1 Departamento de Microbiología II, Facultad de Farmacia, Universidad
Complutense de Madrid, 28040 Madrid, Spain
2 Centro de Citometría de Flujo y Microscopía Confocal,
Universidad Complutense de Madrid, 28040 Madrid, Spain
* Author for correspondence (e-mail: jarroyo{at}farm.ucm.es )
Accepted 10 April 2002
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Summary |
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Key words: Saccharomyces cerevisiae, Glycosidase, Cell wall, Crh2p, Polarity, GFP
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Introduction |
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During vegetative growth, the initiation of bud development comprises
diverse molecular steps: recognition of the appropriate site for budding,
polarisation of the actin cytoskeleton, secretion and cell wall synthesis. The
site for bud formation is determined in a cell-type-dependent fashion. In
haploid strains, mother cells select a bud site immediately adjacent to their
previous daughter, and the daughter cells bud next to the birth site (axial
budding pattern). In diploids, mother cells select bud sites adjacent to their
daughter cells or on the opposite end of the cell (bipolar budding pattern)
(reviewed by Madden and Snyder,
1998). Ras1-like GTPase Bud1p/Rsr1p and its regulators Bud2p and
Bud5p function in all cell types as the general bud-site selection machinery
that recruits the proteins required for bud formation to the
cell-type-specific landmarks (for reviews, see
Cabib et al., 1998
;
Madden and Snyder, 1998
). In
addition, it is currently well known that the polarity in S.
cerevisiae is under the coordinated control of Rho GTPases and
cyclin-dependent protein kinases. One of the major functions of the essential
Rho GTPase Cdc42p is to regulate the polarisation of the actin cytoskeleton in
yeast, which in turn targets the secretory vesicles and other factors that
support growth (Johnson, 1999
;
Pruyne and Bretscher, 2000a
;
Pruyne and Bretscher,
2000b
).
The biosynthesis and assembly of cell wall components needs to be tightly
controlled both temporally, in order to be perfectly coordinated with other
cellular events such as cell cycle or developmental programs, and spatially,
to be coupled with cytoskeleton dynamics for polarised growth. To date, most
of the work related to the temporal and spatial regulation of cell wall
components has focused on the biosynthesis and deposition of chitin, a
polysaccharide that is synthesised at the plasma membrane. Spatial control of
chitin deposition depends on the actin cytoskeleton and polarity establishment
proteins. Mutants defective in actin
(Novick and Botstein, 1985),
Myo2 (a type V myosin) (Johnston et al.,
1991
) or proteins related to actin function
(Donnelly et al., 1993
;
Haarer et al., 1994
;
Liu and Bretscher, 1992
) or
mutants altered in polarity and bud emergence
(Adams et al., 1990
;
Kim et al., 1991
;
Sloat et al., 1981
) display
altered chitin deposition patterns. Proper chitin location also depends on the
structure formed by the family of septin proteins (Cdc3p, Cdc10p, Cdc11p and
Cdc12p). This family of proteins assembles as a ring at the mother-bud neck,
thereby providing a scaffold for the organisation of proteins at this specific
region of the cell surface (Field and
Kellogg, 1999
; Ford and
Pringle, 1991
; Longtine et
al., 1996
).
The localisation of Chs3p [which is responsible for the bulk of chitin at
the ring between mother and daughter cells and in the lateral cell wall
(Shaw et al., 1991;
Valdivieso et al., 1991
)],
both in time and space, is essential for chitin synthesis during vegetative
growth. Chs3p localises to sites of polarised growth in a cell-cycle-dependent
manner, displaying a similar pattern to that of chitin deposition
(Santos and Snyder, 1997
).
Moreover, the proper targeting of CSIII (Chitin Synthase III) activity depends
on several other genes, such as those encoding the septins and CHS4, CHS5,
CHS6, CHS7, MYO2 and BNI4. In the absence of any of the proteins
encoded by these genes, Chs3p is no longer present in polarised cortical sites
(DeMarini et al., 1997
;
Santos et al., 1997
;
Trilla et al., 1999
;
Ziman et al., 1998
). A model
has emerged in which Chs4p links Chs3p to Bni4p and, hence, to the septins
(DeMarini et al., 1997
).
