Department of Biology, Yale University, New Haven, Connecticut 06520-8103
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Abstract |
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Yeast cells mate by an inducible pathway
that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight
into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging
screen to identify genes whose expression is regulated
by mating pheromone. 91,200 transformants containing
random lacZ insertions were screened for -galactosidase (
-gal) expression in the presence and absence of
factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or
had not previously been known to be pheromone regulated were examined for effects on the mating process.
Mutations in four novel genes, FIG1, FIG2, KAR5/
FIG3, and FIG4 were found to cause mating defects.
Three of the proteins encoded by these genes, Fig1p,
Fig2p, and Fig4p, are dispensible for cell polarization in
uniform concentrations of mating pheromone, but are
required for normal cell polarization in mating mixtures, conditions that involve cell-cell communication.
Fig1p and Fig2p are also important for cell fusion and
conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p
and Fig2p are likely to act at the cell surface as Fig1::
-gal and Fig2::
-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of
eukaryotic proteins that contain a domain homologous
to the yeast Sac1p. Our results indicate that a variety of
novel genes are expressed specifically during mating
differentiation to mediate proper cell morphogenesis,
cell fusion, and other steps of the mating process.
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Introduction |
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THE yeast mating response is an excellent model system for the study of receptor-activated cell differentiation in eukaryotes. Upon encountering appropriate mating pheromones, haploid yeast cells follow a
programmed pattern of cell differentiation in preparation for later events of mating such as cell and nuclear fusion
(Cross et al., 1988; Sprague and Thorner, 1992
; Kurjan,
1993
; Herskowitz, 1995
). Vegetative cells exposed to pheromone stop their progression through the cell cycle and
undergo polarized cell growth to form a specialized structure termed a mating projection. Polarized mating cells
signal one another through their projections, and thereby
direct growth to a mutual site of cell contact and fusion. Cell fusion usually occurs at the tips of the projections,
forming a conjugation tube or bridge. Nuclear congression
and fusion then take place within the conjugation bridge
and the zygote enters the vegetative cell cycle, dividing the
diploid nucleus between itself and its bud. Although the
cytological events of yeast mating have been well described,
the molecular components and mechanisms important for
mating cell morphogenesis, cell fusion, and nuclear fusion
are not well understood.
At the molecular level, mating differentiation is initiated
by the activation of a receptor-coupled signal transduction
cascade. Pheromones are bound by the STE2 and STE3
gene products, which are seven transmembrane segment
receptors located on the surface of MATa and MAT
cells, respectively. These receptors are coupled to a heterotrimeric G protein complex and a cytoplasmic mitogen-activated protein (MAP)1 kinase cascade (Sprague and
Thorner, 1992
). Transduction of the signal by the MAP kinase cascade leads to activation of the transcription factor
Ste12p, which, in turn, promotes the transcription of a set
of genes involved in mating-specific functions. These functions include cell cycle arrest in G1, polarized morphogenesis, agglutination, cell fusion, karyogamy, and adaptation
to the pheromone signal (Sprague and Thorner, 1992
).
Many components of the mating MAP kinase cascade, including the Ste12p transcription factor, have also been
shown to be required in both haploid and diploid cells for
the transition from the normal yeast form of growth to filamentous forms stimulated by nutrient deprivation conditions (Liu et al., 1993
; Roberts and Fink, 1994
). These filamentous forms of polarized growth and unipolar budding
have been proposed to be a mechanism by which cells forage for more favorable nutrient-rich environments (Gimeno et al., 1992
; Kron et al., 1994
).
The mating projection produced by cells exposed to
pheromone serves two important purposes. First, the projection allows the nonmotile yeast cell to extend towards
its mate. The position of the mate is perceived through pheromone gradients emanating from mating partners. This
perception, or partner selection, is accomplished through
the differential activation of mating pheromone receptors
on the surface of the mating cell (Jackson and Hartwell, 1990; Jackson et al., 1991
; Segall, 1993
). Second, growth of
the mating projection is an actin-dependent process that
has been shown to depend on several proteins that also
participate in polarized growth during budding (e.g., Spa2p,
Pea2p, Bem1p, Tpm1p, and Cdc42p) (Herskowitz et al.,
1995
; Pringle et al., 1995
; Roemer et al., 1996
). Recent
studies have demonstrated a physical association between
Cdc24p (the GTP exchange factor for Cdc42p), Bem1p,
actin, and the heterotrimeric G proteins associated with
the pheromone receptors, suggesting a mechanism for linking pheromone pathway activation to localized cell polarization (Leeuw et al., 1995
). However, because these interactions are independent of the state of activation of the
pheromone pathway, the specific mechanism of polarization to sites of pheromone receptor activation remains obscure (Leeuw et al., 1995
; Roemer et al., 1996
).
The second role of the mating projection is to concentrate components involved in cell adhesion (agglutinins),
signaling (pheromones and pheromone receptors), and fusion (Fus1p and Fus2p) to the area of intended cell contact
and fusion. High levels of mating pheromone are required
for normal cell fusion, and several proteins that function
specifically in these processes (a-factor, -factor, Ste2p,
Fus1p, and Fus2p) are all highly localized to projections or
their tips (Trueheart and Fink, 1989
; Jackson et al., 1991
; Sprague and Thorner, 1992
; Elion et al., 1995
). Many cell
polarity genes also function in the cell fusion pathway as
indicated by the increase in cell fusion defects observed
for mutants in a number of such genes (e.g., SPA2, PEA2,
BNI1, RVS161) (Dorer et al., 1997
). These observations
suggest that efficient cell fusion is likely to depend on
proper cell polarity to affect localization of the signaling
and cell fusion components to the projection tip.
In contrast to our extensive knowledge of the components of the mating signal transduction cascade and their
interactions, relatively few proteins are known to be specifically involved in the various downstream events of the
mating process (Sprague and Thorner, 1992; Brill et al.,
1994
; Choi et al., 1994
; Stevenson et al., 1995
). For example, most of the polarity components known to affect mating cell shape and growth also participate in vegetative functions (Herskowitz et al., 1995
; Pringle et al., 1995
; Roemer et al., 1996
). Thus, it is likely that certain mating-specific components remain undescribed that link general polarity proteins to specified sites of cell growth during
mating. Some of these components would be expected to
help direct the growth and shape of the mating projection.
Understanding the downstream events of the mating process, including cell polarization, cell fusion, and nuclear fusion, is of general importance to elucidating these processes in higher eukaryotic cells. The limited number of
downstream genes currently identified as functioning in
these processes suggested that a search for new pheromone-regulated genes might yield additional components
of the mating pathway, and thereby help determine the molecular and cellular mechanisms involved in mating cell
differentiation.
We describe the results of an extensive screen for pheromone-regulated genes. The screen uses a recently developed method of random transposon tagging of yeast genes
to monitor gene expression and investigate mutant phenotypes (Burns et al., 1994). From an initial bank of 189 pheromone-regulated transposon insertions, 45 new pheromone-regulated genes were identified. Among these 45 genes, 30 represent novel genes and 15 encode genes
whose expression was previously unknown to be affected
by pheromone. Furthermore, we find that a subset of
pheromone-induced genes are also induced by conditions
of nitrogen deprivation, suggesting a set of target genes is
shared between the mating and pseudohyphal pathways. Four novel pheromone-induced genes designated Factor-
Induced Gene FIG1, FIG2, KAR5/FIG3, and FIG4 were
determined to be required for different steps of mating
cell differentiation, including the control of mating cell polarity, cell fusion, and nuclear fusion.
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Materials and Methods |
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Yeast Strains and General Methods
The yeast strains used in this study are listed in Table VIII. All strains are
derivatives of Y800 (Burns et al., 1994) and in the S288c background.
Y1406, the diploid strain used in the screen, was constructed by transforming strain Y1400 MATa-cry1 ura3-52 leu2-
98 his3-
200 trp1-
1 with a
PCR fragment (Baudin et al., 1993
) containing the BAR1 gene, in which
the entire protein coding sequence was substituted with the sequence of
the HIS3 gene. The resulting strain, Y1402, was used to construct Y1405 (MATa-cry1/MAT
-CRY1 bar1::HIS3/bar1::HIS3) through backcrossing. A MATa-cry1/MATa-cry1 mitotic recombinant Y1406 was selected from
Y1405 by growth on plates containing crytopleurorine and then confirmed
to be a diploid by transformation of a MAT
plasmid, sporulation, and
tetrad analysis. Y1411, the MATa bar1 haploid strain used for screening is
an ascospore segregant derived in the construction of Y1405. General
cloning procedures are described in Sambrook et al. (1989)
. Yeast media
and methods are presented in Rose et al. (1990)
and Sherman (1986).
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Identification of Pheromone-regulated Genes
The plate assay for detection of pheromone-induced genes was first optimized using two Y1406 strains: one containing a cik1::lacZ fusion carried
on a YCp50 plasmid and the other a FUS1::lacZ fusion carried on a 2 µ-based
plasmid (Trueheart et al., 1987; Page and Snyder, 1992
). Cells were grown
on yeast extract/peptone/dextrose medium (YPD) plates and then replica-plated to two 1-mm filters (Whatman Inc., Clifton, NY). The duplicate filters were incubated for an additional 6 h on YPD medium and then transferred to petri dishes containing 0.8 ml of liquid YPD medium, one of
which contained 5 µg/ml of
-factor (Sigma Chemical Co., St. Louis, MO).
After incubation at 30°C, the filters containing cells were exposed to chloroform vapors to permeabilize the cells, and then the filters were incubated on plates containing X-gal as described previously (Xie et al., 1993
). In
initial optimization experiments, cells were incubated for 6, 8, 10, and 12 h
at 30°C, and then processed for
-gal activity. Optimal signals were observed using pheromone incubation times of 10-12 h, which were used in
screening experiments.
