1 Instituto de Microbiología Bioquímica, Departamento de
Microbiología y Genética, CSIC/ Universidad de Salamanca, Campus
Miguel de Unamuno, 37007 Salamanca, Spain
2 Department of Genetics, University of Debrecen, PO Box 56, 4010 Debrecen,
Hungary
* Author for correspondence (e-mail: cvazquez{at}usal.es)
Accepted 21 January 2003
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Summary |
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Key words: Cell separation, ß-1, 3-glucanase, Ace2p, Cytokinesis, Primary septum hydrolysis
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Introduction |
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Schizosaccharomyces pombe cells are cylindrical, growing by
elongation of their ends and dividing by medial septation followed by cleavage
of the septum, a process known as binary fission (for a review, see
Johnson et al., 1982).
Cytokinesis and cell division are brought about by the action of the
actomyosin ring, whose constriction is perfectly coordinated with the
synthesis of the primary septum. Genetic studies have identified many genes
that are important in the different steps of cytokinesis, such as for the
positioning and assembly of the actomyosin ring, the localization of actin
patches at the site of cell division, and for the physical assembly of the
division septum (Balasubramanian et al.,
1998
; Balasubramanian et al.,
2000
; Chang et al.,
1996
; Gould and Simanis,
1997
; Le Goff et al.,
1999a
; McCollum and Gould,
2001
; Simanis,
1995
). The mid1+, plo1+
and pom1+ genes are required for the division plane to be
established and for correct positioning of the actomyosin ring
(Bähler and Pringle, 1998
;
Bähler et al., 1998a
).
Once the division plane has been established, the medial ring is formed, and
this process is dependent on many genes, including cdc3+,
cdc4+, cdc8+,
rng2+, rng3+ and
myo2+ (reviewed by
Balasubramanian et al., 2000
;
Guertin et al., 2002
;
Le Goff et al., 1999a
).
F-actin patches are subsequently recruited to the medial ring, where they form
the actomyosin-contractile ring. Coordination of ring contraction and the
nuclear cycle requires a network of regulatory proteins that are collectively
referred to as the septation initiation network (SIN). These proteins also
control the formation of the primary septum during constriction of the
actomyosin ring. Genetic studies have indicated that activation of the SIN
pathway might regulate cps1p, a ß-1,3-glucan synthase subunit essential
for the assembly of the division septum
(Le Goff et al., 1999b
;
Liu et al., 2000
). This septum
has a three-layer structure, with a central primary septum (mainly composed of
linear ß-1,3-glucan) surrounded on both sides by two secondary septa
(composed of ß-1,6-branched ß-1,3-glucan and ß-1,6-glucan)
(Humbel et al., 2001
).
Cell separation requires dissolution of the primary septum for the daughter
cells to become two independent entities. Upon completion of mitosis, the
primary septum undergoes rapid autolytic degradation, accompanied by local
erosion of the adjacent regions of the cell wall. Although the mechanism of
actomyosin ring assembly, constriction and formation of the division septum
have received considerable attention, very little is known about how the
cleavage of the cell wall and primary septum is achieved. To address this
question, mutants showing complete or partial defects in cell separation,
resulting in the formation of chains of cells, have been previously isolated
and classified in 16 different groups, named sep1+ to
sep16+ (Grallert et
al., 1999; Sipiczki et al.,
1993
). sep1+ encodes a transcription factor
highly homologous to the HNF-3/forkhead family present in higher eukaryotic
cells and also in other microorganisms
(Ribár et al., 1997
).
Interestingly, another two members of this family of transcription factors
(the Saccharomyces cerevisiae Fkh1p and Fkh2p proteins) have also
been implicated in cell separation
(Hollenhorst et al., 2000
).
sep15+ has recently been cloned and characterized, and
found to encode an essential protein that shows a high degree of similarity to
Med8p, one of the subunits of the mediator complex of S. cerevisiae
RNA polymerase II (Zilahi et al.,
2000
). In addition, recent work has pointed to the importance of
the exocyst complex in cell separation
(Wang et al., 2002
). The
exocyst is an octameric protein complex present in many organisms and is
involved in tethering vesicles to specific sites on the plasma membrane. Based
on the fact that mutants in different subunits show a defect in cell
separation, it has been proposed that this complex might be involved in the
delivery of hydrolytic proteins that are important for cell cleavage.