Targeting of Chs3p to polarised growth sites requires the protein Chs5p, a
protein from the trans-Golgi network
(Santos et al., 1997
;
Santos and Snyder, 1997
). It
has been suggested that Chs5p-containing vesicles target Chs3p to the neck
with the help of the actin cytoskeleton
(Madden and Snyder, 1998
). The
role of two other proteins, namely Chs6p and Chs7p, in the transport of Chs3p
has also been characterised (Trilla et
al., 1999
; Ziman et al.,
1998
). In addition, two Golgi proteins (Sbe2p and Sbe22p) have
recently been identified and associated with the transport of Chs3p as this
protein is mislocalised in sbe2 sbe22 mutants. It has been suggested
that these proteins are also involved in the transport of mannoproteins to the
cell surface (Santos and Snyder,
2000
).
In contrast to the knowledge about the mechanisms that control chitin
synthesis at the cell membrane, little is known about the mechanisms that
control the spatial and temporal distribution of mannoproteins at the cell
wall. The yeast genome encodes approximately 40 different GPI and 4 Pir cell
wall mannoproteins (Smits et al.,
1999). Many of these proteins are expressed in a
cell-cycle-dependent manner (Spellman et
al., 1998
), with most of their encoding genes being transcribed in
the M/G1 phase. Mannoproteins, which are located in an outer layer of the cell
wall, play an important role in determining cell wall porosity, acting either
as structural proteins or as enzymes involved in cell wall construction and
remodelling (Kapteyn et al.,
1999
; Orlean,
1997
).
We have recently described a family of cell wall proteins (Crh1p, Crh2p and
Crr1p); these proteins are involved in cell wall assembly and are differently
expressed during the yeast life cycle
(Rodriguez-Peña et al.,
2000). Crh1p and Crh2p, which are both GPI-anchored cell wall
proteins (Hamada et al.,
1998
), although differently regulated through the cell cycle but
(CRH2 transcript levels are stable throughout the mitotic cycle but
CRH1 has two transcription peaks at G1 and M/G1), localise to
polarised growth sites. The localisation of these proteins is reminiscent of
the distribution of chitin at the cell wall
(Rodriguez-Peña et al.,
2000
), suggesting a probable role for these proteins in the
integration of this polymer within the cell wall matrix. Bearing in mind this
characteristic localisation pattern, in this study we were prompted to
characterise the mechanisms controlling the localisation of the Crh2p cell
wall protein, studying the dependence of this localisation on proteins
involved in polarity selection, transport systems and bud morphogenesis.
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Materials and Methods |
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Yeast manipulations
Yeast transformation was carried out by the lithium acetate protocol
(Gietz and Woods, 1994).
Auxotrophies of the transformants were verified on SD plates (20 g/l glucose,
1.67 g/l yeast nitrogen base without amino acids, 5 g/l ammonium sulphate and
the appropriate amount of amino acids) lacking a particular amino acid or
nitrogen base. The sensitivity/resistance of the segregants to geneticin
(encoded by the KanMX4 module) was tested in 200 mg/l geneticin-YEPD
plates.
Cell polarity and morphogenetic defects were evaluated under fluorescence
microscopy after staining cell wall chitin with Calcofluor White (Sigma), as
described elsewhere (Pringle,
1991).
For studying the dependency of the polarised localisation of Crh2p on the
actin cytoskeleton, cells from the asynchronous wild-type strain 1783
transformed with the plasmid pJV40G were grown to early log phase in YEPD
medium at 24°C, and -factor (Sigma) was added to 10 µg/ml. After
3 hours, more than 80% of cells were observed to have shmoos. At this time
cells were harvested by gentle centrifugation, washed once with fresh medium
and finally resuspended in fresh medium containing Latrunculin-B (LAT-B,
Calbiochem) from a 10 mM DMSO stock to a final concentration of 200 µM or
DMSO as a control. The cells were incubated at 24°C for 1 hour. Samples
corresponding to different steps of the treatment were harvested by gentle
centrifugation, washed twice with PBS buffer, resuspended in PBS and finally
observed under a Fluorescence Microscope Eclipse TE 2000-U (Nikon, Tokyo).
Molecular biology techniques
Standard molecular biology techniques for DNA manipulations and bacterial
transformations were performed as described previously
(Sambrook et al., 1989).
