* Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280; Department of Molecular and
Cellular Biology, University of Arizona, Tucson, Arizona 85721; § Departimento di Biologia and CRIBI Biotechnology Centre,
Universitá degli Studi di Padova, Padova, Italy I-35121; and
Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720
Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.
THE "neck filaments" of the yeast Saccharomyces
cerevisiae were originally identified by electron microscopy (Byers and Goetsch, 1976 Sequence analysis revealed that Cdc3p, Cdc10p, Cdc11p,
and Cdc12p are members of a family of related proteins,
now known as septins (Sanders and Field, 1994 In addition to Cdc3p, Cdc10p, Cdc11p, and Cdc12p, S. cerevisiae also contains three other septins. Two of these,
Spr3p and Spr28p, are expressed only in sporulating cells
and appear to play roles in the extension of the forespore
membrane and/or in the deposition of spore-wall components (De Virgilio et al., 1996 The localization and timing of septin assembly suggest
that these proteins might play a role in the organization of
cell wall assembly at the bud site and, in particular, in the
formation of the chitin ring. This ring forms at the presumptive bud site just before bud emergence. It remains at
the base of the bud as the bud grows and ultimately forms
part of the bud scar marking the division site on the
mother cell. The chitin ring appears to be formed largely
or entirely through the action of chitin synthase III, one of
the three known chitin synthases in S. cerevisiae (Shaw et
al., 1991 Genetic screens have identified several genes that are
required for chitin synthesis by chitin synthase III (Roncero et al., 1988 In this study, we have investigated the role of the septins
in the spatial localization of chitin deposition. The results suggest that Chs3p is normally localized by its assembly
into a complex that involves the septins, Chs4p, and a
novel protein, Bni4p.
Strains, Growth Conditions, and Genetic and
DNA Methods
The yeast strains used in this study are listed in Table I. Yeast media (YM-P
rich liquid medium, YPD rich solid medium, synthetic complete [SC]1 medium, SC lacking specific nutrients, and sporulation medium) have been
described previously (Lillie and Pringle, 1980 Table I.
S. cerevisiae Strains Used in This Study
Table II.
Plasmids Used in This Study
; Byers, 1981
).
These filaments are ~10 nm in diameter and lie immediately subjacent to the plasma membrane in the mother-bud neck; they appear just before or coincident with bud
emergence and disappear during cytokinesis. Temperature-sensitive mutants defective in any of four genes,
CDC3, CDC10, CDC11, and CDC12, fail to form the neck
filaments at restrictive temperature (Byers, B., and L. Goetsch. 1976. J. Cell Biol. 70:35a; Byers, 1981
; Adams, 1984
). These mutants also exhibit hyperpolarized bud
growth and fail to complete cytokinesis (Hartwell, 1971a
;
Adams and Pringle, 1984
; Slater et al., 1985
). Because the
mutants are not blocked in DNA replication or nuclear division, they arrest at restrictive temperature with multiple
elongated buds and multiple nuclei.
; Longtine
et al., 1996
; Cooper and Kiehart, 1996
). Immunofluorescence studies showed that the septins localize to the
mother-bud neck ~15 min before bud emergence and behave as if they are constituents of the neck filaments
(Haarer and Pringle, 1987
; Ford and Pringle, 1991
; Kim et
al., 1991
). Localization of all four septins to the neck is lost
when any one of the four temperature-sensitive mutants is
shifted to restrictive temperature (Ford and Pringle, 1991
;
Kim et al., 1991
; Longtine et al., 1996
), and several other
lines of genetic and biochemical evidence also suggest that
there are direct interactions among these proteins (Longtine et al., 1996
).
; Fares et al., 1996
). The function of the seventh septin (revealed by the genome-sequencing project) is currently under investigation. Septins have
also been identified in a variety of other fungi and animals
(reviewed by Longtine et al., 1996
); they appear to be generally involved in cytokinesis, and protein localization data
suggest that they may also play other roles in the organization of the cell surface. All of the known septins contain a
predicted nucleotide binding site that apparently can bind
GTP (Field et al., 1996
; Longtine, M., and J.R. Pringle, unpublished results), although the role of nucleotide binding
remains unclear. In addition, all but a few of the known
septins contain predicted coiled-coil domains, which might
be involved in the assembly of these proteins into filaments or other higher-order structures (Field et al., 1996
;
Longtine et al., 1996
). Interestingly, although both immunolocalization and cell-fractionation experiments suggest
that the septins are associated with the plasma membrane,
motifs that suggest how these proteins might interact with
the membrane have not been identified.
; Bulawa, 1993
; Orlean, 1997). In contrast, chitin
synthase II appears to be involved in the synthesis of the
chitin in the primary septum that separates mother and
daughter cells during cell division (Shaw et al., 1991
; Bulawa, 1993
; Orlean, 1996
), whereas chitin synthase I may be
involved in cell wall repair (Cabib et al., 1989
, 1992
).
Chitin synthase III synthesizes ~90% of the total cellular
chitin (Bulawa, 1993
; Orlean, 1996
), and is also largely or
entirely responsible for the synthesis of the chitosan (modified chitin) of the spore wall (Briza et al., 1990
, 1994
;
Pammer et al., 1992
; Bulawa, 1993
).
; Valdivieso et al., 1991
; Bulawa, 1992
,
1993
; Pammer et al., 1992
; Kawamoto et al., 1992
; Cabib,
1994
; Orlean, 1997). These include CHS3 (also known as
CSD2, CAL1, CAL4, CAL5, DIT101, or KTI2), CHS4
(also known as CSD4, CAL2, or SKT5), CHS5 (also
known as CAL3), and CHS6 (also known as CSD3).
Chs3p is a transmembrane protein that is thought to be the
catalytic subunit of chitin synthase III (Bulawa, 1993
;
Chuang and Shekman, 1996), and Chs5p may be involved
in the delivery of Chs3p to the budding site (Santos and
Snyder, 1997
). The function of Chs6p has yet to be determined. Chs4p appears to be an activator of chitin synthase
III, as indicated by both in vivo and in vitro analyses (Roncero et al., 1988
; Bulawa, 1992
, 1993
; Trilla et al., 1997
).
The predicted amino acid sequence of Chs4p (GenBank/
EMBL/DDBJ accession number Z23261, ORF YBL0519; Saccharomyces Genome Database ORF YBL061C; Scherens et al., 1993
; Bulawa, 1993
; Trilla et al., 1997
; Greene, J.,
and B. DiDomenico, personal communication; Bulawa,
C., personal communication) contains a potential E-F
hand loop calcium-binding domain and a COOH-terminal CAAX motif preceded by a cluster of basic amino acids,
as in the domains responsible for the membrane localization of some p21ras proteins (Hancock et al., 1990
).
MATERIALS AND METHODS
; Guthrie and Fink, 1991
).
Yeast strains were grown at 30°C except where noted. Escherichia coli
strains DH5
and DH12S (GIBCO BRL, Gaithersburg, MD) and standard media and methods (Sambrook et al., 1989
) were used for plasmid
propagation except where noted. E. coli strain JMB9r
m+
-trpF (Sterner
et al., 1995
) was used to rescue TRP1-containing plasmids from yeast
strain EGY48R; transformants were plated directly onto Vogel and Bonner medium (Davis et al., 1980
) supplemented with 0.2% glucose, 0.5%
casaminoacid hydrolysate, 0.01 M FeCl3, and 75 mg/liter ampicillin.
Standard methods were used for DNA manipulations and yeast genetics (Sambrook et al., 1989; Guthrie and Fink, 1991
) except where noted.