Here we demonstrate that cell separation in S. pombe is an enzymatic process that requires the hydrolysis of certain components of the cell wall. The characterization of eng1+, a gene encoding a protein with endo-ß-1,3-glucanase activity that transiently localizes to the septum region in a ring-like structure, indicated that this protein is involved in cell separation. According to observations obtained from transmission electron microscopy, the eng1p glucanase seems to be required for dissolution of the ß-1,3-glucan material that composes the primary septum.
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Materials and Methods |
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Synchronization of strains carrying the thermosensitive cdc25-22 mutation was achieved by growing the cells at the permissive temperature (25°C) to early log phase (OD595=0.5) and then shifting the cultures to 37°C for 4 hours. Cells were released from arrest by transfer to 25°C, and samples were taken every 20 minutes.
Plasmid and DNA manipulations
The oligonucleotides used for different DNA manipulations are shown in
Table 2. Construction of
plasmid pAB10, carrying the eng1+ coding sequence under
the control of the nmt1+ promoter, was achieved by PCR
amplification of the coding sequence of the endo-ß-1,3-glucanase using
oligonucleotides 423 and 302 (which introduced XhoI and
BglII sites at the ends) and cloning of the resulting fragment
between the XhoI and BamHI sites of plasmid pREP3X.
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Construction of null mutants and GFP-tagged strains
The entire coding sequences of eng1+ (SPAC821.09),
ace2+ (SPAC6G10.12c), and SPAC4G8.13c were deleted to
create the null mutants by replacing the coding sequences by the
ura4+ or kanMX4 cassette. The deletion cassettes
were constructed using the recombinant PCR approach described by Wach
(Wach, 1996). For this
purpose, DNA fragments of 300-500 bp corresponding to the 5' and
3' flanking regions of each gene were PCR amplified using specific
oligonucleotide pairs. The resulting fragments were then fused, by recombinant
PCR, to the kanMX4 cassette (which confers resistance to the G418
antibiotic) or to the ura4+ gene. For
eng1::kanMX4, the oligonucleotide pairs 755-359 and 360-756 were used
to amplify, respectively, specific regions of the 5' and 3' ends,
which were subsequently fused to the KanMX4 cassette obtained from
plasmid pFA-KanMX4 (Wach et al.,
1994
). A similar approach was implemented to construct the
ace2::kanMX4 cassette (using oligonucleotides 675, 676, 677 and 678)
or the SPAC4G8.13c::ura4+ module (with oligonucleotides
679, 680, 681 and 682).
The C-terminally GFP-tagged strain was constructed by direct chromosome
integration of PCR fragments generated using the pFA6a-GFP-kanMX6 plasmid as a
template and oligos 434 and 435
(Bähler et al., 1998b).
The amplified fragment contained the GFP coding region fused in frame to the
last codon of the eng1+ gene and the kanMX6
cassette that was used to select for transformants. Correct integration of the
DNA fragment was verified by PCR.
RNA isolation and northern blot analysis
Cells (1.3x109) were collected at different time intervals
after release from the restrictive temperature (37°C) or from different
mutant strains, and total RNA was prepared using the method described
previously (Percival-Smith and Segall,
1984). For northern blot analysis, 5 µg of RNA was used. The
DNA probes used to detect the different transcripts were:
eng1+, a 534 bp internal fragment (from +1901 to +2435)
obtained by PCR; ura4+, a 1.7 kb
BamHI-HindIII fragment obtained from plasmid
pSK-ura4+; and act1+, a 1.1 kb
fragment containing the whole coding region obtained by PCR.
Microscopy techniques
For light microscopy, cells were fixed in 3.7% formaldehyde and stained
with DAPI (4',6-diamino-2-phenylindole) or Calcofluor White as
previously described (Balasubramanian et
al., 1997). Samples were viewed using a Leica DMRXA microscope
equipped for Nomarski optics and epifluorescence and photographed with a
Photometrics Sensys CCD camera. For time-lapse photography, cells were mounted
on medium containing 0.5% agar. Confocal microscopy was performed on a Zeiss
Axiovert microscope equipped with a LSM510 laser scanning system, and the
images were analysed with LSM510 software.