Restriction enzymes were provided by Boehringer-Mannheim. In the construction
of crh2
strains, the complete ORF was deleted, except for the
start and stop codons. Disruptions were performed by the SFH (Short Flanking
Homology) PCR technique (Wach et al.,
1997
), which allows the replacement of the target ORF by a
selection marker. The strategy for CRH2 deletion using the
KanMX4 marker from the plasmid pFA6a-KanMX4
(Wach et al., 1994
) was
similar to that previously described
(Rodriguez-Peña et al.,
2000
) except that the HIS3 cassette was replaced by
Kanr.
The YCp(CDC42Sc) plasmid containing a wild-type CDC42
gene in a LEU2 centromeric plasmid has been previously described
(Ziman et al., 1991).
GFP constructions
To determine Crh2p localisation, we used the previously described
(Rodriguez-Peña et al.,
2000) internal in-frame fusion with the green fluorescent protein
from Aequorea victoria (GFP) to Crh2p. This cassette was cloned in
centromeric pRS416 URA3 (pJV40U), YCplac111 LEU2 (pJV40L) or
episomic YEp352 URA3 (pJV40G) and YEplac181 LEU2 (pJV40F)
plasmids to fit genetic background requirements. The pJV40G construction has
been described previously
(Rodriguez-Peña et al.,
2000
). pJV40U was obtained by cloning the 3.1 kb
BamHI/HindIII insert from pJV40G into the
BamHI/HindIII-cleaved pRS416 vector
(Sikorski and Hieter, 1989
),
pJV40L by cloning the same insert from pJV40G into the
BamHI/HindIII-cleaved YCplac111 vector
(Gietz and Sugino, 1988
) and
finally, pJV40F was obtained by cloning the 3.1 kb
BamHI/HindIII insert from pJV40G into the
BamHI/HindIII-cleaved YEplac181 vector
(Gietz and Sugino, 1988
).
The Cwp1-GFP fusion protein used in this work was obtained from the
episomic plasmid pAR213 generously donated by Glaxo-Wellcome
(Ram et al., 1998). The 2.6 kb
XbaI/HindIII insert from pAR213 was cloned into the pRS416
vector digested with the same enzymes, giving rise to the pAR214 plasmid.
When necessary, sequence verification of the clones was carried out on an automated DNA sequencer (ABI 377, Applied Biosystems).
Confocal microscopy techniques
Cells were grown overnight in YEPD at 24°C or 28°C and then
transferred to fresh medium. After 3 hours of incubation they were harvested
by gentle centrifugation, washed twice with PBS buffer and finally resuspended
in PBS. In the case of thermosensitive mutants, sample preparation was
identical, except that cells were grown overnight in YED or YEPD at 24°C
and then transferred to fresh medium at the same temperature or at 37°C
when the expression of the mutant phenotype was required (3 hours of
incubation for cdc10-11 and 5 hours for cdc42-1 and
lyt1 mutants). Samples were observed under an Eclipse TE-300 (Nikon,
Tokyo) microscope attached to a Bio-Rad MRC1024 confocal system (BioRad,
Hampstead, UK).
Propidium iodide staining for the detection of lysed cells was performed as
described previously (De la Fuente et al.,
1992).
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Results |
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Additionally, we investigated the distribution of Crh2p in a
cdc42-1ts mutant (DJTD2-16D strain; see
Table 1). Cdc42p is an
essential GTPase necessary for organisation of the actin cytoskeleton and for
cell polarisation (Park et al.,
1997). At the permissive temperature the mutant grows and buds
normally, but at restrictive temperatures the nuclear cycle continues but bud
formation is blocked. The cytoplasmic actin network appears disorganised, and
depolarised growth leads to round unbudded cells
(Adams et al., 1990
). The
cdc42-1 strain was transformed with the pJV40G plasmid. A multicopy
plasmid was used in this case because there was a decrease in the GFP signal
when cells were incubated at 37°C. No changes were observed in the
distribution of the Crh2p-GFP as a function of the plasmid used. When cells
were grown at the permissive temperature (24°C), we did not observe
variations with respect to the wild-type pattern, the protein being present at
the lateral cell wall, incipient budding sites and at the region of the septum
(Fig. 2A). However, when cells
were shifted to the restrictive temperature (37°C for 5 hours), the mutant
phenotype was clearly expressed, with a high percentage of larger, rounded and
unbudded cells. Under these conditions the distribution of Crh2p was
significantly altered, being mainly localised to the lateral cell wall
(Fig. 2B). As expected, the
polarised distribution completely disappeared, suggesting that the
organisation of the actin cytoskeleton is necessary for targeting Crh2p to the
cell wall at sites of polarised growth. To test this possibility, Crh2p
localisation was followed in synchronised cells treated with the actin
inhibitor Latrunculin B (see Materials and Methods). No polarised distribution
of Crh2p was observed in cells treated with this drug (data not shown). The
altered distribution of Crh2p in a cdc42-1 mutant at 37°C was
totally corrected when these cells were co-transformed with a plasmid bearing
a wild-type CDC42 gene [YCp(CDC42Sc)], which confirmed the
results described above (Fig.
