Its8, a Fission Yeast Homolog of Mcd4 and Pig-n, Is Involved in GPI Anchor Synthesis and Shares an Essential Function with Calcineurin in Cytokinesis*

Tomoko Yada, Reiko Sugiura, Ayako Kita, Yuumi Itoh, Yabin Lu, Yeongjin HongDagger , Taroh KinoshitaDagger , Hisato Shuntoh§, and Takayoshi Kuno

From the Department of Pharmacology, Kobe University School of Medicine, Kobe 650-0017, the Dagger  Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, and the § Faculty of Health Science, Kobe University School of Medicine, Kobe 654-0142, Japan

Received for publication, October 11, 2000, and in revised form, December 27, 2000




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In fission yeast, calcineurin is required for cytokinesis and ion homeostasis; however, most of its physiological roles remain obscure. To identify genes that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and high temperature and isolated the mutant its8-1. its8+ encodes a homolog of the budding yeast MCD4 and human Pig-n that are involved in glycosylphosphatidylinositol (GPI) anchor synthesis. Consistently, reduced inositol labeling of proteins suggested impaired GPI anchor synthesis in its8-1 mutants. The temperature upshift induced a further decrease in inositol labeling and caused dramatic increases in the frequency of septation in its8-1 mutants. BE49385A, an inhibitor of MCD4 and Pig-n, also increased the septation index of the wild-type cell. Osmotic stabilization suppressed these morphological defects, indicating that cell wall weakness caused by impaired GPI anchor synthesis resulted in abnormal cytokinesis. Furthermore, calcineurin-deleted cells exhibited hypersensitivity to BE49385A, and FK506 exacerbated the cytokinesis defects of the its8-1 mutant. Thus, calcineurin and Its8 may share an essential function in cytokinesis and cell viability through the regulation of cell wall integrity.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In eukaryotic cells, various proteins are attached to the external leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI)1 anchor (1-4). Cells synthesize the GPI anchor carbohydrate core in the ER by successively adding N-acetylglucosamine, three mannoses, and EtNP onto phosphatidylinositol, thus forming the complete GPI precursor lipid, which is then added to proteins. This core is conserved in all eukaryotes but is modified by various side chains in different organisms. In budding yeast, Saccharomyces cerevisiae (5, 6), and mammalian (7) but not protozoan cells (1), Man1 is modified by EtNP. It has been suggested that Pig-n and Mcd4 are involved in this modification in mammalian and budding yeast cells, respectively (8, 9). BE49385A (also named YW3584), a terpenoid lactone compound, inhibits the modification of Man1 with EtNP (5, 10), and it has been suggested that Pig-n and Mcd4 are targets of this drug (8). MCD4 is essential for growth of S. cerevisiae, and its temperature-sensitive mutant does not generate functional GPI at restrictive temperatures (9). On the other hand, modification of the Man1 by EtNP, mediated by Pig-n, is not essential for attachment of GPI anchors in mammalian cells (8). Thus, BE49385A may be useful for selectively inhibiting the generation of GPI-anchored protein in yeast and other fungi.

Calcineurin, a Ca2+- and calmodulin-dependent protein phosphatase, is a molecular target for specific immunosuppressive drugs such as CsA or FK506 used in organ transplantation (11) and is conserved from yeast to man (12-14). We have studied calcineurin signal transduction pathway in the fission yeast Schizosaccharomyces pombe because this system is amenable to genetic studies and has advantages such as relevance to higher systems. In addition, S. pombe is an excellent model organism in which to study cell division, because it shows the general features of higher eukaryotic cell division. S. pombe has a single gene encoding the catalytic subunit of calcineurin, ppb1+, which is essential for cytokinesis (14, 15). We have previously shown that ppb1+ plays an essential role in maintaining chloride ion homeostasis and acts antagonistically with the Pmk1 MAPK pathway (16-18). To identify gene products sharing an essential function with calcineurin, we have developed a genetic screen using the immunosuppressive drug FK506 for mutants that depend on calcineurin for growth and have identified eight complementation groups (its1-8 for immunosuppressant and temperature-sensitive, to be described elsewhere in detail).

Here we report that its8+ encodes a fission yeast homolog of Mcd4 and Pig-n. Analyses of an immunosuppressant- and temperature-sensitive mutant its8-1, and pharmacological experiments using BE49385A and FK506 revealed that Its8, a GPI anchor-synthesizing protein, and calcineurin appear to share an essential function in cytokinesis and cell viability through the regulation of cell wall integrity.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains, Media, Genetic and Molecular Biology Techniques, and Nomenclature-- Fission yeast strains used in this study are listed in Table I. The complete medium, YPD, and minimal medium, EMM, have been described previously (18). Adenine sulfate (20 µg/ml) was added to YPD when adenine auxotrophs were cultured. FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan), and BE49385A was provided by Banyu Pharmaceutical Co. (Tokyo, Japan). Standard methods for S. pombe genetics were followed according to Moreno et al. (19).