A yeast lacZ fusion library (Burns et al., 1994) was transformed into
strains Y1406 and Y1411. 55,000 Y1406 and 36,200 Y1411 Leu+ transformants were patched on 90-mm petri plates containing synthetic complete
(SC) medium lacking leucine (100 transformants/plate). After growth for
2 or 3 d at 30°C, the cells were replica-plated to two filters and incubated
on YPD plates for an additional 6-12 h. Filters were processed as described above and strains containing potential pheromone-regulated fusions were identified. Individual strains were then retested as single colonies to identify strains that contained reproducibly pheromone-regulated
fusions. 14% of yeast strains expressed
-gal after vegetative growth for a
total of ~13,000 strains. Since there are ~6,500 yeast genes in yeast (Mewes et al., 1997), this corresponds to 2.0 genome equivalents screened.
Therefore, 158 out of 189 pheromone-regulated fusions corresponds to 1.7 genome equivalents analyzed.
The yeast sequence adjacent to the mTn3::lacZ insertion was determined using plasmid rescue procedures described previously (Burns et al.,
1994, 1996
). Briefly, either YIp5 or pRSQ plasmids were integrated into
the mTn3 insertion, and the yeast sequences adjacent to lacZ were recovered as plasmids in Escherichia coli. A primer complementary to the end
of the lacZ sequence was used to determine the sequences of the yeast
DNA adjacent to the mTn3 insertions. The yeast sequences were compared to those in the GenBank database using the BLAST program (Altschul et al., 1990
). These sequences (see Table I) are accessible in GenBank by a search using keywords Pheromone and the fusion number of
interest (e.g., P158). Pheromone response element (PRE) sites were identified by searching sequences using the Fitconsensus program of the
UWGCG package (Devereaux et al., 1984
) and the sites described in (Kronstad et al., 1987
).
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Quantitative -galactosidase (
-gal) Assays
Cells of the indicated strains were grown to midlogarithmic phase (OD600 = 0.4) in SC medium lacking leucine and then divided into two 10-ml aliquots. To determine pheromone induction levels, cells were treated for 2 h
in YPD or YPD + 5 µg/ml of -factor, after which cell lysates were prepared and
-gal activities were measured. Low nitrogen induction levels
were measured by comparison of activities in cell lysates of cells grown for
12 h in SC lacking leucine or SC lacking both leucine and (NH4)2SO4. Cells
were harvested by centrifugation, washed once in Z buffer (60 mM Na2HPO4,
40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM
-mercaptoethanol,
pH 7.0) and the cell pellet frozen at 70°C until use. Cells were prepared for
lysis by resuspending the frozen cell pellet in 0.5 ml of Z buffer, and followed by the addition of 250 µg zymolyase 100T to spheroplast cells (30 min at 30°C). Lysates were made by the addition of ~50 µl of glass beads
(model G-8772; Sigma Chemical Co.), 15 µl of 100 mM PMSF, 7.5 µl of 20%
SDS, and 25 µl of chloroform followed by vortexing for 2 min. Assays of
-gal activity were performed by the addition of 200 µl of the lysate to 0.8 ml of
Z buffer and 200 µl of 4 mg/ml O-nitrophenol-
-D-galactopyranoside. Reactions were stopped with 250 µl of 2 M Na2CO3, and then the activities
were determined as a function of sample absorbance at 420nm, reaction
time, and protein concentration (determined by Bradford assays).
Disruption of FIG1, FIG2, KAR5/FIG3, and FIG4
Complete deletions of the FIG1, FIG2, KAR5/FIG3, and FIG4 genes
were made using a PCR disruption procedure (Baudin et al., 1993). Oligonucleotides containing the 55 bp immediately upstream of the ATG and
downstream of the termination codon of each gene were synthesized with
ends corresponding to sequences A and B below, respectively. Sequences
A and B are complementary to regions that flank the URA3 gene of
pRS316 (Sikorski and Hieter, 1989
). URA3 fragments containing FIG
flanking sequences were amplified by PCR and then transformed into the
diploid strain Y800. Strains containing the correct substitution at the genomic locus were identified by PCR analysis. The resulting heterozygotes
were sporulated and then haploid segregants were analyzed for vegetative
growth and mating defects. Growth rates of all fig
strains were identical to those of wild-type strains at 16, 30 and 37°C. (A) 5
-...AGGCGCGTTTCGGTGATGACGGTG; (B) 5
-...AGGGTGATGGTTCACGTAGTGGGC.
Localization of Fig1::-gal and Fig2::
-gal Proteins
MATa cells of the indicated strains were grown to midlogarithmic phase
(OD600 = 0.3-0.4) in YPD, divided into 10-ml cultures, and then incubated
for 2 h in either the presence or absence of 5 µg/ml -factor. Cells were
harvested, fixed, and processed for immunofluorescence as described
(Gehrung and Snyder, 1990
; Pringle et al., 1991
). To visualize
-gal fusion
proteins, a rabbit anti-
-gal primary antibody (Cappel Laboratories, Malvern, PA) was used at 1:12, followed by a CY3-conjugated sheep anti-rabbit secondary antibody (Sigma Chemical Co.) used at 1:200. All antibodies were preadsorbed against fixed and spheroplasted yeast cells before use.
Analysis of Yeast Mating Defects
Haploid strains containing the lacZ fusion insertion mutations were recovered by transforming the heterozygous MATa/MATa diploid strain with a
YCp50 plasmid containing the MAT gene (gift of F. Cross, Rockefeller
University, NY) followed by sporulation of the transformants. MATa segregants containing the transposon insertion mutation were recovered and
mated to strain Y1402. MATa and MAT
segregants were then obtained
and tested for mating defects. Unilateral matings were carried out between MATa mutant strains and strain Y1408. Bilateral matings were performed between MATa and MAT
segregants carrying either the same
transposon insertion mutation or deletion. Diploids from the matings
were selected on SC medium lacking histidine and tryptophan. Liquid mating reactions were carried out as described in Gehrung and Snyder
(1990)
; agents such as polyethylene glycol (PEG)3350, EGTA,
-factor,
and polymyxin-B sulfate (Sigma Chemical Co.) were added to the tester
strains immediately before addition of the strains whose mating efficiency
was being measured. Relative mating efficiencies given in Table IV represent the mean of two separate assays and are normalized to wild-type levels (1.0% diploid formation). Filter mating assays were performed as described in Sprague (1991)
. Under these conditions, the wild-type
frequency of diploid formation was 51.8 ± 1.3%; similar relative frequencies of mating were observed for fig1
and fig4
strains. fig2
strains exhibit a slight (1.5×) increase in cell number/OD600 of cells. However, this
cannot account for the increase in mating efficiency of fig2
mutants in
unilateral and bilateral matings under liquid conditions: experiments in which the density of cells in wild-type control matings similarly increased,
or fig2
cells decreased, did not show comparable increases in mating efficiency (data not shown).
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Projection formation was analyzed by two methods. To assess projection formation in the presence of isotropic pheromone, cultures of mutant
strains were grown to OD600 = 0.3-0.5 and -factor was then added to final concentrations of 0.5, 1.0, 2.5, and 5 µg/ml. Second, after incubation
for 1, 2, and 4 h, cell morphologies were examined by phase-contrast microscopy. Under these conditions in our strain background, cell cycle arrest is observed at the lowest pheromone concentration and polarized
cells with broad projections are formed at intermediate concentrations,
whereas sharp mating projections are formed at the highest concentration.
Polarized projection formation and zygote morphologies were also analyzed for the fig strains by quantitation of different cell types present in
mating mixes. For these assays, cultures of mutant strains were grown to
OD600 = 0.5, and then 2 ml of each were mixed and pelleted by low-speed
centrifugation. Mating cells were then resuspended in 5 ml of fresh YPAD
and allowed to mate for either 8 h at 30°C or 16 h at 16°C without shaking. After incubation, cells were fixed by the addition of formaldehyde to a final concentration of 3.7% for
1 h, sonicated briefly to disperse cells and
zygotes, and then washed and stored in 1× PBS, 1 M sorbitol. Cell mixtures were prepared for microscopy by pelleting an aliquot of the mating
mix and resuspending in mounting solution containing 4
,6-diamidino-Z-phenylindole (DAPI) (70% glycerol, 30% PBS, 2% wt/vol n-propyl gallate,
0.0225 µg/ml DAPI). The scoring of cell type (round, small, or large polarized) was done by placing an aliquot of the fixed cells in a haemocytometer to facilitate counting. Round cells were scored as unpolarized; polarized cells contained projections and were counted as small-medium (with
an overall length less than that of a typical zygote), or large (equal to or
larger in length than a typical zygote). Quantitation of projection tip
shape was determined by scoring medium to large cells, as these cells have
longer projections, the shape of which (pointed or blunt) are most clearly
differentiated. When indicated, staining of cytoplasmic membranes and
lipids was done after fixation by addition of FM4-64 (Molecular Probes,
Inc., Eugene, OR) to 33 µM final concentration, followed by incubation of
the cells on ice for 30 min. Cells were then washed once in 1× PBS, 1 M sorbitol before resuspending in mounting solution containing DAPI. Under these conditions (i.e., in formaldehyde-fixed cells) FM4-64 uniformly
stains the cytoplasm and nucleus, but is absent from cell wall material as
judged by both the reduced diameter of the staining region relative to the
cell outline (as viewed by differential interference-contrast microscopy
[DIC] optics), and by the absence of staining at sites of cytokinesis in budded cells. Measurements of zygotes comparing fusion bridge width to the
mean width of the parental pair were performed using an intraocular micrometer at a magnification of 1600×. 50 zygotes were measured for each strain.
Electron microscopy of thin sections through zygotes was as described
in Byers and Goetsch (1975), with the following differences. Zygotes were
prepared by mating cells as described above; they were pelleted, washed
once in 2 ml 0.1 M cacodylate buffer, pH 7.4, and then fixed in 0.1 M cacodylate, pH 7.4, containing fresh 3% glutaraldehyde for 30 min at room
temperature. Cells and zygotes were then washed once in 0.1 M cacodylate buffer and then stained with 4% KMnO4, followed by dehydration
through a series of ethanol washes, and then embedded in LR-White
acrylic resin (Polysciences Inc., Warrington, PA) before sectioning. Sections were stained with uranyl acetate and lead citrate and then viewed on
an electron microscope (model EM-109; Carl Zeiss, Inc., Thornwood,
NY) at magnifications of 8-40,000×. Pheromone sensitivity of the different strains was assayed by the halo method using strains Y1450 and Y1451
(Sprague, 1991
).