2C).
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Bud neck localisation of Crh2p depends on the septin ring and septum
integrity during cytokinesis
Previous work from different groups have demonstrated the importance of the
septins in the correct positioning of various proteins that need to be located
at the mother-bud neck, such as Chs3p
(DeMarini et al., 1997). In
view of the localisation pattern of Crh2p in chitin-rich areas of the cell
surface, we investigated the role of septins in the spatial deposition of the
GPI-cell wall protein Crh2p following its localisation in the
temperature-sensitive cdc10-11 septin mutant (VCY1). The septin ring
in this mutant disassembles after a shift from the permissive (24°C) to
restrictive (37°C) temperature. In cells grown at 24°C, Crh2p-GFP
(expressed from pJV40G plasmid) localised to the mother-bud neck and the
lateral cell wall of the cdc10-11 mutant strain, following its usual
localisation pattern (Fig. 3A).
A similar result was also observed in the isogenic wild-type strain grown at
37°C (Fig. 3B). When
cdc10-11 mutant cells were shifted to 37°C for 3 hours, they
developed elongated buds that were unable to complete cytokinesis
(Cid et al., 1998
). Under these
conditions, Crh2p is mainly present in the lateral wall of mother cells
(Fig. 3C-E) but, in accordance
with the loss of bud neck landmarks in this mutant, Crh2p completely
disappeared from the base of the elongated buds
(Fig. 3C-E), in contrast to the
normal strong accumulation of this protein in the septum of wild-type cells,
especially during cytokinesis (Fig.
3A-B). Interestingly, in the elongated buds formed under the
restrictive temperature, Crh2p diffusely marked both sides of the lateral cell
wall at the mother-bud neck. By contrast, no conspicuous Crh2p rings were
formed under these conditions at the base of new buds, including small
(Fig. 3C), medium
(Fig. 3D) and large
(Fig. 3E) sized buds,
suggesting that accurate Crh2p deposition at the mother-bud neck clearly
depends on septin ring integrity. This is consistent with the distribution
pattern of chitin in septin mutants
(DeMarini et al., 1997
) or
mutants with GIN4 deleted
(Longtine et al., 1998
). Gin4p
is a protein kinase involved in septin assembly, in which the chitin detected
by calcofluor staining shows a diffuse band on both sides of the neck whose
intensity decreases with the distance from the mother-bud neck instead of its
usual staining, which is restricted to the mother-cell side of the neck
(Longtine et al., 1998
). The
signal corresponding to the GFP-fusion protein could be occasionally detected
in the neck region of cells that were in the late stages of the cell cycle.
Since the mutation is not `leaky', we interpret that those cells had already
started budding at the time of the temperature shift and thus maintain its
correct localisation (Fig.
3C-E).