                              
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Table I
Fission yeast strains used in this study

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, its8::ura4+). Also, gene disruptions are abbreviated by the gene preceded by Delta  (for example, Delta its8). Proteins are denoted by roman letters and only the first letter is capitalized (for example, Its8).

Isolation of its8-1 Mutants-- The its8-1 mutant was isolated in a screen of cells that had been mutagenized with nitrosoguanidine. Cells of strain HM123 were mutagenized with 300 µM nitrosoguanidine (Sigma) for 60 min (~10% survival) as described by Moreno et al. (19). Mutants were spread onto YPD plates to give ~1,000 cells/plate and were incubated at 27 °C for 4 days. The plates were then replica-plated to 36 °C and to plates containing 0.5 µg/ml FK506. Mutants that showed both temperature sensitivity and FK506 sensitivity were selected. The original mutants isolated were backcrossed three times to wild-type strain HM123 and HM528.

Cloning and Tagging of the its8+ Gene-- To clone the its8+ gene, the its8-1 mutant (KP533) was grown at 27 °C and transformed with an S. pombe genomic DNA library constructed in the vector pDB248 (19). Leu+ transformants were replica-plated to 36 °C, and plasmid DNA was recovered from 14 transformants that showed plasmid-dependent rescue. These plasmids had identical or overlapping inserts as judged from restriction digests, and all 14 complemented both the immunosuppressant sensitivity and temperature sensitivity of the its8-1 mutant.

Its8 was tagged at its C terminus with GFP carrying the S65T mutation (20). To express Its8·GFP under the control of a thiamine-repressible promoter in fission yeast, the coding region for the its8+ gene was amplified using polymerase chain reaction, ligated to the N terminus of GFP, and the resultant construct was subcloned into pREP81 vector (21).

Labeling of Proteins with myo-[2-3H]Inositol-- Wild-type or mutant cells were grown in EMM overnight to mid-log phase, washed twice in EMM-inositol medium and at OD660 = 3, midlog-phase cells were resuspended in 15 ml of EMM-inositol and depleted of inositol for 2 h at 27 °C before the addition of 60 µCi of myo-[2-3H]inositol (American Radiolabeled Chemicals, Inc., St. Louis, MO). Cells were divided into subcultures and labeled for 4 h under various conditions as indicated. Radiolabeled cells were washed twice with 10 mM NaN3 and then resuspended in 150 µl of SDS-polyacrylamide gel sample buffer containing 1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 0.1 mM sodium metabisulfite, chymostatin 0.1 µg/ml, aprotinin 2 µg/ml, pepstatin A 1 µg/ml, phosphoramidon 1 µg/ml, leupeptin 0.5 µg/ml, and antipain 2.5 µg/ml. Glass beads (0.2 g) were then added, and cells were broken mechanically by vortexing for 30 s, after which the tubes were placed in a boiling water bath for 1 min. Vortexing and heating were repeated eight times, after which the glass beads and cellular debris were removed by centrifugation. The extracted 3H-labeled proteins were then separated by SDS-PAGE, processed for fluorography with 1 M sodium salicylate, and exposed to film at -80 °C (22).

In Vitro Mannose Labeling of Microsomes-- Microsomes were prepared from OD660 = 9 of midlog-phase wild-type or mutant cells grown in YPD. Cells were resuspended in 450 µl of ice-cold homogenizing buffer (30 mM Tris-HCl, pH 7.8, 2 mM EDTA, 20% (v/v) glycerol, and 1 mM dithiothreitol containing a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 0.1 mM sodium metabisulfite, chymostatin 0.1 µg/ml, aprotinin 2 µg/ml, pepstatin A 1 µg/ml, phosphoramidon 1 µg/ml, and leupeptin 0.5 µg/ml). Glass beads (0.2 g) were then added, and cells were broken mechanically by vortexing for 30 s, after which the tubes were placed on ice for 30 s. Vortexing and cooling were repeated five times, after which the glass beads and cellular debris were removed by centrifugation at 1,500 × g for 5 min. The supernatant was spun at 100,000 × g for 1 h, and the pellet (microsomes) was suspended in 50 µl of buffer A (50 mM HEPES/NaOH, pH7.4, 25 mM KCl, 0.1 mM 1-chloro-3-tosylamido-7-amino-2-heptanone and 1 µg/ml leupeptin) and stored at -80 °C until use. The microsomes (50 µl) were mixed with 50 µl of buffer B (100 mM HEPES/NaOH, pH7.4, 50 mM KCl, 15 mM MgCl2, 15 mM MnCl2, 3 mM AMP, 30 µM palmitoyl CoA, 3 µg/ml tunicamycin, 0.2 mM 1-chloro-3-tosylamido-7-amino-2-heptanone, and 2 µg/ml leupeptin) and 50 µl (2 µCi) of GDP-[3H]mannose (Amersham Pharmacia Biotech Radiolabeled Chemicals, St. Louis, MO). After the incubation for 1 h at 37 °C, lipids were extracted twice with n-butanol and washed once with water. Mannolipids were separated on Kiesel gel 60 (Merck, Darmstadt, Germany) with a solvent system of chloroform/methanol/water (10:10:3) and detected by exposure to a Fuji BAS1500 image analyzer (Fuji Film Co., Tokyo, Japan) for 3 days. In some cases, the mannolipids were treated with GPI-specific phospholipase D before thin-layer chromatography as described previously (23).

