D), suggesting its function in the cellular accumulation of Mn²⁺ as in S. cerevisiae. Taken together, it is suggested that Pdt1 is localized to the cell membrane and Pdt1 takes up Mn²⁺ from the environment into the cytoplasm.

A specific calcineurin inhibitor FK506 completely blocked the growth of the double mutant cell, and abolished the growth stimulatory effect of Ca²⁺ (Fig. 6B). The addition of Ca²⁺ stimulated cellular calcineurin activity in S. pombe (Hirayama et al. 2003) (Fig. 1) as well as in S. cerevisiae (Stathopoulos-Gerontides et al. 1999; Matheos et al. 1997). This suggests that the growth of the double mutant cell is dependent on calcineurin activity. Consistently, expression of the activated mutant of calcineurin stimulated the growth of pmr1pdt1 cells (Fig. 6C). Interestingly, the addition of Mn²⁺ antagonized the inhibitory effect of FK506 on the double mutant cells (Fig. 6B). Taken together, these results suggest the presence of unknown calcineurin-regulated Mn²⁺ uptake machinery that is different from Pmr1 or Pdt1.

	Experimental procedures

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Strains, media and other related procedures

S. pombe strains used in this study are listed in Table 1. The complete medium, yeast extract, peptone and dextrose (YPD), the minimal medium, Edinburgh minimal medium (EMM), and the phosphate-free EMM were prepared as previously described (Toda et al. 1996). FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). In some experiments, MnSO₄ was removed from the recipe for the EMM medium to prepare the Mn²⁺-deprived EMM medium.

Database searches were performed using the National Center for Biotechnology Information BLAST network service (http://www.ncbi.nlm.nih.gov) and the Sanger Center S. pombe database search service (http://www.sanger.ac.uk).

Cloning of the pmr1⁺ gene

The pmr1⁺ gene was amplified by polymerase chain reaction (PCR) with the genomic DNA of S. pombe as a template. The sense primer used for PCR was 5¢-AA CTGCAGATG AGT GTT CAA TAT GAT GCA TTC AGT G-3¢ (PstI site and start codon underlined), and the anti-sense primer used was 5¢-AA CTGCAGTTA TAC ATT CCT TAG CAG ATA ATT GC-3¢ (PstI site and stop codon underlined). The amplified product was digested with PstI, and the resulting fragment was subcloned into Bluescript SK(+).

Knockout of pmr1⁺ and pdt1⁺ genes

A one-step gene disruption by homologous recombination (Rothstein 1983) was performed. The pmr1::ura4⁺ disruption was constructed as follows. Cloned open reading frame of the pmr1⁺ gene in the Bluescript vector was digested with EcoRI and HindIII, and the resulting fragment containing approximately 70% of the pmr1⁺ coding region was subcloned into the EcoRI/HindIII site of pUC119. Then, a BamHI fragment containing the ura4⁺ gene was inserted into the BamHI site of the previous construct. The fragment containing the disrupted pmr1⁺ gene was transformed into diploid cells. Stable integrants were selected on the medium lacking uracil, and disruption of the gene was checked by genomic Southern hybridization (data not shown). The pdt1::ura4⁺ and pdt1::LEU2 disruptions were constructed similarly by inserting each auxotrophic marker to the BamHI site in the coding region of the pdt1⁺ gene. The pmr1 pdt1 double knockout mutant cells were generated by the genetic cross between pmr1::ura4⁺ and pdt1::ura4⁺ or pdt1::LEU2.

Northern blot analysis

Total RNA was isolated by the method of Kohrer & Domdey (1991). 20 mg of total RNA/lane was subjected to electrophoresis on denaturing formaldehyde 1% agarose gels and transferred to nylon membranes. Hybridization was performed using digoxigenin (DIG)-labelled anti-sense cRNA probes coding for Pmr1 or Pmc1 as previously described (Hirayama et al. 2003). The DIG-labelled hybrids were detected by an enzyme-linked immunoassay using an anti-DIG-alkaline-phosphatase antibody conjugate. The hybrids were visualized by chemiluminescence detection on a light-sensitive film in accordance with the manufacturer's instructions (Roche). Hybridization signals were quantified by using Kodak digital science 1D software (Eastman Kodak Company, New Heaven, CT). Levels of mRNA were normalized to the level of Leu1 mRNA to control for loading error.

Immunoblot analysis of the S. pombe Cpy1 protein

Immunoblot analysis of the Cpy1 was performed by replica-plating freshly grown spots on to nitrocellulose for overnight growth (Black & Pelham 2000). Antibody incubations were carried out using rabbit polyclonal antibody against S. pombe Cpy1 (Tabuchi et al. 1997). Dvps34 (Takegawa et al. 1995) and Dcpy1 (Tabuchi et al. 1997) cells were served as positive and negative controls, respectively.

Acid phosphatase staining

Acid phosphatase from fission yeast was analysed as described by Huang & Snider (1995), with some modifications. The cells were grown in 20 mL EMM medium to mid-log phase at 30 C. To induce the production of acid phosphatase, the cells were centrifuged, washed, resuspended in 20 mL of phosphate-free EMM, and incubated for 12 h at 30 C. The cells were then collected by centrifugation, washed once with 62.5 mM Tris-HCl (pH 6.8), and suspended in 240 mL ice-cold lysis buffer (62.5 mM Tris-HCl, 1 mM EDTA, 2 mM phenylmethylsulphonyl fluoride, 0.1 mM dithiothreitol and 10% glycerol, pH 6.8). Cell lysates were prepared with 0.5-mm glass beads using a Mini BeadBeater (BioSpec Products). The lysates were recovered and centrifuged at 15 000 g for 10 min, and the supernatant were recovered and were mixed with one-third volume of 0.1% bromophenolblue, 15% glycerol and 62.5 mM Tris-HCl (pH 6.8). Samples (10 mg protein) were subjected on 6% native polyacrylamide gel electrophoresis. Electrophoresis and staining of the acid phosphatase activity were performed by the method of Schweingruber et al. (1986).

Microscopic analysis

Cells were grown to exponential phase in the EMM medium and shifted to various conditions as indicated in the figure. In some cases cells were fixed with 3% formaldehyde, washed with phosphate buffered saline (pH 7.0), and stained with Calcofluor to visualize the septum.

Cells were then examined by DIC and fluorescent microscopy using an Axioskop microscope (Carl Zeiss Inc.). Photographs were taken with a SPOT2 digital camera (Diagnostic Instruments Inc.). Images were processed with the CorelDraw software (Corel Corporation Inc.).

	Acknowledgements

 
We thank Susie O. Sio for critical reading of the manuscript. This work was supported by 21st Century COE Program and research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	Footnotes

 
Communicated by: Takashi Toda

^* Correspondence: E-mail: tkuno{at}med.kobe-u.ac.jp

	References

Top Abstract Introduction Results Discussion Experimental procedures References

 
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Received: 15 July 2003
Accepted: 5 November 2003

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