|
The enhanced signal of Crh2p-GFP at cytokinesis in wild-type cells could be
caused by the association of Crh2p with the developing septum, as the septin
mutant, which fails to form septa, clearly lacked Crh2p enrichment at this
region. We next characterised the localisation of Crh2p in yeast cells bearing
the cdc15-lyt1 thermosensitive mutation (mutant L2C24d)
(Jimenez et al., 1998). The
CDC15 gene encodes a protein kinase essential for exit from the M
phase in the S. cerevisiae cell cycle
(Surana et al., 1993
). Cells
carrying the lyt1 mutation are unable to septate at 37°C, but the
septins remain at the mother-daughter neck
(Jimenez et al., 1998
). When
lyt1 cells transformed with the Crh2p-GPF construction (pJV40G
plasmid) were grown at the permissive temperature (24°C), the distribution
of the protein was identical to that of the wild-type
(Fig. 4A). As in the
cdc10-11 mutant, Crh2p was not accumulated at the septal region
between the mother and elongated buds at the restrictive temperature (5 hours
at 37°C). However, in contrast to cdc10-11, in most of these
cells Crh2p localised to the mother-bud neck in a conspicuous ring-like
structure (Fig. 4B). All these
data suggest that the deposition of Crh2p at the neck between mother and
daughter cells during cytokinesis depends both on septins, for proper
localisation of a Crh2p ring structure, and on septum integrity, for the
deposition of Crh2p at the septum structure itself.
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Bud neck localisation of Crh2p depends on Bni4p but not on Chs4p
A model has been suggested for the spatial localisation of CSIII activity,
in which the septin complex localises Bni4p through the interaction of this
protein with Cdc10p and, at the same time, Bni4p localises the chitin synthase
III complex (including Chs3p and Chs4p) through its interaction with Chs4p. In
order to test the requirement of Bni4p for Crh2p distribution, a
bni4 strain (10510A) was transformed with the pJV40U plasmid,
and Crh2p-GFP was monitored by confocal microscopy. As previously described
(DeMarini et al., 1997
), the
majority of bni4 mutant cells showed enlarged bud necks and some
protuberances at previous division sites
(Fig. 5B). As shown in
Fig. 5A, Crh2p localised at
incipient budding sites independently of Bni4p. During cytokinesis, it
localised to both mother and daughter cells at the lateral cell wall and also
in the protuberances at previous division sites. However, no Crh2p was found
at the bud-neck region at the time of cytokinesis
(Fig. 5B), suggesting that the
deposition of Crh2p in this area, late in the cell cycle, depends on the
presence of Bni4p. This result prompted us to test whether, like Chs3p, which
depends both on Bni4p and Chs4p, Chs4p was also necessary for the proper
localisation of Crh2p. To address this, Crh2-GFP was followed in a
chs4
mutant (237 strain) transformed with the pJV40F plasmid.
However, in contrast to Chs3p, Crh2p distribution at the bud neck does not
depend on Chs4p at any stage, early or late, in the cell cycle (data not
shown).
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Crh2p localisation is dependent on Chs5p and Sbe2p/Sbe22p transport
systems
Among the different steps required to achieve correct bud growth, the
secretory machinery plays an essential role, as it is required to specifically
direct new plasma membrane and cell wall material to the growth site
(Igual et al., 1996;
Madden and Snyder, 1998
).
However, the molecular mechanisms that control this process are poorly
understood.
Recently two pathways have been described for the transport of cell wall components to the cell surface. One of them is dependent on Chs5p, and it is required for the transport of Chs3p to the bud neck. The second pathway involves the Sbe2p/Sbe22p redundant proteins, which could be important for cell wall mannoprotein transport.
Previous results (Rodriguez-Peña
et al., 2000), as well as evidence in this work, indicated a
probable functional relationship between Crh2p and chitin deposition.
Therefore, we were prompted to evaluate the dependence of Crh2p localisation
on the aforementioned pathways. To this end, we first analysed the
localisation of the Crh2p-GFP fusion protein in wild-type and chs5
isogenic strains. A chs5 mutant strain (HVY260) and its isogenic
wild-type (15Daub) were transformed with the plasmid pJV40U. However, Crh2p
localisation was difficult to follow in this background, probably because of
the problem of the competition of Crh2p-GFP with the native Crh2p. In an
attempt to solve this problem, the CRH2 gene was deleted in both
strains by means of the SFH-PCR technique (see Materials and Methods). The
double mutant crh2 chs5 (JM96) was viable and did not exhibit any
variation in growth with respect to the strain bearing the single mutation
chs5. We then compared Crh2p-GFP localisation in both isogenic
crh2 (JM95) and crh2 chs5 strains. In the JM95 strain, Crh2p
was mainly observed (85%, (n=50) of the cells with detectable
fluorescence signal) at correct positions and sometimes in vacuoles (this
background is extensively vacuolated) (Fig.