Deletion of the its8+ Gene-- A one-step gene disruption by homologous recombination (24) was performed. The its8::ura4+ disruption was constructed as follows. The HindIII fragment containing the its8+ gene was subcloned into the HindIII site of pGEM-13Zf(+) (Promega). Then, a BamHI-PstI fragment containing the ura4+ gene was inserted into the BamHI-PstI site of the previous construct (Fig. 5A). The HindIII-EcoNI fragment containing the disrupted its8+ gene was transformed into diploid cells (5A/1D). Stable integrants were selected on medium lacking uracil, and disruption of the gene was checked by genomic Southern hybridization (data not shown).

Microscopic Analysis-- For microscopic observation, cells were washed with PBS. In some cases cells were fixed in 1.85% formaldehyde in PBS for 5 min and washed with PBS. Cells were dried on coverslips treated with poly-L-lysine and stained with DAPI and Calcofluor, dissolved in PBS to visualize the DNA or septum, respectively. To label vacuolar membranes, cells were incubated with medium containing 80 µM FM4-64 for 30 min at 27 °C (25). The cells were then centrifuged at 13,000 × g for 1 min, washed by resuspending in new medium to remove free FM4-64, and collected by centrifugation at 13,000 × g for 1 min. Cells were then resuspended in new medium and again incubated for 2-3 h at 27 °C before microscopic observation. The fluorescence in non-fixed or fixed cells was observed using an Axioskop microscope (Carl Zeiss Inc.). Photographs were taken with a SPOT2 digital camera (Diagnostic Instruments Inc.). Images were processed with the CorelDRAW (Corel Corporation Inc.).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of the its8-1 Mutant-- A genetic screen was performed to identify genes that share an essential overlapping function with calcineurin. For this purpose, we have developed a genetic screen using the immunosuppressive drug FK506 for mutants that depend on calcineurin for growth and have identified eight complementation groups (its1-8).

As shown in Fig. 1, the its8-1 mutant grew slightly slower than the wild type at 27 °C, grew very slowly at 33 °C, and grew extremely slowly or failed to grow at 36 °C. It could not grow in the presence of FK506 or CsA on the YPD plates, whereas the wild type could grow well under these conditions (Fig. 1). After longer incubation, small colonies appeared on the plate at 36 °C but not on the plate with FK506 (data not shown).



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Fig. 1.   The immunosuppressant and temperature sensitivity of the its8-1 mutant cells. Wild-type (WT) and its8-1 mutant cells (its8) were streaked onto each plate containing YPD, YPD plus 0.5 µg/ml FK506, or YPD plus 50 µg/ml CsA, and incubated at 27 °C for 4 days, 33 °C for 3 days, or 36 °C for 3 days.

Previous reports showed that calcineurin is an in vivo target of FK506 in fission yeast and inhibition of calcineurin activity by the addition of FK506 to the culture medium resulted in identical phenotypes as those of calcineurin deletion (14, 17). Thus, it could be anticipated that the its8-1 mutant requires functional calcineurin activity for vegetative growth, and tetrad analysis was performed to examine the synthetic lethality with Delta ppb1 mutant. As expected, no double mutant was obtained, indicating that the its8 mutation and calcineurin deletion was synthetically lethal (data not shown). This suggests that calcineurin and Its8 share an essential overlapping function for cell viability.