Most plasmids used in this study are listed in Table II; others are described where appropriate below. Filters containing
clones covering the
yeast genome (Riles et al., 1993
) were purchased from the American Type
Culture Collection (Rockville, MD). Vent DNA polymerase (New England Biolabs, Inc., Beverly, MA) was used in the PCR synthesis of DNA
fragments for cloning; Taq DNA polymerase (Promega Corp., Madison,
WI) was used in other PCR applications. The PCR method of Baudin et
al. (1993)
was used to generate cassettes for deletion of the complete coding regions of CHS4, CDC10, and CHS3, and for deletion of all but the
first 40 nucleotides of the BNI4 coding region. pRS303 and pRS304
(Sikorski and Hieter, 1989
) were used as templates for the HIS3 and
TRP1 markers used in these cassettes. The primers used are listed in Table III. The amplified cassettes were used to transform strain YEF473,
and PCR and/or DNA blot-hybridization analysis was performed to identify transformants that contained the deletions.
Table III. Oligonucleotide Primers Used for PCR-mediated Gene Deletion and Cloning |
Cloning of CHS4 and CHS3
A plasmid shuffle was performed in strain 124Y03A (Table I) to replace
plasmid pB12G with plasmid YEp24(CDC12)N (Table II), thus creating
strain DDY156. This strain was transformed with a yeast genomic DNA
library in plasmid YCp50-LEU2 (kindly provided by F. Spencer and P. Hieter, Johns Hopkins University, Baltimore, MD), and the transformants
were replica-plated to SC-Leu supplemented with 0.1% 5-fluoroorotic
acid (5-FOA) to select against plasmid YEp24(CDC12)N. A complementing plasmid, p196, contained CHS4 (see Results). A 3.4-kb XbaI-HindIII
fragment, which encodes amino acids 1-678 of CHS4 (Scherens et al.,
1993; see Fig. 2 A), was subcloned from p196 into pRS315 (Table II), thus
creating p206. Unidirectional deletions of DNA were made in this plasmid
using appropriate restriction enzymes and a modified exonuclease III procedure (Henikoff, 1984
; Beltzer et al., 1986
), thus creating a series of plasmids containing CHS4 truncated to different extents at the 3
end of the
coding region. Included in this series of plasmids are p377 and p376, which
contain alleles chs4
79 and chs4
560, which encode the NH2-terminal 79 and
560 amino acids of Chs4p, respectively. The complete CHS4 gene was subcloned from p196 into pRS316 as a BglII-EcoRV fragment that contained
~1 kb on either side of the CHS4 open reading frame (ORF; see Fig. 2 A),
thus creating p267. p267 was digested with EcoRI (one site in CHS4 and
the other in vector sequences) and religated to remove the 3
259 nucleotides of the CHS4 ORF and all of the cloned DNA downstream of
CHS4; the resulting plasmid, p357, contains the chs4
610 allele, which encodes amino acids 1-610 of Chs4p. p361 was constructed by simultaneously ligating a 2.7-kb XbaI-EcoRI fragment of p267 (XbaI site from
vector sequences) and a 270-bp EcoRI-XhoI fragment of p348 (see below) into XbaI/XhoI-digested pRS316; this plasmid contains the chs4C693S
allele.
To construct plasmids for two-hybrid analysis, CHS4 was amplified by
PCR using primers CHS4-5 and CHS4-3
(Table III) and plasmid p196 as
template. The BamHI-digested PCR product was ligated into BamHI-
digested pEG202 (Table II), thus fusing full-length CHS4 to the lexA
DNA-binding domain (DBD) sequences and creating p241. In addition,
an EcoRI fragment of p241 was subcloned into EcoRI-digested pEG202;
the resulting plasmid contains the DBD sequences fused to the chs4
610 allele (see above). To construct the chs4C693S allele (encoding Chs4p with
Ser substituted for Cys at amino acid 693), CHS4 DNA was amplified by
PCR using primers CHS4-5
and CHS4-3
C693S and p196 as template.
The BamHI/XhoI-digested PCR product was ligated into BamHI/XhoI-
digested pEG202, thus creating p348, which contains the DBD sequences
fused to the chs4C693S allele. Finally, a fragment encoding amino acids 1-700
of the 1165-amino acid Chs3p (Bulawa, 1992
) was amplified by PCR using primers CHS3F5
and CHS3MR and genomic DNA as template. The
NcoI/XhoI-digested PCR product was ligated into NcoI/XhoI-digested
pEG202 and pJG4-5PL, thus creating plasmids containing chs3
700 fused
to the DBD sequences and activation domain (AD) sequences, respectively.
To construct a fusion gene encoding full-length Chs4p fused to the
green fluorescent protein (GFP) (Chalfie et al., 1994), a fragment containing GFP-encoding sequences was amplified by PCR using primers that
contained both EcoRI and NotI restriction sites and pS65T-C1 (Clontech,
Palo Alto, CA) as template. The PCR product was digested with EcoRI
and ligated into EcoRI-digested pUC18, thus creating pUC18GFP. CHS4
was subcloned from p267 in an XbaI-KpnI fragment (both sites from the
polylinker) into XbaI/KpnI-digested pALTER (Promega Corp.), and a
NotI site was introduced immediately upstream of the CHS4 ORF using the procedures recommended by Promega Corp., thus creating
pANCHS4. The NotI fragment containing GFP-encoding sequences was
isolated from pUC18GFP and ligated into NotI-digested pANCHS4,
thus fusing the GFP and CHS4 open reading frames in plasmid pANGFPCHS4. The XbaI-KpnI fragment that contained the GFP-CHS4 fusion was isolated from pANGFPCHS4 and ligated into XbaI/KpnI- digested pRS316. The ligation mixture was transformed directly into
strain DDY172-2A (chs4-
1; Table I), and Ura+ transformants were
screened for normal chitin deposition by Calcofluor staining. One such
transformant, DDY172-2AX, was considered to contain the desired GFP-CHS4 plasmid, p326. Attempts to recover this plasmid in E. coli were unsuccessful.
To construct plasmid p408, the CHS3-containing plasmid pHV7
(Valdivieso et al., 1991) was digested with EcoRI and SalI (both sites from
vector sequences). The CHS3-containing fragment was ligated into
EcoRI/SalI-digested pBSKS+ (Stratagene Inc., La Jolla, CA), thus creating pBSKSCHS3. This plasmid was digested with SalI and NotI, and the
CHS3-containing fragment was ligated into SalI/NotI-digested pRS315.
The ligation mixture was transformed directly into strain DDY244 (chs3-
1/chs3-
1 chs4-
1/chs4-
1 [p326]; Table I). Leu+ Ura+ transformants
were selected and screened by Calcofluor staining. Several transformants
displayed normal Calcofluor staining and were thus considered to contain
p408. Attempts to recover this plasmid in E. coli were unsuccessful.
Cloning of BNI4
A yeast genomic DNA library was constructed by ligating BamHI-
digested YEp13 to partially Sau3AI-digested yeast genomic DNA from
strain JC01A ( cdc12-5 met1; S288C background) and transforming the
ligation mixture into E. coli. Approximately 5.5 × 104 transformants were
obtained, of which ~95% contained cloned DNA. A DNA fragment from
p279 (Table II) was used to screen this library by colony hybridization. A
BNI4-containing plasmid, p356, was isolated (see Fig. 2 A). BNI4 was subcloned from p356 by digesting with SphI (one site in the vector sequences,
one site downstream of BNI4), treating with T4 DNA polymerase, and ligating into SmaI-digested pRS425 and pRS315, thus creating high-copy
(p365) and low-copy (p366) BNI4 plasmids. p367 and p368 were constructed by digesting p365 and p366, respectively, with BglII (which cuts
uniquely in the BNI4 ORF), treating with T4 DNA polymerase, and religating; the resulting bni4
508 allele encodes amino acids 1-508 of Bni4p
followed by 12 missense amino acids. In addition, a BNI4-containing
HindIII-SacI fragment (both sites from the polylinker) was subcloned
from p365 into YEp351, thus creating p372. This plasmid was digested
with SalI and religated, thus creating p374, in which the BNI4 open reading frame is truncated after codon 20.