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Results |
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Isolation of Pheromone-regulated Genes
To identify genes specifically regulated during yeast mating, a random lacZ insertional mutagenesis scheme was used.
This method uses a library of yeast DNA fragments containing mini-Tn3::lacZ::LEU2 insertions (Burns et al., 1994).
The lacZ gene lies near one end of the insertion and lacks
an ATG initiator methionine codon; therefore, expression
in yeast is primarily expected to occur because of in-frame
insertion into yeast genes to produce yeast protein::
-gal
fusions. The library was introduced into either a diploid
MATa/MATa leu2
/leu2
bar1
/bar1
or a haploid MATa
leu2
bar1
yeast strain, and then transformants that exhibited enhanced or reduced expression of
-gal in the presence of the
-factor mating pheromone were identified. The use of a diploid strain allows for the isolation of
pheromone-regulated genes that are essential for vegetative growth, whereas the use of strains that lack the Bar1
protease degrades
-factor increases the responsiveness of
the cells to pheromone under our screening conditions. To
facilitate screening large numbers of transformants, an X-gal
plate assay for identifying pheromone regulated
-gal fusions was developed and then optimized using two yeast
strains expressing
-gal fusions with known pheromone-induced proteins, Fus1p and Cik1p (refer to Materials and
Methods; Trueheart et al., 1987
; Page and Snyder, 1992
).
55,000 transformants of a diploid strain and 36,200 transformants of a haploid strain were screened for -gal
expression in the presence and absence of
-factor. 186 strains were identified that reproducibly exhibited increased
-gal activity after pheromone treatment; three
strains displayed decreased activity after treatment. Examples of the pheromone regulated-
-gal expression levels
observed for lacZ fusions in the four novel FIG genes further characterized in this study, and an example of the
class of pheromone-repressed genes are presented in Fig. 1.
To determine the identity of the pheromone-regulated
yeast genes producing the -gal fusion proteins, the yeast
genomic DNA adjacent to the lacZ insertions was plasmid-rescued into E. coli and then sequenced for 158 fusion
strains (Burns et al., 1994
). A summary of these results and
the relative levels of vegetative and pheromone-induced
(or -repressed) expression for the different pheromone-regulated genes identified in this study is presented in Table I. Based on the combined criteria of expression pattern and sequence identity, the fusions occur in genes that can
be classified into five major categories: (a) known pheromone-induced genes; (b) previously characterized genes
not reported to be induced by pheromone; (c) novel pheromone-induced genes; (d) pheromone-repressed genes;
and (e) pheromone- and nitrogen-regulated genes.
Comparison of the number of genes identified by our
screen to the total number of reported pheromone-
induced genes (~22, Sprague and Thorner, 1992; Table I),
along with the observation that many genes are represented by only one or two transposon fusions, indicates
that our screen is not yet saturated. However, many genes
are represented by multiple independent insertions. Extrapolating from the number of different genes identified,
54, and the 1.7 genome equivalents screened and analyzed
(refer to Materials and Methods), we estimate there are
~67 different pheromone-regulated genes in yeast. This
number is probably an underestimate because our transposon mutagenesis procedures have certain biases as shown by the overrepresentation of fusions to SPO11 and HOG1
(Burns et al., 1994
). A larger and probably more accurate
figure of 132 genes is obtained if we extrapolate from the
number of pheromone-induced genes identified in our
screen, nine, with those already known. Thus, we conclude
there are ~67-132 pheromone-induced genes in yeast,
thereby comprising 1-2% of all yeast genes.
Several Types of Genes Respond to Mating Pheromone
65 insertions reside in nine known pheromone-induced
genes including STE6, FUS2, PCL2, CIK1, AFR1, KAR4,
and Ty elements (see Table I for references). Ty1, Ty2,
and Ty3 were previously known to be pheromone-induced
(Boeke and Sandmeyer, 1991; Sprague and Thorner, 1992
;
Kurihara et al., 1996
); our study indicates that the expression of Ty5 elements is also induced. Ty elements and their
long terminal repeats (LTRs) are abundant in the genome (Olson, 1991
), and comprise a large fraction (50 out of
158) of the pheromone-induced fusions identified in this
screen. Additionally, some of the genes identified in this
study are located adjacent to known pheromone-induced
genes (see Table I). Examples include fusion P313B, which
lies in an open reading frame (ORF) adjacent to AFR1,
and the fusions in YFL027c (P28) and the HOG1 gene
(P423A), which lie next to STE2 and a Ty LTR delta sequence, respectively. It is likely that the nearby regulatory
sequences affect the expression of these genes as documented previously for Ty elements (Van Arsdell et al.,
1987
; Company et al., 1988
). Some of these cross-regulated genes may also perform functions in the mating pathway.
In addition to known pheromone-induced genes, many
genes (13) had been identified previously, but were not
known to be pheromone-induced (Table I B). These include SPO11, HOG1, CKI3/YCK3, and RVS161. SPO11 is
a sporulation-induced gene required in the early steps of
meiosis. HOG1 is a MAP kinase homologue that regulates
the osmotic stress response (Brewster et al., 1993). CKI3/
YCK3 is a homologue of the yeast casein kinase I-related
genes YCK1, YCK2, and HRR25, and has recently been
identified as a high copy suppressor of gcs1 mutants, which
are defective in exit from stationary phase (Wang et al.,
1996
). RVS161, previously characterized as playing a role
in actin cytoskeletal functions and cell polarity, has recently been described as important for efficient cell fusion and mating under certain conditions (Crouzet et al., 1991
;
Dorer et al., 1997
). The induced expression of these genes
during pheromone response suggests that many of these
genes may function in the mating process.
A surprising subset of the pheromone-induced genes
identified in this study include genes which are known, or
can be expected to participate in pseudohyphal growth
and/or in nitrogen metabolism, a determinant of pseudohyphal growth (Gimeno et al., 1992; Ljungdahl et al., 1992
).
These genes include PHD1, YFL056c, GAP1, AMD1, DUR1,2, and potentially YGR111w. PHD1 was originally
isolated as a gene that, when present in multiple copies,
promotes pseudohyphal growth (Gimeno and Fink, 1994
).
YFL056c encodes a protein with 57% amino acid identity
over the first 174 of its 212 residues to an aryl alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. In that organism, the gene is induced by
nitrogen starvation conditions, and its product is implicated in lignin degradation (Reiser et al., 1994
). The degradation of lignins, an important constituent of plant cell
walls, facilitates fungal invasion into host plant tissues.
GAP1 and AMD1 encode a general amino acid permease
and AMD1 encodes a putative amidase. YGR111w encodes a probable lysine N6-acetyltransferase, an enzyme
involved in the degradation of lysine. DUR1,2 encodes a
urea amidolyase that converts urea to ammonia. The functions of these last four genes are likely to permit the efficient
use of alternative nitrogen sources such as those provided by
amino acids. PHD1, GAP1, AMD1, and DUR1,2 (Table I E) are each induced by nitrogen starvation (Table III), as
has been shown previously for DUR1,2 and GAP1 (Jauniaux and Grenson, 1990
; Stanbrough and Magasanik, 1995
).
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Another class of pheromone-regulated genes display decreased expression in pheromone-treated cells. The three
pheromone-repressed genes we identified include: PHO81,
FOX2, and a novel gene, QOR1 (refer to Fig. 1 for the
pheromone-dependent repression of FOX2 expression).
PHO81 encodes a repressor of the Pho85 CDK-G1 kinase complex (Ogawa et al., 1995), FOX2 functions in peroxisome biogenesis (Kunau and Hartig, 1992
), and QOR1 has
strong similarity to quinone oxidoreductases, suggesting a
function in oxidative respiration in mitochondria. The relatively limited number of pheromone-repressed genes
identified may be the result of the long half-life (~20 h) of
-gal in yeast (Bachmair et al., 1992
); this could make many
pheromone-repressed genes difficult to identify in the 12-h pheromone incubation used in our screen. Surprisingly,
the FOX2 and QOR1
-gal fusions are not in-frame. However, it is likely that these out-of-frame fusions reflect the
normal regulation patterns of these genes. In a separate
study, we have prepared an in-frame fusion in the QOR1
gene (Minehart, S., S. Erdman, and M. Snyder, unpublished data). Although the absolute levels of expression
for the original out-of-frame fusion strain were lower, as
expected, both the in- and out-of-frame fusions exhibited similar relative levels and kinetics of
-gal induction (expression of QOR1 is induced by carbon source changes at
the diauxic shift) and pheromone repression. Interestingly,
each of the pheromone-repressed genes is likely to be subject to glucose repression; possible mechanisms to explain
their regulation by the pheromone pathway are presented
in the Discussion.
A large number of novel genes was also identified, and further characterization of four of these genes, FIG1-4, is presented below. Some novel genes encode proteins that have homologues in higher eukaryotes, whereas others are predicted to encode proteins that lack extensive homology to other known proteins in the databases (Table I C). Nonetheless, many of the unique proteins have distinctive sequence features. For example, many of the novel pheromone-regulated proteins contain regions predicting their insertion into, or association with, cellular membranes (examples include Fig1p, Fig2p, Yar027wp, andYpl156cp).
Finally, in several cases the lacZ fusion resided either in
short ORFs, out-of-frame, reverse orientation, or in regions flanking genes (although most fusions were found to
be in-frame with ORFs). These results indicate that sequences in addition to long ORFs can be expressed as protein in vivo, and are corroborated both by our previous
study that found that short ORFs outside of predicted coding sequences are often expressed, and by recent analyses
of the yeast transcriptome using SAGE techniques (Burns
et al., 1994; Velculescu et al., 1997
). For two genes, FUS2
and GAP1, out-of-frame fusions were found in addition to
several in-frame fusions. For both genes, in- and out-
of-frame fusions were regulated similarly. Many of the insertions obtained in the HOG1 region are either out-of-frame, in reverse orientation, or in flanking regions; nonetheless, all exhibit similar levels of pheromone induction consistent with responses to the same regulatory elements in each case. Thus, we presume that in many, if not
most, cases, the regulation that is observed for any particular lacZ fusion reflects the expression of the transcript for
the ORF into which the lacZ is inserted, an interpretation
supported by our studies with QOR1 insertions. One
mechanism to account for the expression of out-of-frame
fusions is translational frameshifting.