Isolation of the its8+ Gene and Identification of a Mutation Site-- The its8+ gene was cloned by complementation of the temperature-sensitive growth defect (Fig. 2A). It also complemented the FK506-sensitive growth defect (Fig. 2A). Nucleotide sequencing of the cloned DNA fragment revealed that the its8+ gene was SPBC24E9.08c. A BLAST search of protein sequence databases revealed that its8+ encodes a 935-amino acid homolog of budding yeast Mcd4 (39%) (9) and human Pig-n (34%) (8). It has been suggested that Mcd4 and Pig-n catalyze the addition of EtNP to Man1 of the GPI anchor core (8). A single base change (C to T transition at the 1014th nucleotide from the start codon) was detected in the its8+ gene when the genomic DNA sequence from the its8-1 mutant was analyzed. This mutation resulted in replacement of a proline with a serine residue at position 288. This amino acid residue is conserved among Its8, Mcd4, and Pig-n proteins, but not among phosphodiesterases or nucleotide pyrophosphatases that share three highly conserved motifs (Ref. 9; Fig. 2B). The predicted topology of Mcd4 based on sequence analysis suggested that proline at 291 (corresponds to Pro-288 of Its8) is located in the large, hydrophilic N-terminal domain extending into the lumen of the ER, which is highly conserved in Its8, Mcd4, and Pig-n (9).



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Fig. 2.   Cloning of the its8+ gene and characterization of the its8-1 mutant. A, cloning of the its8+ gene. Wild-type (WT) and its8-1 mutant cells transformed with a control vector (pDB248) or the vector containing the its8+ gene were streaked onto each plate containing YPD and incubated for 3 days at 36 °C or 4 days at 27 °C in the presence of 0.5 µg/ml FK506. B, comparison of the sequences around the mutation site of the its8-1 mutant and other related enzymes. Amino acid sequences of Its8, Mcd4, Pig-n, YLL031c, YJL062w, NucPdeAse (Oryza sativa nucleotide phosphodiesterase), HsPdAse (human phosphodiesterase Ialpha ), and PC-1 (mouse nucleotide phosphodiesterase) are shown. Asterisk points to proline 288, which is mutated to serine in S. pombe 1ts8-1. Motif 3 is one of the three conserved motifs found in phosphodiesterases and pyrophosphatases (9). Identical amino acids are marked by filled boxes with white letters. Alignments were performed using ClustalW (42). C, incorporation of inositol into proteins was defective in its8-1 mutant. Wild-type (lanes 1-3) and its8-1 mutant (lanes 4-6) cells were labeled with myo-[2-3H]inositol at 27 °C (lanes 1, 3, 4, 6) or at 36 °C (lanes 2, 5) for 4 h. Cells were labeled in the presence (lanes 3, 6) or absence (lanes 1, 2, 4, 5) of 0.5 µg/ml FK506. Total protein extracts from OD660 = 1 of mid-log phase cells were resolved by SDS-PAGE. Positions of molecular mass markers are shown to the left.

Incorporation of Inositol into Proteins Was Defective in the its8-1 Mutant-- To confirm that Its8 is a functional homolog of Mcd4 and Pig-n and to study the role of calcineurin for GPI anchor synthesis, we examined inositol incorporation into proteins in wild-type cells and its8-1 mutants. This is because the GPI anchor contains an inositol moiety (26, 27), and GPI-anchored proteins are the only proteins known to be covalently attached to inositol (28-33). Cells were incubated at 27 °C in the absence of inositol for 2 h and labeled with myo-[2-3H]inositol at 27 °C or at 36 °C for 4 h. To examine the effects of calcineurin inhibition, FK506 was added to the labeling medium (0.5 µg/ml) and incubated at 27 °C for 4 h. Labeled samples were then processed for SDS-PAGE.

its8-1 mutant cells showed a decreased inositol labeling of proteins, and this was further reduced upon temperature upshift (Fig. 2C). On the other hand, temperature upshift had no effect on the inositol labeling of the wild-type cells, and FK506 treatment had no effect on the inositol labeling of both its8-1 mutant and wild-type cells (Fig. 2C). These results are consistent with the idea that the P288S mutation does not completely abolish the enzyme activity of Its8, but causes a severe loss of function especially at the restrictive temperature.

Impaired GPI Biosynthesis in its8-1 Mutant Cells-- We examined in vitro mannose labeling of microsomes to see whether Its8 is involved in GPI biosynthesis in S. pombe. Multiple mannolipids were generated by incubation of microsomes from either wild-type or its8-1 mutant with GDP-[3H]mannose. Some of them were not GPI intermediates, because they were resistant to GPI-specific phospholipase D. Membranes of its8-1 mutant generated one major mannolipid that was not seen in the wild-type (Fig. 3, arrow). This lipid appeared to be a GPI species because it was sensitive to GPI-specific phospholipase D (Fig. 3, lanes 3 and 4). Although we could not pinpoint a step of GPI biosynthesis in which Its8 was involved, these results suggest that impairment of Its8 function resulted in an accumulation of one GPI species in the mutant.



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Fig. 3.   Requirement of Its8 for GPI synthesis revealed by in vitro mannose labeling of microsomes. Microsomes from either wild-type or its8-1 cells were labeled with GDP-[3H]mannose. After the incubation, lipids were extracted and treated with GPI-specific phospholipase D (GPI-PLD) or reaction buffer. Arrow indicates a major mannolipid in its8-1 mutant. DPM, dolichol-phosphate-mannose.

