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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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).
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
(for example,
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.).
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RESULTS |
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.
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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
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
I ), 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.
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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.
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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.
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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
its8 strain showed extremely slow cellular growth under all conditions tested (Fig. 5B).
its8 strain could not grow in the presence of FK506 (Fig.
5B) and the growth of the
its8 strain was
further inhibited by temperature upshift (data not shown). Microscopic
observation revealed that most of the
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 its8. Wild-type
(WT) and 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
its8. Wild type (WT) and 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.
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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
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
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.
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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
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.
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As calcineurin regulates 1,3-
-D-glucan synthesis and
FK506 treatment inhibits the glucan synthesis in budding yeast (34, 35), we hypothesized that FK506 inhibits 1,3-
-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-
-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-
-D-glucan synthase mutant, cps1 (Ref.
37; data not shown). These observations are consistent with a close relationship between GPI anchoring and 1,3-
-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 (
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
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
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
-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
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
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 pmk1 strains
by -glucanase. Cells exponentially growing in EMM medium were
harvested and incubated with -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%).
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DISCUSSION |
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
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-
-D-Glucan is a major structural polymer of yeast
cell wall and is synthesized from UDP-glucose by the multisubunit
enzyme 1,3-
-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-
-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-
-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,
pmk1 mutant, which showed a severe
defect in cell wall integrity, did not show FK506 sensitivity, and the
pmk1
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.