6A). By contrast, the localisation pattern of Crh2p changed in the
JM96 strain (chs5 mutant), where most of the Crh2p protein
accumulated in internal vesicles (90% of cells, n=50)
(Fig. 6B), and only
occasionally did a residual signal of the protein reached the expected sites
(Fig. 6C). These results
pointed out that Crh2p localisation is dependent on Chs5p and suggest that the
transport systems for Chs3p and Crh2p are to, a certain extent, coincident.
Interestingly, the transport of another GPI cell wall protein, Cwp1p, does not
depend on this transport system as the Cwp1p-GFP localisation pattern was
similar to one previously described (Ram
et al., 1998
) and did not vary between chs5
(Fig. 6D) and its isogenic
wild-type (data not shown).
|
Two proteins (Sbe2p and Sbe22p) from the Golgi apparatus have recently been
implicated in the transport of Chs3p. Although the secretion of invertase and
exoglucanase was not affected in the sbe2 sbe22 mutant, the
participation of Sbe2p and Sbe22p in the transport of other mannoproteins has
been suggested, as deduced from the reduced mannoprotein layer of the sbe2
sbe22 strain (Santos and Snyder,
2000). To investigate the possible role of the Sbe2p and Sbe22p
proteins in the transport of Crh2p to the cell surface, we followed Crh2-GFP
localisation in an sbe2 sbe22 background (Y1949) and compared this
with its distribution in the isogenic wild-type strain (Y603), both of which
were transformed with the pJV40U plasmid. Crh2p-GFP was correctly localised in
the wild-type strain grown at 24°C, marking the lateral cell wall,
although mainly accumulating in the mother-bud neck and septum during
cytokinesis as well as in bud scars (Fig.
7A). However, in the absence of Sbe2p and Sbe22p, Crh2p was
completely delocalised. Instead of its usual localisation pattern, the whole
cargo of the protein was retained, and it accumulated in an internal
compartment of the cell, being unable to reach the cell surface either at the
lateral cell wall or at polarised growth sites
(Fig. 7B). Interestingly, and
in accordance with the above described non-dependence of Cwp1p transport on
Chs5p, the transport and correct localisation of the GPI cell wall protein
Cwp1p does not depend on Sbe2p and Sbe22p, as judged from the similar pattern
of fluorescence observed in both the sbe2 sbe22 mutant
(Fig. 7C) and its isogenic
wild-type strain (data not shown). Taken together, these results confirm
previous observations that indicated that Chs5p and Sbe2p/Sbe22p play a
selective role in transport of proteins to the cell wall. Our observations
clearly show that these two transport systems are not specific for chitin
synthesis (Chs3p) but are also required for other aspects of the biogenesis or
modification of the cell wall.
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Discussion |
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Our results represent new insights into the mechanisms that control the
spatial localisation of proteins associated with the cell wall matrix to
polarised growth sites. We have specifically studied the cell wall
mannoprotein Crh2p, a protein involved in cell wall assembly and covalently
attached to the cell wall glucan (Hamada
et al., 1998) (J.M.R.-P., C.R., A.A. et al., unpublished). Correct
localisation of Crh2p at the mother-bud neck is first controlled by the
cellular machinery responsible for the selection of the new budding site. In
the absence of Bud1p, the cell polarity establishment protein complex is
recruited by the cell to the mother-bud neck, but it is not recruited to the
correct place. Crh2p is deposited following the random budding pattern of
bud1 cells. Therefore, Crh2p recruitment relies on other cell
polarity cues rather than on the recognition of bud site selection elements.
In fact, correct Crh2p localisation to polarised growth sites is completely
dependent on Cdc42p. Both actin cytoskeleton polarisation and organisation of
the septin ring are disrupted in a cdc42ts mutant under
the restrictive temperature. The lack of Crh2p polarisation in
Latrunculin-B-treated cells demonstrates that the actin cytoskeleton is
necessary for targeting Crh2p to the cell wall at sites of polarised growth.