Localization of Its8-GFP-- To investigate the intracellular distribution of Its8, Its8·GFP protein was expressed from a multicopy vector containing an attenuated version of the thiamine-repressible nmt1 promoter. Its8·GFP was fully functional as demonstrated by its complementation of its8-1 mutant phenotypes (data not shown). Its8-GFP expressed in fission yeast exhibited a light double ring-like signal surrounding both the nucleus (defined by DAPI) and the cell surface (Fig. 4). Most of the cells exhibited the double ring-like signal, and a few cells exhibited signals in the vacuolar membrane (defined by FM4-64). This pattern demonstrated the localization of Its8 protein in the ER and is consistent with the previous reports showing ER localization of Mcd4 (9) and Pig-n (8).



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Fig. 4.   Subcellular localization of Its8·GFP. Mid-log phase wild-type cells expressing GFP-tagged Its8 (Its8-GFP) were grown in EMM medium and labeled with DAPI (to visualize the DNA) and FM4-64 (to visualize the vacuolar membrane) and examined by differential interference contrast (DIC) and fluorescent microscopy. The bar indicates 10 µm.

Disruption of the its8+ Gene-- To analyze Its8 function, the its8+ gene was knocked out in a diploid by homologous recombination using the ura4+ marker gene (Fig. 5A). Southern blotting of the genomic DNA of one such transformant confirmed that the wild-type gene had been replaced by the derivative containing the ura4+ insertion (data not shown). Tetrad analysis revealed that the its8+ gene was non-essential. However, the Delta its8 strain showed extremely slow cellular growth under all conditions tested (Fig. 5B). Delta its8 strain could not grow in the presence of FK506 (Fig. 5B) and the growth of the Delta its8 strain was further inhibited by temperature upshift (data not shown). Microscopic observation revealed that most of the Delta its8 cells were enormously enlarged and round-shaped (Fig. 5C). Multiseptated cells were also frequently observed. These microscopic findings suggest that Its8 is implicated in the establishment of cytokinesis and cell polarity.



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Fig. 5.   Disruption of the its8+ gene. A, a restriction map of the genomic its8+ locus, and the plasmid construct used for the its8+ gene disruption. The arrow indicates the extent and direction of the its8+ ORF, which encodes 935 amino acids. B, slow growth phenotype of Delta its8. Wild-type (WT) and Delta its8 cells were streaked onto each plate containing YPD and incubated at 27 °C in the presence or absence of 0.5 µg/ml FK506. Photographs were taken at the 4th and 8th day of the incubation. C, morphological phenotypes of Delta its8. Wild type (WT) and Delta its8 cells cultured on YPD plates at 27 °C for 4 days were shown. The cells were stained with DAPI (to visualize the DNA) and Calcofluor (to visualize cell wall and septum) and examined by differential interference contrast (DIC) and fluorescent microscopy. The bar indicates 10 µm.

A Shift to the Restrictive Temperature or Addition of FK506 to the Culture Medium Dramatically Impaired Cytokinesis of its8-1 Mutant Cells-- As described above, a shift to the higher temperature inhibited myo-[2-3H]inositol incorporation into proteins (Fig. 2C), suggesting that various phenotypes of the its8-1 mutant found at the restrictive temperature are due to the lowered synthesis of GPI-anchored protein caused by an impairment of Its8 function. Also, analysis of the Delta its8 strain is difficult because of its extremely slow growth. Then, we examined the phenotypic changes upon temperature upshift and upon FK506 treatment in its8-1 mutant to search the physiological function related to Its8 (Fig. 6). Cells grown to mid-log phase at 27 °C in liquid YPD medium were subjected to a shift to 33 °C or 36 °C, or to the medium containing FK506. Even at the permissive temperature of 27 °C, 20-25% of the its8-1 mutant cells had a division septum, compared with the 6-12% seen in a wild-type population (Fig. 6, A and C). Upon temperature upshift to 33 °C, the frequency of septated cells significantly increased (up to 65%), and cells with two or more septa were frequent (Fig. 6A). These results suggest that an impairment of Its8 function and reduced GPI anchor synthesis caused defects in cytokinesis in its8-1. Upon temperature upshift to 36 °C, enormously enlarged and round-shaped cells similar to Delta its8 cells were observed in addition to septated cells (data not shown).