Two-hybrid Analyses
Two-hybrid analysis (Fields and Sternglanz, 1994) was performed using
strain EGY48R and the system described by Gyuris et al. (1993)
. Screening was performed using CHS4 plasmid p241 as the bait, and a yeast genomic DNA library in pJG4-5. Leu+ transformants were screened for galactose-dependent
-galactosidase activity by filter assays (Shapira et al.,
1983
), and quantitative
-galactosidase assays were then performed in duplicate or triplicate on promising candidates essentially as described previously (Ausubel et al., 1993). Directed two-hybrid analyses were quantitated similarly. Fusions of full-length CDC3, CDC10, CDC11, and CDC12
to the AD sequences in pJG4-5 and the DBD sequences in pEG202 have
been described previously (De Virgilio et al., 1996
).
Antibodies and Protein Procedures
Cdc11p-specific and Chs3p-specific antibodies have been described previously (Ford and Pringle, 1991; Chuang and Schekman, 1996
). To prepare
Bni4p-specific antibodies, a malE-BNI4 fusion was constructed by subcloning an EcoRI-SalI fragment encoding Bni4p amino acids 65-497 (see
Fig. 2 A; the EcoRI site was in the vector) from p279 into pMAL-c2 (New
England Biolabs). The fusion protein produced by the resulting plasmid
was largely degraded upon induction (data not shown). Therefore, this
plasmid was digested with NsiI and SalI (see Fig. 2 A), treated with T4
DNA polymerase, and religated, thus creating plasmid p353, which encodes MalE fused to amino acids 65 to 288 of Bni4p. This fusion protein
was not degraded extensively during induction or extraction (data not
shown). In addition, the same fragment of BNI4 was subcloned from p353
(using sites from the vector) into pGEX1 (Pharmacia LKB Biotechnology
Inc., Piscataway, NJ), thus creating p363, which encodes glutathione-S-transferase (GST) fused to amino acids 65-288 of Bni4p. MalE-Bni4p was
extracted from E. coli containing p353 as described by Maina et al. (1988)
,
purified on an amylose resin column (New England Biolabs), and used to
raise antibodies by standard procedures (Cocalico Biologicals, Reamstown, PA). To affinity-purify Bni4p-specific antibodies, GST-Bni4p was
extracted from E. coli containing p363 as described by Ausubel et al.
(1993), purified on a glutathione-agarose column (Thiol Picky Gel; Molecular Probes, Inc., Eugene, Oregon), electrophoresed on SDS gels (Laemmli, 1970
), and blotted to nitrocellulose. Affinity purification was then performed as described by Ford and Pringle (1991)
.
Yeast proteins were isolated for Western analysis by boiling cells in
2.5× Laemmli buffer (Laemmli, 1970). Western analysis was performed as
described previously (Haarer and Pringle, 1987
). Blots were stained with
Ponceau S to ensure equal protein loading.
Morphological Observations
Cells to be observed were grown in liquid media except for those containing GFP-Chs4p (see below). Overall cell morphologies were observed using an oil-immersion ×60 objective with differential-interference-contrast (DIC) optics. Immunofluorescence and visualization of chitin deposition by Calcofluor staining were performed as described previously (Pringle et
al., 1989). The localization of GFP-Chs4p was visualized by growing cells
containing plasmid p326 on SC-Ura or SC-Ura-Leu solid medium for 16-
20 h at 23°C. Cells were scraped from the plates into ice-cold 70% ethanol
and incubated for 10 min on ice. The cells were then collected by centrifugation, resuspended in water, and observed by epifluorescence microscopy using an FITC filter set. The localization of GFP-Chs4p was visualized similarly in temperature-sensitive mutant cells, except that some
plates were shifted to 37°C for 20 min before collecting the cells in 70%
ethanol. All microscopy was performed using a Microphot SA microscope
(Nikon Inc., Melville, NY).
Delocalization of Chitin Deposition in Septin Mutants
The septin proteins form a ring at the presumptive bud site
shortly before the chitin ring is formed in the overlying cell wall (see above). These observations suggested that the
septins might be involved in the localization of chitin deposition. To address this possibility, temperature-sensitive
septin mutants (cdc3, cdc10, cdc11, and cdc12) were
shifted from permissive to restrictive temperature, and cells
were examined by fluorescence microscopy after staining
with Calcofluor. In each mutant, the chitin rings and bud
scars formed during growth at permissive temperature appeared normal (Fig. 1, A-D), and their appearance did not
change after the shift to restrictive temperature (Fig. 1, E-H).
The first buds formed during growth at restrictive temperature, however, did not have discrete chitin rings at their
bases. Instead, the Calcofluor-stainable material formed a
diffuse band whose intensity decreased with distance from
the mother-bud neck (Fig. 1, E-H). In contrast, several
other temperature-sensitive cell-cycle mutants (cdc7, cdc13,
cdc21, cdc31) displayed apparently normal chitin rings at
the bases of the buds formed during cell-cycle arrest at restrictive temperature (data not shown). Of particular interest was the cdc4 mutant, which forms abnormally elongated buds like those of the septin mutants (Hartwell,
1971b) but maintains its septin organization (Byers, B.,
and L. Goetsch. 1976. J. Cell Biol. 70:35a; Haarer and Pringle, 1987
; Kim et al., 1991
) and forms normal-looking chitin
rings (Fig. 1 I) at restrictive temperature. Thus, it appears
that the delocalization of chitin deposition in the septin
mutants results specifically from the loss of septin function
and is not simply a consequence of cell-cycle arrest or abnormal bud shape.
Synthetic Lethality Between cdc12-5 and chs4
A genetic link between the septins and chitin synthesis was uncovered during a screen for mutations that are synthetically lethal with the cdc12-5 septin mutation. This screen identified several complementation groups,2 one of which, defined by strain 124Y03A, required at least two additional mutations in combination with cdc12-5 to produce the synthetic-lethal phenotype. To identify the genes defined by these mutations, strain DDY156 (a derivative of 124Y03A containing a URA3-marked CDC12 plasmid; see Materials and Methods) was transformed with a genomic DNA library in a low-copy, LEU2-marked vector, and candidate plasmids were selected on the basis of their ability to allow growth on medium containing 5-FOA, which selects against the URA3-marked plasmid. Two of the ~8,000 transformants screened contained plasmids that complemented the synthetic lethality. One plasmid, p196, complemented well, and the other, p197, complemented poorly.
To begin characterizing p196 and p197, portions of the
cloned DNAs were used as probes for DNA-DNA blot-hybridization analyses. The p196 and p197 DNAs did not
hybridize to each other or to CDC12 DNA. In addition,
the p196 DNA hybridized to DNA from the left arm of
chromosome II ( clone ATCC numbers 70693, 70310, and 71168), whereas the p197 DNA hybridized to DNA
from the left arm of chromosome V (
clone ATCC numbers 70355 and 70842). CDC12 is on the right arm of chromosome VIII (Mortimer and Schild, 1980
). Thus, p196 and
p197 contain different genes, and neither contains CDC12.
Because p196 allows DDY156 to grow relatively well in
the presence of 5-FOA (i.e., in the absence of plasmid-borne CDC12), it presumably complements the mutation
that is most deleterious in combination with cdc12-5. This
plasmid was therefore chosen for further characterization.
To identify the gene of interest in p196, various fragments were subcloned into pRS315 (low copy, LEU2) or
pRS316 (low-copy, URA3) and transformed into strain
DDY156 or 124Y03A. A 3.4-kb XbaI-HindIII fragment
(Fig. 2 A, plasmid p206) was sufficient for complementation of the synthetic lethality. Partial sequence analysis of
this fragment (data not shown) and comparison to the full
sequence of this chromosome region (Scherens et al., 1993; accession number Z23261) revealed that the fragment
contains two truncated ORFs. One of these contains all
but the COOH-terminal 18 codons of YBL0519 or CHS4/
CSD4/CAL2/SKT5 (Bulawa, 1992
, 1993
; Kawamoto et al.,
1992
; Trilla et al. 1997
; Greene, J., and B. DiDomenico,
personal communication; Bulawa, C., personal communication), a gene involved in chitin synthesis (see introduction), and the other contains the NH2-terminal 272 codons
of YBL0517, which encodes a 687-amino acid protein of
unknown function. A 3.8-kb BglII-EcoRV fragment (Fig.