Pheromone-regulated Gene Expression
Based on the results of quantitative -gal assays presented
in Table II, the levels of induced expression upon pheromone treatment are ~1.3->700-fold for most of the pheromone-induced fusions. In cases where these levels have
been measured, the figures reported here agree closely
with those found previously (e.g., FUS2 and CIK1; Page
and Snyder, 1992
; Elion et al., 1995
).
|
An upstream regulatory element termed the PRE has
been identified as mediating the pheromone-induced transcription of a number of genes involved in the mating response (e.g., FUS2, CSH1, MFA2, STE6, STE2, BAR1, Ty
elements and CIK1) (Van Arsdell et al., 1987). These sequences represent potential binding sites for Ste12p, the
transcription factor that mediates pheromone-induced
transcription, and are generally found upstream of pheromone-induced protein coding sequences (Kronstad et al.,
1987
; Errede and Ammerer, 1989
; Page and Snyder, 1992
).
We searched the regions immediately upstream of the four
novel FIG genes characterized in this study and found sequences matching the PRE consensus (Fig. 2). Since several of these genes are pheromone dependent for their expression yet contain only PRE sites that differ from the
consensus, these results indicate that variant PRE sites are
likely to be important for Ste12p-dependent regulation of
some genes (e.g., FIG1 and FIG4, Fig. 2). An additional search for Mcm1p binding sites, which can be found near
PRE sites of a subset of pheromone-induced genes such as
FUS1 (Herskowitz et al., 1992
), failed to identify sequences
in the upstream regions of the FIG1-4 genes closely matching the consensus binding site.
|
Mating pheromone treatment of cells causes cell cycle
arrest in G1, and it has been proposed that this arrest may
influence the expression of some genes that would be indirectly controlled by activation of the mating pathway
(Stetler and Thorner, 1984; Price et al., 1991
). We tested
whether the pheromone-induced expression of the four
FIG genes characterized in the present study is a consequence of direct or indirect regulation by the pheromone- response pathway. MATa strains carrying lacZ fusions of the four FIG genes were crossed to a MAT
cdc28-1 strain,
and MATa cdc28-1 fig::lacZ progeny were tested for induction of gene expression after cell cycle arrest in the absence of mating pheromone treatment (cdc28-1 strains
shifted to the restrictive temperature arrest in G1). No increase in gene expression was observed for any of the four
genes in the absence of pheromone treatment, nor was any
expression observed in a/
cells (data not shown). In addition, mating-induced expression of the four genes was observed in both a and
cell types as monitored by the mating of strains of either cell type carrying lacZ fusions in
these genes to yeast strains of the opposite mating type.
These data, combined with the presence of upstream sites
similar to the PRE consensus sequence in the four FIG
genes, strongly suggest that the pheromone-induced expression of these genes in haploid cells of both mating
types is because of direct regulation by Ste12p.
Four Novel Pheromone-induced Genes Are Important for Yeast Mating
To begin the characterization of the pheromone-regulated
genes identified from our screen, the mating phenotypes
of 20 haploid mutant strains carrying different transposon
insertions were analyzed (Table I). Haploid strains containing the lacZ insertions were derived from MATa/MATa
diploid parental insertion strains and examined for defects
in (a) viability; (b) cell cycle arrest and polarized growth in
response to pheromone; (c) pheromone sensitivity and adaptation; (d) pheromone production in each cell type; and
(e) mating efficiency in both unilateral and bilateral matings (i.e., a lacZ insertion strain × wild-type or a lacZ insertion strain ×
lacZ insertion strain, respectively). No
defects in viability, cell cycle arrest, polarized projection formation, adaptation, or pheromone production were detected for the strains that were examined. Evaluation of
mating efficiencies under conditions of reduced cell densities, however, did identify three mutant strains, fig1::lacZ,
fig2::lacZ, and fig3::lacZ that were each altered in mating
efficiency relative to a wild-type strain.
The roles of FIG1, FIG2, and KAR5/FIG3 in yeast mating were investigated in detail using a variety of mating
conditions. Because of its striking pheromone-induced expression pattern and its homology to the yeast Sac1p, a
known effector of actin cytoskeletal dynamics (Cleves et al.,
1989; Novick et al., 1989
), the role of FIG4 in mating was
also examined. Although initial studies failed to reveal a
mating defect in fig4::lacZ strains, it is possible that the transposon insertion allele that was tested, P403A-2, may
encode a fusion protein that retains some level of Fig4p
activity, as it contains 90% of the Sac1p homology domain
(see below). To ensure that null phenotypes were analyzed, strains in which the entire protein coding sequence
of each of these genes was substituted with URA3 were
constructed by PCR (Baudin et al., 1993
). The fig1
, fig2
,
kar5
/fig3
, and fig4
strains grew at rates identical to
those of wild-type cells, and no vegetative growth defects were apparent at 16°, 25°, 30°, and 37°C.
As observed with the transposon insertion alleles, fig1,
fig2
, kar5
/fig3
, and fig4
mutants appeared normal
for cell cycle arrest and recovery, pheromone sensitivity,
and projection formation at all pheromone concentrations
tested (Fig. 3 for mating projection results; refer to Materials and Methods). However, the fig
strains each exhibited quantitative mating defects, and the severity of the defect differed depending upon the mating condition (Table IV). At 30°C, absence of Fig1p, Kar5/Fig3p, or Fig4p results in a bilateral mating defect that reduces mating efficiency 2.5-, 77.4-, and 2.9-fold, respectively, relative to that
of a wild-type strain. In contrast, loss of Fig2p reproducibly increases the mating efficiency 3.2-7.2-fold in both
unilateral and bilateral matings. Increased mating efficiency through the loss of a gene product in otherwise
wild-type cells is a novel phenotype for a gene that functions in mating. The increased mating efficiency for fig2
strains is likely because of their enhanced agglutination relative to wild-type cells (see below). The mating phenotypes of the fig1
, fig2
, and kar5
/fig3
strains were the
same as their respective transposon insertion mutants. We
also tested the relative mating efficiencies of fig1
, fig2
,
and fig4
mutants using mating conditions that concentrate cells on filters (Sprague, 1991
). Under these conditions, the relative mating efficiencies of fig 1
and fig4
were similar to those observed by liquid conditions. The
increased mating efficiency of fig2
strains was no longer observed; instead we observed a 6.6-fold decrease in mating efficiency relative to wild-type strains. We presume
that in contrast to liquid mating conditions that require
cells to agglutinate to mate efficiently (Kurjan, 1993
), the
close packing of cells caused by collection on filters reduces or eliminates the need for agglutination in the filter-mating assays. As noted below, the increased mating efficiency of fig2
strains in liquid assays is likely due to the
hyperagglutination activity of these cells; this activity is no
longer expected to be important in filter-mating assays.
|
We also investigated the effects of different conditions
on the mating efficiencies of fig 1, fig2
, and fig4
mutants (Table IV); the severe effect of the kar5
/fig3
mutation on mating efficiency precluded its accurate measurement under these conditions. At 16°C, the mating
efficiencies of both fig1
and especially fig2
bilateral
matings are impaired relative to wild-type strains (1.4- and
18-fold, respectively). The bilateral matings involving
fig1
and fig2
mutants are also inhibited more strongly
than wild type by polymyxin B sulfate, a membrane-disrupting agent. The effects of PEG and EGTA on the mutant matings revealed additional differences between the
fig1
and fig2
strains. While PEG is a potent (5.2-7.6-fold) enhancer of mating efficiency for wild-type, fig1
,
and fig4
strains, it has a much smaller effect on the mating efficiency of fig2
strains. Interestingly, the mating efficiency of fig1
bilateral matings is more sensitive to
EGTA, exhibiting a 3.1-fold decrease relative to wild-type
strains. The relative mating efficiency of fig4
mutants
was affected to similar degree as the mating efficiency of
wild-type strains by the different conditions. In summary,
the differing effects of the conditions of cold temperature, PEG, and EGTA on the mating efficiencies of fig 1
, fig2
,
and fig4
strains suggest that Fig1p, Fig2p, and Fig4p play
distinct roles in mating, and may provide insights into their
molecular functions (see Discussion).
fig2 and kar5/fig3
Mating Cells Hyperagglutinate
and Form Small Colonies, Respectively
After the discovery that fig mutants exhibit altered mating efficiencies, we sought to determine the phenotypic basis of these effects. Two macroscopic phenotypes were observed in matings involving fig2
and kar5/fig3
mutants.
