Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca and Departamento de Microbiología y Genética, Universidad de Salamanca 37007 Salamanca, Spain
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Saccharomyces cerevisiae CHS7 gene encodes an integral membrane protein located in the ER which is directly involved in chitin synthesis through the regulation of chitin synthase III (CSIII) activity. In the absence of CHS7 product, Chs3p, but not other secreted proteins, is retained in the ER, leading to a severe defect in CSIII activity and consequently, to a reduced rate of chitin synthesis. In addition, chs7 null mutants show the yeast phenotypes associated with a lack of chitin: reduced mating efficiency and lack of the chitosan ascospore layer, clear indications of Chs7p function throughout the S. cerevisiae biological cycle.
CHS3 overexpression does not lead to increased levels of CSIII because the Chs3p excess is retained in the ER. However, joint overexpression of CHS3 and CHS7 increases the export of Chs3p from the ER and this is accompanied by a concomitant increase in CSIII activity, indicating that the amount of Chs7p is a limiting factor for CSIII activity. Accordingly, CHS7 transcription is increased when elevated amounts of chitin synthesis are detected.
These results show that Chs7p forms part of a new mechanism specifically involved in Chs3p export from the ER and consequently, in the regulation of CSIII activity.
Key words: chitin synthesis; chitin synthase III; endoplasmic reticulum export; morphogenesis; cell wall ![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CHITIN is a structural polymer present in most fungal
cell walls. Although its amount varies from 1-30%
of the cell wall, it has been shown to be indispensable for the maintenance of the fungal cell (reviewed in
Cabib et al., 1996). In Saccharomyces cerevisiae, chitin accounts for only 3% of the cell wall mass. However, some
evidence indicates that S. cerevisiae cannot survive without
chitin (Bowers et al., 1974
; Shaw et al., 1991
). S. cerevisiae contains three different chitin synthases (CSs)1 responsible for the synthesis of this polymer: CSI acts as a repair
enzyme at the time of cytokinesis (Cabib et al., 1989
, 1992
); CSII makes the chitin disk of the primary septum
that separates mother and daughter cells (Sburlati and
Cabib, 1986
; Shaw et al., 1991
); and CSIII makes 90-95%
of the cellular chitin, including the chitin synthesized
during mating and sporulation (Roncero et al., 1988b
;
Valdivieso et al., 1991
; Pammer et al., 1992
). Each CS contains at least one catalytic subunit encoded respectively by
CHS1, CHS2, and CHS3 gene products (reviewed in Bulawa, 1993
; Cabib et al., 1996
). These genes show significant similarity, but the differences among them should
account for the different kinetics and regulatory requirements of each CS (Choi and Cabib, 1994
).
The fact that each CS has a different cellular function
suggests that the activity of each enzyme must be spatially
and temporally regulated. In the case of CSI and CSII, it
has been proposed that these would be regulated at the
transcriptional (Pammer et al., 1992) and posttranslational
(Choi et al., 1994a
; Uchida et al., 1996
) levels, the latter
mechanism achieved through proteolytic processing (reviewed in Cabib et al., 1996
). By contrast, CSIII is not
proteolytically regulated and transcriptional regulation of
CHS3, although it does exist (Pammer et al., 1992
), does
not control CSIII activity (Choi et al., 1994a
; Chuang and
Schekman, 1996
; Cos et al., 1998
). In addition to CHS3,
three other genes, CHS4, CHS5, and CHS6, have been
shown to be required for CSIII activity (reviewed in Bulawa, 1993
). Chs4p has a dual function: it is a direct activator of CSIII activity (Trilla et al., 1997
) and at the same
time is responsible for anchoring, through Bni4p, of Chs3p
to the septin ring (DeMarini et al., 1997
). Chs5p is involved in the polarized transport of Chs3p in specialized
vesicles (Santos and Snyder, 1997
). However, its function
is not restricted to Chs3p transport, since it is also involved
in the transport of other proteins during mating (Santos et al.,
1997
). Chs6p is required for Chs3p transport in a later step
than Chs5p (Ziman et al., 1998
). It also has been shown
that Chs3p is subject to endosomal internalization (Ziman
et al., 1996
), although the biological meaning of this process is not known. It therefore seems clear that the function of CSIII mainly depends on proper Chs3p transport and localization.
In addition to temporally and spatially regulated chitin
synthesis during vegetative growth, there are several circumstances in the life cycle of S. cerevisiae where chitin
synthesis is altered. Chitin synthesis is significantly increased during mating and Calcofluor treatment and in
both instances this increase depends on CSIII activity
(Roncero et al., 1988a,b). During sporulation, CSIII is required to make chitin the precursor of chitosan, a component of the outermost layer of the ascospore wall (Pammer et al., 1992
). However, it is not yet known if chitin/chitosan synthesis is increased during this process. Despite all these findings, there is still no clear picture of how CHS genes
can regulate chitin synthesis in vivo, and to date only
Chs4p can be envisaged as a direct regulator of CSIII activity.
This paper addresses CHS7, a new S. cerevisiae gene required for CSIII activity. CHS7 is involved in the regulation of CSIII activity by controlling Chs3p export from the ER. The significance of this specific control mechanism is discussed within the framework of the hierarchy of proteins involved in the control of CSIII activity.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains, Growth Conditions, and Genetic Methods
Yeast strains used in this study are listed in Table I. S. cerevisiae strains
were grown in YEPD (1% Bacto yeast extract, 2% peptone, 2% glucose)
or SD medium (2% glucose, 0.7% Difco yeast nitrogen base without
amino acids, 0.07% amino acids solid mix). Growth supplements were added
to SD medium when required. Escherichia coli was grown in LB medium
(1% bactotriptone, 0.5% bacto yeast extract, 1% NaCl) supplemented
with 100 µg/ml ampicillin. Standard methods were used for DNA manipulations (Sambrook et al., 1989) and yeast genetics (Rose et al., 1990
).
|
Calcofluor resistance was tested by a plate assay (Valdivieso et al.,
1991) in SD medium buffered with 50 mM potassium hydrogen phthalate,
pH 6.1, and supplemented with different Calcofluor concentrations (50-
1,000 µg/ml; Bayer Industrial Corporation). Calcofluor staining was observed after growing cells in the presence of 75 µg/ml Calcofluor for 2 h at
30°C.