This polarisation is also dependent (as explained below) on the presence of a
functional septin ring. To our knowledge, this is the first cell wall protein
whose localisation depends on the polarity signalling pathway. The importance
of the actin cytoskeleton in chitin deposition has also been reported
previously. Chitin is delocalised in temperature-sensitive actin mutants
(Novick and Botstein, 1985
),
and cdc42 mutants also show defects in correct chitin deposition
(Adams et al., 1990
). In
accordance with the important role for the actin cytoskeleton in the correct
organisation and construction of the cell wall, an aberrant cell wall is
formed over the surface of the isodiametrically growing act1 mutant
cells (Gabriel and Kopecka,
1995
). In this context, our data indicate that proteins localised
extracytoplasmically and involved in cell wall construction, such as Crh2p,
are unable to reach their correct destination at the cell surface in the
absence of an organised actin cytoskeleton.
The data reported here clearly demonstrate that Crh2p, a GPI cell wall
mannoprotein localised in the extracellular matrix, depends on the septin ring
for proper deposition at the mother-bud neck. Many other proteins that
function at the mother-bud neck, such as Chs3p, Bni4p, Chs4p
(DeMarini et al., 1997), Bud3p
(Chant and Pringle, 1995
) or
Bud4p (Sanders and Herskowitz,
1996
), depend on septins for their localisation. Septins provide a
scaffold for the organisation of these proteins (and probably other proteins
still unidentified) needed at this specific region of the cell surface
(Field and Kellogg, 1999
),
some of them being required for chitin deposition. However, to date no other
proteins directly involved in cell wall construction itself other than those
involved in chitin deposition (such proteins are associated with the plasma
membrane and not with the cell wall structure) have been shown to be organised
by the septin scaffold. Our results clearly extend the previous role of
septins in chitin localisation to other proteins involved in cell wall
construction and suggest that septins could play an important role in the
organisation of the whole cell wall assembly process at the mother-bud neck.
Moreover, proper localisation of Crh2p at this region during cytokinesis
depends on septum integrity. In wild-type cells, Crh2p accumulates at the
septum during the cytokinesis stage, probably because the cell needs the
protein there at this time for the formation of the secondary septum. However,
in cdc10-11 and cdc15-lyt1 mutants, which are unable to
septate, Crh2p is not accumulated at this region. It is likely that in the
absence of specific unknown septum landmarks, proteins involved in septum
formation, including Crh2p, are not recruited to this site.
In the absence of Bni4p, Crh2p is not able to localise to the mother-bud
neck, in particular to the septum in cells undergoing cytokinesis. DeMarini et
al. have offered evidence that Bni4p provides a link between septins and Chs3p
through its interaction with Chs4p, at least in the periods immediately before
and during bud emergence (DeMarini et al.,
1997). More recently it has been proposed that Bni4p is required
for bud-neck localisation of Chs4p at bud emergence but not at cytokinesis,
suggesting the existence of other chitin targeting mechanisms late in the cell
cycle (Kozubowsky et al., 2001). The data reported here suggest that Bni4p
might be not only related to the Chs3p distribution but also involved in the
interaction between septins and other proteins involved in cell wall
construction, such as Crh2p. In contrast to Chs3p, proper localisation of
Crh2p does not depend on Chs4p either in bud emergence or during cytokinesis.
Bni4p could therefore be part of the mechanisms involved in the localisation
and recruitment of the cell wall protein Crh2p to bud neck late in the cell
cycle, at the time of cytokinesis, when this protein needs to be accumulated
at this region for proper septum formation. Supporting this idea, Bni4p has
been localised as a ring on both sides of the neck at cytokinesis (Kozubowsky
et al., 2001). Probably Bni4p has a more general function as a protein
scaffold for proteins required for polarised growth at the bud-neck region,
early at bud emergence and later during cytokinesis.