During mating, wild-type cells gather into clusters through
agglutination. fig2
strains exhibit a hyperagglutination
phenotype in which mating cells aggregate to a greater extent than wild-type cells. This phenotype is observed by
both uni- and bilateral crosses using settling assays (Fig. 4
A), and microscopic examination of mating cells (data not
shown). Hyperagglutination caused by the fig2
mutation
is an interaction specific to mixtures of mating cells; fig2
mutant strains of either mating type do not aggregate during vegetative growth or when mixed with cells of the
same mating type. Hyperagglutination of fig2
strains during mating was observed at both 30° and 16°C, indicating
that the cold sensitivity of fig2
mutant matings is caused
by a defect independent of agglutination.
|
The second macroscopic mating phenotype occurs in bilateral crosses of kar5/fig3 mutants. Matings of wild-type
and all other fig
mutant strains gave rise to uniformly-sized diploid colonies after 1.5 d of incubation at 30°C. In
contrast, matings of kar5/fig3
mutants produced many
small, irregular colonies as shown in Fig. 4 B. The number
of smaller colonies approximates that of the total number
of colonies formed in matings involving wild-type cells. Cells from both large and small colonies were fixed and
then stained with Hoechst to examine their nuclear contents. Budding cells, cells with mating projections, anucleate and multinucleate cells, and zygotes were observed in
each case. Progeny from both classes of colonies mated
with both MATa and MAT
tester strains. These phenotypes are consistent with nuclear fusion failures in kar5
/
fig3
prezygotes (see below). Such failures would be expected to lead to unstable heterokaryons, which, in turn,
produce haploid progeny.
fig1, fig2
, and fig4
Strains Exhibit Defects in
Mating Cell Morphology
The mating properties of the fig mutant strains were investigated further by examining the morphology and distribution of nuclei in cells and zygotes in wild-type and bilateral fig
mating mixtures (Fig. 5). Cell shape and
degree of polarization (unpolarized, small-medium polarized, and large polarized cells and zygotes) were quantified (Table V). Three of the fig
mutations, fig1
, fig2
,
and fig4
, each alter the morphologies of mating projections and zygotes in distinct ways.
|
|
fig1, and to a lesser extent fig4
, mating mixtures have
fewer medium and large polarized cells than wild-type or
fig3
matings (Fig. 5; Table V). Many of the fig1
and
fig4
cells that are polarized possess mating projections
with tips that are broader and less focused than those of
wild-type cells; for these strains the percentage of large
cells with pointed projections was less than half that of
wild-type cells or other fig
mutants (Fig. 5, insets; Table
V). In addition, in the case of fig4
cells, we often observe
multiple bumps around the cell periphery of unpolarized but enlarged cells, suggestive of failures in the intial establishment of mating cell polarity. We also examined the distribution of actin in these strains by rhodamine conjugated-phalloidin staining (Fig. 6). The pattern of actin
staining at the mating projection tip is typically less intense
and more dispersed in both fig1
and fig4
cells compared
to that of wild-type cells, whereas actin polarization remains normal in fig2
cells. Thus, whereas FIG1 and FIG4
are dispensible for forming normal projections in isotropic levels of mating pheromone, in mating mixtures these
genes are important both for the execution of cell polarization and the development of mating projection shape
(see Discussion). Although the effects of the fig 1
and
fig4
mutations on cell polarization are similar, differences in zygote morphologies between these two mutants
suggest they perform different functions in the mating process; fig 1
, but not fig4
, zygotes display cell fusion defects (Fig. 5, and see below).
|
The morphological alterations in mating projection formation caused by the fig2 mutation are distinct from
those generated by the fig1
and fig4
mutations. fig2
cells form hyperpolarized mating projections that are often narrower and longer than those of wild-type cells (Fig.
5). A consequence of the hyperpolarization of the fig2
mating projection is the formation of zygotes possessing narrow fusion bridges (the central portion of zygotes
formed by fusion between the polarized tips of mating
cells) (Fig. 5). Measurement of the ratio of fusion bridge
width/average parental cell pair width for 50 wild-type,
fig1
, and fig2
zygotes supports this observation; for
wild-type and fig1
zygotes these ratios are 0.52 and 0.51, respectively, whereas for fig2
zygotes the value is 0.30. Thus, FIG2 is important for mating cell projection shape and conjugation bridge diameter.
While preparing this manuscript, we learned that FIG3
corresponds to the previously identified KAR5 gene, whose
molecular characterization has not been reported. Analysis of cell polarization and zygote formation in fig3 mutant cells indicated that cell polarization and zygote morphology is normal, unlike that of fig1
, fig2
, and fig4
mating cells. Instead, kar5
/fig3
zygotes displayed nuclear fusion defects in which nuclei lie within close proximity but fail to fuse (Fig. 5). This result is consistent with
that reported previously for kar5 mutant alleles (Kurihara
et al., 1994
; Fig. 5, this study).
FIG1 and FIG2 Function in Cell Fusion and Nuclear Migration
To help understand the functions of FIG1 and FIG2 in the
differentiation of wild-type mating cells, we examined the
cell morphologies and nuclear positions of prezygotes and
zygotes formed by wild-type, fig1, and fig2
strains
mated at 16°C; this condition enhances the mating defects
of the mutant strains. As observed for fig1
strains at
30°C, fig1
and fig2
zygotes formed at 16°C display cell
fusion defects. These defects were quantified by examining prezygotes and zygotes using DIC microscopy, DAPI
staining (to examine nuclear fusion and morphology), and
staining with the lipophilic dye FM4-64 which decorates lipids and membranes, but not cell wall material (Fig. 7). As
shown in Table VI, the incidence of partial and complete
failures in cell fusion is increased markedly in fig1
zygotes (ninefold), and more modestly in fig2
zygotes (approximately twofold).
|
|
When fig2 strains mate at both 30° and 16°C, a high
frequency (84%) of zygotes show the hyperpolarization/
narrow fusion bridge phenotype. As shown in Fig. 7, a
number of defects appear to be caused by the narrow fusion bridge phenotype of fig2
mutants. The most prevalent phenotype, observed in ~80% of fig2
zygotes, is a
novel nuclear morphology that suggests a failure to comple the late steps of nuclear fusion. Normally, nuclear fusion proceeds by the microtubule-dependent congression
of nuclei, followed by nuclear membrane fusion (Kurihara
et al., 1994
). The fused haploid nuclei then form a contiguous, elliptical or quasispherical diploid nucleus. In wild-type zygotes possessing a bud, the nucleus is often located
near the site of bud emergence, or can be seen to be segregating or to have segregated between the zygote and bud (Fig. 7; top two rows). In fig2
zygotes, the newly fused
nucleus nearly always has an abnormal shape, and in zygotes possessing a bud it is frequently observed to lie in
abnormal positions, suggesting difficulties in nuclear migration to the bud site or in subsequent segregation events
(Fig. 5; Fig. 7, bottom two rows; Table VI). fig2
zygotes
appear delayed in rounding up of the nucleus, as judged by
the presence of contiguous DAPI staining material across
the fusion bridge region (Fig. 7, Table VI). In the majority
of these nuclear configurations, two interconnected DAPI
staining regions are observed on either side of the fusion bridge, whereas less frequently a single DAPI staining region is observed to be contiguous with nuclear material remaining in the fusion bridge (Table VI). For each of these
cases, the majority of these altered nuclear configurations
occur in fig2
zygotes displaying the narrow bridge phenotype shown in Figs. 5 and 7.
To further examine the cell and nuclear fusion defects
visualized by light microscopy, we performed electron microscopic analysis on thin section preparations of wild-type,
fig1, and fig2
zygotes (Fig. 8). Inspection of micrographs
of the fig1
zygotes confirms the presence of undissolved
cell wall materials and membrane causing both partial and
complete fusion defects (Fig. 8, B and C; this is particularly
evident in higher magnification micrographs; data not
shown). Moreover, examination of the partial fusion defects by both fluorescent microscopic techniques and electron microscopy indicates that nuclear fusion is a robust
process, capable of being executed through very small regions of cytoplasmic continuity (for example, Fig. 7, fig1
center panel; Fig. 8 C; and Table VI, partial fusion defect
column). Analysis of fig2
zygotes revealed elongated nuclear morphologies consistent with those visualized by
DAPI staining of whole zygotes. In summary, these different data demonstrate that fig1
and fig2
zygotes exhibit
both cell fusion and nuclear morphology defects.
|
FIG1, FIG2, and FIG4 Function in at Least Two Different Mating Cell Differentiation Pathways Required for Cell Shape and Polarity
The different effects of nonoptimal mating conditions on
the mating efficiencies of fig1, fig2
, and fig4
strains
suggested that these mutants are defective in different
pathways involved in mating cell differentiation (Table IV).
To investigate this further, we examined the epistatic relationships of the fig
mutations by characterizing the mating cell projection and zygote morphologies of double mutant strains mated at 30° and 16°C; bilateral matings of
MATa and MAT
fig1
fig2
, fig1
fig4
, and fig2
fig4
mutant strains were examined (Fig. 9). For most of the
double mutant strains, the phenotype of any single mutation was never completely epistatic to that of another (Table VII). All double mutants carrying the fig1
mutation
displayed reductions in the fraction of cells producing
pointed mating projections and increases in the rate of cell
fusion defects. Similarly, double mutants involving the
fig2
mutation displayed a narrow conjugation bridge and
the aberrant nuclear morphology phenotypes; these mutants also hyperagglutinated at both 30° and 16°C. All double mutants involving the fig4
mutation displayed a reduction in the percentage of cells with pointed projections.
Thus, the morphological phenotypes of the fig1
fig2
and
fig2
fig4
double mutants represent a combination of
those observed in each of the corresponding single mutants, suggesting that Fig2p functions in a distinct pathway
from that of either Fig1p or Fig4p (Table VII).
|
|
There are exceptions to these independent epistasis relationships. The fig1 and fig4
mutations did not produce additive effects in cell polarization, suggesting that
these mutants may function in the same or significantly
overlapping pathways for this particular process (Table
VII; however, see Discussion). In addition, the fraction of
cells with a pointed projection tip in the fig1
fig2
and
fig2
fig4
mutants was reduced relative to that of fig2
mutants alone (Table VII). This suggests that hyperpolarization caused by the absence of Fig2p function may partly
require the function of the polarization pathway(s) in which Fig1p and Fig4p function. We are cautious, however, in interpreting this relationship as one that applies to
normal mating cell polarization, since hyperpolarization is
a consequence of the loss of FIG2 function and not a polarization event normally occurring in wild-type mating cells. In summary, these results indicate that the FIG1,
FIG2, and FIG4 genes encode proteins that are components of at least two distinct mating cell differentiation
pathways required for projection shape and polarity.