Construction of Plasmids and Strains
Gene replacement was performed basically as described by Rothstein
(1983). The
chs7-1 disruption was constructed by replacing the 1.2-kb
NdeI-NdeI fragment containing the CHS7 coding region (from nucleotide
+24-297, downstream from the stop codon) with the HIS3 gene, thus creating pTM10. The NdeI downstream site is still 724 bp ahead of the next
ATG, therefore this deletion should not affect the YHR143 open reading
frame (ORF). Strains W303-1A and Y1306 were transformed with a linear
3.5-kb EcoRV-EcoRV fragment from pTM10, containing the chs7::HIS3
gene, giving rise to strains JAY6 and JAY27. The
chs7-2 disruption was
constructed by replacement of the 1.2-kb NdeI-NdeI fragment with the
HIS2 gene, affording pTM100. By digestion of pTM100 with SacI, a 4.1-kb
fragment containing the chs7::HIS2 gene was used to transform strains
HVY374 and HVY376, creating strains JAY25 and JAY26. The
chs6-1
disruption was constructed using a pGEM-CHS6 plasmid, in which the
1.6-kb BalI-EcoRI internal fragment of CHS6 was replaced with the
URA3 gene, thus creating pTM12. By digestion of pTM12 with SalI-SphI,
a 3.0-kb fragment containing the chs6::URA3 gene was used to transform
strain Y1306, obtaining JAY28. Correct replacement of the CHS7 and
CHS6 loci was determined by PCR analysis and tests of Calcofluor resistance. To construct a fusion gene encoding the full-length Chs7p fused to
the green fluorescent protein (GFP; Martin-Chalfie et al., 1994
), a NotI
site was created by directed mutagenesis at the end of the CHS7 coding
region to give pTM14. A 0.7-kb NotI-NotI fragment from pTM13 containing the GFP-encoding sequence (Fernandez-Abalos et al., 1998
) was
ligated into NotI-digested pTM14, thus creating pTM15 (pRS316::CHS7-GFP). CHS7::3XHA construction (pTM16) was achieved by inserting a
115-bp NotI-NotI DNA fragment containing three copies of the hemagglutinin epitope (HA) at the same NotI site of pTM14. The functionality
of the hybrid proteins was determined by complementation of the phenotypes associated with
chs7. For intracellular localization of Sec63p,
15Daub cells were transformed with plasmid pRS315::SEC63-MYC,
kindly provided by R. Schekman (University of California, Berkeley, CA;
Lyman and Schekman, 1997
).
Multicopy plasmids pRS423::CHS3 or pRS423::CHS3-3XHA were obtained by inserting the EcoRI-SalI fragment of pHV7 that contains either
CHS3 or CHS3-3XHA genes into pRS423 (Cos et al., 1998). pRS425::
CHS4 was done by cloning the CHS4 gene as a BamHI fragment into
pRS425 (Trilla et al., 1997
). Similarly, for construction of pRS426::CHS7,
CHS7 gene was cloned as a EcoRV-StuI fragment (pTM11; this work)
into pRS426. Plasmids pRS423, pRS425, and pRS426 have been previously described (Christianson et al., 1992
).
Chitin and Chitin Synthase Activity Determinations
Chitin measurements were performed as described (Bulawa et al., 1986)
using chitinase from Serratia marcescens (Sigma Chemical Co.). GlcNAc
release was determined colorimetrically by the method of Reissig et al.
(1955)
. Total amounts of chitin are expressed as nanomoles of GlcNAc
liberated per 100 mg of cells. For CS activity assays, total cell membranes
were prepared from 250 ml of exponentially growing cells (2 × 107 cells/ml)
as described by Valdivieso et al. (1991)
. CS activity was measured essentially as described in Choi and Cabib (1994)
: CSI activity was assayed in
50 mM Tris-HCl, pH 6.5, and 5 mM magnesium acetate; CSII activity was
assayed in 50 mM Tris-HCl, pH 8.0, and 5 mM cobalt acetate; and CSIII
was assayed in 50 mM Tris-HCl, pH 8.0, with 5 mM cobalt acetate and
nickel acetate. For the proteolytic activation step, 2 µl of trypsin (1-3 mg/ml)
was added to the reaction medium and proteolysis activation was stopped
after 15 min of incubation by adding 2 µl of soybean trypsin inhibitor solution. 1.1 mM UDP[14C]GlcNAc (Nycomed Amersham; 400 cpm/nmol)
was used as substrate for the reaction. Newly synthesized chitin was determined by measuring the radioactivity incorporated into the insoluble material after the addition of 10% trichloroacetic acid and filtration through glass fiber filters (Whatman Inc.; Choi and Cabib, 1994
). Specific activity
is expressed as nanomoles of GlcNAc incorporated per hour per milligram of protein.
Direct and Indirect Immunofluorescence
Localization of Chs7p-GFP was observed in exponentially growing cells
containing pTM15. After mounting, images were captured with a Zeiss laser-confocal microscope (LSM 510) with a 63× objective and processed
with Adobe Photoshop software. Localization of Chs3p-3XHA by indirect immunofluorescence was carried out as described in Bähler and Pringle (1998) with slight modifications. Spheroplasts were obtained by treatment with 5 µg/ml Zymolyase 100T (Seikagaku) for 30-45 min at 30°C in
PEMS (100 mM Pipes, 1 mM EGTA, 1 mM MgSO4, 1.2 M sorbitol, pH
6.9) buffer. Mouse HA.11 anti-HA mAb (Berkeley Antibody Co.) was
used at a 1:100 dilution for 16 h at 4°C. Cy3-conjugated anti-mouse secondary antibody (Sigma Chemical Co.) was used at a 1:300 dilution for 45 min at 25°C. After mounting, images were captured with a Zeiss laser-confocal microscope (LSM 510) with a 63× objective. For these experiments,
an epitope-tagged version of Chs3p in cells whose chromosomal locus had
been replaced with CHS3-3XHA was used (Strain Y1306; Santos and Snyder, 1997
). All strains used for immunofluorescence were derived from
Y1306. Sec63p-Myc was immunolocalized with a similar protocol, but using supernatants of 9E10 hybridome at 1:100 dilution as primary antibodies (Evan et al., 1985
).
Northern Blot Analysis
For Northern blot analysis, total RNA was prepared as described by Sambrook et al. (1989) from cells grown under different conditions. 12.5 µg of
total RNA was loaded per lane and after electrophoresis, transferred to
Hybond membranes (Nycomed Amersham). Hybridization was carried
out as described (Sambrook et al., 1989
) using a 1.2-kb NdeI-NdeI fragment from CHS7 as a probe. For quantitative analysis, Northern blots
were exposed to PhosphorImager screens (BASS 1500; FujiFilm).