2 A, plasmid p267), which contains all of CHS4 but only
103 codons of YLB0517, was also sufficient for complementation of the synthetic lethality, suggesting that CHS4
was the gene responsible for the complementation. To
confirm this conclusion, restriction enzyme digestion and
an exonuclease procedure (see Materials and Methods)
were used to truncate CHS4 further. Plasmids that contained as little as 560 codons of CHS4 still complemented well (Fig. 2 A, plasmid p376), but a plasmid that contained
only 79 codons of CHS4 (together with the same portion
of YBL0517) failed to complement (Fig. 2 A, plasmid
p377). Thus, CHS4 indeed appears to be responsible for
the observed complementation. Because the two reported
sequences for the CHS4 region differ (Kawamoto et al.,
1992
; Scherens et al., 1993
), we resequenced the regions of discrepancy (nucleotides
20-80 and 1655-1755 of
YBL0519). Our sequence agrees with that of Scherens et
al. (1993)
, which predicts that CHS4 encodes a protein of
696 amino acids that contains a putative calcium-binding
domain (amino acids 237-250) and terminates in a probable prenylation site (CAAX motif) (Fig. 3 A).
Because of the complicated genetics of strain 124Y03A,
we asked if cdc12-5 and a mutant allele of CHS4 (csd4-3::
LEU2; Table I, note ) were synthetically lethal in the absence of other known mutations. Strain JF2 (cdc12-5) was
mated to strain 70.2A (csd4-3::LEU2), and random spores
from the resulting diploid were analyzed at 23°C. From a
total of 100 viable segregants recovered, 42 were Leu+,
and thus contained the csd4-3::LEU2 mutation. Of these
42, 20 were unable to grow at 37°C, indicating that they
also contained the cdc12-5 mutation. Of these 20, 8 were
also inviable at 30°C, a temperature at which strain JF2
can grow. In addition, at 23°C, all of the putative double
mutants displayed the morphological abnormalities characteristic of both single mutants when grown at 37°C (i.e.,
elongated buds for cdc12-5 [Adams and Pringle, 1984
] and
high vacuole content for csd4-3::LEU2 [Bulawa, C., personal communication]). Thus, cdc12-5 and csd4-3::LEU2
do have a synthetic phenotype, but whether the double
mutants are viable or not depends on the growth temperature and the genetic background, consistent with the observations on strain 124Y03A itself.
Two-hybrid Analysis of Chs4p
The data described above suggested that Chs4p might interact directly with Cdc12p or one of the other septins. To
test this possibility, we performed directed two-hybrid
analysis using LexA DBD fusions of full-length Chs4p, a
truncated Chs4p that contains amino acids 1-610 (the
product of chs4610), and Chs4p with an altered CAAX
motif (the product of chs4C693S), together with AD fusions
of full-length Cdc3p, Cdc10p, Cdc11p, and Cdc12p (see
Materials and Methods). None of the combinations displayed significant interaction (data not shown). These results suggested that the Chs4p-septin interaction might be
indirect. Therefore, to identify other potentially relevant
proteins, a two-hybrid screen was performed using DBD-Chs4p (from plasmid p241) as the bait (see Materials and
Methods). 200 Leu+ colonies were picked from a total of
~8 × 106 transformants; of these 200, 38 proved to be galactose-dependent for both leucine prototrophy and
-galactosidase activity, indicating that the plasmids in these
strains encoded proteins that interacted with DBD-Chs4p.
Sequencing of the vector-insert junctions revealed that the
38 plasmids defined 10 genes. To identify genes whose products might be involved in septin-dependent chitin
deposition, representative plasmids were tested in two-
hybrid assays against DBD fusions of full-length Cdc3p,
Cdc10p, Cdc11p, and Cdc12p. One plasmid, p279, containing a portion (denoted bni4E) of a gene we named BNI4
(bud neck involved), showed interaction with DBD-Cdc10p as well as with DBD-Chs4p (Table IV), and was thus chosen for further analysis (see below). Other Chs4p interactors will be described elsewhere.
Table IV. Two-hybrid Interactions among Chs4p, Bni4p, the Septins, and Chs3p |
Because Chs4p does not require its COOH-terminal region to complement the chitin synthesis defect caused by
the chs4-1 allele (see below), we also tested DBD-Chs4p
610 and DBD-Chs4pC693S against the AD-Bni4pE
isolate. A strong signal was observed with DBD-Chs4pC693S,
but no interaction was observed with DBD-Chs4p
610 (Table IV). Therefore, the two-hybrid interaction between
DBD-Chs4p and AD-Bni4pE appears to require the
COOH-terminal 86 amino acids of Chs4p, but not the
CAAX motif. (The increased two-hybrid signal with
DBD-Chs4pC693S, relative to DBD-Chs4p, probably reflects a lack of prenylation that allows more of the protein
to enter the nucleus.)
Because Chs4p is an activator of chitin synthase III (Bulawa, 1993), we also performed two-hybrid analyses using
the Chs4p, Bni4p, and septin constructs together with
DBD and AD fusions to a fragment containing the NH2-terminal 700 amino acids of Chs3p (the catalytic subunit of
chitin synthase III). The Chs3p fragment interacted with
Chs4p, Chs4pC693S, and itself, but not (or only very weakly)
with Chs4p
610, Bni4pE, or any of the septins (Table IV).
Taken together, the two-hybrid results suggest that Chs4p may interact directly with Chs3p and indirectly with the septins through Bni4p.
Cloning, Mapping, and Sequence Analysis of BNI4
The BNI4 fragment from the original two-hybrid clone
was used to obtain the full-length gene by colony hybridization of E. coli harboring a yeast genomic-DNA library
in plasmid YEp13 (see Materials and Methods). One plasmid, p356, was found to contain the complete BNI4 ORF
plus at least 0.6 kb of DNA on either side (Fig. 2 A). A
BNI4-containing fragment extending from the SphI site of
YEp13 to the SphI site immediately downstream of BNI4 (Fig. 2 A) was subcloned from p356 into the low-copy vector pRS315. The resulting plasmid (p366) complemented
the bni4-1 allele (see below). BNI4 (accession number
Z69381, ORF N1146 [Pandolfo et al., 1996
]; Saccharomyces Genome Database ORF YNL233W) has an open reading frame of 892 codons predicted to encode a 100,588-Mr protein with a net charge of
44 (Fig. 3 B). Bni4p is predicted (Lupas et al., 1991
) to contain a coiled-coil domain
(probability >90%) from amino acids 106-139 (Fig. 3 B);
no other motifs or homologies have been identified. The
region upstream of BNI4 (nt
290 to
285) contains an
MluI restriction site, suggesting that this gene might be
regulated in a cell-cycle-dependent fashion (Andrews and
Mason, 1993
; Koch and Nasmyth, 1994
; Breeden, 1996
). Hybridization to a
clone (ATCC number 70396; Riles et
al., 1993
) agreed with the sequence analysis in placing
BNI4 on the left arm of chromosome XIV between ZWF1
and MDG1.