Fig1p and Fig2p Localize to the Cell Periphery
To gain further insight into the function of the different
pheromone-regulated genes, the subcellular localizations
of -gal fusion proteins in 14 strains carrying lacZ fusions
to different genes, including fig1::lacZ, fig2::lacZ, fig3::lacZ,
and fig4::lacZ, were analyzed using anti-
-gal antibodies
and indirect immunofluorescence. Fusion proteins encoded by fig3::lacZ and fig4::lacZ, along with those from
10 other strains, failed to localize in a discrete pattern or at
a level above background. Presumably, some of these fusion proteins may lack sequences required for their stability or subcellular localization. Two strains, fig1::lacZ and
fig2::lacZ, were, however, found to exhibit strong
-gal
staining at discrete sites in pheromone-treated cells (Fig.
10). In each case the
-gal fusion proteins appeared to be
abundant, based on staining intensity, and localized to the
cell periphery in 100% of the cells (n > 400). The Fig1::
-gal
and Fig2::
-gal fusion proteins were often slightly polarized toward the projection tips, but did not appear to be as
sharply concentrated at the tips as reported previously for
the Fus1 and Fus2 proteins (Trueheart et al., 1987
; Elion et
al., 1995
). For both Fig1::
-gal and Fig2::
-gal fusions
(~10%), in a small fraction of cells perinuclear staining
was observed (Fig. 10). Such staining was also observed in
rare cells (<3%) that were not treated with pheromone
and the staining was very weak (Fig. 10). This perinuclear
staining may represent low levels of the fusion proteins
contained in the endoplasmic reticulum. The localization
of Fig1p and Fig2p suggests they may perform their functions at the cell periphery.
|
The FIG1, FIG2, KAR5/FIG3, and FIG4 Proteins Contain Distinct Sequence Features
The four genes characterized in detail in this study, FIG1, FIG2, KAR5/FIG3, and FIG4, are predicted to encode proteins of 298-, 1609-, 504-, and 879-amino acids, respectively. Each of these proteins is predicted to contain domains suggestive of a structure, localization, or function of the proteins. (Fig. 11 A).
|
FIG1 and FIG2 are predicted to encode membrane-
associated proteins. Fig1p contains four predicted transmembrane (TM) domains with a loop between the first
and second TM segments that is expected to be extracellular and contain several potentially glycosylated residues
(Fig. 11 A). The protein has several features in common with members of the four transmembrane (4TM) superfamily of proteins (Wright and Tomlinson, 1994), including the transmembrane segments, the potential extracellular glycosylated loop, and the location of polar and charged
residues at conserved points within two of the TM domains (N23 in TMD1 and D255 in TMD4) (Wright and
Tomlinson, 1994
). Fig2p contains a predicted signal peptide at its amino terminus and potential glycosyl phosphatidylinositol (GPI) anchor sequence at its carboxy terminus. The protein is serine/threonine rich (44.5% serine
or threonine) as are many extracellular proteins, and contains many potential N-linked glycosylation sites (Klis, 1994
; Cid et al., 1995
).
The sequence of KAR5/FIG3 is predicted to encode a
protein capable of containing several long coiled-coil domains (i.e., helical regions with heptad repeats of hydrophobic residues) in the center of the protein. A protein of
577 amino acids in length and possessing limited sequence,
but high structural similarity is also present in the Schizosaccharomyces pombe genome (EMBL/GenBank/DDBJ accession number D87337).
FIG4 encodes a protein that belongs to a family of proteins characterized by a domain that is similar in sequence
to the yeast Sac1 protein. Mutations in SAC1 affect actin
cytoskeletal and secretory functions, possibly through alterations in phospholipid metabolism (Cleves et al., 1989;
Novick et al., 1989
; Whitters et al., 1993
). The alignment of
the protein sequences of selected members of this multigene family is shown in Fig. 11 B. In S. cerevisiae there are
five genes encoding proteins that belong to this multigene family, SAC1, FIG4, YNL106c, YOR3231w, and YIA2, and
several members also exist in other organisms (Majerus,
1996
; McPherson et al., 1996
; Mewes et al., 1997). Fig4p
and Sac1p share 25% identity over 530 amino acids, including three highly conserved domains of 14/17, 9/13, and
10/12 identical amino acids (Fig. 11 B). Interestingly, Fig4p
displays highest sequence identity within the Sac1 domain (29-34%) to a subgroup of the Sac1 domain-containing
proteins that includes a Caenorhabditis elegans and human
homologue of Fig4p. This subgroup of proteins is distinguished, in part, by a 19-amino acid extension of the region of homology shared by the proteins past the carboxy-terminal border of the domain of Sac1p similarity (Fig4p
residues 596-614, Fig. 11 B). Since each of the proteins
contains a unique carboxy terminus, it is possible that the
carboxy-terminal domains confer different specificities on
the proteins toward the execution of their cellular functions.
![]() |
Discussion |
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Identification of Pheromone-regulated Genes
In this study, a lacZ transposon insertion screen was used to identify genes whose expression is regulated in response to mating pheromone. We recovered fusions in known pheromone-regulated genes, previously characterized genes not known to be pheromone regulated, and many novel genes. Based on the frequency with which pheromone-regulated lacZ fusions were found, we estimate that 1-2% of all yeast genes are pheromone regulated.
At least two earlier attempts to identify pheromone-regulated sequences in yeast have been made using differential hybridization screens (Stetler and Thorner, 1984; McCaffrey et al., 1987
). Specific genes were not reported in
one study, and the other was limited to the description of a
single gene, FUS1. One advantage of the lacZ insertion
approach is that it allows the direct identification of pheromone-regulated genes and permits rapid further analysis of multiple target genes.
Previously Characterized Genes and Novel Genes Provide Additional Insights into the Mating Process and Gene Function
Whereas our screen isolated an expected group of known
pheromone-induced genes, a set of previously characterized genes was also found to be under pheromone regulation. The most frequently identified pheromone-regulated
gene of this class was SPO11. SPO11 is induced in meiosis,
and Spo11p is found covalently attached to the ends of
meiotic double-strand beaks in genomic DNA (Atcheson et al., 1987; Keeney et al., 1997
). Our data suggests that
SPO11 is expressed in haploids and exhibits enhanced expression upon pheromone treatment. Consistent with pheromone regulation is the presence of two consensus PRE
sites upstream of the SPO11 coding sequence. SPO11 does
not promote recombination during mating, as matings of
MATa spo11
his4-280 cells to MAT
spo11
his4-260 cells did not reveal any change in the number of His+ prototrophs relative to control matings with wild-type cells (data not shown). Spo11p, which localizes to the nucleus
(Burns et al., 1994
), might play a role in the organization
of the diploid nucleus after nuclear fusion. This organization may occur soon after nuclear fusion as homologous
chromosomes appear associated during vegetative growth
(Weiner and Kleckner, 1994
).
Several other pheromone-regulated genes that may function in mating were also identified by our screen. Two genes
encoding proteins that might function in the cell cycle regulatory events that mediate pheromone-induced arrest
and recovery are HOG1 and CKI3/YCK3. A recent study
has found that the HOG1 osmoregulatory MAP-kinase
pathway negatively regulates the activity of the mating pathway, based in part on the finding that pheromone sensitivity was greater in a hog1 strain than in a wild-type
strain (Hall et al., 1996
). CKI3/YCK3 encodes one of the
four casein kinase I homologous proteins found in S. cerevisiae (Robinson et al., 1992
; Wang et al., 1996
). CKI3/
YCK3 has recently been identified as a high copy suppressor of gcs1 mutants that fail to reenter the cell cycle from
stationary phase (Wang et al., 1996
). Thus, CKI3/YCK3 encodes a pheromone-induced protein that has activity as a
cell cycle regulator.
Our study also found the cell polarity gene RVS161 to
be pheromone regulated. Rvs161p has recently been shown
to be important for proper cell fusion and mating under
certain conditions (Dorer et al., 1997). Mutations in the
RVS161 gene affect actin-dependent processes including
diploid bud site selection and endocytosis (the gene is allelic to END6), and a mammalian homologue of Rvs161p, amphiphysin, has been predicted to be involved in synaptic vesicle recycling (Crouzet et al., 1991
; David et al.,
1994
; Munn et al., 1995
; McPherson et al., 1996
). Perhaps
Rvs161p acts to maintain the proper positioning and/or organization of the actin cytoskeleton to directly or indirectly control the local regulation of cell surface membranes and receptors. A possible role for Rvs161p in the
regulation of pheromone receptors is particularly interesting in light of the common requirement for the function of
these proteins in the process of cell fusion (see below; Brizzio et al., 1996
; Elia and Marsh, 1996
; Dorer et al., 1997
).
Another interesting group of genes that we found to be
regulated by mating pheromone are the pheromone-
repressed genes. The novel gene QOR1, as well as FOX2
and PHO81, have been suggested or demonstrated to be
subject to glucose repression (Minehart, S., S. Erdman, and
M. Snyder, unpublished data; Johnston and Carlson, 1992;
Kunau and Hartig, 1992
; Timblin et al., 1996
). The common property of glucose regulation suggests that the decreased expression levels of these genes in cells exposed to
pheromone may occur through a common mechanism. Activation of the pheromone pathway may curb the expression of these genes directly by affecting a common regulator, as would be the case if glucose repression is established
and/or maintained at the G1 phase of the cell cycle. Arrest
of the cell cycle in G1 by pheromone would, in turn, reduce expression of these genes. Alternatively, repression by the
pheromone pathway might work indirectly by altering the
expression of upstream genes that participate in the sensing
of nutrient levels, which then regulate the expression of the
pheromone-repressed genes (Johnston and Carlson, 1992
).
Some Pheromone-induced Genes Such as PHD1 Are Induced by Both Mating and Pseudohyphal Differentiation Signals
We found that several genes are induced by both pheromone and pseudohyphal growth-promoting conditions (Tables I), suggesting that a common set of target genes
may be involved in both processes. One of these genes is
PHD1, whose overexpression enhances pseudohyphal growth in yeast, and whose product is a transcription factor capable of activating the expression of at least five potential target genes (Gimeno, 1994; Gimeno and Fink,
1994
). A potential PHD1 target, CDC91, was found in our
screen, and a second target corresponds to the DAL81
gene, which, like several of the genes identified in our
screen (i.e., GAP1, DUR1,2, AMD1, and YGR111w), is involved in nitrogen metabolism (Magasanik, 1992
). Thus,
induction of Phd1p by low nitrogen conditions or pheromone may, in turn, activate a common set of target genes
important for the processes of pseudohyphal growth and
mating under certain conditions (see below).