-Factor treatment for Northern blot experiments was carried out in exponentially growing cells using YEPD medium supplemented with 5 µM
-factor (Choi et al., 1994a
).
Subcellular Fractionation Experiments
Cell lysates were prepared (as described in Santos and Snyder, 1997) from
a JAY25 strain transformed with pRS315::SEC63-MYC and pRS316::
CHS7-3XHA plasmids. In brief, exponentially growing cells in SD medium (1.5 g wet wt), were resuspended in 5 ml of 17% sucrose (wt/vol) in
50 mM Tris-HCl, pH 7.5, and 1 mM EDTA containing the protease inhibitor cocktail (1 mM phenylmethanesulfonil fluoride and 1 mg/ml each of
leupeptin, pepstatin, and aprotinin), and broken by vortexing with glass
beads. Lysated cells were centrifuged at 1,500 g for 10 min. The cleared
supernatant was layered on top of 33 ml of a linear sucrose gradient (10-
65% wt/vol) in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and centrifuged in
a SW28 rotor at 25,000 rpm for 20 h at 4°C. 1.25 ml fractions were collected from the bottom using a peristaltic pump. Equal volumes of each fraction were examined for their content of Pma1p, Kre2p, Sec63p-MYC, and Chs7p-HA, determined by SDS-PAGE and immunoblot with their respective antibodies. Rabbit polyclonal antibodies (1:30,000 dilution;
kindly provided by Dr. R. Serrano, Universidad de Valencia, Valencia,
Spain) were used to detect Pma1p, a typical plasmatic membrane marker.
Kre2p, a medial-Golgi compartment protein (Lussier et al., 1995
), was detected using polyclonal antibodies (1:1,000 dilution) kindly supplied by H. Bussey (McGill University, Montreal, Qúebec, Canada). Sec63-MYC, an
ER marker, was detected using monoclonal anti-MYC antibodies (supernatants of 9E10 hybridome, 1:5,000 dilution). Mouse HA.11 anti-HA
mAb (Berkeley Antibody Co.; 1:2,000 dilution) was used to detect Chs7p-HA. In all cases, Western blots were developed using ECL (Nycomed
Amersham).
Western Blot Analysis of Chs3p
Chs3p-3XHA expression levels were determined in total cellular extracts
by Western blot analysis as described in Cos et al. (1998). Blotted proteins
were incubated with mouse HA.11 anti-HA antibody (Berkeley Antibody
Co.; 1:2,000 dilution) and developed using ECL (Nycomed Amersham).
Endoglycosidase H (EndoH) treatment was carried out on Chs3p-3XHA after immunoprecipitation under denaturing conditions as described by Cos et al. (1998). The immunoprecipitated complex was subjected to Western blot analysis.
Other Methods
Protein was measured by the method of Lowry et al. (1951). Yeast strains
were mated quantitatively as described in Santos et al. (1997)
. The frequency of diploid formation was estimated as the number of diploids
formed out of the total number of cells. Exoglucanase activity was assayed
as described by San Segundo et al. (1993); the method is based on the release of reducing sugar groups from laminarin, which were quantified by
the method of Somogyi (1952)
and Nelson (1957)
. The
-factor secretion
assay was developed as described in Michaelis and Herskowitz (1988)
.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of CHS7
A new screening for S. cerevisiae mutants resistant to Calcofluor was carried out to identify new genes involved in
chitin synthesis. Ethyl methanesulfonate-mutagenized JAY1
cells (~20% of survival) were plated in YEPD media supplemented with 1 mg/ml Calcofluor exactly as described in
Roncero et al. (1988b). After 4 d of incubation, 53 bona
fide independent clones which were able to grow were selected (mutation frequency 1.1 × 10
5). These mutants
were back-crossed with previously known Calcofluor resistant and chitin-deficient mutants (chs3, chs4, and chs5). Diploid analysis indicated that most mutants belonged to
chs3 (83%), chs4 (5.7%), or chs5 (5.7%) complementation
groups. Only three of them, cwr6, cwr12, and crw19 (Calcofluor white resistant), defined new complementation
groups. Further genetic testing indicated that mutants
cwr12 and cwr19 belonged to the same group. A preliminary characterization of these mutants indicated that all
showed defects in chitin synthesis.
The genes affected in cwr12 and cwr6 mutants were
cloned by complementation of the Calcofluor resistant
phenotype after transformation with a centromeric library
(Rose et al., 1987). Complementation was confirmed to be
dependent on the presence of plasmid and complementing plasmids were isolated in E. coli for further work. Originally isolated plasmids were subjected to endonuclease restriction mapping and the results obtained indicated that
the plasmid complementing cwr12 contained the previously described CHS6/CSD3 gene (Bulawa, 1992
). Therefore, its study was discontinued. The DNA fragment that
complemented the cwr6 mutant contains several ORFs
(Fig. 1 A). Subcloning experiments indicated that the minimum fragment able to complement was the StuI-EcoRV
fragment included in pTM11. With these results, we were
able to obtain a preliminary identification of YHR0142w
as the gene that complements cwr6 mutation. Due to the
defect in CS observed in cwr6 mutant, we named this gene
CHS7 following accepted nomenclature (Cabib, 1994
).
|
CHS7 encodes a protein of 316 amino acids that contains six or seven putative transmembrane domains (Fig. 1, B and C). It has no significant homology with any known protein, although the search programs showed limited homologies with other transmembrane regions.
Characterization of chs7 Mutants
We made several chs7 null mutants by replacing the CHS7
ORF by different auxotrophic markers (Materials and
Methods). Table II shows some of the characteristic phenotypes associated with this mutation. chs7 null mutants
had only 13.6% of the wild-type chitin, a defect that was
associated with a comparable decrease in CSIII activity.
Due to this defect in chitin synthesis, chs7 cells were resistant to 1 mg/ml Calcofluor and they did not show enlarged
septa after Calcofluor treatment. In addition, chs7/
chs7 diploids showed compact ascospores, a defect associated
with the absence of the chitosan layer (Pammer et al.,
1992
). chs7 mutations also reduced their mating efficiency
to a level similar to that of chs3 mutants (Table II). It
should be noted that the original cwr6 mutants showed
similar, but less pronounced phenotypes. As expected,
wild-type CHS7 (YHR0142w) in a centromeric plasmid
complemented the phenotypes observed in the null (Table
II, last column) or point (not shown) chs7 mutants. Once
we had constructed null mutants, we confirmed the allelism between YHR142w and CHS7 genetically. Diploid
strain yhr142::HIS3/cwr6 was resistant to Calcofluor and analysis of 18 tetrads in this cross showed a Calcofluor
resistance segregation of 4:0. Therefore, CHS7 and YHR142w must be the same gene.
|
Analysis of CHS7 Expression
To analyze CHS7 expression levels, we determined CHS7
mRNA levels in cells harvested after growth under different conditions. CHS7 was detected in all conditions (Fig.