For scanning electron microscopy (SEM), cells were harvested, washed in 0.1
M sodium phosphate buffer, pH 7.4, prefixed with glutaraldehyde (5%
glutaraldehyde in phosphate buffer) for 1 hour, washed twice in buffer, and
placed in 1% osmium tetraoxide for 1 hour at 4°C. The material was
subsequently washed in distilled water and dehydrated in a graded acetone
series. The dehydrated cells were mounted on specimen holders, air-dried,
coated with gold, and examined under a Zeiss DSM 940 scanning electron
microscope. For transmission electron microscopy (TEM), the cells were stained
with potassium permanganate according to the protocol described previously
(Johnson et al., 1973).
Electron photomicrographs were taken with a Jeol Jam-1010 electron
microscope.
Assay for ß-glucanase activity
ß-1,3-glucanase activity was assayed in cell extracts or in culture
supernatants as previously described
(Baladrón et al., 2002).
Determination of the reducing sugars released in the reactions was performed
by the methods of Somogyi (Somogyi,
1952
) and Nelson (Nelson,
1957
). One unit of activity was defined as the amount of enzyme
that catalysed the release of reducing sugar groups equivalent to 1 µmol of
glucose per hour, and specific activity was expressed as units per milligram
of protein or per milligram of dry cell weight. For activity against PNPG, the
amount of p-nitrophenol released was determined
spectrophotometrically by measuring optical density at 410 nm. One unit of
enzyme catalyzed the release of 1 µmol of p-nitrophenol per hour
under the reaction conditions used.
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Results |
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To analyse whether the protein identified on the basis of sequence
similarity also showed endoglucanase activity, the coding region was
PCR-amplified using specific oligonucleotides and was cloned under the control
of the strong inducible promoter nmt1+, which is regulated
by thiamine (Forsburg, 1993).
The resulting plasmid (pAB10) was introduced into strain h20 and
ß-glucanase activity was assayed on cell extracts using laminarin (a
ß-1,3-glucan polymer) as substrate. The results showed that in presence
of thiamine (nmt1+ promoter repressed) the level of
ß-glucanase activity was similar to that found in cells carrying the
vector alone (pREP3X). However, in cells that had been growing for 16 hours in
the absence of thiamine (nmt1+ promoter induced), a four-
to fivefold increase in ß-glucanase activity was observed
(Fig. 1B). This result
indicates that the S. pombe eng1p has ß-glucanase activity.
Substrate specificity was tested under the same conditions described above,
using two other substrates: pustulan (a linear ß-1,6-glucan) and
p-nitrophenyl-ß-D-glucopyranoside (PNPG), a synthetic compound that is
only cleaved by glucanases with an exo-hydrolytic mode of action. As shown in
Fig. 1C, no activity was
detected against these two compounds, confirming that eng1p, as previously
described for other GFH81 proteins
(Baladrón et al., 2002),
is specific for ß-1,3-glucans and that it acts by cleavage of the
internal bonds of the polymer chains.
eng1p is involved in cell separation in S. pombe
To investigate the function of eng1p endo-ß-1,3-glucanase during the
cell cycle of fission yeast, eng1 cells were generated using a
PCR-based system (Wach, 1996
).
eng1
mutants were viable at all temperatures and showed no
apparent growth defect in either rich or minimal medium. When the morphology
of mutant cells was analysed by microscopic observation, a defect in cell
separation was apparent: most of the mutant cells in the culture were
clustered in groups of four cells (95% of cells; n=210 cells)
compared with the wild-type strain (3% of cells; n=234 cells).