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Fig. 6.   Defective cytokinesis in the its8-1 mutant. A, wild-type (WT) and its8-1 cells grown to mid-log phase at 27 °C in liquid YPD medium were subjected to a shift to 33 °C for 8 h and stained with DAPI (to visualize DNA) and Calcofluor (to visualize cell wall and septum). The bar indicates 10 µm. B, cells grown to mid-log phase at 27 °C in liquid YPD medium were shifted to the medium containing 0.5 µg/ml FK506 and stained with DAPI and Calcofluor. The bar indicates 10 µm. C and D, its8-1 mutant showed a dramatic increase in the septation index upon shift to the restrictive temperature (C) or by addition of FK506 (D). Overnight cultures of wild-type cells (open circle), or its8-1 mutant cells (filled circle), were diluted with fresh YPD or YPD supplemented with 0.5 µg/ml FK506, and incubated at indicated temperatures. Percentage of septated cells was measured hourly with Calcofluor staining.

To better understand the relationship between Its8 function and calcineurin signaling, we examined the effect of FK506 on the phenotypes of the its8-1 mutant. Strikingly, nearly 80% of its8-1 mutant cells were septated 8 h after the addition of FK506 at 27 °C, whereas 30% of wild-type culture displayed septated cells (Fig. 6, B and D). The frequency of multiple-septated cells in the its8-1 mutant was unusually high, considering that the cells with multiple septa were rarely seen in wild-type cells under the same culture condition (Fig. 6B). In addition, branched cells were also very frequent in the its8-1 mutant (Fig. 6B). These results strongly suggest that loss of calcineurin activity accentuated the defective cytokinesis and cell polarity control of its8-1 mutant cells and that Its8 and calcineurin play an overlapping function in cytokinesis and cell polarity control.

Its8 Is a Molecular Target of BE49385A and the Calcineurin-deletion Mutant Is Hypersensitive to BE49385A-- It has been shown that Mcd4 and Pig-n are molecular targets of BE49385A (8). To study whether Its8 is also a molecular target of BE49385A, we examined the effect of its8+ overexpression on the sensitivity to BE49385A in wild-type cells. The full-length its8+ sequence was cloned in the multicopy vector pDB248 (19). Cells transformed with pDB248-its8+ could grow well on plates containing 2 µg/ml BE49385A, whereas cells transformed with the empty vector grew very slowly on this plate. The wild-type cells exhibited impaired growth on plates containing 0.2 µg/ml BE49385A (Fig. 7A). These results suggest that Its8 is also a target of BE49385A. The above results are very similar to those of Mcd4 overexpression in S. cerevisiae (8). Consistently, BE49385A inhibited inositol incorporation into proteins in fission yeast cells (Fig. 7C). The drug concentration required to inhibit the cell growth of fission yeast is much lower than that of budding yeast; however, very high concentrations of BE49385A could not completely inhibit the growth of fission yeast and inositol incorporation (Fig. 7, A and C). This is in good agreement with the result that its8+ gene disruption was not lethal but resulted in an extremely slow growth. Addition of low or medium level concentrations of BE49385A to the wild-type cell culture dramatically increased the septation index (Fig. 7, D and E). Higher concentrations of BE49385A resulted in enlarged and round cell morphology similar to Delta its8 (data not shown). Again, these results indicate that impairment of Its8 function causes defects in cytokinesis and cell polarity.



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Fig. 7.   BE49385A specifically inhibited Its8 function and caused cytokinesis defects in the wild-type cells. A, Its8 is a molecular target of BE49385A. Serial 10-fold dilutions starting with OD660 = 0.3 of log-phase cells carrying a multicopy vector containing its8+ or an empty vector were spotted (5 µl) on solid medium with or without added drugs as indicated. Plates were incubated at 27 °C and photographed at the 4th day. B, calcineurin deletion and its8-1 mutants are hypersensitive to BE49385A. Serial 10-fold dilutions starting with OD660 = 0.3 of log-phase wild-type or mutant cells were spotted as described above. C, incorporation of inositol into proteins was inhibited by BE49385A. Wild-type cells were labeled with myo-[2-3H]inositol in the presence (lane 2) or absence (lane 1) of 0.5 µg/ml BE49385A at 27 °C for 4 h. Samples were analyzed as described in Fig. 2. D, morphological defects caused by BE49385A. Wild-type cells grown to mid-log phase at 27 °C in liquid YPD medium were shifted to the medium containing 0.2 µg/ml BE49385A and stained with DAPI and Calcofluor 8 h after the medium change. The bar indicates 10 µm. E and F, BE49385A caused a dramatic increase in the septation index in the wild-type cells (E) and in the calcineurin-deletion mutants (F). Overnight cultures of wild-type cells (E) or calcineurin-deletion mutant cells (F) were diluted with fresh YPD (open circle) or YPD supplemented with BE49385A (filled circle, 0.2 µg/ml (E) or 50 ng/ml (F)), and incubated at 27 °C. Percentage of septated cells was measured hourly with Calcofluor staining.