2) as a single band of ~1.1 kbp, in clear agreement with sequence data. Therefore, CHS7 is expressed constitutively.
However, as shown in Fig. 2 A, -factor treatment (mimicking the mating process) increased CHS7 expression 3.5 times after only 15 min; shortly after, vegetative levels
were resumed. Calcofluor treatment also increased CHS7 expression twofold (Fig. 2 A, right). Despite its constitutive expression during vegetative growth, CHS7 mRNA
levels were strongly increased during sporulation, reaching
a maximum (~24-fold more) after 10 h of sporulation
induction. Thereafter, CHS7 mRNA levels slowly decreased. The typical expression pattern observed resembled that of a middle sporulation gene (Krisak et al., 1994
;
Hepworth et al., 1998
). These results allowed us to conclude that the expression of CHS7 is transcriptionally regulated. In addition, CHS7 transcription increased under all
growth conditions in which chitin synthesis was increased.
|
Chs7p Is a Membrane Protein Localized in the ER
Chs7p was tagged at the COOH terminus either with the
3XHA epitope or GFP (Materials and Methods) to determine the subcellular localization of Chs7p. Chimeric proteins Chs7p-3XHA or Chs7p-GFP complemented the Calcofluor resistance phenotype of chs7 null mutant (JAY6
strain). In addition, the chimeric proteins restored wild-type levels of chitin synthesis in this strain, as determined by chitin staining with Calcofluor (Cos et al., 1998). It can be concluded that Chs7p-3XHA and Chs7p-GFP are fully
functional. Western blot analysis indicated that Chs7p-3XHA runs as a 35-kD protein that is localized in the particulate fraction (data not shown). Molecular size and
subcellular localization are in clear agreement with sequence predictions. Logarithmically growing cells expressing Chs7p-GFP from its own promoter showed the fluorescent staining depicted in Fig. 3 A. Chs7p-GFP was
uniformly localized in the nuclear periphery and in discrete patches associated with the cytoplasmic membrane.
This pattern coincides with that of Sec63p-Myc (Fig. 3 B),
a typical ER marker (Sadler et al., 1989
). The vacuolar
staining observed seems to be an artifact because it does
not appear in all the cells and it does not show the typical green color of GFP (data not shown). A similar distribution in the ER was observed by indirect immunofluorescence in cells expressing Chs7p-3XHA (data not shown).
No polarized distribution of Chs7p was observed in either
case. To confirm the immunofluorescence results a subcellular fractionation experiment was carried out. Total cellular extract were loaded into the top of a linear sucrose density gradient and after centrifugation, Pma1p, Sec63p-MYC, Kre2p, and Chs7p-3XHA distribution was analyzed
by Western blot as described in Materials and Methods.
Fig. 3 C shows that membrane protein Pma1p is localized
at the bottom of the gradient, mainly between fraction 1 to
4. Sec63p is localized from fractions 5 to 17, with a significant peak between the 11 to 15 fractions. Chs7p distribution is quite similar with that of Sec63, also showing maximum accumulation between 11 to 15 fractions. Golgi
compartment distribution, marked by Kre2p (Lussier et
al., 1995
), is displaced to lighter fractions. Therefore, it can
be concluded that Chs7p is an ER membrane protein.
|
Chs7p Is Required for Chs3p Export from the ER
Functional CSIII activity depends on the appropriate synthesis and transport of Chs3p, its catalytic subunit. Chs7p
does not seem involved in the synthesis of Chs3p, since
Chs3p levels in chs7 mutants are similar to those observed in wild-type (Fig. 4 A). Therefore, Chs7p could be
involved in the transport of Chs3p, a process that is mediated by Chs4p, Chs5p, and Chs6p. Following a strategy
similar to that used in the characterization of these genes,
we localized an HA-tagged version of Chs3p (Santos and Snyder, 1997
) in wild-type and chs7-null mutants. As previously reported (Chuang and Schekman, 1996
; Santos
and Snyder, 1997
), Chs3p-3XHA localized in the base of
the emerging bud in wild-type strains (Fig. 4 C, a). However, in
chs7 mutants all the Chs3p-3XHA remained in
the ER, showing a perinuclear localization with partial association with the plasma membrane (Fig. 4 C, b, compare
to Fig. 3 A). No polarized distribution of Chs3p-3XHA
was observed in
chs7 mutants. Higher magnification
(Fig. 4 C, c) indicated that the perinuclear staining was
punctuated, suggesting possible Chs3p aggregation. In addition, it is important to notice that although retained in
the ER, Chs3p-3XHA is correctly core-glycosylated in
chs7 mutants (Fig. 4 B). Therefore, it can be concluded
that Chs7p is required for Chs3p export from the ER and
Chs7p must be the initial step in the hierarchy of proteins
required for CSIII activity. To confirm this hypothesis, we
analyzed the localization of Chs3p-3XHA in
chs4
chs7,
chs5
chs7, or
chs6
chs7 double mutants (Fig. 5, B, D,
and F). In all cases, Chs3p was retained in the ER and did
not show the localization described for
chs4,
chs5, or
chs6 single mutants. As previously reported (DeMarini et al., 1997
), the distribution of Chs3p in
chs4 mutant was
polarized (Fig. 5 A). However, it was not as densely
packed at the base of the bud as in the wild-type (Fig. 4 C,
a). In
chs5 and
chs6 mutants, Chs3p was localized in internal vesicles (Fig. 5, C and E) without polarized distribution (Santos and Snyder, 1997
; Ziman et al., 1998
). From
these results, we conclude that the chs7 mutation is epistatic to the chs mutations and that Chs7p is necessary for
Chs3p export from the ER.
|
|
Chs7p Is Not Required for the Transport of other Proteins
Chs7p could play a general role in secretion or protein
sorting rather than being specific for chitin synthesis. To
test this hypothesis, we analyzed the behavior of several
secreted or sorted proteins in chs7 mutants.