Staining with Calcofluor, a dye that in S. pombe shows greater
affinity for the ß-1,3-glucans present in the primary septum that
separates the two sisters cells, revealed that a septum had been assembled
between the cells (Fig. 2). In
addition, DAPI staining of DNA indicated that each cell contained a single
nucleus (not shown). These results therefore indicate that
eng1
cells are able to complete nuclear segregation and
cytokinesis normally but that they have a defect in cell separation, resulting
in the formation of groups of cells.
|
To analyse the separation defect in greater detail, cell growth was
monitored using time-lapse differential interference contrast (DIC)
microscopy. In wild-type cells, cell division produced two equivalent cells
that separated immediately after cell division
(Fig. 3A). In contrast,
eng1 cells failed to complete septum dissolution and cell
separation (Fig. 3B, black
arrows). However, even under these conditions, they were able to reinitiate
polarized growth and undergo a new round of mitosis and cell division,
constructing new septa (white arrows) that resulted in the formation of groups
of four cells (see supplementary movies:
http://jcs.biologists.org/supplemental).
A similar morphology, four connected cells, was observed when cells were
prepared for scanning electron microscopy
(Fig. 3C). All of these results
therefore suggest that the protein encoded by eng1+ is
involved in cell separation.
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eng1p is required for degradation of the primary septum
To further assess the nature of the separation defect of
eng1 mutants, transmission electron microscopy was used to
compare the morphology of the septum region between the wild-type and the
mutant strain. In wild-type cells, the three-layer structure of the septum was
apparent, with a clear primary septum surrounded by two darker layers
corresponding to the secondary septum (Fig.
4A). In these cells, it was observed that the primary septum was
being degraded centripetally, from the cortex to the midpoint of the septum,
and no remnants of this structure were seen in the region from which the two
cells had already detached themselves. Inspection of the septal region in
mutants lacking the eng1p endo-ß-1,3-glucanase also revealed the typical
three-layer structure, indicating that the septum had been normally assembled.
However, in this case it was evident that cell separation had not proceeded in
the usual way, because abundant cell wall material that had not been correctly
degraded was present between the two sister cells (indicated by arrows in
Fig. 4B,C). Interestingly, most
of the extra material clearly corresponded to the ß-1,3-glucan-rich
primary septum. These observations therefore indicate that cell separation in
S. pombe requires enzymatic degradation of the primary septum, and
that the endo-ß-1,3-glucanase eng1p is required for this process.
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The endo-ß-1,3-glucanase eng1p localizes to the septal
region
To determine the subcellular localization of eng1p, the coding sequence of
the green fluorescent protein (GFP) was fused inframe before the
eng1+ stop codon using a PCR based approach
(Bähler et al., 1998b).
The C-terminus was chosen because a putative signal sequence is present at the
N-terminal end of the protein, which could be important for entry into the
secretory pathway and for the proper localization of this ß-glucanase.
All the resulting strains contained the fusion under the control of the native
eng1+ promoter and the fusion protein was functional.
Since eng1p may exert its function in a cell-cycle-regulated manner during cytokinesis, the localization of the eng1p-GFP protein was first analysed in a synchronous population of S. pombe cells. To this end, a strain containing a cdc25-22 thermosensitive allele, which arrests at the G2/M boundary, and the eng1p-GFP fusion was constructed. Cells were arrested by incubation at the restrictive temperature for 4 hours and then transferred to the permissive temperature. The fluorescence corresponding to the eng1p-GFP appeared 90 minutes after release, coinciding with the first peak of synchronous septation (Fig. 5). Interestingly, in all the cells observed, eng1p-GFP was localized to the region of the cell where polarized growth occurs at this time point (i.e. the septum). Thus, eng1p appears to be synthesized periodically during the cell cycle and seems to accumulate in the septum region, in agreement with the proposed role in cell separation.
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The localization of the eng1p-GFP fusion was further analysed in cells that had also been stained with Calcofluor to assess septum formation. The localization of eng1p-GFP was found to be almost coincident with the position of the primary septum, as shown when the fluorescence from Calcofluor and GFP were overlain (Fig. 6A). Interestingly, the green fluorescence observed in cells carrying the eng1p-GFP fusion reporter was more intense in a circumference surrounding the primary septum (indicated by the Calcofluor fluorescence) rather than in the septum itself, suggesting that the protein could be localized in a ring structure. To confirm this observation, confocal microscopy was used. The results of the 3D reconstruction of the green fluorescence found in cells carrying the eng1p-GFP indicated that in the cell wall this ß-glucanase is localized to a ring-like structure that completely surrounds the primary septum (Fig. 6B; see supplementary movies online).