As calcineurin regulates 1,3-beta -D-glucan synthesis and FK506 treatment inhibits the glucan synthesis in budding yeast (34, 35), we hypothesized that FK506 inhibits 1,3-beta -D-glucan synthesis thereby causing severe defects in cell wall integrity in its8-1 mutant cells in which GPI anchor synthesis is impaired. To test this hypothesis, we examined the effect of aculeacin A, an inhibitor of 1,3-beta -D-glucan synthase (36), on its8-1 mutant. As expected, its8-1 mutant cells were sensitive to aculeacin A (data not shown). Consistently, its8-1 mutant cells also showed synthetic growth defect with a 1,3-beta -D-glucan synthase mutant, cps1 (Ref. 37; data not shown). These observations are consistent with a close relationship between GPI anchoring and 1,3-beta -D-glucan synthesis in preserving cell wall integrity and function.

Notably, very low concentrations (50 ng/ml) of BE49385A could completely inhibit the growth of the calcineurin-deletion mutant (Delta ppb1) (Fig. 7B), further strengthening the notion that GPI anchoring and calcineurin-mediated signaling are sharing an essential overlapping function in cytokinesis and cell viability. Likewise, treatment of wild-type cells with FK506 or aculeacin A caused hypersensitivity to BE49385A (data not shown). BE49385A could further increase the septation index of Delta ppb1, suggesting that Its8 and calcineurin do not function in a single process of cytokinesis (Fig. 7F).

its8-1 mutant was hypersensitive to BE49385A, although not as sensitive as Delta ppb1 (Fig. 7B) and is consistent with the idea that its8-1 mutation does not completely abolish the enzyme activity of Its8 but causes a considerable loss of its function.

Defective Cell Wall Integrity of its8-1 Mutant Causes Abnormal Cytokinesis-- Addition of 1.2 M sorbitol to the growth medium as an osmotic stabilizer suppressed the temperature- and FK506-sensitive phenotypes of the its8-1 mutant cells (Fig. 8A). Microscopic observation revealed that morphological defects caused by high temperature and FK506 were also osmo-remedial (Fig. 8B). These results suggest that the its8-1 mutant has defects in cell wall integrity (18). To confirm this hypothesis, we performed beta -glucanase treatment (18) of the its8-1 mutant and compared with wild-type cells. its8-1 mutant cells lysed much faster than wild-type cells (Fig. 8C). The sensitivity of the its8-1 mutant is as severe as that of the Delta pmk1 mutant (Fig. 8C), which lacks a MAPK-regulating cell wall integrity of fission yeast (18). These data suggest that the its8-1 mutant cells were defective in cell wall synthesis and that the morphological defective phenotypes of its8-1 mutants were, at least in part, attributable to defects in cell wall integrity. Consistently, the osmotic stabilizer suppressed the morphological defects caused by low or medium-level concentration of BE49385A, and the osmotic stabilizer only partially improved the slow growth or morphological defects of Delta its8 (data not shown).



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Fig. 8.   Immunosuppressant- and temperature-sensitive phenotypes of its8-1 are osmo-remedial, and its8-1 mutant cells are defective in cell wall integrity. A, rescue of the its8-1 mutants with high osmolarity. Wild-type (WT) and its8-1 mutant cells were streaked onto a rich YPD plate containing 1.2 M sorbitol with or without 0.5 µg/ml FK506, and incubated at 27 °C for days or 36 °C for 3 days. B, morphological defects caused by high temperature and FK506 were osmo-remedial. its8-1 cells grown to mid-log phase at 27 °C in liquid YPD medium were shifted to restrictive temperature (33 °C) for 8 h or added with FK506 for 8 h in the presence of 1.2 M sorbitol and stained with DAPI and Calcofluor. Bar, 10 µm. C, cell wall digestion of wild-type, its8-1 mutant, and Delta pmk1 strains by beta -glucanase. Cells exponentially growing in EMM medium were harvested and incubated with beta -glucanase (Zymolyase) at 30 °C with vigorous shaking. Cell lysis was monitored by measuring optical density at 660 nm (the value before adding the enzyme was taken as 100%).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In an attempt to identify genes that may have overlapping function with calcineurin, we isolated a fission yeast gene its8+. Three lines of evidence suggest that Its8 is involved in GPI anchor synthesis in fission yeast. First, its8+ encodes a protein with high sequence similarity to the budding yeast Mcd4 and mammalian Pig-n that catalyze the addition of EtNP to Man1 of the GPI anchor core (8). Second, overexpression of Its8 conferred resistance to BE49385A, a specific inhibitor of Mcd4 and Pig-n. Third, incorporation of inositol into proteins, as an indicator of GPI anchor synthesis (9), was defective in its8-1 mutant.