A critical step in proper localisation of proteins involved in polarised
growth, including those necessary for cell wall construction, is the transport
of these proteins to their respective sites of action. The secretory pathway
must deliver all the proteins required for these events to discrete growth
sites at the cell surface. Myo2p, a yeast class V myosin, has been implicated
in the transport of a class of secretory vesicles, from the mother to the bud,
that facilitate the transport of a specific set of proteins
(Govindan et al., 1995). These
authors proposed that the cargo of the vesicles that are rapidly accumulated
in a myo2-66 mutant could be the components necessary for cell wall
assembly, such as chitin synthases, chitinase or endoglucanases. Which
specific proteins are transported in these vesicles is now beginning to be
understood. Work by Santos and Snyder
(1997
) demonstrated that Chs3p
is one of these proteins (Santos and
Snyder, 1997
). The localisation of Chs3p requires Myo2p and the
actin cytoskeleton in addition to Chs5p. Chs5p, a protein from the trans-Golgi
network, is necessary for the proper secretion of Chs3p, and the possibility
that this protein might be associated with the outside of secretory vesicles,
facilitating their interaction with either the transport machinery or
components at the bud site and bud-neck region, has been suggested
(Santos and Snyder, 1997
). We
wondered whether Chs5p might be involved in the delivery of other proteins
that contribute to cell wall construction. The work reported here demonstrates
the dependence on Chs5p for the proper localisation of Crh2p to polarised
growth sites and particularly to the mother-neck region. Therefore,
Chs5p-containing vesicles may not represent, as previously thought, a specific
and unique subset of vesicles involved in the transport of Chs3p but may also
be involved in the localisation of other proteins that participate in the
process of cell wall assembly, such as Crh2p. Interestingly, the transport of
other secreted proteins such as Exg1p, - an extracellular exo-ß 1,3
glucanase - is not affected in chs5 mutants
(Santos et al., 1997
).
Likewise, the localisation of the GPI cell wall Cwp1p does not depend on
Chs5p, as reported here.
Another pathway for the transport of cell wall components to the cell
surface has recently been proposed by Santos and Snyder
(Santos and Snyder, 2000).
This pathway, involving two Golgi proteins - Sbe2p and Sbe22p - must be
interconnected with the one based on Chs5p, as Chs3p is also mislocalised in
the sbe2/sbe22 mutant. A substantially diminished outer layer of
mannoproteins has been observed in this mutant. However, no specific
mannoproteins have been associated so far with this pathway. Neither the
secretion of invertase nor that of exoglucanase is defective in this mutant
(Santos and Snyder, 2000
).
Moreover, preliminary results from this work suggested that transport of other
two cell wall proteins (Cwp1p and Pir2p) do not depend on this pathway. Here
we demonstrate that the transport and localisation of Cwp1 does not depend on
this pathway. However, the transport of Crh2p to the cell wall at the sites of
polarised growth and even to the lateral cell wall is completely impaired in a
sbe2 sbe22 mutant, meaning that transport of this mannoprotein is
dependent on the Sbe2p/Sbe22p pathway. Our data point to the notion that Crh2p
and the machinery for chitin synthesis, in particular Chs3p, are clearly
associated. The localisation of Crh2p is not only reminiscent of and resembles
the distribution of chitin in the cell wall, but also depends on the
mechanisms already described for the transport of Chs3p; at least partially
for Chs5p and completely for Sbe2p and Sbe22p. However, the localisation of
Crh2p does not requires chitin deposition, since its localisation is
maintained in both chs2 and chs3 mutants
(Rodriguez-Peña et al.,
2000
). Interestingly, however, and in accordance with the
association of Crh2p deposition with sites of chitin localisation, Crh2p is
strongly accumulated in the aberrantly thickened (and very chitin-rich) septa
seen in a chs2 mutant
(Rodriguez-Peña et al.,
2000
).
From many studies it is now becoming clear that the secretion of proteins
through the secretory pathway involves different vesicles that transport
specific sets of cargo proteins (Chuang and
Schekman, 1996; Govindan et
al., 1995
; Madden and Snyder,
1998
). The data presented here support this hypothesis and offer
new insight into the cargo of these vesicles. Why is the transport of Crh2p
dependent on the Chs5p and Sbe2p/Sbe22p pathways, whereas other cell wall
proteins such as Cwp1p, Exg1 and Pir2p, are not? Cwp1p and Crh2p are both GPI
cell wall proteins (containing a glycosylphosphatidylinositol-derived
structure) covalently linked to ß-1,6 glucan. In contrast, Pir2p, which
does not contain a GPI anchor site, is covalently linked to ß-1,3 glucan,
and Exg1p is mainly secreted to the culture supernatant. Therefore, the
differences in the dependence on the mechanisms of transport described above
between the cell wall proteins Crh2p, Cwp1p, Pir2p and Exg1p cannot be
explained in terms of their association with different structural components
of the cell wall. An attractive hypothesis to account for these results is the
existence of specific transport systems that could be involved in the
localisation of functionally related cell wall proteins required for cell wall
construction at specific sites of the cell surface and/or in a particular
stage of cell growth.
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References |
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