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eng1+ expression and ß-1,3-glucanase
activity peak during the septation process
The above results suggested that the endoglucanase-encoding gene
eng1+ is expressed periodically during the cell cycle. To
confirm this point, the expression of this gene was monitored by northern blot
analysis in cdc25-22 mutant cells that had been synchronized by
arrest-release. When the level of eng1+ mRNA was examined,
a periodic cell cycle variation was found, maximum accumulation being observed
20-40 minutes before the peak of septation
(Fig. 7A), in good agreement
with the results obtained from the protein localization experiments. As a
control for RNA loading in all the lanes, a gene that displayed no cyclic
variation such as ura4+ was used.
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ß-glucanase activity against laminarin was also assayed in
cdc25-22 synchronised cells at the same time intervals as those used
for northern analysis. The results, shown in
Fig. 7B, indicated the
existence of a periodic increase in ß-glucanase activity, that slowly
rose to reach a maximum coincident with the septation peak. The maximum
activity was detected 20-40 minutes after the time of maximum mRNA
accumulation (compare the septation index of both experiments), suggesting
that the protein accumulates until the separation process is completed. To
check that the increase in activity was indeed due to the product of
eng1+, this gene was deleted in the cdc25-22
mutant strain. When ß-glucanase activity was measured in the double
cdc25-22 eng1 mutant, only a basal level of activity (mainly
due to the protein encoded by the eng2+ gene, data not
shown) was detected in all samples. Thus, ß-1,3-glucanase activity in
S. pombe periodically oscillates during the cell cycle, maximum
accumulation coinciding with the septation and cell separation processes, in
good agreement with the expression pattern of the eng1+
gene.
eng1+ expression is regulated by the
ace2+ transcription factor
In budding yeast, ScAce2p regulates the expression of a group of genes
involved in cell separation, such as chitinase (CTS1),
endo-ß-1,3-glucanase (ENG1), and YHR143w
(Dohrmann et al., 1992;
Doolin et al., 2001
;
Baladrón et al., 2002
),
and for this reason ace2 mutants display a cell separation defect,
forming large aggregates of cells. BLAST searches of the S. pombe
genome revealed the presence of two proteins with significant similarity to
ScAce2p: namely, the products of the SPAC6G10.12c (e value=-21) and
SPAC4G8.13c (e value=-10) ORFs. However, this latter protein is more similar
to the S. cerevisiae Crz1p transcription factor (e value=-23),
suggesting that the former could be the functional homolog of ScAce2p. To test
this possibility, both genes were independently deleted in a wild-type
background by replacing the coding region with the kanMX4 (for the
SPAC6G10.12c ORF) or ura4+ (for SPAC4G8.13c) marker genes.
The resulting strains, LE25 (lacking SPAC6G10.12c, which will be referred to
as ace2+ based on the homologies to ScAce2p, see below)
and LE26 (lacking SPAC4G8.13c), were used to analyse eng1+
expression and to check the morphological appearance of the cells.
Northern analysis was performed in the two mutant strains and in the
isogenic wild-type strain to test whether either of the two putative
transcriptional regulators were controlling the expression of
eng1+ in S. pombe. As can be seen in
Fig. 8A, the accumulation of
eng1+ mRNA is slightly reduced in the LE26 strain (lacking
SPAC4G8.13c) but was almost absent in the ace2 mutant. This
observation therefore clearly indicates that eng1+
expression requires ace2p, which is similar to what has been described for the
S. cerevisiae ENG1 gene
(Baladrón et al., 2002
),
although a minor contribution of the SPAC4G8.13c protein to its regulation
cannot be ruled out.
|
The morphology of ace2 cells was also analysed by
microscopic inspection (Fig.
8B). In contrast to wild-type cells (panel 1) or mutants lacking
SPAC4G8.13c (panel 2), ace2
-null mutants displayed a severe
cell separation defect (panel 3), resulting in the formation of mycelial cells
with a branched morphology, as has been reported for other genes such as
sep1+ and spl1+
(Sipiczki et al., 1993
). Thus,
the product of the SPAC6G10.12c ORF appears to be a functional homolog of
ScAce2p, regulating the expression of a group of genes required to complete
cell separation in S. pombe (one of which is the
endo-ß-1,3-glucanase encoded by eng1+).