-factor and
exo-
(1-3)glucanase are two extracellular S. cerevisiae
proteins that follow the normal secretion route. Fig. 6
shows that the expression levels of these two proteins are
unaffected in
chs7 mutants. Similarly, CSI and CSII, the
other two CS activities present in S. cerevisiae, showed wild-type levels in
chs7 mutants. Apparently, Chs7p is
not required for secretion or sorting of other cellular proteins.
|
In addition to the regular secretion mechanism, S. cerevisiae contains several other mechanisms involved in the
specific assembly and transport of certain proteins. Sorting
of vacuolar ATPase (v-ATPase) or several amino acid
permeases to cytoplasmic membrane has been shown to
depend on specific mechanisms of export from the ER (reviewed in Kaiser et al., 1997), raising the possibility of the
involvement of Chs7p is such processes. However, chs7
null mutants grew in glycerol and did not show synthetic
lethality with other amino acid auxotrophies, clear indications that v-ATPase and amino acid permeases function
properly in
chs7 mutants.
Chs7p Is a Limiting Factor in the Constitution of Functional CSIII Activity
So far we have shown that Chs7p is required for Chs3p export. However, from Fig. 2 it is also apparent that CHS7
expression increases when more chitin is synthesized.
These observations suggest that the level of expression of
CHS7 could be involved in the control of CSIII activity. If
so, this would explain why overexpression of CHS3 does
not increase CSIII levels. To confirm this possibility, we
measured CSIII activity in strains overexpressing CHS
genes in different combinations. Overexpression of CHS3
(Cos et al., 1998) or CHS7 (Fig. 7) alone had no significant
effect on CSIII. As previously reported (Trilla et al.,
1997
), overexpression of CHS4 increased CSIII activity
and reduced its trypsin dependence. Joint overexpression
of CHS4 and CHS3 or CHS4 and CHS7 did not alter the
effect of CHS4 on CSIII (Fig. 7). Cooverexpression of CHS3 and CHS7 increased total CSIII almost four times.
However, basal activity was only increased 1.3 times and,
as a consequence, the activation factor by trypsin treatment was almost five times, in comparison with 1.6-2
times, for the controls. Joint overexpression of CHS4,
CHS3, and CHS7 did not further increase total CSIII activity. However, basal activity was increased more than
twofold. Therefore, activation by trypsin treatment was reduced to 2.5 in this case. Taken together, these results indicate that the amounts of Chs7p and Chs3p act as limiting
factors to total CSIII activity. In addition, Chs4p levels are
also limiting in the reconstitution of an in vitro fully active
CSIII. Overexpression of CHS3, CHS4, and CHS7 genes
also induced a modest, but significant, increase in chitin
synthesis in vivo because a strain overexpressing these three genes contained ~68 ± 11% more chitin than the
corresponding wild-type. This increase in chitin synthesis
was not observed if only CHS3 and CHS7 were overexpressed (not shown).
|
These results clearly suggest that the amount of Chs7p is
a limiting factor in vivo for CSIII activity and therefore,
could be the reason for the absence of an increase in CSIII
activity after CHS3 overexpression. In fact, immunostaining of cells overexpressing CHS3-3XHA alone revealed a
severe retention of Chs3p in the ER even in the presence
of normal amounts of Chs7p (Fig. 8 A). Cooverexpression of CHS3 with CHS7 dramatically reduced Chs3p accumulation in the ER (Fig. 8 B) and only residual staining was
observed at ER level. In this case, Chs3p was mainly localized in discrete patches in the cytoplasm, which in some
cells showed a polarized distribution (Fig. 8 B, arrows).
This distribution resembled an intermediate stage between chs6,
chs5, and
chs4 mutants (Fig. 5, A, C, and E). In sum, Chs3p excess is not retained in the ER in the
presence of high levels of Chs7p and hence, higher levels
of functional CSIII activity are achieved.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chitin is a minor, but essential polymer in the S. cerevisiae
cell wall. Despite its low abundance, three different CS activities have been described in this yeast (Cabib et al.,
1996). From a quantitative point of view, CSIII activity is
the most important, since it is involved in the synthesis of
90-95% of cell wall chitin during vegetative growth (Roncero et al., 1988b
; Valdivieso et al., 1991
; Bulawa, 1992
). In
addition, it is required for the synthesis of new chitin that
takes place during the mating and sporulation processes
(Roncero et al., 1988b
; Pammer et al., 1992
).
Recently, it has been shown that the transcriptional regulation of CHS3, the gene that encodes the catalytic subunit of CSIII, is not the control mechanism for CSIII activity (Choi et al., 1994a; Chuang and Schekman, 1996
; Cos
et al., 1998
). Therefore, the identification of genes involved in the control of CSIII and chitin synthesis gains interest. Three proteins, Chs4p, Chs5p, and Chs6p, have
been identified, so far, as factors involved in the control of
CSIII activity. This report deals with the isolation of a new
gene, CHS7, also involved in the control of CSIII activity. CHS7 was isolated by complementation of a mutant resistant to Calcofluor, showing that this strategy still has potential, although most (83%) of the mutants isolated belong to the chs3 complementation group.
chs7 mutants have reduced levels of CSIII activity and
chitin in their cell walls (Table II). This defect is comparable to that observed in chs3 null mutants (Cos et al., 1998
)
and stronger than that detected in chs4 (Trilla et al., 1997
),
chs5 (Santos et al., 1997
), or chs6 mutants (Bulawa, 1993
),
underscoring the relevance of this gene in the control of
CSIII activity. CHS7 encodes a small protein with six or
seven putative transmembrane domains (Kyte and Doolittle, 1982
). Repeated database searches using different algorithms did not reveal any protein with significant homology to this protein. Some Chs7p regions with similarity
to other integral membrane proteins probably reflect the
hydrophobicity of the membrane-spanning domains rather
than protein conservation. Chs7p colocalized with Chs3p
in a crude particulate cell fraction and we therefore expected to find colocalization of Chs3p and Chs7p in the
plasma membrane. However, Chs7p was localized exclusively in the ER (Fig. 3), showing the typical localization
of other ER proteins such as Sec63p (Sadler et al., 1989
;
Fig. 3 B). This localization does not seem to be an artifact
of the chimeric Chs7p-GFP because similar results were
obtained with Chs7p-3XHA either in immunofluorescence (data not shown) or in subcellular fractionation (Fig.
3 C) experiments.
A functional explanation to account for this localization,
unique among Chs proteins, became apparent when we
observed that Chs3p accumulates at the ER in the absence
of Chs7p (Fig. 4 C, b). However, the Chs3p protein accumulated in the chs7 mutant is correctly core-glycosylated
(Fig. 4 B), a clear indication that Chs3p is translocated and
correctly localized in the ER compartment in this mutant.