Genetic Analysis of CHS4 and BNI4
To examine the phenotypes resulting from the loss of
CHS4 and/or BNI4 function, these ORFs were deleted
(Fig. 2 A) in the same genetic background. DNA-DNA
blot-hybridization analyses confirmed the success of these
deletions (Fig. 2 B). For both DDY172 (CHS4/chs4-1) and DDY173 (BNI4/bni4-
1) (Table I), viability segregated 4+:0
, and TRP1 segregated 2:2 at 22, 30, 37, and
39°C, indicating that neither CHS4 nor BNI4 is essential
for viability. chs4-
1 and bni4-
1 haploids were mated,
and the resulting diploid (DDY176) was subjected to tetrad analysis. Segregants that contained both mutations
were viable at temperatures from 22-37°C and had growth rates similar to those of their CHS4 BNI4 siblings. DIC
microscopy revealed that some chs4-
1 cells had enlarged
bud necks and that many had protuberances at previous
division sites (Fig. 4 B; cf. wild-type cells in Fig. 4 A), as
noted previously for chs1 chs3 mutants by Shaw et al.
(1991)
. Similarly, the majority of bni4-
1 cells had enlarged bud necks, and some had protuberances at previous division sites (Fig. 4 C), and the majority of chs4-
1 bni4-
1 cells had both enlarged bud necks and protuberances
(Fig. 4 G). The double-mutant cells also were extensively
vacuolated.
Consistent with previous evidence that Chs4p is essential for normal chitin synthase III activity (Roncero et al.,
1988; Bulawa, 1992
, 1993
), Calcofluor staining showed that
the chs4-
1 mutants and chs4-
1 bni4-
1 double mutants
lacked bud scars, although some cells did display Calcofluor-staining material at the narrowest point of constriction in the mother-bud neck (Fig. 4, E and J; cf. Fig. 4 D
and Fig. 1, A-D), presumably due to chitin synthesized by
chitin synthase I and/or II (Bulawa, 1993
; Orlean, 1997). In contrast, examination of bni4-
1 strains revealed that Calcofluor-staining material was abundant but partially delocalized (Fig. 4 F). This phenotype and those described below were complemented by a low-copy plasmid containing
full-length BNI4 (p366) but not by a plasmid that contained BNI4 truncated after codon 508 (p368) (data not shown). Thus, Bni4p appears to be required for normal localization of chitin deposition, and the COOH-terminal
portion of Bni4p is required for its function. In addition,
because the Calcofluor-staining material seen in the
mother-bud necks of chs4-
1 bni4-
1 double-mutant cells
(Fig. 4 J) is indistinguishable from that seen in chs4-
1 cells (Fig. 4 E), it appears that Bni4p acts specifically in the chitin synthase III pathway.
Several truncated alleles of CHS4 complemented the
synthetic lethality between cdc12-5 and chs4 (see above
and Fig. 2 A). Therefore, we determined if these truncated
alleles and chs4C693S could also complement the chitin synthesis defect of a chs4-1 strain (DDY174). Interestingly,
plasmids p361 (chs4C693S), p206 (chs4
678), p357 (chs4
610),
and p376 (chs4
560) all rescued the chitin synthesis defect
as assayed by Calcofluor staining, although p377 (chs4
79)
did not (Fig. 2 A). In the cases of chs4C693S, chs4
678, and
chs4
610, the chitin synthesized was localized normally, but
in the case of chs4
560, chitin was delocalized (data not shown).
To determine the effects of Bni4p overexpression, a
high-copy BNI4 plasmid (p356) was transformed into
BNI4 (YEF473) and bni4-1 (DDY175) strains. The resulting transformants were viable at 23, 30, and 37°C.
However, the majority of cells grew with multiple, elongated buds with enlarged bud necks and displayed delocalized chitin deposition (Fig. 4, H and K). This phenotype
was similar to that of cdc10-
1 mutants (Fig. 4, I and L;
Flescher et al. [1993]). Immunoblot analysis (see Fig. 5)
confirmed that Bni4p indeed accumulates to higher-than-normal levels when plasmid p356 is present. To confirm
that the abnormal morphology was indeed due to BNI4
overexpression (and not to some other element in p356), a
high-copy BNI4 plasmid (p372) and a derivative that contained the same DNA except for a fragment internal to the
BNI4 ORF (p374) were constructed and transformed into
wild-type strain YEF473. Transformants that carried p372
had the abnormal morphology, whereas those that carried
p374 had normal morphology (data not shown). In addition, a high-copy plasmid that encodes only the NH2-terminal 508 amino acids of Bni4p (p367) caused the same
morphology as did the plasmids carrying full-length BNI4,
although Bni4p
508 was unable to complement the bni4-
1
allele when expressed from either low-copy (p368) or
high-copy (p367) plasmids (data not shown). Thus, the abnormal morphology seen in strains carrying multiple copies of BNI4 is in fact due to overaccumulation of Bni4p,
which suggests that excess Bni4p can titrate a factor or factors required for normal chitin deposition and cytokinesis. The NH2-terminal 508 amino acids of Bni4p appear to be
sufficient for this titration.
Localization of Bni4p in Wild-type and Mutant Strains
To determine the intracellular localization of Bni4p, we
raised antibodies against a MalE-Bni4p fusion protein and
affinity-purified them using a GST-Bni4p fusion protein
(see Materials and Methods). In extracts of wild-type cells,
the purified antibodies recognized primarily a polypeptide
of approximately the Mr predicted for Bni4p (Fig. 5, lane
2). This polypeptide was absent in extracts of a bni4-1
strain (Fig. 5, lane 1) and present at a higher level (along
with several probable breakdown products) in extracts of a strain containing a high-copy BNI4 plasmid (Fig. 5, lane
3), confirming that the antibodies are specific for Bni4p.
In immunofluorescence experiments on wild-type cells
grown in rich medium, Bni4p was detected as a single ring
on most unbudded cells and as a single ring on the mother-cell side of the mother-bud neck in nearly all small-budded
cells and most medium-budded cells (Fig. 6 A). In some
medium-budded and many large-budded cells, Bni4p appeared as a double ring in the mother-bud neck, although with the signal on the bud side generally weaker than that
on the mother-cell side (Fig. 6 A, arrowheads). The similarity in appearance between most of the Bni4p rings seen
on unbudded cells and those seen at the necks of small-budded cells suggests that the former represent sites of incipient bud emergence. However, some of the Bni4p rings
on unbudded cells appear to represent residual Bni4p at
the preceding division site, a hypothesis supported by the
observation of some cells with Bni4p rings near both poles (Fig. 6 A, arrows). In wild-type cells grown in synthetic
complete medium, the Bni4p signal was also asymmetric,
but the signal on the bud side typically appeared earlier in
the cell cycle and subsequently reached an intensity apparently equivalent to that on the mother-cell side (Fig. 6 B).
No signal was observed in a bni4-1 strain (Fig. 6 C), confirming the specificity of the staining. Thus, Bni4p appears
to localize to a ring at the presumptive bud site before bud
emergence and is present asymmetrically on the mother-cell side of the neck early in the cell cycle, a pattern that
correlates well with the localization and timing of bud-scar chitin deposition. Later in the cell cycle, Bni4p appears to
accumulate also on the bud side of the neck, and a symmetric distribution of Bni4p across the neck was typical
even in cells with small buds in a Bni4p-overproducing
strain (Fig. 6 D). The overproducing cells also appeared to
have Bni4p distributed throughout the cell or plasma
membrane, and some unbudded cells displayed an intensely staining disc or ring (Fig. 6 D, arrow). Localization
of Bni4p to the mother-bud neck is consistent with the
two-hybrid data showing its interactions with Cdc10p and
Chs4p, two other proteins that localize to this region (see
introduction and below).
To determine if mutations in functionally related genes
affect Bni4p localization, we examined chs4-1, chs3-
1,
cdc10-
1, and cdc12-6 mutants. (The cdc12-6 allele was
used because of its rapid loss of the septin ring upon shift
to nonpermissive temperature [Adams, 1984
; Haarer and
Pringle, 1987
; Chant et al., 1995
].) Bni4p localization appeared normal in the chs4-
1 and chs3-
1 strains (Fig. 6,
E and F), but the immunofluorescence signal was completely or almost completely absent in the cdc10-
1 strain (Fig. 6 G). In addition, Bni4p localized normally in a
cdc12-6 strain grown at 23°C (Fig. 6 H) but was undetectable after shift to nonpermissive temperature for 5 or 60 min (Fig. 6 I, and data not shown), paralleling the loss in
septin localization (Fig. 6, J and K). Taken together, these
results show that Bni4p localization depends upon the septins but not upon Chs4p or Chs3p.