In the present study, we showed that impairment of Its8 function resulted in failure of cytokinesis and cell polarity control through defective cell wall integrity, which will eventually block cell proliferation. Also, inhibition of calcineurin activity further exacerbates these phenotypes.

Synthetic Lethal Interaction between Its8 and Calcineurin-- The its8-1 mutant carries a single base pair mutation in the its8+ gene that leads to an impairment of GPI anchor synthesis as shown by decreased inositol labeling. Here, we have shown that Delta ppb1 its8-1 double mutant is inviable. These findings of strong genetic interactions between Its8 mutation and Ppb1 calcineurin provide in vivo support for a functional interaction between GPI anchor and calcineurin.

1,3-beta -D-Glucan is a major structural polymer of yeast cell wall and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta -D-glucan synthase. In budding yeast, two catalytic subunits of the glucan synthase, Fks1 and Fks2, have been reported (34, 35). Simultaneous disruption of FKS1 and FKS2 is lethal, suggesting that Fks1 and Fks2 are alternative subunits with essential overlapping function. Analysis of FKS2 expression revealed that FKS2 is induced by the addition of Ca2+ to the growth medium, and this induction is completely dependent on calcineurin, suggesting calcineurin-dependent transcription of FKS2. Consistently, disruption of FKS1 resulted in the immunosuppressant-sensitive phenotype (34). In fission yeast, two genes encoding catalytic subunits of 1,3-beta -D-glucan synthase, cps1+ and gls2+, have been described (37, 38). It has been reported that the cps1-12 mutation confers hypersensitivity to CsA (37), suggesting a similar calcineurin-mediated regulatory mechanism. Thus, we hypothesized that calcineurin deletion or FK506 inhibits 1,3-beta -D-glucan synthesis in fission yeast, thereby causing cell wall weakness. In budding yeast, GPI-anchored proteins play an important role in cell wall biogenesis, for cell wall glycoproteins can be transferred into the cell wall upon cleavage of the GPI glycan and formation of a cross-link to cell wall glucans (39). Although it is not known whether this mechanism for incorporation of glycoproteins into the cell wall is used in fission yeast, our present data showed that the its8-1 mutant has defects in cell wall integrity (Fig. 8C), suggesting GPI anchor synthesis is also important for cell wall integrity in fission yeast. We speculate that loss of calcineurin activity further exacerbates the defect in cell wall integrity as described above, which in turn results in synthetic lethality. However, Delta pmk1 mutant, which showed a severe defect in cell wall integrity, did not show FK506 sensitivity, and the Delta pmk1Delta ppb1 double mutant grew normally although it showed abnormal morphology (16, 17). Thus, our present study together with these previous data have shown that at least two types of defects occur in the cell wall integrity of fission yeast on the basis of FK506-sensitivity: FK506-sensitive type and FK506-insensitive type. However, the underlying mechanism remains undefined.

Impairment of GPI Anchor Synthesis Caused Defects in Cytokinesis and Control of Cell Polarity through the Regulation of Cell Wall Integrity-- As shown in Fig. 7, inhibition of GPI anchor synthesis caused specific morphological defects similar to the its8-1 mutant including abnormal cytokinesis and cell polarity control. These observations indicate that GPI anchor proteins are implicated for efficient cytokinesis and cell polarity control. The most notable observation was that addition of FK506 to the culture medium further exacerbated these morphological defects. This result suggests that calcineurin and GPI-anchored proteins act synergistically in cytokinesis and cell polarity control.

Interestingly, Mcd4, a budding yeast homolog of Its8, was isolated in a screen to isolate mutants defective for bud emergence and polarized growth independent of the requirement for the actin cytoskeleton in those processes (9, 40, 41). In addition, many GPI anchoring-specific budding yeast mutants also exhibit similar bud emergence defects as the mcd4 mutant. Gaynor et al. (9) suggested the cell wall as a likely common denominator linking the two processes of bud emergence and GPI anchoring, because cell wall remodeling and integrity are both required in order for bud emergence to proceed normally. In the present study, we clearly show that morphological phenotypes such as abnormal cytokinesis and polarity in the its8-1 mutant were osmo-remedial, indicating that these phenotypes could be attributable to defective cell wall integrity.


    ACKNOWLEDGEMENT

We thank Susie O. Sio for critical reading of the manuscript.


    FOOTNOTES

* This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan, and a grant from the Novartis foundation (Japan).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: 81-78-382-5441; Fax: 81-78-382-5459; E-mail: tkuno@kobe-u.ac.jp.

Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M009260200


    ABBREVIATIONS

The abbreviations used are: GPI, glycosylphosphatidylinositol; ER, endoplasmic reticulum; EtNP, phosphoethanolamine; Man1, first mannose; CsA, Cyclosporin A; MAPK, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein; PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; CsA, cyclosporin A.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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