The reason why induction of genes involved in nitrogen
metabolism might be advantageous for mating is unclear.
Perhaps the induction of these genes is a fortuitous vestige
of their common activation mechanism. Previous studies
have shown that several components of the Ste20p MAP-
kinase signaling pathway (Ste20p, Ste11p, Ste7p) and the
transcription factor, Ste12p, are used by both the mating
and pseudohyphal pathways (Liu et al., 1993; Roberts and
Fink, 1994
). Nonetheless, the common induction of several
genes by both pathways suggests they may share some
common features and mechanisms. Consistent with this
hypothesis, we have recently found that yeast cells exposed to low levels of mating pheromone form filamentous arrays similar to pseudohyphal cells (Erdman, S., and
M. Malczynski, unpublished results). Similarly, the pheromone induction of genes that permit the use of alternative nitrogen sources may enable mating cells to grow through
different environments in search of mates.
Although our work suggests a specific group of target
genes shared by the pseudohyphal and pheromone-
responsive pathways, others are unique to either of the
two pathways. A previous study has shown that FUS1 is
not activated in cells undergoing haploid invasive growth
and FUS2, AFR1, ADP1, FIG1, FIG2, and KAR5/FIG3 are
only induced by mating pheromone (Erdman, S., unpublished data; Roberts and Fink, 1994). Furthermore, a recent
study has identified a gene, TEC1, whose product, along
with Ste12p, appears to mediate the transcriptional induction of genes specific to pseudohyphal differentiation
(Madhani and Fink, 1997
). Thus, there remain many genes
that are induced only during pseudohyphal growth or mating, presumably contributing to the unique aspects of
these processes.
FIG1, FIG2, KAR5/FIG3, and FIG4 Identify New Components and Steps of the Yeast Mating Pathway
Four novel pheromone-regulated genes characterized in this study play distinct roles in yeast mating. Strains in which the FIG genes are deleted display vegetative growth properties identical to those of wild-type strains, consistent with their specific involvement in the mating process. Analyses of the cell polarization behavior in mating mixtures of mutants revealed that Fig1p, Fig2p, and Fig4p are each necessary for normal mating projection formation; Kar5p/Fig3p is important for nuclear fusion.
Thus far, two major classes of proteins have been shown
to be required for normal mating cell polarization: (a) general polarity components common to both budding and
mating processes; and (b) pheromone-regulated components such as the pheromone receptor (Ste2p), Far1p, and
Afr1p (Konopka et al., 1988; Konopka, 1993
; Herskowitz
et al., 1995
; Konopka et al., 1995
; Pringle et al., 1995
; Roemer et al., 1996
). Fig1p, Fig2p, and Fig4p, like the latter
group of proteins, are induced by mating pheromone and appear to function in the specification and/or organization
of pheromone receptor-dictated sites of polarized growth.
However, a significant difference exists between the phenotypes of mutants in the FIG1, FIG2, and FIG4 genes
and those in STE2, FAR1, and AFR1. The fig mutants arrest and polarize normally in the presence of uniform concentrations of mating pheromone, but exhibit polarization defects only in mating mixtures; ste2, far1, and afr1 mutants exhibit defects under both conditions (Konopka et al.,
1988
; Chang and Herskowitz, 1990
; Konopka, 1993
; Chenevert et al., 1994
; Dorer et al., 1995
; Konopka et al., 1995
;
Valtz et al., 1995
). One principal difference between these
two conditions is that in the presence of isotropic concentrations of pheromone, cells use axial and distal bud site
polarity landmarks to specify sites of mating projection
formation, whereas in mating mixtures cells choose projection sites on the basis of the pheromone receptors detecting gradients of mating pheromones that emanate from
potential mating partners (Jackson and Hartwell, 1990
;
Madden and Snyder, 1992
; Segall, 1993
; Valtz et al., 1995
).
These different conditions also produce distinct cell morphologies and may reveal differing aspects of the mating
process (Figs. 3 and 5; Madden and Snyder, 1992
; Rose
and Marsh, 1997
). Interestingly, strains lacking either FIG1
or FIG2 also possess cell fusion defects; the cell fusion process has also been speculated to be regulated by cell-cell contact (Rose and Marsh, 1997
). Thus, from the specific
requirements for FIG1, FIG2, and FIG4 for normal mating
cell projection polarization and shape in mating mixtures,
we conclude that the products of these genes perform cell
polarity functions associated with polarized growth sites
that are dictated by cell-cell communication events.
The mating projection defects of the fig mutants provide
three important insights into the process of mating projection formation. First, a number of proteins in distinct pathways participate in cell polarity events governing mating
projection growth. Differences observed in the sensitivities of fig1, fig2
, and fig4
mutants in the genes to specific mating conditions suggests that the Fig1, Fig2, and
Fig4 genes function primarily in distinct pathways. Phenotypic analyses of the mating cell polarization and cell fusion properties of double mutants clearly indicate that
FIG2 functions in a different pathway from that of FIG1
and FIG4. Genetic arguments suggest that FIG1 and FIG4
function in the same or extensively overlapping pathways
based on their lack of additive effects on projection tip
shape. However, mutants in these genes display a number
of distinct properties (e.g., EGTA sensitivity and cell fusion defects in fig1
mating cells), suggesting they may
contribute to cell polarization in different ways. The epistatic relationship of the FIG1 and FIG4 genes will be ascertained more precisely after additional molecular characterization of the functions of these genes.
Second, although a number of gene products participate
in cell polarity events that form the mating projection,
only a subset of these polarity components also function in
cell fusion. FIG1 and FIG4 are both required for mating
projection polarization, whereas only FIG1 has a role in
cell fusion. A recent study has also found that many general polarity components (e.g., Spa2p, Pea2p, Rvs161p,
and Bni1p) are required for cell fusion, whereas another
(Bem1p) is not (Dorer et al., 1997). Perhaps fusion-specific polarity components recruit and localize cell signaling
components (i.e., pheromones, receptors, and transporters), which, in turn, target cell fusion proteins (Brizzio et al.,
1996
; Elia and Marsh, 1996
; Dorer et al., 1997
). Alternatively, cell polarity components might directly localize both
signaling and cell fusion components to cell-cell contact sites.
Finally, the defects observed in fig2 zygotes possessing
narrow conjugation bridges reveal a prerequisite of proper
conjugation bridge shape for the efficient execution of
subsequent events of nuclear fusion and segregation. This
requirement further emphasizes the importance of components that regulate the spatial control of mating projection
morphogenesis, which, in turn, determines the shape of
the conjugation bridge. Since the mating defect of fig2
strains is normally revealed only by quantitative mating assays and low temperature conditions (conditions not
commonly used in genetic screens), it will be interesting to
examine mutants in other cell wall components for similar
morphological phenotypes.
FIG1p Is a Novel Transmembrane Protein That Functions in Cell Polarization and Fusion
A number of lines of evidence suggest that Fig1p functions
in organization of polarized growth sites during mating.
Analysis of the mating morphology of fig1 mutant cells
indicates a role for Fig1p in cell polarization. fig1
mutants are also defective in cell fusion, which depends upon
pheromone signaling and actin-interacting proteins (Brizzio et al., 1996
; Elia and Marsh, 1996
; Dorer et al., 1997
).
Fig1p could participate in pheromone receptor-directed or
cell fusion-targeted polarity functions in a number of different ways. Fig1p might directly or indirectly associate
with known components of signaling or fusion complexes
located at growth sites initially established by activated
pheromone receptors (Madden and Snyder, 1992
; Valtz
et al., 1995
). Alternatively, Fig1p might act by controlling
ion flux (see below), which, in turn, affects organization of
the actin cytoskeleton. Finally, fig1
mutants share many
properties (e.g., cold sensitivity, EGTA supersensitivity, bilateral cell fusion defects) with mutants defective in FUS1, which encodes a type I transmembrane protein (McCaffrey
et al., 1987
; Trueheart et al., 1987
; Elion et al., 1995
). Thus,
Fig1p and Fus1p may function in the same pathway(s).
The Fig1p is a predicted 4 TM domain-containing protein, and localization of a Fig1::-gal fusion protein shows
that it is likely to reside in the plasma membrane within
close proximity to other membrane-associated polarity and
cell fusion components, such as the Ste2p and Ste3p transmembrane pheromone receptors, the G-protein subunits,
and Fus1p (for review see Sprague and Thorner, 1992
).
Fig1p also possesses similarities to the mammalian 4 TM
superfamily of proteins. Members of this superfamily have been implicated in cell polarity functions in the mammalian immune system (Wright and Tomlinson, 1994
). Some
of these proteins have been shown to act as effector molecules, coupling G-proteins to their cognate receptors (Wright
and Tomlinson, 1994
). Fig1p may act in a similar manner
by helping recruit general polarity components to sites of
activated G-proteins (Leeuw et al., 1995
; Madden and Snyder, 1992
; Valtz et al., 1995
).
Features of the Fig1p sequence suggest it may act as an
ion transporter or channel. The presence of charged amino
acids at conserved locations within their transmembrane
segments has led to speculation that 4 TM superfamily
proteins may function as ion transporters (Wright and
Tomlinson, 1994). A second 4 TM superfamily of genes,
the connexins, also possess such charged domains within their transmembrane segments (Goodenough et al., 1996
).
Connexins and ductins, a structurally related group of proteins, function as ion transporters at gap junctions, which
are sites of cell-cell contact and communication in higher
eukaryotes (Finbow et al., 1994
; Goodenough et al., 1996
;
Bryant, 1997
). Fig1p contains charged residues within two
predicted transmembrane segments and mating of fig1
mutants is supersensitive to the addition of EGTA, a chelator of calcium ions. The quantitative mating defect of
fig1
mutants is also rescued by Ca2+ supplementation of
the medium (Erdman, S., unpublished data). It is known
that yeast cells exposed to mating pheromone undergo a
large calcium influx (Ohsumi and Anraku, 1985
). Thus, Fig1p might serve a role in mediating or coupling this ion influx
to the activity of other morphogenetic proteins (e.g., Cdc24p;
Miyamoto et al., 1987
). Although the exact mechanism of its
function in mating cell polarization and fusion is not known,
Fig1p represents the first component specific to mating
cells involved in both these processes. Further study of
Fig1p will likely yield significant insights into the nature of
the link between cell polarization and fusion during mating.