Therefore, Chs7p should be required for Chs3p export
from the ER. Moreover, the chs7 mutation is epistatic
over other chs mutations, suggesting that Chs7p is the initial element in the hierarchy of proteins involved in CSIII
activity. These results explain the stronger defect in CSIII
activity observed in chs7, as compared with
chs4,
chs5,
or
chs6 mutants. It should be noted that Chs4p, Chs5p, or
Chs6p is not required for Chs3p export from the ER (Fig.
5). At this point, all the data points to the specific involvement of Chs7p in the export of Chs3p from the ER. However, the concomitant participation of other CHS products
in this export cannot be ruled out. Despite this, preliminary evidence indicates that Chs5p and Chs4p are efficiently exported in
chs7 mutants (data not shown) and
therefore, their participation is rather unlikely.
To date, there is no clear relationship between the levels
of expression of different CHS genes and chitin levels, although regulation of chitin synthesis has been observed
during the yeast life cycle (Roncero et al., 1988a,b; Pammer et al., 1992
). It should especially be stressed that overexpression of CHS3 several times (~30) did not increase
CSIII activity or chitin levels (Cos et al., 1998
) and that
Chs3p levels remain stable during vegetative growth (Choi
et al., 1994a
; Chuang and Schekman, 1996
). Under these
circumstances, only Chs4p can be envisaged as a controller of CSIII activity (Bulawa, 1993
; Trilla et al., 1997
). Might
Chs7p also be involved in the control of CSIII? Fig. 2
shows some results in favor of this hypothesis, since CHS7
is the only CHS gene whose transcription is increased under all conditions in which chitin synthesis is seen to be increased: mating, Calcofluor treatment, and sporulation.
The increase in CHS7 expression during sporulation is especially relevant and its pattern of induction resembles that of a middle-late sporulation gene (Hepworth et al.,
1998
). The timing of expression places CHS7 among
the sporulation genes involved in spore wall formation
(Krisak et al., 1994
; Hepworth et al., 1998
). Further and
more direct confirmation of this hypothesis was achieved
after joint overexpression of CHS3 and CHS7 genes. CSIII
activity rose dramatically after the overexpression (Fig. 7),
reaching values several-fold higher than those of the control strain. This increase depends on joint overexpression,
suggesting the requirement of a balanced expression of
both genes.
Biochemically, we have shown that Chs7p acts as a limiting factor when Chs3p levels are increased, but the reason
at an intracellular level for this limitation remains unknown. As shown in Fig. 8 A, the cells cannot process an
excess of Chs3p and therefore, overexpression of CHS3
leads to Chs3p retention in the ER. However, if CHS7 is
overexpressed in strains with high levels of Chs3p, this
protein is released from the ER (Fig. 8 B). In addition, the
localization of Chs3p in this strain resembles that observed in chs6 (Ziman et al., 1998
) or
chs5 (Santos and Snyder,
1997
) mutants (bright internal spots) with partial polarization (Fig. 8 B, arrows), as in chs4 mutants (Fig. 5 B; DeMarini et al., 1997
). These results indicate that although
Chs3p is efficiently exported from the ER, its transport to
its physiological site of action is impaired, probably due to
some other limiting factors. In this condition, the high
amount of active CSIII detected suggests that the limiting
factor, if it exists, should appear at the time of or after
CHS6 participation, because mutations in genes that function early on the pathway show severe defects in CSIII activity (Bulawa, 1993
; Santos et al., 1997
).
Chitin synthesis increases in S. cerevisiae during -factor
treatment and this process is associated with the increase
in CSIII activity (Roncero et al., 1988a
). Recently, it has
been shown that during
-factor treatment Chs3p levels
increase significantly (Cos et al., 1998
). However, no
Chs3p is localized at the ER (Santos and Snyder, 1997
).
We have shown that the effect of increased Chs3p levels
on chitin synthesis depends mainly on correct Chs3p export from the ER. Therefore, the increase in CHS7 transcription observed during
-factor treatment (Fig. 2 A)
guarantees the export of excess Chs3p and the increase in
chitin synthesis required during this process.
Overexpression of CHS3 and CHS7 leads to a significant increase in CSIII activity. However, the activity
obtained under these conditions was highly zymogenic.
When we increased Chs4p levels in the same experiment,
the CSIII dependence on trypsin decreased significantly.
Therefore, it seems that Chs4p is also a limiting factor for
this activity. We cannot be sure if Chs4p function is mediated by proteolytic processing of CSIII, but it does seem clear that in vitro trypsin treatment of CSIII resembles the
activation carried out in vivo by Chs4p. Moreover, the
strain carrying the triple overexpression also showed a significant increase in cellular chitin levels (68% higher than
controls) indicating that these genes not only participate in
the control of CSIII, but also in the in vivo control of chitin
synthesis. We cannot expect a perfect correlation between
CSIII and chitin levels, since there could be as yet undescribed cellular determinants participating in this process.
In addition, problems of interference could arise in the
replication of three independent plasmids that could mask quantitative interpretation of these data. By contrast,
strains overexpressing only CHS3/CHS7, although they
showed high CSIII activity, did not show any increase in
chitin levels. This result is the first experimental evidence
that Chs4p is an in vivo regulator of chitin synthesis because until now, the Chs4p function has only been determined in CSIII measurements in vitro (Bulawa, 1993; Choi
et al., 1994b
; Trilla et al., 1997
).
With this evidence in mind, it can be concluded that the extent of CSIII activity in vivo depends on a delicate balance between the levels of Chs3p, Chs4p, and Chs7p proteins, a balance that ensures the level of chitin synthesis required at each moment of the S. cerevisiae life cycle, besides the specific roles of CSI and CSII activities.