Localization of Chs4p in Wild-type and Mutant Strains
To localize Chs4p, we constructed plasmid p326, a low-copy plasmid encoding a GFP-Chs4p fusion protein. chs4-1 strains harboring this plasmid (DDY172-2AX and
DDY197) displayed normal Calcofluor staining and normal morphology (data not shown), indicating that the
GFP-Chs4p fusion protein can supply Chs4p function. Examination of DDY197 cells by fluorescence microscopy
revealed that GFP-Chs4p localized to a patch or ring at
the presumptive bud site in many unbudded cells (Fig. 7 A,
small arrows), to a ring at the base of the bud in most (if
not all) cells with tiny buds (Fig. 7 A, arrowheads), and to
both sides of the neck in many cells with large buds (Fig. 7
A, asterisk); localized signal was rarely detectable in cells
with medium-sized buds (Fig. 7 A, large arrow). Some of
the GFP-Chs4p fluorescence on unbudded cells appeared
to represent residual protein at the preceding division site,
a hypothesis supported by the observation that some cells
displayed patches or rings of fluorescence near both poles
(Fig. 7 A and G, small arrows). The localization observed
for GFP-Chs4p is very similar to that observed for Chs3p
(Chuang and Schekman, 1996
; Santos and Snyder, 1997
;
also see below) in that both proteins are localized to the
mother-bud neck early and late in the cell cycle but appear to be absent from the neck during intermediate stages of
the cell cycle.
To explore the roles of other proteins in Chs4p localization, we examined chs4-1, p326-containing cells that
were also bni4-
1, chs3-
1, or cdc12-6, or that overproduced Bni4p. bni4-
1 cells with or without a control plasmid showed little or no localization of GFP-Chs4p to presumptive bud sites or the necks of tiny buds (Fig. 7 B;
Table V; and data not shown). As expected, introduction of a low-copy BNI4 plasmid restored normal GFP-Chs4p
localization (Fig. 7 C; Table V). In contrast, overexpression of Bni4p from a high-copy plasmid led to mislocalization of GFP-Chs4p to bud tips, to the daughter-cell side of
the neck in small-budded cells, and throughout growing
buds (Fig. 7 D). A chs3-
1 strain also displayed little or no
normal localization of GFP-Chs4p (Fig. 7 E; Table V), although this protein was occasionally mislocalized to the
tips of tiny buds (Fig. 7 E, arrowhead). As expected, introduction of a low-copy CHS3 plasmid restored normal
GFP-Chs4p localization (Fig. 7 F; Table V). Finally, shift
of a cdc12-6 strain from 23 to 37°C led to a loss of normal
GFP-Chs4p localization (Fig. 7, G and H; Table V). As expected, when a low-copy CDC12 plasmid was introduced,
GFP-Chs4p remained localized after shift to 37°C (Fig. 7, I
and J; Table V). Taken together, these results suggest that
normal Chs4p localization depends upon the septins,
Bni4p, and Chs3p.
Table V. Localization of GFP-Chs4p in Wild-type and Mutant Strains* |
Localization of Chs3p in chs4, bni4, and Septin Mutants
The evidence presented above suggests that the localization of Chs3p (and hence of chitin deposition) may depend
upon Chs4p, Bni4p, and the septins. To test this hypothesis, Chs3p-specific antibodies (Chuang and Schekman,
1996) were used to localize this protein in wild-type, chs4-
1, bni4-
1, chs4-
1 bni4-
1, Bni4p-overexpressing, and
septin mutant strains. As found previously (Chuang and
Schekman, 1996
; Santos and Snyder, 1997
), Chs3p was observed in wild-type cells as a patch (presumably at the incipient bud site) in many unbudded cells (Fig. 8 A, arrow),
as a distinct ring on the mother-cell side of the mother-bud
neck in many tiny-budded cells (Fig. 8 A, arrowheads), and
as a band on both sides of the neck in some cells with large
buds (data not shown). In addition, punctate staining,
which was also seen (although to a lesser extent) in chs3-
1 control cells (Fig. 8 B), was visible throughout the cells. In contrast, in chs4-
1, bni4-
1, and chs4-
1 bni4-
1
strains, Chs3p was typically found to be localized diffusely
throughout the bud and in the vicinity of the mother-bud
neck in tiny-budded cells, but it did not form a distinct ring
at the neck like those seen in wild-type cells (Fig. 8, C-E).
In addition, some unbudded cells of the mutant strains displayed patches of staining resembling, but typically less
distinct than, those seen in wild-type cells (Fig. 7, C-E, arrows). In Bni4p-overexpressing and cdc10-
1 strains, Chs3p
was observed in a ring in a few cells (Fig. 8, F and G, arrowheads) but was more commonly found diffusely throughout the cells. In a cdc12-6 strain grown at 23°C, Chs3p localized normally (Fig. 8 H), but normal localization was
lost within 5 min after a shift to restrictive temperature
(Fig. 8 I), paralleling the loss of septin localization (Fig. 8,
J and K). Taken together, the data suggest that the normal
localization of Chs3p to its major site of action (i.e., the
site of chitin-ring formation) is dependent upon Chs4p,
Bni4p, and the septins, and that the localization of Chs3p
and Chs4p is interdependent (see also above). In the absence of Chs4p and/or Bni4p, the cell appears to recruit Chs3p to the correct general location but fails to assemble
it into the well-defined ring seen in wild-type cells.
Localization of Septins in chs4, bni4, and chs3 Mutants
The results presented above indicate that the localization
of Bni4p, Chs4p, and Chs3p depends upon the septins. To
determine if the localization of the septins depends upon
these other proteins, chs4-1, bni4-
1, chs4-
1 bni4-
1,
chs3-
1, and Bni4p-overexpressing strains were examined
by immunofluorescence using Cdc11p-specific antibodies.
Cdc11p localized nearly normally in the mutant strains (Fig. 9). However, the band of septin staining appeared
somewhat narrower (not extending as far into either
mother cell or bud) in the mutants (compare Fig. 9, B-E,
to Fig. 9 A). This effect was not apparent in the Bni4p-overexpressing strain (Fig. 9 F). It is not clear whether the
altered appearance of the septin band is a real change in
the structure of the septin assembly or a visual artifact resulting from a change in neck shape and/or dimensions. In
any case, it appears that Chs4p, Bni4p, and Chs3p have, at most, a modest influence on septin organization and are
not required to localize the septins to the mother-bud
neck.
A Model for the Spatial Localization of Chitin Synthase III Activity
The results described above suggest a model in which a hierarchic assembly of proteins based on the septins is responsible for the spatial localization of cell-wall chitin deposition by chitin synthase III. In this model (Fig. 10 A), the
septins are recruited to and anchored at the presumptive
bud site and mother-bud neck by mechanisms independent of the other proteins considered here. The septin
complex then localizes Bni4p through the interaction of
this protein with Cdc10p, and Bni4p, in turn, localizes the chitin synthase III complex (including Chs3p and Chs4p)
through its interaction with Chs4p.