Fig2p Is a Putative GPI-anchored Cell Surface Protein That Affects Cell Adhesion and Shape
The sequence of Fig2p predicts it to be an extensively glycosylated protein that is GPI-anchored to the cell surface.
The protein is also rich in serine and threonine, similar to
many cell surface proteins of yeast (Klis, 1994; Cid et al.,
1995
). These properties suggest that Fig2p, like other
pheromone-regulated cell wall components such as chitin
synthase I and a subunit of 1,3-
-D-glucan synthase (encoded by CHS1 and FKS2, respectively), probably function to provide structural support to the newly deposited cell wall of the growing mating projection (Appeltauer and
Achstetter, 1989
; Mazur et al., 1995
).
The role of Fig2p in mating cells provides insights into how
cell surface components are coupled to polarized growth
processes in the underlying cell cortex and how these components affect cell-cell communication events. Loss of
Fig2p function removes a component of the cell wall and
produces enhanced apical growth of the mating projection.
Thus, disruptions in extracellular components can influence the spatial program of polarized growth in the underlying cell cortex. Perhaps the absence of Fig2p activates a
checkpoint monitoring projection growth, analogous to
that described for bud morphogenesis (Lew and Reed,
1995). Loss of Fig2p function also causes a novel mating
phenotype of hyperagglutination and increased mating
efficiency at 30°C under liquid mating conditions. Two
hypotheses to account for these effects are either that the absence of Fig2p alters cell wall structure such that agglutinins become more exposed on the cell surface, increasing
their effectiveness, or that the pool of GPI anchors or pathway components attaching them to these proteins is limiting so that the absence of one (Fig2p) increases the number
of other proteins (e.g., Aga1p or Ag
1p) receiving anchors.
Despite their increased efficiency of mating at 30°C, fig2
mutants are still cold sensitive for mating (18-fold reduced
relative to wild-type cells). These mutants also hyperagglutinate during mating at 16°C, indicating their mating defect
lies in a cellular process other than agglutination. A striking property of fig2
mutant cells is their narrow mating
projections relative to wild-type cells, and consequently,
an unusually narrow conjugation bridge formed between
mating cells. Presumably this long and narrow bridge impedes the movement(s) of nuclei within the zygote. The
nuclear movements involved in migration and segregation
are dependent upon microtubules making contact with appropriate cortical sites; these sites may be less accessible in
fig2
zygotes (Palmer et al., 1992
; Farkasovsky and Kuntzel, 1995
). Taken together, these phenotypes of fig2
zygotes reveal a requirement for proper mating projection
shape (and thus fusion bridge shape) for the normal execution of later steps of mating, including nuclear migration and segregation.
Fig4p Belongs to a Gene Family Whose Members Possess Homology to Sac1p and Regulate Actin-dependent Processes
Like fig1 mutants, fig4
mutants are also impaired in
mating cell morphogenesis. Fig4p displays extensive sequence similarity to the yeast Sac1p. Mutations in SAC1
alter actin cytoskeletal and secretory pathway dynamics
and cause auxotrophy for inositol (Whitters et al., 1993
).
Loss of Sac1p function suppresses growth defects caused
by certain conditional actin mutations and bypasses secretory defects imposed by mutations in the SEC14 gene
(Cleves et al., 1989
). These effects in sac1
mutants are
thought to be the result of altered levels of phosphitidylinositol derivatives, which, in turn, may influence the activity of actin regulatory proteins responsive to inositol triphosphate (InsP3) (Whitters et al., 1993
; De Camilli et al., 1996
).
Thus, one potential role for Fig4p in pheromone-induced
morphogenesis is in the regulation of effector molecules of
the actin cytoskeleton. Such a role for Fig4p is supported
by the observation that the distribution of actin in mating
projection tips of fig4
cells is usually dispersed and less
intense relative to that in wild-type cells.
Although both proteins play roles in actin-dependent
processes, an important issue that remains is whether
Fig4p is performing Sac1p-related functions in mating cells
or carries out a novel function. At least some conservation
of biological activity exists between the two diverged proteins, since multiple copies of FIG4 are capable of suppressing some phenotypes of a sac1 mutant (Kearns et al.,
1997
; Gedvilaite, A., and V. Bankiatis, personal communication). Although Fig4p does not appear to be present in
vegetative cells, Sac1p is likely to still be present in fig4
cells during mating, and may, thus, functionally overlap
with Fig4p under these circumstances. We are currently
examining the mating properties of sac1
mutants and
fig4
sac1
mutants to address this issue.
Fig4p belongs to a growing family of proteins whose
members contain a Sac1p homology domain; based on sequence comparisons this family is composed of several distinct groups (this study; Majerus, 1996). Two Sac1p family
members, a human and a C. elegans protein, display the
highest identity to Fig4p and are likely to be orthologues.
Other mammalian members of the Sac1p family have been
found to localize to neuronal synapses, which are highly polarized structures (McPherson et al., 1996
). These proteins are thought to participate in the actin-dependent endocytosis of membranes from the nerve terminus (De
Camilli et al., 1996
). The activity performed by the Sac1p homology domain in any of these proteins remains undetermined. Further study of Fig4p and the family of Sac1p-
related proteins should yield important insights into the
functions of a large family of proteins that are conserved throughout eukaryotes and play key roles in cell polarity.
KAR5/FIG3 Encodes a Pheromone-regulated Protein Functioning in Nuclear Fusion
Mutations in KAR5/FIG3 display the strongest quantitative
mating defect of the four genes characterized in this study.
This defect was also distinct from those of fig1, fig2
,
and fig4
in that a large number of small and irregularly
shaped colonies were generated from the kar5
/fig3
× kar5
/fig3
bilateral matings. These small colonies contain unstable heterokaryons that arise though failures in
nuclear fusion. This observation is consistent with the finding that FIG3 corresponds to the KAR5 gene (Beh et al.,
1997). KAR5 was identified in a screen for bilateral mating mutants (Kurihara et al., 1994
).
The biochemical activity of Kar5p/Fig3p during nuclear
fusion is not known. Kar5p contains extended coiled-coil
regions, suggesting it either associates with itself or another protein to function in nuclear membrane fusion.
Since many structural proteins such as intermediate filaments contain large coiled-coil segments (Steinert and
Roop, 1988), Kar5p may play some structural role during
nuclear fusion. Kar5p might act in conjunction with the previously described KAR2/BiP gene product, which is also
involved in nuclear fusion and has mutant phenotypes
very similar to kar5 (Rose et al., 1989
). Like Kar2p/BiP, an
epitope-tagged Kar5p localizes in an endoplasmic reticulum-like pattern in yeast (Erdman, S., and M. Snyder, unpublished data). Unlike KAR5, KAR2/BiP is not induced
by pheromone (Rose et al., 1989
). The process of nuclear
membrane fusion, however, displays a requirement for
pheromone stimulation (Rose et al., 1986
). Kar5p may
thus represent the component of the process of nuclear
membrane fusion that confers its pheromone requirement.
Expression-based Screens and the Identification of Gene Function
Although many pheromone-induced genes were identified
in this screen, mating phenotypes were not observed for
most of the insertion mutant strains. A number of recently
described pheromone-regulated components incur only subtle defects upon loss of function (Konopka, 1993; Iida et al.,
1994
; Gammie et al., 1995
; Konopka et al., 1995
). Additionally, mutations in many of the genes characterized in
this study (e.g., FIG1, FIG2, and FIG4) caused relatively
mild quantitative mating defects while producing significant morphological defects. Thus, the failure to identify
requirements for the other genes tested in our study
through measurements of mating efficiency and halo assays of pheromone sensitivity may not be surprising. The
functions performed by many of these genes may also be
partially or completely overlapped by other genes within
the pheromone response pathway or in related parallel
pathways that function in mating-specific processes. Identification of such redundant genes by expression-based methods, nonetheless, provides a valuable first step in the
determination of the function of these genes. Further analysis of many of these types of genes will be necessary to
obtain a complete understanding of the components and
mechanisms that function in yeast mating.
![]() |
Footnotes |
---|
Received for publication 14 August 1997 and in revised form 14 November 1997.
S. Erdman was supported by an American Cancer Society Postdoctoral Fellowship. This research was supported by National Institutes of Health grants (HD32637 and GM36494).We are grateful to F. Cross for the MAT plasmid and B. Andrews for the
sst2 strains. We thank A. Gedviliate and V. Bankaitis; and C. Beh and M. Rose for communication of results before publication. The technical support of A. Sheehan and especially L. Umansky is gratefully acknowledged. B. Piekos provided expert assistance with electron microscopic
techniques. We thank Y. Barral, B. Manning, P. Ross-Macdonald, and S. Vidan for critical comments on the manuscript and members of the Snyder lab for useful discussions. S. Minehart prepared the in-frame QOR1::
-gal fusion.
![]() |
Note Added in Proof |
---|
The molecular characterization of KAR5 has recently been reported: Beh, C., V. Brizzio, and M.D. Rose. 1997. KAR5 encodes a novel pheromone-inducible protein required for homotypic nuclear fusion. J. Cell Biol. 139:1063-1076.
![]() |
Abbreviations used in this paper |
---|
-gal,
-galactosidase;
DAPI, 4
,6-diamidino-Z-phenylindole;
DIC, differential interference-contrast microscopy;
FIG, factor-induced gene;
MAP, mitogen-activated kinase;
ORF, open reading frame;
PEG, polyethylene glycol;
PRE, pheromone response element;
SC, synthetic complete medium;
TM, transmembrane;
TMD, transmembrane domain;
YPD, yeast extract/peptone/dextrose medium.
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