At this point, we are still unable to pinpoint the exact
role of Chs7p in the export of Chs3p. However, we can
compare this system with the assembly of v-ATPase or the
transport of amino acid permeases, the two previously reported cases in S. cerevisiae that involve specific proteins
in their exit from the ER (reviewed in Kaiser et al., 1997).
v-ATPase mature complex is assembled in the ER with
the help of several proteins, such as Vma12p or Vma22p,
which do not leave the ER and therefore do not participate in the active v-ATPase complex (Graham et al.,
1998
). Likewise, Shr3p is required for the release of several amino acid permeases from the ER (Ljungdahl et al.,
1992
). If this protein is not present, the amino acid permeases cannot be loaded into COPII vesicles (Kuehn et al.,
1996
, 1998
). We can exclude the participation of CHS7 in
any of these processes because chs7 mutants did not show phenotypes associated with the lack of v-ATPase or the
amino acid permeases. The role of Chs7p in the export of
Chs3p could be similar to that reported in any of these
cases. In the absence of Chs7p, the CSIII complex, including Chs3p, is either not assembled or not loaded into
secretory vesicles. There is indirect evidence suggesting
that Chs7p could have a similar role to Shr3p. First of all,
Shr3p and Chs7p, although lacking significant homology, have a very similar secondary structure with several transmembrane domains. This type of secondary structure is
typical of resident ER proteins (outfitters) rather than
proteins loaded into cargo vesicles (escorts or guides; Herrmann et al., 1999
). Chs7p also lacks the protein sequences
involved in COPI mediated retrograde transport. In addition, we have been unable to detect Chs7p outside of
the ER compartment. However, we cannot exclude that
Chs7p is rapidly recycled between the ER and Golgi compartments. The answer to this question will have to await
the description of the nature of the secretory vesicles involved in the transport of Chs3p.
The results reported here indicate that CHS7 is part of a specific mechanism for CS export and its presence in vertebrates is therefore unlikely. This opens a new possibility in the design of an antifungal agent that selectively inhibits chitin synthesis.
![]() |
Footnotes |
---|
Address correspondence to Cesar Roncero, Departamento de Microbiología y Genética. CSIC/Universidad de Salamanca, Edificio Departamental, R-219, Avda Campo Charro s/n, 37007 Salamanca, Spain. Tel.: 34-923-294733. Fax: 34-923-224876. E-mail: crm{at}gugu.usal.es
Received for publication 21 December 1999 and in revised form 7 May 1999.
We thank M.H. Valdivieso, B. Santos, and R. Schekman for plasmids and strains, A. Pandiella and members of the A. Durán laboratory for critical comments on the manuscript, and N. Skinner for language revision. Special thanks are due to D. Baggot (R. Schekman Laboratory) for informing us that Chs3p is retained in the ER when overexpressed and to H. Bussey for providing the anti-Kre2p antibody.
This work has been supported by a Ministerio de Educacion y Ciencia predoctoral fellowship to J.A. Trilla and Comision Interministerial de Ciencia y Tecnologia grants BIO95-0500 and BIO98-0814 to C. Roncero.
![]() |
Abbreviations used in this paper |
---|
CS, chitin synthase; cwr, Calcofluor white resistant; GFP, green fluorescent protein; HA, hemagglutinin epitope; ORF, open reading frame; v-ATPase, vacuolar ATPase.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Bähler, J., and
J.R. Pringle.
1998.
Pom1p, a fission yeast protein kinase that
provides positional information for both polarized growth and cytokinesis.
Genes Dev.
12:
1356-1370
|
2. | Bowers, B., G. Levin, and E. Cabib. 1974. Effect of polyoxin D on chitin synthesis and septum formation in Saccharomyces cerevisiae. J. Bacteriol. 119: 564-575 |
3. | Bulawa, C.E.. 1992. CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis. Mol. Cell. Biol. 12: 1764-1776 [Abstract]. |
4. | Bulawa, C.E.. 1993. Genetics and molecular biology of chitin synthesis in fungi. Annu. Rev. Microbiol. 47: 505-534 |
5. | Bulawa, C.E., M. Slater, E. Cabib, J. Au-Young, A. Sburlati, W.L. Adair Jr., and P.W. Robbins. 1986. The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo. Cell. 46: 213-225 |
6. | Cabib, E. 1994. Nomenclature of genes related to chitin synthesis. Yeast Newsl. XLIII:58. |
7. | Cabib, E., A. Sburlati, B. Bowers, and S.J. Silverman. 1989. Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae. J. Cell Biol. 108: 1665-1672 [Abstract]. |
8. | Cabib, E., S.J. Silverman, and J.A. Shaw. 1992. Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae. J. Gen. Microbiol. 138: 97-102 |
9. | Cabib, E., J.A. Shaw, P.C. Mol, B. Bowers, and W.J. Choi. 1996. Chitin biosynthesis and morphogenetic processes. In The Mycota. Biochemistry and Molecular Biology, Vol. III. R. Brambl, G.A. Marzluf, editors. Springer-Verlag, Berlin. 243-267. |
10. | Choi, W., and E. Cabib. 1994. The use of divalent cations and pH for the determination of specific yeast chitin synthases. Anal. Biochem. 219: 368-372 |
11. | Choi, W., B. Santos, A. Duran, and E. Cabib. 1994a. Are yeast chitin synthases regulated at the transcriptional or the posttranslational level? Mol. Cell. Biol. 14: 7685-7694 [Abstract]. |
12. | Choi, W., A. Sburlati, and E. Cabib. 1994b. Chitin synthase 3 from yeast has zymogenic properties that depend on both the CAL1 and CAL3 genes. Proc. Natl. Acad. Sci. USA. 91: 4727-4730 [Abstract]. |
13. | Christianson, T.W., R.S. Sikorski, M. Dante, J.H. Shero, and P. Hieter. 1992. Multifunctional yeast high-copy-number shuttle vectors. Gene. 110: 119-122 |
14. | Chuang, J.S., and R.W. Schekman. 1996. Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p. J. Cell Biol. 135: 597-610 [Abstract]. |
15. | Cos, T., R.A. Ford, J.A. Trilla, A. Duran, E. Cabib, and C. Roncero. 1998. Molecular analysis of Chs3p participation in chitin synthase III activity. Eur. J. Biochem. 256: 419-426 [Abstract]. |
16. |
DeMarini, D.J.,
A.E.M. Adams,
H. Fares,
C. De Virgilio,
G. Valle,
J.S. Chuang, and
J.R. Pringle.
1997.
A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell
wall.
J. Cell Biol.
139:
75-93
|
17. | Evan, G.I., G.K. Lewis, G. Ramsay, and J.M. Bishop. 1985. Isolation of monoclonal antibodies specific for human c-myc proto-oncogen product. Mol. Cell. Biol. 5: 3610-3616 |
18. | Fernandez-Abalos, J.M., H. Fox, C. Pitt, B. Wells, and J.H. Doonan. 1998. Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans. Mol. Microbiol. 27: 121-130 |
19. |
Graham, L.A.,
K.J. Hill, and
T.H. Stevens.
1998.
Assembly of the yeast vacuolar H+-ATPase occurs in the endoplasmic reticulum and requires a
Vma12p/Vma22p assembly complex.
J. Cell Biol.
142:
39-49
|
20. |
Hepworth, S.H.,
H. Friesen, and
J. Segall.
1998.
NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes
in Saccharomyces cerevisiae.