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Discussion |
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Cell separation in S. pombe requires enzymatic hydrolysis of
the primary septum
The goal of cytokinesis is common in all organisms: to physically separate
a mother cell into two daughter cells. This is achieved by a common set of
mechanisms and involves the use of an actomyosin contractile ring that
provides the mechanical force required for the separation process. However,
fungi and yeast cells synthesize a division septum behind the ring as it
constricts, generating new cell wall material between the daughter cells.
Thus, cell separation in these organisms requires dissolution of both the
primary septum and the cylinder of cell wall that surrounds it
(Robinow and Hyams, 1989).
In budding yeast, the chitin-rich primary septum is synthesized by the
action of chitin synthase II [the ScChs2p protein
(Schmidt et al., 2002;
Shaw et al., 1991
)], after
which a secondary septum is laid down at both sides. Cell separation in S.
cerevisiae requires partial hydrolysis of the primary septum between the
mother cell and the new daughter cell, a process that is mediated by the
action of the endo-chitinase encoded by CTS1 (for a review, see
Cabib et al., 2001
). In
addition, other cell wall components, such as glucans, must be hydrolysed for
cell separation to be completed, and the endo-ß-1,3-glucanase ScEng1p has
recently been shown to participate in this process
(Baladrón et al., 2002
).
Interestingly, both these proteins localize to the mother-daughter neck region
during the time of cell separation, but in an asymmetrical manner, according
to the division pattern of budding yeast. They are only present at the
daughter side of the septum because the genes that encode them are expressed
only in newborn cells (Baladrón et
al., 2002
; Colman-Lerner et
al., 2001
).
In fission yeast, the primary septum, which is rich in linear
ß-1,3-glucan (Horisberger and
Rouver-Vauthey, 1985; Humbel
et al., 2001
), is the first part to be laid down in a centripetal
fashion until it completely closes and compartmentalizes the two daughter
cells. Cps1p, an integral membrane protein known as the putative catalytic
subunit of ß-1,3-glucan synthase, is essential for division septum
assembly and it localizes to the division site
(Ishiguro et al., 1997
;
Le Goff et al., 1999b
;
Liu et al., 1999
;
Liu et al., 2002
;
Cortés et al., 2002
)
Following this, each daughter contributes cell wall material to its own side
of the primary septum, building a secondary septum mainly composed of
ß-1,6-branched ß-1,3-glucan and ß-1,6-glucan
(Horisberger and Rouver-Vauthey,
1985
; Humbel et al.,
2001
). Separation of the sister cells requires two degradative
processes: erosion of the surrounding cylinder of cell wall at its junction
with the septum and dissolution of the primary septum
(Robinow and Hyams, 1989
). It
has been proposed that degradation of the primary septum might be a mechanical
process triggered by rupture of the cell wall of the mother cell
(Sipiczki and Bozsik, 2000
).
Here we have shown that cell separation requires enzymatic hydrolysis of the
primary septum in a process that is mediated by the endo-ß-1,3-glucanase
encoded by eng1+. The fact that this protein is present in
a ring-like structure surrounding the septum before cell separation has
started suggests that eng1p could perhaps be involved in the dissolution of
the cell wall material that surrounds the separation septum, a process that
may trigger cell separation. Once the cell wall has been dissolved, the
phenotype of eng1
mutants clearly supports the idea that the
endo-ß-1,3-glucanase eng1p would be involved in the degradation of the
fibrillar ß-1,3-glucans that are so abundant in the primary septum of
fission yeast. However, we were unable to detect the constriction of the
fluorescent ring concomitant with the disappearance of the primary septum,
perhaps due to the fact that the eng1p-GFP fluorescence is very faint and
rapidly disappears or, alternatively, because they do not occur in a
synchronic manner. In the absence of eng1p, cell separation is delayed in
comparison with wild-type cells, and may be achieved either through
participation of other hydrolytic enzymes or through mechanical rupture of the
cell wall in a process in which the primary septum is not completely
hydrolysed. The observation that the phenotype of eng1
cells
is much less severe than that of other previously identified mutants (such as
sep1
or spl1
) or that brought about by the
ace2 deletion clearly suggests that additional proteins (perhaps
other hydrolytic activities) would also be involved in cell separation. In
this context, and similar to the case of S. cerevisiae, other
proteins with sequence similarity to glucanases are present in the S.