Several lines of evidence support the central role of the
septins in this model. The septins form a ring at the presumptive bud site ~15 min before bud emergence (Ford
and Pringle, 1991; Kim et al., 1991
), and the chitin ring,
formed by chitin synthase III (Shaw et al., 1991
; Bulawa,
1993
; Orlean, 1997), appears in the immediately overlying
cell wall somewhat later (just before bud emergence) (Hayashibe and Katohda, 1973
; Kim et al., 1991
). Thus, the
septins are in the right place at the right time to play a role
in localizing chitin synthase III activity. In addition, conventional electron microscopic observations on the septin-associated filaments (Byers and Goetsch, 1976
; Byers,
1981
), immunofluorescence and immunoelectron microscopy (Haarer and Pringle, 1987
; Kim et al., 1991
; Ford and
Pringle, 1991
; Mulholland, J., D. Preuss, and D. Botstein,
personal communication), and cell-fractionation experiments (Healy, A., M. Longtine, and J.R. Pringle, unpublished results) all suggest that the septins are closely associated with the plasma membrane, so that a role in the
localization of the plasma membrane-associated chitin
synthase III complex is plausible. Moreover, temperature-sensitive septin mutants are unable to form normal chitin
rings at restrictive temperature (Fig. 1). Although a variety of cell-cycle mutants display delocalized chitin deposition after extended incubations at restrictive temperature
(Sloat et al., 1981
; Roberts et al., 1983
; Shaw et al., 1991
),
the failure to form a chitin ring at the base of the first bud
produced at restrictive temperature was unique to the septin mutants among those examined, suggesting that this effect results specifically from the loss of septin function.
Finally, the septin mutants are also defective in the localization of Bni4p, Chs4p, and Chs3p (Figs. 6-8).
The mechanisms by which the septins themselves are recruited to and anchored at the presumptive bud site and
mother-bud neck remain obscure. Septin localization appears to depend on a signal from the Cdc42p GTPase, but
the effectors of this signal are not known (Pringle et al.,
1995). Although it initially seemed possible that the transmembrane protein Chs3p or the (presumably) prenylated protein Chs4p might be involved in anchoring the septins
to the plasma membrane, deletion of CHS3, CHS4, or
BNI4 has little or no effect on septin localization, at least
as judged by immunofluorescence. As the septins themselves have no motifs that suggest how membrane association might occur (Longtine et al., 1996
), there is presumably at least one protein involved in this association that
has yet to be identified.
Several lines of evidence also suggest that Bni4p provides a link between the septins and the chitin synthase III
complex. The two-hybrid data show that Bni4p can interact with both Cdc10p and Chs4p. In addition, except for its
asymmetric concentration on the mother-cell side of the
neck (as considered further below), Bni4p colocalizes with
the septins, and Bni4p localization is lost in septin mutants. (In this regard, the residual Bni4p localization observed in some cdc10-1 cells probably just reflects residual septin function in this severely compromised but viable
strain.) Interaction of Bni4p with the septins is further supported by the observations that bni4 mutant cells often
have enlarged bud necks and that overproduction of
Bni4p (or a Bni4p fragment) causes a phenotype similar to
that of septin mutants, presumably because excess Bni4p
titrates some factor from the neck that is required for septin function. Finally, either a loss of Bni4p or its overproduction causes a partial delocalization of chitin deposition,
which parallels an inability to achieve normal localization
of either Chs4p or Chs3p. In contrast, the localization of
Bni4p itself is not obviously affected by the loss of either
Chs4p or Chs3p. Bni4p appears to function specifically in
the chitin synthase III pathway, because the amount and
localization of residual chitin (presumably synthesized by
chitin synthase I and/or II) appear the same in a chs4 bni4 strain as in a chs4 BNI4 strain.
Several lines of evidence also suggest that Chs4p links Chs3p to Bni4p, and hence to the septins. The two-hybrid data show that Chs4p can interact with both Bni4p and Chs3p. In addition, Chs4p and a fraction of the Chs3p colocalize with Bni4p and the septins for at least a portion of the cell cycle, and this localization is lost in both bni4 and septin mutants. Although the loss of chitin synthase III activity in a chs4 mutant makes it impossible to ask directly where chitin would be deposited, immunofluorescence observations show that the normal localization of Chs3p is also lost in a chs4 mutant. Importantly, the data also suggest that Chs3p is not just a passive passenger but instead plays a key role in the assembly of the complex of which it is a part. In particular, the two-hybrid data show that Chs3p can interact with itself as well as with Chs4p, and the localization of Chs4p is lost in a chs3 mutant.
An attractive feature of the model described above is
that it helps to explain how the chitin ring can form asymmetrically on the mother-cell side of the neck. In particular, although the ring of septin proteins (and septin-associated filaments) appears to be symmetric across the neck
(Byers and Goetsch, 1976; Byers, 1981
; Haarer and Pringle, 1987
; Kim et al., 1991
; Ford and Pringle, 1991
), Bni4p
is concentrated asymmetrically on the mother-cell side of
the neck, especially early in the cell cycle. This presumably restricts the chitin synthase III complex (and hence chitin
deposition) to the mother-cell side of the neck, as indeed
observed experimentally using GFP-Chs4p and antibodies
to Chs3p. It is not yet clear how Bni4p itself becomes
asymmetrically localized, but one possibility is suggested
by the presence in the BNI4 upstream region of an MluI
restriction site (and hence a potential MCB-type transcription-control element), which suggests that BNI4 may be
transcribed specifically early in the cell cycle (Andrews
and Mason, 1993
; Koch and Nasmyth, 1994
; Breeden, 1996
),
as indeed are a variety of other genes whose products are
involved in cell-wall synthesis (Igual et al., 1996
). Thus,
most Bni4p may be synthesized and become bound to the
septins before bud emergence, when the septins exist only
as a ring at the surface of the mother cell. Subsequent low-level synthesis of Bni4p could account for its gradual accumulation in association with the septins on the bud side of
the neck; however, by this point, chitin synthase III components may have been retrieved from the plasma membrane by endocytosis (Chuang and Schekman, 1996
) or
otherwise inactivated, so that no chitin deposition on the
bud side results.
The analyses reported here leave several interesting issues unresolved. For instance, the model of Fig. 10 A does
not address the mechanisms involved in the initial delivery
of the chitin synthase components to the presumptive bud
site or their later retrieval from the plasma membrane
(see, however, Chuang and Schekman, 1996; Santos and
Snyder, 1997
). In this regard, it is interesting that Chs3p
showed some localization to the incipient bud site in bni4,
chs4, and septin mutants, although it was not organized into the normal tight band at the base of the emerging bud.
Similarly, GFP-Chs4p also sometimes appeared concentrated at the incipient bud site in bni4, chs3, and septin
mutants, although the localization rarely (if ever) appeared normal. These observations parallel the findings
that chitin deposition is not completely delocalized in bni4
and septin mutants. Thus, it appears that there is a mechanism for the delivery of Chs3p and other chitin synthase
III components to the general region of the presumptive
bud site that is independent of the septins, Bni4p, and
Chs4p. This mechanism might simply be the polarization
of the actin cytoskeleton and secretory system, which is expected to result in the polarized delivery to the cell surface
of integral membrane proteins such as Chs3p (Drubin and Nelson, 1996
). In the absence of the restraint offered by
the septin/Bni4p/Chs4p complex, Chs3p and associated
proteins might simply diffuse through the membrane away
from their site of initial insertion (Fig. 10 B), resulting
(when Chs4p and other components necessary for chitin
synthase activity are present) in the delocalized chitin deposition that is observed.
In addition, some questions remain about the interactions among Chs4p, Bni4p, and Chs3p. The truncated
Chs4p encoded by the chs4610 allele failed to show interaction in two-hybrid tests either with Bni4p or with Chs3p,
although Chs4p
610 could still support not only chitin synthase III activity but also the normal localization of the
chitin synthesized. Although these observations appear at
first sight to violate the model of Fig. 10, we note that negative results in two-hybrid tests can be misleading (for example, if the fusion proteins are awkwardly folded) and
that the tests in question were performed with truncated alleles of both BNI4 and CHS3, whereas the analysis of
chitin deposition was performed in a strain that contained
chs4
610 but was wild-type for BNI4 and CHS3. Thus, given
the weight of other evidence supporting the model of Fig.