Mol. Cell. Biol.
18:
5750-5761
|
21. | Herrmann, J.M., P. Malkus, and R. Schekman. 1999. Out of the ER: outfitters, escorts and guides. Trends Cell Biol. 9: 5-7 . |
22. | Kaiser, C.A., R.E. Gimeno, and D.A. Shaywitz. 1997. Proteins secretion, membrane biogenesis, and endocytosis. In The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol. III. J. Pringle, J.R. Broach, E.W. Jones, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 91-228. |
23. | Krisak, L., R. Strich, R.S. Winters, J.P. Hall, M.J. Mallory, D. Kreitzer, R.S. Tuan, and E. Winter. 1994. SMK1, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cerevisiae. Genes Dev. 8: 2151-2161 [Abstract]. |
24. | Kuehn, M.J., R.W. Schekman, and P.O. Ljungdahl. 1996. Amino acid permeases require COPII components and the ER resident Shr3p for packaging into transport vesicles in vitro. J. Cell Biol. 135: 585-595 [Abstract]. |
25. | Kuehn, M.J., J.M. Herrmann, and R. Schekman. 1998. COPII-cargo interactions direct protein sorting into ER-derived transport vesicles. Nature. 391: 187-190 |
26. | Kyte, J., and R.F. Doolittle. 1982. A simple method for displaying the hydropathy of a protein. J. Mol. Biol. 157: 105-132 |
27. | Ljungdahl, P.O., C.J. Gimeno, C.A. Styles, and G.R. Fink. 1992. SHR3: a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast. Cell. 71: 463-478 |
28. |
Lowry, O.H.,
J. Rosebrough,
A.L. Farr, and
R.J. Randall.
1951.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275
|
29. |
Lussier, M.,
A. Sdicu,
T. Ketela, and
H. Bussey.
1995.
Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1 ![]() |
30. | Lyman, S.K., and R. Schekman. 1997. Binding of secretory precursor polypeptides to a translocon subcomplex is regulated by BiP. Cell. 88: 85-96 |
31. | Martin-Chalfie, Y.T., G. Euskirchen, W.W. Ward, and C.D. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science. 263: 802-805 |
32. | Michaelis, S., and I. Herskowitz. 1988. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol. Cell. Biol. 8: 1309-1318 |
33. | Nelson, M.J.. 1957. Colorimetric analysis of sugars. Methods Enzymol. 3: 85-86 . |
34. | Pammer, M., P. Briza, A. Ellinger, T. Schuster, R. Stucka, and M. Brteintenbach. 1992. DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls. Yeast. 9: 1089-1099 . |
35. |
Reissig, J.L.,
J.L. Strominger, and
L.F. Leloir.
1955.
A modified colorimetric
method for the estimation of N-acetyl-aminosugars.
J. Biol. Chem.
217:
959-966
|
36. | Richardson, H.E., C. Wittenberg, F.R. Cross, and S.I. Reed. 1989. An essential G1 function for cyclin-like proteins in yeast. Cell. 59: 1127-1133 |
37. | Roncero, C., M.H. Valdivieso, J.C. Ribas, and A. Duran. 1988a. Effect of Calcofluor white on chitin synthases from Saccharomyces cerevisiae. J. Bacteriol. 170: 1945-1949 |
38. | Roncero, C., M.H. Valdivieso, J.C. Ribas, and A. Duran. 1988b. Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white. J. Bacteriol. 170: 1950-1954 |
39. | Rose, M.D., P. Novick, J. Thomas, D. Bostein, and G. Fink. 1987. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 60: 237-243 |
40. | Rose, M.D., F. Wisnton, and P. Hieter. 1990. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. |
41. | Rothstein, R.J.. 1983. One-step gene disruption in yeast. Methods Enzymol. 101: 202-211 |
42. | Sadler, I., A. Chiang, T. Kurihara, J. Rothblatt, J. Way, and P. Silver. 1989. A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ and Escherichia coli heat shock protein. J. Cell Biol. 109: 2665-2675 [Abstract]. |
43. | Sambrook, J., E.F. Fritsch, and T. Manniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. |
44. | San Segundo, P., J. Correa, C.R. Vazquez de Aldana, and F. del Rey. 1993. SSG1, a gene encoding a sporulation-specific 1,3-B-glucanase in Saccharomyces cerevisiae. J. Bacteriol. 175: 3823-3837 [Abstract]. |
45. |
Santos, B., and
M. Snyder.
1997.
Targeting of chitin synthase 3 to polarized
growth sites in yeast requires Chs5p and Myo2p.
J. Cell Biol.
136:
95-110
|
46. | Santos, B., A. Duran, and M.H. Valdivieso. 1997. CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae. Mol. Cell. Biol. 17: 2485-2496 [Abstract]. |
47. |
Sburlati, A., and
E. Cabib.
1986.
Chitin synthase 2, a presumptive participant in
septum formation in Saccharomyces cerevisiae.
J. Biol. Chem.
261:
15147-15152
|
48. | Shaw, J.A., P.C. Mol, B. Bowers, S.J. Silverman, M.H. Valdivieso, A. Duran, and E. Cabib. 1991. The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J. Cell Biol. 114: 111-123 [Abstract]. |
49. |
Somogyi, M..
1952.
Notes on sugar determination.
J. Biol. Chem.
195:
19-23
|
50. | Trilla, J.A., T. Cos, A. Duran, and C. Roncero. 1997. Characterisation of CHS4 (CAL2), a gene of Saccharomyces cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4. Yeast. 13: 795-807 |
51. | Uchida, Y., O. Shimmi, M. Sudoh, M. Arisawa, and H. Yamada-Okabe. 1996. Characterization of Chitin synthase 2 of Saccharomyces cerevisiae II: both full size and processed enzymes are active for chitin synthesis. J. Biochem. 119: 659-666 [Abstract]. |
52. | Valdivieso, M.H., P.C. Mol, J.A. Shaw, E. Cabib, and A. Duran. 1991. CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae. J. Cell Biol 114: 101-109 [Abstract]. |
53. | Ziman, M., J.S. Chuang, and R.W. Schekman. 1996. Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway. Mol. Biol. Cell. 7: 1909-1919 [Abstract]. |
54. |
Ziman, M.,
J.S. Chuang,
M. Tsung,
S. Hamamoto, and
R. Schekman.
1998.
Chs6p-dependent anterograde transport of Chs3p from the chitosome to the
plasma membrane in Saccharomyces cerevisiae.
Mol. Biol. Cell.
9:
1565-1576
|