pombe genome. Thus, a protein related to eng1p has recently been reported
in S. pombe [the product of the SPAC23D3.10c ORF
(Baladrón et al.,
2002
)]. This protein, named eng2p, also exhibits
endo-ß-1,3-glucanase activity, but eng2
mutants do not
have a cell separation defect (A.B.M.-C. and F.d.R., unpublished), which
suggests that eng2p is not involved in cell separation. Additionally, three
genes that show similarity to yeast exo-ß-1,3-glucanases and two proteins
related to fungal
-glucanases are also present in the S. pombe
genome, although no data about their putative involvement in morphogenetic
processes are yet available.
The spatial regulation of hydrolases during cell separation is also
important for the process to be achieved successfully. Unlike cell separation
in S. cerevisiae, which is an asymmetric process resulting in the
formation of two cells of different size, S. pombe cells divide by
medial septation, to generate two equivalent cells. Asymmetries in S.
pombe cells have been found only in spindle-pole bodies
(Sohrmann et al., 1998),
mating-type switching (Klar,
1990
), and in for3 mutants, which lack a formin required
for symmetry to be maintained during cell growth
(Feierbach and Chang, 2001
).
This difference is also reflected in the localization of the proteins involved
in septum degradation: while in S. cerevisiae CTS1 and ENG1
expression occurs only in the daughter cell and the proteins are located at
the daughter side of the septum
(Baladrón et al., 2002
;
Colman-Lerner et al., 2001
),
in fission yeast the protein appears to be localized in a symmetrical fashion
in a ring surrounding the primary septum. Thus, whereas in budding yeast it is
the daughter cell that separates from the mother, in fission yeast both sister
cells seems to contribute equally to the cell separation process.
It has recently been proposed that the exocyst, a multiprotein complex
involved in the late steps of the exocytic pathway, is essential in S.
pombe for the delivery of proteins that are important for cell cleavage,
including putative hydrolytic enzymes
(Wang et al., 2002). Mutants
deficient in any of the subunits of this complex accumulate 100 nm vesicles,
although the content of these vesicles has not yet been characterized. Since
eng1p localizes to the septum region at the time of cell cleavage, it is
therefore possible that it could be one of the components being transported in
such vesicles.
Temporal regulation of eng1+ expression requires
the ace2p protein
In addition to the tight spatial regulation observed for the eng1p protein,
we also observed a strict temporal regulation of ß-1,3-glucanase activity
during the cell cycle, the maximum being reached at the time of cell
separation. Furthermore, the variation in activity is in good agreement with
the expression pattern of eng1+, indicating that this
activity is regulated at transcriptional level and not by any other
post-transcriptional mechanism. The expression of eng1+
requires the product of the SPAC6G10.12c gene, a protein that shows strong
sequence similarity to the ScAce2p especially in the DNA-binding region,
although the product of the SPAC4G8.13c ORF could have a minor role in its
regulation. Similarly to what has been described for ScAce2p
(Dohrmann et al., 1992),
deletion of the S. pombe protein results in a severe cell separation
defect because null mutants are unable to separate and show a hyphal and
branched pattern of growth similar to that of sep1
or
spl1
mutants (Grallert et
al., 1999
; Ribár et
al., 1999
; Sipiczki et al.,
1993
). Based on these functional and sequence similarities, we
have named the SPAC6G10.12c ORF ace2+. The phenotype of
ace2
mutants is much more severe than that observed in
eng1
mutants, which indicates that ace2p might regulate the
expression of other genes also involved in the cell separation process,
including additional hydrolytic enzymes required for the dissolution of other
cell wall components.
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Acknowledgments |
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Footnotes |
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References |
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