10, we think it most likely that Chs4p
610 does interact with
full-length Bni4p and Chs3p under normal circumstances in vivo. This interpretation is supported by the observation
that Chs4p
610 does not support chitin synthesis in a bni4-
1 strain (DeMarini, D.J., unpublished observations).
Finally, it should be noted that the model of Fig. 10 addresses specifically the localization of the proteins of interest in the period just before and during bud emergence; the mechanisms involved in the apparent relocalization of Chs3p, Chs4p, and (perhaps) associated proteins to the mother-bud neck late in the cell cycle may be different.
The Roles of Chs4p and Chs3p
Our results taken together with those of previous studies
(Roncero et al., 1988; Bulawa, 1992
, 1993
) suggest that
Chs4p has at least two roles in relation to chitin synthase
III. One role is to activate Chs3p, and the second is to anchor Chs3p to the septins via Bni4p. Interestingly, these
roles appear to be separable, as judged from the occurrence of delocalized chitin deposition in a bni4 mutant but
not in a bni4 chs4 double mutant, the ability of a CHS4
plasmid to restore (delocalized) chitin deposition to the
latter strain, and the ability of a chs4
560 plasmid to restore
chitin synthesis, but not normal chitin localization, to a
chs4 mutant strain. The mechanism by which Chs4p activates Chs3p remains unclear, although the two-hybrid
data suggest that the two proteins may interact directly.
A surprising result is that the presumed membrane-
attachment domain of Chs4p (the CAAX motif and preceding cluster of basic amino acids) is apparently not required for either of the roles just discussed. Presumably,
protein-protein interactions are sufficient to localize Chs4p
appropriately in the absence of direct membrane attachment. It is possible that the membrane-attachment domain is simply unnecessary for any aspect of Chs4p function, but
it also seems possible that this domain is important for
some as-yet-undefined role of the protein. In this regard, it
is of interest to ask why Chs4p and Chs3p reappear at the
mother-bud neck late in the cell cycle, given that chitin
synthase II appears to synthesize the chitin of the primary
septum (Shaw et al., 1991; Bulawa, 1993
; Orlean, 1997).
One possibility is that chitin synthase III may function as a
redundant system for the synthesis of septal chitin. This
possibility is supported by the observations that a chs2 strain is viable and produces an abnormally thick, chitin-based septum and that chs2 and chs3 mutations are synthetically lethal (Shaw et al., 1991
). However, it also seems
possible that Chs4p and/or Chs3p are involved in the recruitment and/or spatial organization of other proteins
that participate in cell-wall synthesis or other events at the
division site. There are several other observations consistent with this possibility. For example, it is not clear that
either the formation of protuberances at previous division sites by chs4 and bni4 mutants or the synthetic lethality of
chs4 and cdc12 mutations can be explained in terms of the
role of Chs4p in chitin synthase III activity. In addition, we
observed that mutation of the putative Ca2+-binding site
of Chs4p (a double D238R, D240R mutation; see Fig. 3 A)
had no obvious effect on chitin synthase III activity, as judged by Calcofluor staining (DeMarini, D.J, unpublished observations). Finally, it is intriguing that Fks1p,
one of the catalytic subunits of the enzyme that synthesizes the major cell-wall constituent 1,3-
-D-glucan (Douglas et al., 1994
), also localizes to the mother-bud neck just
before cytokinesis (Qadota et al., 1996
).
Except for the apparent noninvolvement of the putative
membrane-attachment and Ca2+-binding domains in the
action of chitin synthase III at the beginning of the cell cycle,
little can be said as yet about structure-function relationships
in Chs4p. However, the observation that the chs4560 allele
could support chitin synthesis, but not the proper localization of the chitin synthesized, does suggest that the COOH-terminal portion of Chs4p is required for its interaction with Bni4p.
A General Model for Septin Function
Proteins in the septin family have now been identified in a
wide variety of fungal and animal cells (Cooper and Kiehart, 1996; Longtine et al., 1996
). In every cell type examined to date, the septins appear to be involved in cytokinesis and/or septum formation, but the precise role of the
septins in these processes remains obscure, a problem
compounded by the fact that the mechanisms of cytokinesis in fungal and animal cells otherwise appear quite different. In addition, the septins are found localized in ways that suggest that they have important roles in processes
distinct from cytokinesis. For example, in mating yeast
cells, the septins are concentrated near the bases of mating
projections (Ford and Pringle, 1991
; Kim et al., 1991
) and
appear to interact with other proteins involved in the polarized morphogenesis that occurs in response to mating
pheromones (Konopka et al., 1995
). In sporulating yeast
cells, the septins appear to be concentrated at the leading edge of the extending forespore membrane during the early
stages of spore formation and subsequently are found surrounding the entire developing spore before spore-wall
deposition (De Virgilio et al., 1996
; Fares et al., 1996
). In
Drosophila, septins are highly concentrated at the leading
edge of the furrows during cellularization of the embryo,
in specific surface domains of polarized epithelial cells, and
in neurons of the embryonic central nervous system (Neufeld and Rubin, 1994
; Fares et al., 1995
). A major challenge
is to understand what general principles of septin function
may explain their roles in all these different contexts.
In this study, an amalgam of genetic, two-hybrid, and
protein-localization data have led us to a model for septin
function in localizing the action of chitin synthase III, an
enzyme responsible for the synthesis of a particular component of the cell wall. In particular, as discussed in detail
above, it appears that the septins provide a template upon
which the proteins of the chitin synthase III complex assemble. Previous studies had suggested a similar role for
the septins in the function of Bud3p and Bud4p, two proteins that are required for the selection of bud sites in the
axial pattern (Chant et al., 1995; Sanders and Herskowitz, 1996
). In particular, Bud3p and Bud4p assemble at the
mother-bud neck in a septin-dependent fashion during
each cell cycle; they then remain at the division sites on
mother and daughter cells long enough to direct the assembly of a new bud site to an adjacent location in the
next cell cycle. Extrapolation from these examples and
consideration of the patterns of septin localization described above suggest that the septins may function generally in the spatial organization of cell surface proteins that
are involved in signaling, localized membrane insertion, or
the synthesis of components of the cell wall or extracellular matrix. In this context, it is interesting to note that
chitin synthase III is also responsible for the synthesis of
the chitosan layer of the S. cerevisiae spore wall (Pammer
et al., 1992
; Bulawa, 1993
; Orlean, 1997) and that a homologue of Chs4p, but not Chs4p itself, is expressed in sporulating cells (Bulawa, 1993
). If this general view of septin
function is correct, then progress in understanding septin
function in particular contexts will depend on identifying
the particular proteins with which the septins interact.
Both genetic and biochemical approaches, in appropriate
systems, should contribute to such identification.
Received for publication 7 April 1997 and in revised form 17 June 1997.
The present address of Douglas J. DeMarini is Department of Comparative Genetics, Smithkline Beecham Pharmaceuticals, King of Prussia, PA 19406.We thank C. Bulawa, B. Osmond, and P. Robbins for strains, helpful discussions, and the communication of unpublished results; B. DiDomenico and J. Greene for communicating unpublished results; members of the Pringle laboratory, especially E. Bi and M. Longtine, for antibodies, strains, plasmids, and valuable discussions; P. Watts for the library of AD fusions; C. Roncero for plasmid pHV7; and S. Whitfield for providing outstanding photographic services.
This work was supported by National Institutes of Health Grant GM31006 (to J.R. Pringle), funds from the RJEG Trust, fellowships from the L. and Th. LaRoche Stiftung and the Ciba-Geigy-Jubilaeums-Stiftung (to C. De Virgilio), and a Howard Hughes Medical Institute predoctoral fellowship (to J. Chuang).
5-FOA, 5-fluoroorotic acid; AD, activation domain; DBD, DNA-binding domain; DIC, differential-interference-contrast; GFP, green fluorescent protein; GST, glutathione-S-transferase; ORF, open reading frame; SC, synthetic complete.
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